Seed Shattering in a North American Oryzeae grain: Developmental and Genomic Signatures of Early Domestication

Seed Shattering in a North American Oryzeae grain: Developmental and Genomic Signatures of Early Domestication | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Seed Shattering in a North American Oryzeae grain: Developmental and Genomic Signatures of Early Domestication Reneth Millas, Lillian McGilp, Alan Mickelson, Maybell Banting, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7032638/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Northern Wild Rice (NWR; Zizania palustris L.) is an aquatic grain endemic to North America and a member of the Oryzeae tribe. As an outcrossing crop with a short breeding history, domestication progress in cultivated NWR (cNWR) is ongoing and seed shattering remains a major barrier to yield stability. In this study, we investigated the developmental and genetic mechanisms underlying seed retention by integrating phenotypic, anatomical, and molecular analyses across wild and cultivated populations. Time-course phenotyping using four methods revealed a ~ 90% reduction and two-week delay in shattering in cNWR relative to wild populations. Histological analysis indicated anatomical reorganization of the abscission layer in cNWR, consistent with selection for seed retention. Comparative genomic analyses identified multiple NWR orthologs of key Oryza sativa shattering genes, revealing lineage-specific gene duplication, pseudogenization, and divergence from Z. latifolia . Expression profiling of candidate genes via RT-qPCR across floret developmental stages suggested a regulatory role for ZpSh5c , a putative OsSh5 ortholog, in modulating seed shattering. In cNWR, delayed and reduced expression of ZpSh5c mirrored observed differences in shattering timing, highlighting its potential involvement in abscission zone development. Together, these findings provide new insights into the anatomical and molecular basis of seed shattering in NWR and demonstrate the utility of comparative frameworks for accelerating trait improvement in emerging, non-model crops. Zizania palustris seed retention abscission layer Oryza sativa gene expression multiple sequence alignment domestication Figures Figure 1 Figure 2 Figure 3 PLAIN LANGUAGE SUMMARY Northern Wild Rice (NWR, Zizania palustris ) has been grown in irrigated paddies in the United States since the 1950s. Seed shattering, that is, when ripe seed separates from the plant pedicel, reduces grain yields in cultivated NWR (cNWR) production. However, little is known about this process. This study compared the seed shattering of two cNWR populations to a wild, unselected population. We found that cNWR seed retention increased ~90% compared to the time when the wild type shatters, and cNWR began shattering about two weeks later than the wild type. Imaging of a layer of cells that lead to shattering at the pedicel, where the seed and stem meet, also revealed differences between cNWR and the wild type. Being closely related to white rice, we leveraged available white rice genomic resources to identify potential NWR genes that may be associated with seed shattering. We found that the gene, ZpSh5c , is expressed in the same pattern as shattering occurs in both wild and cultivated plants, suggesting its possible role in the regulation of shattering in NWR. INTRODUCTION Seed shattering is a natural seed dispersal mechanism widespread among wild plant species, including the wild ancestors of many modern crops (Konishi et al., 2006 ; Li & Gill, 2006 ). As species were domesticated, traits that favored seed retention were selected, leading to the replacement of wild type alleles with mutations that reduced or eliminated shattering (Konishi et al., 2006 ; Lin et al., 2012 ). A key target of selection was a developmentally regulated abscission event involving the formation and breakdown of an abscission layer, a specialized cell layer, at the seed–pedicel junction (Oba et al., 1995 ; Fuller & Allaby, 2009 ; Watanabe et al., 2003 ). Disruption or loss of this abscission layer increases seed retention, significantly improving yields and harvest efficiency, which are critical milestones in crop domestication. Numerous genes and quantitative trait loci (QTL) underlying seed shattering have been identified in crop species, particularly in Oryza sativa L. and related species (Oba et al., 1995 ; Cai and Morishima, 2000 ; Thomson et al., 2003 ; Lin et al., 2007 ; Konishi et al., 2006 ; Ishikawa et al., 2010 ; Lee et al., 2016 ; Wu et al., 2017 ). Several of these have been cloned or fine-mapped, revealing their roles in regulating the development and function of the abscission layer. Notable examples include the sh4 gene, which encodes a MYB3 transcription factor, (Li et al., 2006 ; Purugganan & Fuller, 2009 ; Zhou et al., 2012 ); qSH1 , which encodes a BEL1-type homeobox gene (Konishi et al., 2006 ); Sh5 , a highly homologous gene of qSH1 (Yoon et al., 2014 ); SHAT1 , an APETALA2 (AP2) transcription factor (Zhou et al., 2012 ); and SH1 , a YABBY transcription factor (Lin et al., 2012 ). Together, these genes can modulate the development and degradation of the abscission zone and have been repeatedly targeted during the independent domestication of cereal crops. Across crops, studies have revealed that seed shattering pathways are generally conserved, with many genes either orthologous or functionally analogous across divergent lineages (Dong & Wang., 2015; Ishikawa et al., 2022 ). This pattern of evolutionary convergence reflects similar selection pressures during domestication that were used to increase seed retention (Doebley, 2006; Lenser and Theißen, 2013 ; Olsen & Wendel, 2013 ; Purugganan & Fuller, 2009 ; Tranbarger et al., 2017 ). Comparative genomic studies have helped to shape our understanding of these convergent evolutionary patterns and are increasingly applied to identify candidate domestication genes in wild and understudied crop species (Kahler et al., 2014 ; Fu et al., 2019 ; Liu et al., 2022 ). For example, recent studies, have reported that SH1 likely plays a role in seed shattering across a wide range of grass species, including Lolium perenne (Perennial ryegrass), Setaria italica (Foxtail millet), and Zizania latifolia (Manchurian wild rice) (Fu et al., 2019 ; Liu et al., 2022 ; Odonkor et al., 2018 ; Yu et al., 2023 ). By leveraging such comparative frameworks, researchers can identify conserved regulatory genes and causal variants for key traits, ultimately accelerating the improvement of emerging, non-model crops. One emerging crop with strong potential for comparative genomics is Northern Wild Rice (NWR, Zizania palustris L .), an annual aquatic grass and Crop Wild Relative (CWR) of O. sativa within the Oryzeae tribe. Endemic to the Great Lake Region of North America, NWR production in irrigated, diked paddies began in the region in the 1950s. In 1968, the first cultivated NWR (cNWR) variety was released, featuring improved seed retention compared to its wild counterparts (Oelke, 1982 ). Like Oryza species (Jin et al., 1993; Jin et al., 1982 , 1990 , 1995 ; Jin and Inouye, 1982a , b , 1985 ), seed shattering in NWR is associated with development of an abscission layer at the seed-pedicel junction composed of parenchyma and sclerenchyma cells, where variation in cell thickness and number contributes to genotype-specific differences (Hanten et al., 1980 ). Previous genetic studies have suggested that seed shattering in NWR is a recessive, quantitative trait regulated by at least two or three genes (Woods & Clark, 1976 ; Elliott & Perlinger, 1977 ; Kennard et al., 2002 ). Despite ongoing efforts, modern cNWR varieties and breeding germplasm still exhibit some degree of seed shattering, primarily due to the species’ outcrossing nature and limited breeding history (Elliot, 1980). The trait’s persistence contributes to substantial yield losses, particularly after late-summer storms or high winds (Imle, 2001 ). As such, NWR offers a valuable system for exploring the genetic basis of seed shattering and applying insights from domesticated relatives to accelerate trait improvement in a semi-domesticated species. Improving seed retention in cNWR requires a better understanding of the developmental, anatomical, and genetic factors that regulate seed shattering. Despite its agronomic importance, key aspects of this trait remain underexplored. Given the close phylogenetic relationship between O. sativa and NWR, comparative genomics offers a powerful framework for candidate gene discovery (Haas et al., 2021 ). Therefore, this study aimed to: (1) Assess the phenotypic variation in seed shattering using four quantitative phenotyping methods; (2) Examine the abscission layer anatomy of two cNWR populations and one wild NWR population using histological analysis; (3) Identify and compare the protein sequences of putative O. sativa orthologs in NWR and Z. latifolia ; and (4) Evaluate the expression profiles of putative NWR orthologs of O. sativa seed shattering-related genes across stages of panicle development and seed maturation. MATERIALS AND METHODS Plant materials. Three NWR populations were used in this study, including a wild type population exhibiting a profuse shattering habit, collected from Sullivan Lake, Minnesota (MN), USA in 2020 (46.149519 N; − 93.936795 W); a cNWR breeding line, ‘FY-C20’ developed for seed length; and a cNWR variety, ‘Itasca-C12’, released in 2007 (Porter et al., 2008 ), which served as the industry’s standard for seed retention at the time of this study. Seeds were removed from cool storage (3℃) (McGilp et al., 2023 ), rinsed three times, and placed in new plastic bags containing fresh water on a lab benchtop. After 10 days, germinated seedlings were transplanted in the greenhouse. Plant growth conditions. Experiments were conducted at the University of Minnesota (UMN) Plant Growth Facilities in Saint Paul, MN, in the spring of 2021. Greenhouse conditions were maintained at 22°C (± 2°C) with a 16-hour photoperiod. Cone-tainers (112.98 cm 2 ) were filled with a steamed soil mix supplemented with 0.14 g urea (20 lb/acre) and 0.14 g of iron chelate (1 lb/ 1000 ft 2 ), then placed in aluminum aquaponic tanks (66 cm x 183 cm x 70 cm). Tanks were filled with water (13℃) up to 2–4 cm above the surface of the cone-tainers one week before seedlings were planted. To limit algae growth, water was circulated under 5 psi using a 1056GPH 276W submersible pump (Simple Deluxe, Duarte, California, USA) and treated twice weekly with Algaefix (1 ml/10 gallons of water; API, Chalfont, Pennsylvania, USA). After germination, one seedling was transplanted per cone-tainer. Plants were top-dressed with urea (20lb/acre) after tillering every 2–3 weeks. Seed shattering phenotypic data collection. The degree of seed shatter was measured on the main stem panicles of three individual plants per population per time point over the course of NWR seed ripening at the Principal Phenological Stage (PPS) 8 (Duquette & Kimball, 2020 ) using four different methods. Individual plants were collected at their respective days after anthesis (DAA), including 21, 28, 35, and 42 DAA. The first method was a visual estimation of the percentage of shattered seed at the time of data collection, ranging from 0 to 100%, where 0 = no shatter and 100% = complete shatter. The second method was a drop test, whereby a panicle was dropped three times from a height of 60.96 cm, and the proportion of shattered seeds was calculated relative to the total number of seeds per panicle (Altendorf et al., 2021 ). The third and fourth methods measured breaking tensile strength (BTS) using a Chatillon digital force gauge (Ametek, Inc, Berwyn, Pennsylvania), either by pulling the seed from the pedicel (BTS-P) or bending (BTS-B). For BTS-P, the seed was gently gripped with forceps attached to the gauge, and force was applied in a straight, downward direction until detachment occurred. The maximum force required to remove the seed was recorded in grams-force (g), a unit of force where 1g ≈ 0.0098 Newtons. For BTS-B, force was applied laterally to the seed at the point of attachment and gently bent until detachment occurred. The force required to break the seed from the pedicel was also recorded in g. Histology of the seed-pedicel junction . Seed-pedicel junctions of five plants per population were sampled before reaching the hard dough stage of seed ripening (PPS 85), which corresponded to 21 DAA for the wild type population’s individuals and 28 DAA for the cultivated line’s individuals. Seeds were bisected, placed in a vacuum chamber, and fixed in a mixture of formalin-acetic acid-alcohol (FAA; 53:5:10:32) solution containing 0.1% Triton X-100. Fixed samples were stored in 70% ethanol at 4°C before being processed at the Histology and Research Laboratory, UMN. Upon arrival, the samples were fixed in 2% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.0), dehydrated in an incremental ethanol and ethanol-xylene series (3:1, 1:1, 1:3, 100% xylene), and embedded in Paraplast Plus (Leica Biosystems, Wetzlar, Germany). Sections (10 µm) were cut using a rotary microtome, stained with 0.05% toluidine blue O in 0.1 M phosphate buffer (pH 6.8), and imaged using an Axio Scan.Z1 slide scanner (Zeiss Group, Oberkochen, Baden-Württemberg, Germany). Plant tissue collection . Three plant tissues were collected from three individual plants per population in liquid nitrogen as independent biological replicates. The sampled tissues included flag leaf at the heading stage (PPS 51); male florets at 7 DAA at PPS 66 when all flowering was complete and before pollen shed (PPS 69); and female florets at 1, 7, 18, 28, and 35 DAA throughout seed development (PPS 7) and maturation (PPS 8). In the case of the wild type population, female florets were collected only up to 18 DAA, as most seeds had shattered by the next time point. Tissue samples were stored at -80°C until further use. RNA isolation and cDNA synthesis. Total RNA was extracted using a RNeasy® Plant Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. RNA concentration and purity were measured using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). To eliminate DNA contamination, RNA samples were treated with the DNA-free™ DNA Removal Kit (Thermo Fisher Scientific, Waltham, MA). Complementary DNA (cDNA) was synthesized from total RNA using the SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen, Thermo Fisher Scientific, Waltham, MA), following the manufacturer’s protocol. Synthesized cDNA was stored at − 20°C until further use. Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays . Gene expression quantification was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA) on 96-well plates. Each 20 µL reaction contained 50 ng of cDNA template, 10 µL of iTaq™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA), and 0.2 µL (10 mM) each of forward and reverse primers (Supplemental Table 1) using standard thermal cycling conditions (95°C for 20 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s). Each gene was evaluated using three biological replicates and two technical replicates per biological replicate, tissue type, and time point. Negative controls of no-template and water as template were used in all runs. Selection and evaluation of candidate reference genes for RT-qPCR assays. To identify suitable internal reference genes for RT-qPCR normalization, five NWR genes were selected as candidates based on their stable expression in other plant species (Kozera & Rapacz, 2013 ; Joseph et al., 2018 ) and across eight NWR tissue types (Haas et al., 2021 ). These included putative homologs of actin 1 (ACT1), actin 4 (ACT4), eukaryotic translation initiation factor 5-like (EIF-5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBI). Primers were designed using PrimerQuest (Integrated DNA Technologies; Coralville, Iowa, USA) based on exon sequences extracted from the NWR reference genome v1.0 (Haas et al., 2021 ) using in-house R script. The amplification efficiency and expression stability of each primer set were assessed in leaf, male floret, and female floret tissues across all time points. Standard curves were generated using a 5-fold serial dilution of cDNA (200, 40, 8, 1.6, and 0.32 ng/µL). Sterile DNase- and RNase-free water was used as a control in all reactions. Evaluation and selection of putative seed shattering orthologs . Protein sequences of well-characterized O. sativa seed shattering genes, SH1 , qSH1 , sh4 , Sh5 , and SHAT1 , were retrieved from the O. sativa ssp. japonica reference genome (GenBank accession: GCF_034140825.1). Putative orthologs identified in Z. latifolia in Yan et al. 2022 were obtained from the Chinese wild rice ‘Jaiobai’ genome (GCA_0483537475.1; Guo et al., 2015 ). These O. sativa and Z. latifolia protein sequences were used as queries in BLASTp searches against the Z. palustris ‘Itasca-C12’ reference genome assembly v1.0 (GCA_019279435.1; Haas et al., 2021 ) using the NCBI BLASTp tool ( www.ncbi.nlm.nih.gov/ ). Candidate orthologs in NWR were identified based on percent protein identity with a cut-off rate of 70%. Multiple sequence alignments (MSA) were performed using CLUSTALW v2.0 (Larkin et al., 2007 ) and a maximum likelihood (ML) phylogenetic tree with 100 bootstraps was calculated with the randomized accelerated ML (RAxML v8.2.11; Stamatakis, 2014 ) software. The resulting alignments and phylogram were visualized with ETE3 3.1.2 (Huerta-Cepas et al., 2016 ) via GenomeNET ( https://genome.ip/tools/ete/ ) , using default parameters. Protein sequence alignments for qSh1 and Sh5 orthologs were also conducted using NCBI’s COBALT (Constraint-based Multiple Alignment Tool) with default parameters to visualize sequence divergence among homologs. Orthogroups were inferred using OrthoFinder version 2.5.4 (Emms & Kelly, 2015 ) and protein domain annotation was performed using Pfam version 37.4 (Finn et al., 2014 ) via the InterProScan web server ( https://www.ebi.ac.uk/interpro/ ) to identify conserved domains in the predicted protein sequences. Based on these analyses, gene-specific primers (Supplemental Table 2) were designed for selected orthologs of SH1 and Sh5 for downstream RT-qPCR validation of gene expression, following the experimental protocol described above. Data analysis. To assess variation in seed shattering, an analysis of variance (ANOVA) was conducted in R version 4.3.1 (R Core Team, 2021) for each of the four phenotyping methods, across four developmental timepoint. Genotype and timepoint were both considered fixed effects. A statistical significance threshold of 0.05 was used in all analyses. Tukey’s Honestly Significant Difference (HSD) tests were performed using the Agricolae package version 1.3.7 (de Mendiburu, 2021 ) to identify pairwise differences among groups. To compare phenotyping methods, Pearson’s correlation coefficients were calculated with the Hmisc package version 5.2.2 (Harrell, 2023 ). For reference gene selection, primer efficiency was assessed for each candidate reference gene primer pair by constructing linear regression curves based on the logarithm of the cDNA and the threshold curve cycle (C T ). Primer efficiency (E) was calculated from the slope (S) of the standard curves generated from serial dilutions, using the equation E= [10 (1/−S)− 1] × 100% (Rutledge & Cote, 2003). The coefficients of determination (R 2 ) were also computed. Expression stability across tissue types and timepoints were evaluated using RefFinder (Xie et al., 2023 ), which also utilizes geNorm (Vandesompele et al., 2002 ; Hellemans et al., 2008 ) and NormFinder (Andersen et al., 2004 ). The best candidate reference genes with amplification efficiencies within the optimal range (100% ± 10%) and minimal variation in expression were selected for assessing gene expression of candidate shattering genes. Gene expression levels of seed shattering-related genes in female and male tissues were quantified using the 2 −∆∆CT method (Livak & Schmittgen, 2001 ). For each sample, threshold cycle (C T ) values of the target gene were first normalized to the selected reference gene to obtain CT values. Relative expression was then calculated by comparing normalized CT values of each tissue sample to those of the same genes expressed in flag leaf tissue, which was used as the baseline calibrator. The data was transformed using log 2 and presented as relative gene expression. This approach enabled quantification of fold changes in gene expression relative to a consistent non-reproductive tissue control. To evaluate differential expression between the wild type population and the cNWR populations, ANOVA and Tukey’s HSD were also performed. RESULTS Seed shattering variability in Northern Wild Rice populations To evaluate genetic and phenotypic variation in seed shattering, we quantified this trait in three Z. palustris populations using four phenotyping methods at 21, 28, 35, and 42 days after anthesis (DAA). These methods included visual ratings, a drop test, and two biomechanical tension force methods (BTS-P and BTS-B). Statistical analyses (ANOVA, Tukey’s HSD; Table 1 – 2 ) revealed visual ratings and the drop test results were significantly influenced by both genotypes and DAA, while BTS-P had significant variation by genotype and BTS-B had no statistical differences (Table 1 ). In general, visual and drop test scores showed increasing seed shattering over time, while BTS-B and BTS-P values, reflecting resistance to detachment, decreased (Table 2 ). Importantly, differences in the shattering phenotype were not due to variation in developmental timing, as all populations reached comparable phenological stages within one to three days of each other (Figure S1). Table 1 Analysis of Variance (ANOVA) of four phenotyping methods to characterize seed shattering in Northern Wild Rice (NWR; Zizania palustris ) post-anthesis. Method Source of Variation Df Sums of Squares Mean Squares F-value p- value Visual DAA 3 635.42 211.81 5.45 < 0.01 Genotype 2 69,705.56 34,852.78 896.21 < 0.0001 DAA:Genotype 6 266.67 44.44 1.14 0.37 Residuals 24 933.33 38.89 Drop Test DAA 3 2,527.16 842.39 16.01 < 0.0001 Genotype 2 36,198.54 18,099.27 343.97 < 0.0001 DAA:Genotype 4 189.28 47.32 0.90 0.48 Residuals 20 1,052.39 52.62 BTS-P DAA 2 18,193.62 9,096.81 3.50 0.06 Genotype 2 33,073.60 16,536.80 6.36 < 0.01 DAA:Genotype 2 5,465.79 2,732.90 1.05 0.38 Residuals 14 36,419.62 2,601.40 BTS-B DAA 2 0.00 0.00 0.70 0.51 Genotype 2 0.00 0.00 2.05 0.17 DAA:Genotype 2 0.00 0.00 1.10 0.36 Residuals 14 0.00 0.00 Legend. BTS-P: Breaking tensile strength by pulling; BTS-B: breaking tensile strength by bending; DAA: days after anthesis; Df: degrees of freedom Table 2 Tukey’s mean separations analysis for four phenotyping methods to characterize seed shattering in Northern Wild Rice (NWR; Zizania palustris) over time and populations. Visual (%) Drop Test (%) BTS_Pulling (g) BTS_Bending (g) Genotype / DAA 21 28 35 42 21 28 35 42 21 28 35 42 21 28 35 42 Itasca-C12 0 b 0 b 0 b 8.33 b 0 d 0 d 6.25 cd 23.23 bc NA 206.78 a 168.67 ab 142.93 ab NA 0.014 a 0.023 a 0.01 a FY-C20 0 b 0 b 1.67 b 13.33 b 0 d 0 d 10.42 cd 35.27 b NA 185.67 ab 139.67 ab 44.20 b NA 0.027 a 0.014 a 0.005 a Wild type 86.67 a 98.33 a 100 a 100 a 91.67 a 98.33 a 100 a 100 a NA 91.33 ab NA NA NA 0 a NA NA Legend: Days after anthesis, DAA; not applicable, NA; grams, g; Same letters indicate no statistically significant difference (P = 0.05) Methods: A visual rating, 0-100% shattering; a drop test, 0-100% shattering; breaking tensile strength by pulling, BTS-P, and breaking tensile strength by bending (BTS_B) were measured in force (g) For visual ratings, the wild type population consistently exhibited greater seed shattering than both cNWR populations, with no significant differences found between Itasca-C12 and FY-C20. Shattering in the wild type increased from 86.67% at 21 DAA to 100% by 35 DAA (Table 2 ). FY-C20 began shattering at 35 DAA, reaching 13.3% by 42 DAA, while Itasca-C12 showed delayed onset, with 8.3% shattering by 42 DAA (Table 2 ). The drop test also effectively differentiated the wild type from both cultivated populations across all time points (Table 1 ; Table 2 ). The visual and drop test methods were highly correlated with one another (r = 0.99, p > 0.001) across all time points (Supplementary Table 3). For BTS-P, only one time point could be assessed for the wild type, which yielded 91.3 g of force at 28 DAA, compared to 185.7 g for FY-C20 and 206.8 g for Itasca-C12 (Table 2 ). By 42 DAA, Itasca-C12 required greater force to detach seeds than FY-C20 (144 g vs. 44 g; Table 2 ). Although BTS-P differences among populations were not statistically significant, Itasca-C12 exhibited higher BTS-P values compared to FY-C20 across all time points. BTS-P was negatively and significantly correlated with both visual and drop test results across time points, except with the visual method at 35 DAA (Supplementary Table 3). BTS-B displayed non-significant negative correlations with the visual and drop test methods across timepoints. BTS-B was also non-significantly positively correlated with BTS-P results across timepoints, with one significant correlation between BTS-B and BTS-P at 35 DAA (Supplementary Table 3). Histological analysis of the seed-pedicel abscission layer. We examined the anatomical structure of the abscission layer in a wild type population, characterized by high levels of seed shattering, and two cultivated populations, Itasca-C12 at 28 DAA and FY-C20 at 21 DAA, which exhibit reduced seed shattering. All three populations developed a distinct abscission layer at the seed-pedicel junction (Fig. 1 ). In the wild type population (Fig. 1 b), the abscission layer was well-defined, consisting of densely packed and structurally organized parenchyma cells. In contrast, the cultivated populations appear to have more loosely arranged cells and larger intercellular spaces disrupting the continuity of the layer. Among the cultivated populations, FY-C20 showed a more compact and organized abscission layer than Itasca-C12, which had a more diffuse structure and well-defined vascular bundle (Fig. 1 c and 1 d, respectively). Putative relationships of seed shattering-related genes in Oryzeae. Protein sequences of five O. sativa genes involved in seed abscission layer development, SH1 ( OsSH1 ), qSH1 ( OsqSH1 ), Sh5 ( OsSh5 ), sh4 ( Ossh4 ), and SHAT1 ( OsSHAT1 ), were queried against the Z. palustris genome in NCBI GenBank using the BlastP tool (Supplementary Table 4). Multiple orthologs were identified in NWR, consistent with a whole-genome duplication (WGD) event in the Zizania lineage following its divergence from Oryza approximately 26 to 30 million years ago (Guo et al., 2015 ; Haas et al., 2021 ; Yan et al., 2022 ). Previously reported candidate orthologs (Haas et al., 2021 ) were confirmed as tops hits based on percent sequence similarity and conserved domain structure, except for OsSHAT1 , where ZPchr0004g38673 ( ZpSHAT1a ) and ZPchr0458g2256 ( ZpSHAT1b ) were stronger candidates than the previously reported ZPchr0013g34051 (Haas et al., 2021 ). Additionally, ZPchr0458g22823 ( Zpsh4c ) was identified as a novel candidate of Ossh4 , based on high protein sequence similarity to ZpSh4a, ZpSh4b , and Ossh4 (Table 3 ). A percent protein identity (PI) matrix was generated to evaluate the sequence similarity among O. sativa , Z. palustris , and Z. latifolia seed shattering potential orthologs (Table 3 ). The highest PI between O. sativa and Z. palustris was observed for SH1 (89.34%), while the lowest PI was for Sh5a (71.23%; Table 3 ). The PI comparisons between NWR and Z. latifolia ranged from 70.00% for Sh5a to 98.84% for SH1 . Table 3 Percent protein identity (PI) of seed shattering-related genes in Oryza sativa ( Os ) and putative Zizania palustris ( Zp ) and Zizania latifolia ( Zl ) orthologs. O. sativa (Os) Z. palustris (Zp) Z. latifolia (Zl) Percent Identify Comparisons between Species Gene description Gene GenBank ID Candidate Gene GenBank ID Candidate genes GenBank IDs (a and b, respectively) Zp vs. Os Zp vs. Zl a/b Os vs. Zl a/b OsSH1 NP_001389121.1 ZpSH1 ZPchr0003g18426 ZlSH1a / ZlSH1b ABZP36_005173 / ABZP36_018673 89.84 91.98 / 98.84 91.94 / 91.91 YABBY transcription factor (Lin et al., 2012 ) OsSh5 XP_015637721.1 ZpSh5a ZPchr0001g31104 ZlSh5a ABZP36_013876 71.23 78.85 / 70.00 89.56 / 83.92 Bel1-type homeobox (Yoon et al., 2014 ) ZpSh5b ZPchr0005g15825 84.07 90.05 / 95.11 ZpSh5c ZPchr0010g10516 ZlSh5b ABZP36_030161 87.5 95.64 / 89.25 ZpSh5d ZPchr0010g7757 85.51 95.64 / 89.25 OsqSH1 XP_015641948.1 ZpqSH1a ZPchr0001g31318 ZlqSH1a ABZP36_025045 83.42 85.44 / 91.43 85.66 Bel1-type homeobox (Konishi et al., 2006 ) ZpqSH1b ZPchr0007g5872 ZlqSH1b ABZP36_016879 83.74 90.86 / 83.28 85.07 OsSHAT1 XP_015636848.1 ZpSHAT1a ZPchr0004g38673 ZlSHAT1a ABZP36_003055 83.65 90.32 / 88.45 85.74 AP2 transcription factor (Zhou et al., 2012 ) ZpSHAT1b ZPchr0458g22566 ZlSHAT1b ABZP36_007933 84.69 90.30 / 96.67 84.02 Ossh4 XP_015635337.1 Zpsh4a ZPchr0004g38781 Zlsh4a / Zlsh4b ABZP36_008339 / ABZP36_004261 80 78.05 / 80.18 76.47 / 80.12 Trihelix transcription factor (Li et al., 2006 ) Zpsh4b ZPchr0458g22499 79.17 87.41 / 92.78 Zpsh4c ZPchr0458g22823 80.17 86.36 / 84.21 Legend: Zl a and b genes identified in Yan et al., 2022 ; Zl a and b genes GenBank ID in the ‘Jaiobai’ genome (GCA_0483537475.1; Guo et al., 2015 ); See Supplementary Table 4 for protein sequences A cluster analysis of O. sativa shattering genes and putative Z. palustris and Z. latifolia orthologs identified two main clusters in the cladogram, each with moderate bootstrap support of 75%. The first main cluster consisted of two subclusters separating SH1 (BS = 98%) from qSH1 and SH5 (BS = 75%). The second cluster (BS = 75%) consisted of sh4 and SHAT1 subclusters, supported by BS values of 92% and 93%, respectively (Fig. 2 ). Within the SH1 subcluster, OsSH1 formed a distinct branch from Zizania sequences (BS = 98%), while the ZlSH1b sequence was the closest ortholog to ZpSH1 (BS = 65%; 98.84% PI) (Fig. 2 ; Table 3 ). In the qSH1 subcluster (BS = 75%), ZpqSH1a and ZlqSH1b grouped together (BS = 98%; 91.43% PI). OsqSH1 , albeit with a weak bootstrap (52%), grouped more closely with ZpqSH1b and ZlqSH1a , who were 90.86% identical and in a clade with a BS of 98%. Within the sh5 subcluster (BS = 66%), OsSh5 separated from Zizania genes, which grouped with a BS of 82%. ZpSH5b clustered closely with ZlSH5b (BS = 100%; 95.11% PI), while ZpSh5a , ZpSh5c , and ZpSh5d clustered with ZlSh5a (BS = 91%; and PI ranging from 78.85 to 95.64%), although ZlSh5a formed an independent branch separated from the ZpSH5 orthologs (BS = 71%). The variation in PI among the ZpSh5 orthologs and across species was largely attributed to ZpSh5a . Within the second main cluster, SHAT1 subcluster was well supported (BS = 93%), integrated by low supported groups (BS = 39%) containing OsSHAT1 , ZlSHATb , and ZpSHAT1b , and another (BS = 58%; 90.32% PI) of ZlSHAT1a and ZpSHAT1a (Fig. 2 ; Table 3 ). The sh4 subcluster (92% BS) exhibited a stepwise branching pattern, starting with Zlsh4b , followed by Zpsh4a (BS = 13%), Ossh4 (BS = 37%), and Zlsh4a (BS = 92%), with Zpsh4b and Zpsh4c forming a well-supported terminal cluster (BS = 99% BS). Among the five genes evaluated, Zizania orthologs of Ossh4 showed some of the most divergent protein sequences, with PI percentages ranging from 76.47–80.17% (Table 3 ). Orthogroup and Pfam analyses provided additional support for several conserved shattering-related orthologs (Supplementary Table 5 and Supplementary Table 6). ZpSH1 was identified as an ortholog of OsSH1 and assigned to orthogroup OG0010120, with similar sequences in Z. latifolia (Supplementary Table 5). All orthologs contained a YABBY protein domain (Supplementary Table 6). For Sh5 , OrthoFinder analysis indicated that ZpSh5b was the strongest orthogonal candidate for OsSh5 within orthogroup OG0013611, which also contained ZlSh5a and ZlSh5b (Supplementary Table 5). Two closely related sequences ZpSh5c and ZpSh5d grouped within orthogroup OGOO26538, with no clear orthologs in O. sativa or Z. latifolia . ZpSh5a could not be assigned to an orthogroup. Pfam analysis found that all Sh5 orthologs contain a homeobox domain and a POX domain, except for ZpSh5a (Supplementary Table 6). MSA revealed that ZpSh5a exhibited a truncated protein sequence compared to those of other orthologs (Supplementary Fig. 2). The alignment of sequences also revealed that OsSh5 and all ZpSh5 genes had 100% protein identities for their homeobox domains but differed in their POX domains, within which OsSh5 differed from ZpSh5b and ZpSh5c by four and eight amino acids, respectively (Supplementary Table 7). Comparison of OsqSH1 identified ZpqSH1b as a likely ortholog and it was placed in orthogroup OG0008858, along with ZlqSH1a and ZlqSH1b (Supplementary Table 5), while ZpqSH1a was not assigned an orthogroup. However, all Z. palustris orthologs in this group, including ZpqSH1a , have homeobox and POX domains (Supplementary Table 6). For OsSHAT1 , ZpSHAT1b was the strongest orthogonal candidate and grouped in orthogroup OG0003844 with ZlSHAT1a and ZlSHAT1b (Supplementary Table 5), while ZpSHAT1a was not assigned to an orthogroup. Pfam searches indicated that all SHAT1 ortholog sequences contained two tandem AP2/ERF domains (Supplementary Table 6). Lastly, Ossh4 along with Zpsh4a, Zpsh4b, and Zpsh4c genes were grouped within the orthogroup OG0004851 (Supplementary Table 5). All Z. palustris ortholog sequences for sh4 contained a Myb/SANT-like DNA-binding domain 4 (Supplementary Table 6). Reference gene selection for RT-qPCR experiments. Five commonly used reference genes in plant gene expression studies, ACT1, ACT4, EIF-5, UBI, and GAPDH, were evaluated across leaf, female floret, and male floret tissues to identify those with the most stable expression for normalizing RT-qPCR expression data (Supplementary Table 1). Primer efficiencies ranged from 106–113% with R 2 values between 0.997 and 0.998 (Table 4 ). Stability rankings using RefFinder consistently identified ACT1 as the most stable and ACT4 as the least stable expressed gene (Table 4 ). Based on these results, ACT1 was selected as the internal reference gene for downstream expression analyses. Table 4 Primer efficiency and stability of reference gene candidates for gene expression analyses in male and female floret tissues of Northern Wild Rice (NWR; Zizania palustris ) based on RefFinder, which includes geNorm and NormFinder analyses. Gene Z. palustris gene Chr. Position (bp) Description R2 %E RefFinder geNorm NormFinder ACT1 ZPchr0012g19383 11 23,232,269 Actin 1 0.998 110 1.19 0.89 0.94 ACT4 ZPchr0010g7718 10 55,029,365 actin-related protein 4 0.998 113 5 4.9 8.9 EIF-5 ZPchr0006g44672 6 52,111,321 eukaryotic translation initiation factor 5-like 0.997 107 1.41 0.89 1.47 GADPH ZPchr0008g11968 8 9,259,023 glyceraldehyde-3-phosphate dehydrogenase 0.997 106 3 1.31 1.74 UBIQ ZPchr0007g6160 7 6,416,073 ubiquitin-conjugating enzyme E2-17 kDa isoform X1 0.998 106 4 2.14 2.13 Legend: primer efficiency, %E; RefFinder (Xie et al., 2023 ); geNorm (Vandesompele et al., 2002 ; Hellemans et al., 2008 ); NormFinder (Andersen et al., 2004 ); lower values indicate higher stability and rank Expression of SH1 and Sh5 orthologs in Z. palustris female florets. We analyzed the relative expression of NWR candidate orthologs of OsSH1 and OsSh5 , ZpSH1 and ZpSh5a, ZpSh5b , and ZpSh5c in NWR female floret tissues (including the seed-pedicel junction) across a time course following anthesis (Supplementary Table 2). ZpSH1 was selected due to its high expression in female panicle tissue (Haas et al., 2021 ) and its conserved role in seed shattering across grass species (Maity et al., 2021). Among four ZpSh5 putative paralogs, ZpSh5a , ZpSh5b , and ZpSh5c were included based on their relatively high expression levels (Haas, et al., 2021 ) and to explore their potential roles in the regulatory pathways of seed shattering, considering their putative diversification following the WGD. ZpSh5d was omitted from RT-qPCR assays due to its low expression in 8 NWR tissues (Haas et al., 2021 ). To characterize candidate gene expression, female florets were collected at 1, 7, 18, 28, and 35 DAA for RT-qPCR analysis. Data from the wild type population were unavailable at 28 and 35 DAA due to near-complete seed shattering (98%) by 28 DAA (Table 2 ). The RT-qPCR gene expression time course profiles of female florets are summarized across genes in Fig. 3 and by population in Supplementary Fig. 3. ZpSH1 was upregulated compared to leaf tissue, except for FY-C20 at 35 DAA (Fig. 3 a). Across DAA, the expression of ZpSH1 in the wild type was high on 1 DAA, peaked at 7 DAA, and declined at the last timepoint of the population’s collection, 18 DAA. FY-C20’s expression was lower than the wild type from 1 to 18 DAA but with peaks at 7 and at 28 DAA and was slightly downregulated by 35 DAA. Itasca-C12 had higher expression than the wild type from 1 to 18 DAA, peaking at 1 DAA and then declining through the rest of the timepoints. For ZpSh5a , expression in female floret tissues was downregulated at 1 and 7 DAA for all populations compared to leaf tissue (Fig. 3 b). Expression of the wild type was slightly upregulated at 18 DAA, its final timepoint. The expression of ZpSh5a in FY-C20 after 7 DAA was upregulated with a peak at 28 DAA, and undetectable at 35 DAA. In Itasca-C12, the gene was downregulated from 1 DAA through 18 DAA, undetectable at 28 DAA, and upregulated at 35 DAA. The expression of ZpSh5b in female floret tissue was downregulated compared with leaf tissue for all populations and timepoints except for the wild type at 1 DAA (Fig. 3 c). The expression of ZpSh5c in female floret tissue was upregulated for all populations and timepoints compared with leaf tissue (Fig. 3 d). The expression in the wild type was highest at 1 DAA, then declined through 18 DAA. Expression in FY-C20 and Itasca-C12 was higher at 1 DAA than 7 and 18 DAA, after which expression peaked at 28 DAA for both populations and then declined at 35 DAA (Fig. 3 d). However, the expression of FY-C20 throughout the time course was higher relative to Itasca-C12. ANOVA conducted on a per-gene basis revealed that genotype was not significant for any of the genes (Table 5 ). Expression of ZpSH1 and ZpSh5a was influenced by DAA; ZpSH1 expression at 7 DAA was different from 35 DAA and ZpSh5c expression at 1 and 7 DAA was different from 28 DAA (Supplementary Table 8). No significant differences between populations or time points were observed for candidate genes ZpSh5b and ZpSh5c . ANOVA conducted on a per population basis revealed significant differences between gene expression for all populations, where ZpSH1 and ZpSh5c had higher expression than ZpSh5a and ZpSh5b (Table 5 ; Supplementary Table 8). For FY-C20, there were also significant differences between timepoints; 28 DAA was significantly different from 1 DAA and 35 DAA. For Itasca-C12, the interaction between gene and DAA was significant. Overall, ZpSH1 had higher expression, with 1 and 7 DAA being significantly higher than ZpSh5b at 7, 18, and 28 DAA and also, ZpSh5a at 1 and 7 DAA. The expression for all timepoints of ZpSh5c was intermediate and did not differ significantly from any other genes (Supplementary Table 8). Table 5 Analysis of Variance (ANOVA) analyses of the relative gene expression of four Northern Wild Rice (NWR; Zizania palustris ) candidate orthologs of Oryza sativa genes related to seed shattering in three NWR populations. Gene Source of Variation Df Sum of Squares Mean Squares F-value P-value ZpSH1 DAA 4 83.44 20.86 2.87 0.044 Genotype 2 12.93 6.46 0.89 0.424 Replication 1 10.69 10.69 1.47 0.237 DAA:Genotype 6 23.95 3.99 0.55 0.766 Residuals 25 181.9 7.28 ZpSh5a DAA 4 94 23.5 6.66 0.001 Genotype 2 23.35 11.68 3.31 0.054 Replication 1 4.13 4.13 1.17 0.29 DAA:Genotype 6 28.58 4.76 1.35 0.275 Residuals 24 84.73 3.53 ZpSh5b DAA 4 6.26 1.56 0.44 0.777 Genotype 2 6.58 3.29 0.93 0.415 Replication 1 1.02 1.02 0.29 0.6 DAA:Genotype 5 20.97 4.19 1.18 0.36 Residuals 16 56.69 3.54 ZpSh5c DAA 4 8.35 2.09 0.4 0.806 Genotype 2 16.89 8.44 1.62 0.221 Replication 1 20.25 20.25 3.89 0.062 DAA:Genotype 6 22.38 3.73 0.72 0.641 Residuals 21 109.36 5.21 Genotype Source of Variation Df Sum of Squares Mean Squares F-value P-value Wild type DAA 2 18.91 9.46 2.35 0.118 Genes 3 201.23 67.08 16.66 < 0.001 Rep 1 0.09 0.09 0.02 0.882 DAA:Genes 6 32 5.33 1.32 0.286 Residuals 23 92.61 4.03 FY-C20 DAA 4 85.9 21.48 3.51 0.018 Genes 3 121.08 40.36 6.59 0.001 Rep 1 19.06 19.06 3.11 0.088 DAA:Genes 11 36.53 3.32 0.54 0.859 Residuals 31 189.89 6.13 Itasca-C12 DAA 4 4.99 1.25 0.27 0.892 Genes 3 245.14 81.71 18 < 0.001 Rep 1 26.69 26.69 5.88 0.021 DAA:Genes 12 117.75 9.81 2.16 0.04 Residuals 33 149.82 4.54 *Degrees of freedom, df; days after anthesis, DAA Expression of SH1 and Sh5 orthologs in male florets. To investigate potential overlap in the regulatory mechanisms between male and female floret shattering, we analyzed expression in male florets at 7 DAA. Similar to the expression patterns in female florets, ZpSh1 and ZpSh5c were upregulated in all populations (Fig. 3 ). Expression was highest in Itasca-C12 for ZpSH1 and in the wild type for ZpSh5c . At 7 DAA, the expression of ZpSh5a in male florets was downregulated for all populations, consistent with the early DAA in female florets. For ZpSh5b , wild type male florets were upregulated while the cultivated populations were downregulated, consistent with the expression patterns of female florets at 1 DAA (Fig. 3 ). When averaged across genes and populations the male floret expression at 7 DAA was significantly correlated with 1 (0.82), 7 (0.80), and 18 (0.38) DAA in the female floret tissue (Supplementary Table 9). DISCUSSION Selection-driven changes in cultivated Northern Wild Rice seed retention Seed shattering is a key adaptive trait in wild grasses, enabling seed dispersal, but it presents a major obstacle in grain domestication. In cNWR, strong selection pressure for seed retention has modified the timing and structure of seed abscission, despite a short domestication history. Our results show that cultivated populations exhibited a 90% reduction and 2-week delay in shattering relative to the wild type (Table 2 ), possibly due to alteration of the abscission zone. Histological observations support this, where cultivated populations displayed less organized and compact abscission layers, consistent with delayed or disrupted cell separation processes (Fig. 1 ). These changes parallel those seen in domesticated Oryza species (Jin and Inouye, 1981 ; Oba et al., 1995 ; Ji et al., 2006 ; Lin et al., 2007 ; Thurber et al., 2013 ; Li et al., 2024 ) and suggest that similar cellular mechanisms are being targeted in NWR. The observed changes in tensile strength and shattering progression, particularly in the higher seed-retaining Itasca-C12 (Table 2 ), suggest population-specific modifications to abscission layer timing or robustness, reflecting the ongoing selection for this trait. While this study identified a potential relationship between seed shattering and the development of cNWR’s abscission layer, further investigation into the timing of abscission layer development and degradation is needed, as histological assessments were limited to a single time point per population. Gene duplication and divergence underlie shattering variation Gene duplication and divergence are central to trait evolution following WGD events. Our comparative genomic analyses revealed that NWR harbors multiple orthologs, similar to Z. latifolia (Yan et al., 2022 ), of known O. sativa seed shattering genes, including sh4 , SHAT1 , qSH1 , and Sh5 . This redundancy, arising from a WGD and subsequent gene duplications in NWR (Haas et al., 2021 ), suggests that regulatory divergence rather than gene presence/absence may drive variation in NWR seed shattering. For example, SHAT1 orthologs showed evidence of divergence in NWR. ZpSHAT1b clustered with known functional orthologs, while ZpSHAT1a did not cluster with any known orthogroup, possibly reflecting neofunctionalization or regulatory repurposing (Wang et al., 2012 ). The exception was a single functional ortholog of OsSH1 , ZpSh1 , which retained a YABBY transcription factor and consistently clustered with orthologs from O. sativa and Z. latifolia (Supplementary Table 5), indicating its conserved role in abscission regulation. In particular, ZpSh5 , a Bel1-homeobox domain, exhibited paralog divergence among four strong Sh5 candidates. ZpSh5a ’s loss of key homeobox and POX domains, for example, suggests it may be transitioning toward pseudogenization (Panchy et al., 2016 ). The retention of multiple Sh5- like genes, with varying degrees of structural and functional integrity, reflects the history of WGD and subsequent segmental or transposon-mediated duplications (Fedoroff, 2012 ; Huang et al., 2022 ; Leister, 2004 ; Wang et al., 2012 ). The differential expression of ZpSh5 paralogs also supports a role for subfunctionalization or regulatory partitioning in NWR genes following duplication. While some copy number differences between Z. palustris and Z. latifolia could be due to assembly artifacts, others likely reflect true genomic expansion, consistent with the larger genome size of Z. palustris . This complexity highlights the challenge of linking genotype to phenotype in recently duplicated genomes and the need for functional assays to dissect roles of individual paralogs. Developmental timing of gene expression differentiates shattering phenotypes Developmental regulation of gene expression is a key determinant of phenotypic variation in domestication traits such as seed shattering. In this study, we identified a general trend of higher expression of ZpSH1 and ZpSh5c relative to the other tested genes. ZpSH1 exhibited higher expression in the early DAA compared to later DAA (Fig. 3 a), consistent with SH1 ’s known role in promoting abscission layer formation in grasses (Lin et al., 2012 ; Yu et al., 2023 ) and supports the hypothesis that ZpSH1 functions as a conserved regulator of shattering in NWR. The expression of ZpSh5c , by contrast, was reduced in cultivated populations compared to the wild type (Fig. 3 d). The delay in peak expression of FY-C20 (28 DAA) aligned with FY-C20’s shattering time course, consistent with Sh5 ’s known role in regulating abscission zone differentiation and downstream enzymes (Yoon et al., 2014 ) and suggests that altered expression timing could contribute to extended seed retention in NWR. A similar pattern of delay has been observed in U.S. weedy rice ( O. sativa ), which has re-evolved its shattering ability, compared to its wild progenitor, Oryza rufipogon (Thurber et al., 2011 ). These findings support a model in which selection for seed retention in NWR has acted on regulatory changes, such as the timing and magnitude of gene expression, and that population-specific responses to artificial selection vary in cNWR. While sample size was limited, strong correlations between male and female floret expression in the early DAA also suggest that these tissues may have similar shattering mechanisms. In NWR, male floret shattering occurs prior to female floret shattering and has been used as an early, indirect selection tool for improving seed retention (Kennard et al., 2002 ). These results reinforce the utility of this selection method in breeding programs. Overall, the developmental and regulatory changes identified in this study resemble patterns observed in Oryza , where domestication involved sequential, quantitative shifts in expression across multiple shattering-related loci (Ishikawa et al., 2022 . We cannot rule out the unknown interactions between these genes and others not included in this study that can modulate seed shattering in NWR and should be subjects of future research to understand mechanisms of seed shattering in this native North American species. CONCLUSIONS Despite the short breeding history of cNWR, the reduction of seed shattering is already evident at the anatomical, physiological, and gene expression levels. Delayed abscission layer development, increased tensile strength, and altered expression of key regulatory genes collectively support the hypothesis that selection has reshaped the developmental and genetic control of shattering in cNWR. Comparative genomic analyses revealed duplicated orthologs of O. sativa shattering genes, consistent with the WGD history and genomic complexity of Z. palustris . Among these, ZpSH1 and ZpSh5c showed biologically relevant expression variation across populations, suggesting roles in modulating abscission zone formation and seed retention. ZpSh5c ’s delayed expression in one of the cultivated populations aligns with its delayed shattering phenotype and may reflect a target of selection. Together, these findings highlight the potential of cNWR as a model for investigating the early stages of domestication. Its phylogenetic proximity to Oryza and ongoing artificial selection offer a rare opportunity to study the molecular evolution of domestication traits in real time. Further integrative studies across broader germplasm, incorporating functional assays and regulatory network analysis, will be key to resolving the complex genetic basis of seed retention in this North American cereal. Abbreviations analysis of variance, ANOVA breaking tensile strength, BTS breaking tensile strength by pulling, BTS-P breaking tensile strength by bending, BTS-B chromosome, ch complementary DNA, cDNA cultivated Northern Wild Rice, cNWR days after anthesis, DAA Northern Wild Rice, NWR Oryza sativa , Os principal phenological stage, PPS quantitative trait loci, QTL reverse transcription-quantitative polymerase chain reaction, RT-qPCR University of Minnesota, UMN whole genome duplication, WGD Zizania latifolia , Zp Zizania palustris , Zl Declarations Ethics Approval and Consent to participate Not applicable Consent for publication Not applicable Availability of data and materials Most data is provided within the manuscript or supplementary information files. Other raw data files associated with this project have also been submitted to the Data Repository for the University of Minnesota (DRUM) and can be accessed via doi XXXX. Competing interests The authors declare no conflicts of interest Funding This work was supported by the State of Minnesota, Agricultural Research, Education, Extension and Technology Transfer program. Author contributions R. M.: data generation, expression data generation, figure generation, writing; A. M.: histology data generation; M. B.: data generation, writing, editing; L. M.: figure generation, writing, editing; C. C. M.: data analysis and interpretation; editing; J. K.: original writing, editing, figure generation Acknowledgements The authors would like to thank the University of Minnesota’s Clinical and Translational Science Institute (https://ctsi.umn.edu/services/specimen/histology-digital-imaging) for their histological imaging of the seed-pedicel abscission layer. References Altendorf, K. R., DeHaan, L. R., Larson, S. R., & Anderson, J. A. (2021). 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The YABBY gene SHATTERING1 controls activation rather than patterning of the abscission zone in Setaria viridis . New Phytologist, 240 (2), 846–862.https://doi.org/10.1111/nph.19157 Zhou, Y., Lu, D., Li, C., Luo, J., Zhu, B. F., Zhu, J., & Han, B. (2012). Genetic control of seed shattering in rice by the APETALA2 transcription factor Shattering Abortion1 . The Plant Cell, 24 (3), 1034–1048.https://doi.org/10.1105/tpc.111.094383 Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTALTABLESANDFIGURES.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7032638","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":482709369,"identity":"aac66508-05da-4807-9eb3-223e1d319d4f","order_by":0,"name":"Reneth Millas","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Reneth","middleName":"","lastName":"Millas","suffix":""},{"id":482709370,"identity":"961645f9-43d6-4de6-9038-cbfadb589668","order_by":1,"name":"Lillian McGilp","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Lillian","middleName":"","lastName":"McGilp","suffix":""},{"id":482709371,"identity":"99a1c0e1-72bb-42e6-8c6d-a84ea81af955","order_by":2,"name":"Alan Mickelson","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Alan","middleName":"","lastName":"Mickelson","suffix":""},{"id":482709372,"identity":"d15ec72a-ab57-4046-9cec-eccec08b8b2b","order_by":3,"name":"Maybell Banting","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Maybell","middleName":"","lastName":"Banting","suffix":""},{"id":482709373,"identity":"ac7f314d-4881-427b-a2d0-bb0dfadee3ac","order_by":4,"name":"Claudia Castell-Miller","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Castell-Miller","suffix":""},{"id":482709374,"identity":"2d4ddee5-2b8e-46ed-84ab-0c7102117d87","order_by":5,"name":"Jennifer Kimball","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYBACxh7GBmYGBgsZfgifmWgtEjySDcRqYeABK5PgMThArBbmnsOtmwtqJHiMb2SnPWCosE5sIOiw3sa22zOOSfCY3cjdbsBwJp0ILf2Mbbd52MBatkkwth0mVss/oMNmgLT8I0YLyGG8bUDvS4C0NBCjpedg2+2ZfRI8EmfebpNIOJZuTFCLYU/6s9sF32zk+NuBtnyosZYlrAVFRQIh5SAgT4yiUTAKRsEoGOEAAFL7PLhAGq87AAAAAElFTkSuQmCC","orcid":"","institution":"University of Minnesota","correspondingAuthor":true,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Kimball","suffix":""}],"badges":[],"createdAt":"2025-07-02 21:38:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7032638/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7032638/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86661732,"identity":"03c1e03e-71d3-4d99-a0a4-31663ab231ea","added_by":"auto","created_at":"2025-07-14 10:39:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":867040,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology and abscission layer histology of the seed-pedicel junctions in Northern Wild Rice (NWR; \u003cem\u003eZizania palustris\u003c/em\u003e) populations at the hard dough seed phenological stage.\u003c/p\u003e\n\u003cp\u003eLegend. a. Representative image of the NWR seed and pedicel at early seed developmental stage; b. Unselected, highly shattering wild type genotype; c. Shattering resistant FY-C20 genotype and d. Higher shattering resistance Itasca-C20 genotype. AL: abscission layer; VB: vascular bundle.\u003c/p\u003e\n\u003cp\u003eHistological images were observed at a 20X magnification at the plant principal phenological stage 85 (hard dough seed) which corresponded to 21 days after anthesis (DAA) for the wild type, and 28 DAA for the cultivated populations.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7032638/v1/7a64a2fe58ab2cd5e7b5407f.png"},{"id":86659467,"identity":"72943a14-d678-4f0f-af5b-3a02bc28b78c","added_by":"auto","created_at":"2025-07-14 10:31:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":188189,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of seed shattering \u003cem\u003eOryza sativa \u003c/em\u003eputative orthologs in \u003cem\u003eZizania palustris\u003c/em\u003e and \u003cem\u003eZizania latifolia\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eLegend. \u003cem\u003eOs\u003c/em\u003e = \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eZp\u003c/em\u003e = \u003cem\u003eZizania palustris\u003c/em\u003e, \u003cem\u003eZl\u003c/em\u003e = \u003cem\u003eZ. latifolia\u003c/em\u003e; \u003cem\u003eSH1\u003c/em\u003e = \u003cem\u003eShattering1\u003c/em\u003e (Lin et al., 2012) , \u003cem\u003eqSH1\u003c/em\u003e = QTL of seed shattering in chromosome 1 (Konishi et al., 2006) , \u003cem\u003eSHAT1\u003c/em\u003e = \u003cem\u003eShattering Abortion1 \u003c/em\u003e(Zhou et al, 2012), \u003cem\u003esh4 \u003c/em\u003e=(Li et al, 2006) , \u003cem\u003eSh5\u003c/em\u003e(Yoon et al, 2014). Midpoint rooted cladogram constructed with aligned protein sequences of shattering genes using the ETE3 pipeline from Genome Net (www.genome.jp) and 100 bootstraps. Branch length and bootstrap values (in square brackets) are located at branches.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7032638/v1/bf8140076f5e88deea4fbea4.png"},{"id":86661733,"identity":"49124aaa-65fa-4ff8-a128-9db151b2b3a6","added_by":"auto","created_at":"2025-07-14 10:39:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16709,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of candidate seed-shattering genes in female and male floret tissues of Northern Wild Rice (NWR; \u003cem\u003eZizania palustris\u003c/em\u003e) post-anthesis across a wild type and two cultivated populations.\u003c/p\u003e\n\u003cp\u003eLegend: \u003cem\u003eZp\u003c/em\u003e = \u003cem\u003eZizania palustris\u003c/em\u003e, \u003cem\u003eqSH1\u003c/em\u003e = QTL of seed shattering in chromosome 1 (Konishi et al., 2006), \u003cem\u003eSh5 \u003c/em\u003e= (Yoon et al, 2014). Log2 relative gene expression was measured in male florets collected at 7 days after anthesis (DAA) (indicated in gray) and in female florets collected at 1, 7, 18, 28, and 35 DAA across 4 genes\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7032638/v1/883922d30e7423b0dc026803.png"},{"id":87787768,"identity":"02ed35bd-a8bc-43ad-bbdb-9157cc71df19","added_by":"auto","created_at":"2025-07-29 04:32:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2560313,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7032638/v1/0dc9eb0c-aa24-42c7-9857-d312b3876252.pdf"},{"id":86661734,"identity":"82826c45-079d-42f4-9cd5-26fd5fa1e07a","added_by":"auto","created_at":"2025-07-14 10:39:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":901716,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTALTABLESANDFIGURES.docx","url":"https://assets-eu.researchsquare.com/files/rs-7032638/v1/778a3660a17aaaa70c002a2c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seed Shattering in a North American Oryzeae grain: Developmental and Genomic Signatures of Early Domestication","fulltext":[{"header":"PLAIN LANGUAGE SUMMARY","content":"\u003cp\u003eNorthern Wild Rice (NWR, \u003cem\u003eZizania palustris\u003c/em\u003e) has been grown in irrigated paddies in the United States since the 1950s. Seed shattering, that is, when ripe seed separates from the plant pedicel, reduces grain yields in cultivated NWR (cNWR) production. However, little is known about this process. This study compared the seed shattering of two cNWR populations to a wild, unselected population. We found that cNWR seed retention increased ~90% compared to the time when the wild type shatters, and cNWR began shattering about two weeks later than the wild type. Imaging of a layer of cells that lead to shattering at the pedicel, where the seed and stem meet, also revealed differences between cNWR and the wild type. Being closely related to white rice, we leveraged available white rice genomic resources to identify potential NWR genes that may be associated with seed shattering. We found that the gene, \u003cem\u003eZpSh5c\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eis expressed in the same pattern as shattering occurs in both wild and cultivated plants, suggesting its possible role in the regulation of shattering in NWR.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eSeed shattering is a natural seed dispersal mechanism widespread among wild plant species, including the wild ancestors of many modern crops (Konishi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Li \u0026amp; Gill, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). As species were domesticated, traits that favored seed retention were selected, leading to the replacement of wild type alleles with mutations that reduced or eliminated shattering (Konishi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A key target of selection was a developmentally regulated abscission event involving the formation and breakdown of an abscission layer, a specialized cell layer, at the seed\u0026ndash;pedicel junction (Oba et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Fuller \u0026amp; Allaby, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Watanabe et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Disruption or loss of this abscission layer increases seed retention, significantly improving yields and harvest efficiency, which are critical milestones in crop domestication.\u003c/p\u003e\u003cp\u003eNumerous genes and quantitative trait loci (QTL) underlying seed shattering have been identified in crop species, particularly in \u003cem\u003eOryza sativa\u003c/em\u003e L. and related species (Oba et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Cai and Morishima, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Thomson et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Konishi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ishikawa et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Several of these have been cloned or fine-mapped, revealing their roles in regulating the development and function of the abscission layer. Notable examples include the \u003cem\u003esh4\u003c/em\u003e gene, which encodes a MYB3 transcription factor, (Li et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Purugganan \u0026amp; Fuller, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2012\u003c/span\u003e); \u003cem\u003eqSH1\u003c/em\u003e, which encodes a BEL1-type homeobox gene (Konishi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e); \u003cem\u003eSh5\u003c/em\u003e, a highly homologous gene of \u003cem\u003eqSH1\u003c/em\u003e (Yoon et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); \u003cem\u003eSHAT1\u003c/em\u003e, an \u003cem\u003eAPETALA2\u003c/em\u003e (AP2) transcription factor (Zhou et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2012\u003c/span\u003e); and \u003cem\u003eSH1\u003c/em\u003e, a YABBY transcription factor (Lin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Together, these genes can modulate the development and degradation of the abscission zone and have been repeatedly targeted during the independent domestication of cereal crops.\u003c/p\u003e\u003cp\u003eAcross crops, studies have revealed that seed shattering pathways are generally conserved, with many genes either orthologous or functionally analogous across divergent lineages (Dong \u0026amp; Wang., 2015; Ishikawa et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This pattern of evolutionary convergence reflects similar selection pressures during domestication that were used to increase seed retention (Doebley, 2006; Lenser and Thei\u0026szlig;en, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Olsen \u0026amp; Wendel, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Purugganan \u0026amp; Fuller, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tranbarger et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Comparative genomic studies have helped to shape our understanding of these convergent evolutionary patterns and are increasingly applied to identify candidate domestication genes in wild and understudied crop species (Kahler et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Fu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For example, recent studies, have reported that \u003cem\u003eSH1\u003c/em\u003e likely plays a role in seed shattering across a wide range of grass species, including \u003cem\u003eLolium perenne\u003c/em\u003e (Perennial ryegrass), \u003cem\u003eSetaria italica\u003c/em\u003e (Foxtail millet), and \u003cem\u003eZizania latifolia\u003c/em\u003e (Manchurian wild rice) (Fu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Odonkor et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). By leveraging such comparative frameworks, researchers can identify conserved regulatory genes and causal variants for key traits, ultimately accelerating the improvement of emerging, non-model crops.\u003c/p\u003e\u003cp\u003eOne emerging crop with strong potential for comparative genomics is Northern Wild Rice (NWR, \u003cem\u003eZizania palustris L\u003c/em\u003e.), an annual aquatic grass and Crop Wild Relative (CWR) of \u003cem\u003eO. sativa\u003c/em\u003e within the Oryzeae tribe. Endemic to the Great Lake Region of North America, NWR production in irrigated, diked paddies began in the region in the 1950s. In 1968, the first cultivated NWR (cNWR) variety was released, featuring improved seed retention compared to its wild counterparts (Oelke, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Like \u003cem\u003eOryza\u003c/em\u003e species (Jin et al., 1993; Jin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1982\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Jin and Inouye, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1982a\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003eb\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), seed shattering in NWR is associated with development of an abscission layer at the seed-pedicel junction composed of parenchyma and sclerenchyma cells, where variation in cell thickness and number contributes to genotype-specific differences (Hanten et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). Previous genetic studies have suggested that seed shattering in NWR is a recessive, quantitative trait regulated by at least two or three genes (Woods \u0026amp; Clark, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Elliott \u0026amp; Perlinger, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Kennard et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Despite ongoing efforts, modern cNWR varieties and breeding germplasm still exhibit some degree of seed shattering, primarily due to the species\u0026rsquo; outcrossing nature and limited breeding history (Elliot, 1980). The trait\u0026rsquo;s persistence contributes to substantial yield losses, particularly after late-summer storms or high winds (Imle, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). As such, NWR offers a valuable system for exploring the genetic basis of seed shattering and applying insights from domesticated relatives to accelerate trait improvement in a semi-domesticated species.\u003c/p\u003e\u003cp\u003eImproving seed retention in cNWR requires a better understanding of the developmental, anatomical, and genetic factors that regulate seed shattering. Despite its agronomic importance, key aspects of this trait remain underexplored. Given the close phylogenetic relationship between \u003cem\u003eO. sativa\u003c/em\u003e and NWR, comparative genomics offers a powerful framework for candidate gene discovery (Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, this study aimed to: (1) Assess the phenotypic variation in seed shattering using four quantitative phenotyping methods; (2) Examine the abscission layer anatomy of two cNWR populations and one wild NWR population using histological analysis; (3) Identify and compare the protein sequences of putative \u003cem\u003eO. sativa\u003c/em\u003e orthologs in NWR and \u003cem\u003eZ. latifolia\u003c/em\u003e; and (4) Evaluate the expression profiles of putative NWR orthologs of \u003cem\u003eO. sativa\u003c/em\u003e seed shattering-related genes across stages of panicle development and seed maturation.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cem\u003ePlant materials.\u003c/em\u003e Three NWR populations were used in this study, including a wild type population exhibiting a profuse shattering habit, collected from Sullivan Lake, Minnesota (MN), USA in 2020 (46.149519 N; \u0026minus;\u0026thinsp;93.936795 W); a cNWR breeding line, \u0026lsquo;FY-C20\u0026rsquo; developed for seed length; and a cNWR variety, \u0026lsquo;Itasca-C12\u0026rsquo;, released in 2007 (Porter et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), which served as the industry\u0026rsquo;s standard for seed retention at the time of this study. Seeds were removed from cool storage (3℃) (McGilp et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), rinsed three times, and placed in new plastic bags containing fresh water on a lab benchtop. After 10 days, germinated seedlings were transplanted in the greenhouse.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePlant growth conditions.\u003c/em\u003e Experiments were conducted at the University of Minnesota (UMN) Plant Growth Facilities in Saint Paul, MN, in the spring of 2021. Greenhouse conditions were maintained at 22\u0026deg;C (\u0026plusmn;\u0026thinsp;2\u0026deg;C) with a 16-hour photoperiod. Cone-tainers (112.98 cm\u003csup\u003e2\u003c/sup\u003e) were filled with a steamed soil mix supplemented with 0.14 g urea (20 lb/acre) and 0.14 g of iron chelate (1 lb/ 1000 ft\u003csup\u003e2\u003c/sup\u003e), then placed in aluminum aquaponic tanks (66 cm x 183 cm x 70 cm). Tanks were filled with water (13℃) up to 2\u0026ndash;4 cm above the surface of the cone-tainers one week before seedlings were planted. To limit algae growth, water was circulated under 5 psi using a 1056GPH 276W submersible pump (Simple Deluxe, Duarte, California, USA) and treated twice weekly with Algaefix (1 ml/10 gallons of water; API, Chalfont, Pennsylvania, USA). After germination, one seedling was transplanted per cone-tainer. Plants were top-dressed with urea (20lb/acre) after tillering every 2\u0026ndash;3 weeks.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSeed shattering phenotypic data collection.\u003c/em\u003e The degree of seed shatter was measured on the main stem panicles of three individual plants per population per time point over the course of NWR seed ripening at the Principal Phenological Stage (PPS) 8 (Duquette \u0026amp; Kimball, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) using four different methods. Individual plants were collected at their respective days after anthesis (DAA), including 21, 28, 35, and 42 DAA. The first method was a visual estimation of the percentage of shattered seed at the time of data collection, ranging from 0 to 100%, where 0\u0026thinsp;=\u0026thinsp;no shatter and 100% = complete shatter. The second method was a drop test, whereby a panicle was dropped three times from a height of 60.96 cm, and the proportion of shattered seeds was calculated relative to the total number of seeds per panicle (Altendorf et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The third and fourth methods measured breaking tensile strength (BTS) using a Chatillon digital force gauge (Ametek, Inc, Berwyn, Pennsylvania), either by pulling the seed from the pedicel (BTS-P) or bending (BTS-B). For BTS-P, the seed was gently gripped with forceps attached to the gauge, and force was applied in a straight, downward direction until detachment occurred. The maximum force required to remove the seed was recorded in grams-force (g), a unit of force where 1g\u0026thinsp;\u0026asymp;\u0026thinsp;0.0098 Newtons. For BTS-B, force was applied laterally to the seed at the point of attachment and gently bent until detachment occurred. The force required to break the seed from the pedicel was also recorded in g.\u003c/p\u003e\u003cp\u003e\u003cem\u003eHistology of the seed-pedicel junction\u003c/em\u003e. Seed-pedicel junctions of five plants per population were sampled before reaching the hard dough stage of seed ripening (PPS 85), which corresponded to 21 DAA for the wild type population\u0026rsquo;s individuals and 28 DAA for the cultivated line\u0026rsquo;s individuals. Seeds were bisected, placed in a vacuum chamber, and fixed in a mixture of formalin-acetic acid-alcohol (FAA; 53:5:10:32) solution containing 0.1% Triton X-100. Fixed samples were stored in 70% ethanol at 4\u0026deg;C before being processed at the Histology and Research Laboratory, UMN. Upon arrival, the samples were fixed in 2% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.0), dehydrated in an incremental ethanol and ethanol-xylene series (3:1, 1:1, 1:3, 100% xylene), and embedded in Paraplast Plus (Leica Biosystems, Wetzlar, Germany). Sections (10 \u0026micro;m) were cut using a rotary microtome, stained with 0.05% toluidine blue O in 0.1 M phosphate buffer (pH 6.8), and imaged using an Axio Scan.Z1 slide scanner (Zeiss Group, Oberkochen, Baden-W\u0026uuml;rttemberg, Germany).\u003c/p\u003e\u003cp\u003e\u003cem\u003ePlant tissue collection\u003c/em\u003e. Three plant tissues were collected from three individual plants per population in liquid nitrogen as independent biological replicates. The sampled tissues included flag leaf at the heading stage (PPS 51); male florets at 7 DAA at PPS 66 when all flowering was complete and before pollen shed (PPS 69); and female florets at 1, 7, 18, 28, and 35 DAA throughout seed development (PPS 7) and maturation (PPS 8). In the case of the wild type population, female florets were collected only up to 18 DAA, as most seeds had shattered by the next time point. Tissue samples were stored at -80\u0026deg;C until further use.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRNA isolation and cDNA synthesis.\u003c/em\u003e Total RNA was extracted using a RNeasy\u0026reg; Plant Mini Kit (Qiagen, Valencia, CA) following the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were measured using a NanoDrop\u0026trade; 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). To eliminate DNA contamination, RNA samples were treated with the DNA-free\u0026trade; DNA Removal Kit (Thermo Fisher Scientific, Waltham, MA). Complementary DNA (cDNA) was synthesized from total RNA using the SuperScript\u0026trade; III First-Strand Synthesis SuperMix (Invitrogen, Thermo Fisher Scientific, Waltham, MA), following the manufacturer\u0026rsquo;s protocol. Synthesized cDNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further use.\u003c/p\u003e\u003cp\u003e\u003cem\u003eReverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays\u003c/em\u003e. Gene expression quantification was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA) on 96-well plates. Each 20 \u0026micro;L reaction contained 50 ng of cDNA template, 10 \u0026micro;L of iTaq\u0026trade; Universal SYBR\u0026reg; Green Supermix (Bio-Rad, Hercules, CA), and 0.2 \u0026micro;L (10 mM) each of forward and reverse primers (Supplemental Table\u0026nbsp;1) using standard thermal cycling conditions (95\u0026deg;C for 20 s, followed by 40 cycles of 95\u0026deg;C for 3 s and 60\u0026deg;C for 30 s). Each gene was evaluated using three biological replicates and two technical replicates per biological replicate, tissue type, and time point. Negative controls of no-template and water as template were used in all runs.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSelection and evaluation of candidate reference genes for RT-qPCR assays.\u003c/em\u003e To identify suitable internal reference genes for RT-qPCR normalization, five NWR genes were selected as candidates based on their stable expression in other plant species (Kozera \u0026amp; Rapacz, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Joseph et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and across eight NWR tissue types (Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These included putative homologs of actin 1 (ACT1), actin 4 (ACT4), eukaryotic translation initiation factor 5-like (EIF-5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBI). Primers were designed using PrimerQuest (Integrated DNA Technologies; Coralville, Iowa, USA) based on exon sequences extracted from the NWR reference genome v1.0 (Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) using in-house R script. The amplification efficiency and expression stability of each primer set were assessed in leaf, male floret, and female floret tissues across all time points. Standard curves were generated using a 5-fold serial dilution of cDNA (200, 40, 8, 1.6, and 0.32 ng/\u0026micro;L). Sterile DNase- and RNase-free water was used as a control in all reactions.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEvaluation and selection of putative seed shattering orthologs\u003c/em\u003e. Protein sequences of well-characterized \u003cem\u003eO. sativa\u003c/em\u003e seed shattering genes, \u003cem\u003eSH1\u003c/em\u003e, \u003cem\u003eqSH1\u003c/em\u003e, \u003cem\u003esh4\u003c/em\u003e, \u003cem\u003eSh5\u003c/em\u003e, and \u003cem\u003eSHAT1\u003c/em\u003e, were retrieved from the \u003cem\u003eO. sativa\u003c/em\u003e ssp. \u003cem\u003ejaponica\u003c/em\u003e reference genome (GenBank accession: GCF_034140825.1). Putative orthologs identified in \u003cem\u003eZ. latifolia\u003c/em\u003e in Yan et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e were obtained from the Chinese wild rice \u0026lsquo;Jaiobai\u0026rsquo; genome (GCA_0483537475.1; Guo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These \u003cem\u003eO. sativa\u003c/em\u003e and \u003cem\u003eZ. latifolia\u003c/em\u003e protein sequences were used as queries in BLASTp searches against the \u003cem\u003eZ. palustris\u003c/em\u003e \u0026lsquo;Itasca-C12\u0026rsquo; reference genome assembly v1.0 (GCA_019279435.1; Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) using the NCBI BLASTp tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Candidate orthologs in NWR were identified based on percent protein identity with a cut-off rate of 70%. Multiple sequence alignments (MSA) were performed using CLUSTALW v2.0 (Larkin et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and a maximum likelihood (ML) phylogenetic tree with 100 bootstraps was calculated with the randomized accelerated ML (RAxML v8.2.11; Stamatakis, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) software. The resulting alignments and phylogram were visualized with ETE3 3.1.2 (Huerta-Cepas et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) via GenomeNET (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ip/tools/ete/\u003c/span\u003e\u003cspan address=\"https://genome.ip/tools/ete/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, using default parameters. Protein sequence alignments for \u003cem\u003eqSh1\u003c/em\u003e and \u003cem\u003eSh5\u003c/em\u003e orthologs were also conducted using NCBI\u0026rsquo;s COBALT (Constraint-based Multiple Alignment Tool) with default parameters to visualize sequence divergence among homologs. Orthogroups were inferred using OrthoFinder version 2.5.4 (Emms \u0026amp; Kelly, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and protein domain annotation was performed using Pfam version 37.4 (Finn et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) via the InterProScan web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify conserved domains in the predicted protein sequences. Based on these analyses, gene-specific primers (Supplemental Table\u0026nbsp;2) were designed for selected orthologs of \u003cem\u003eSH1\u003c/em\u003e and \u003cem\u003eSh5\u003c/em\u003e for downstream RT-qPCR validation of gene expression, following the experimental protocol described above.\u003c/p\u003e\u003cp\u003e\u003cem\u003eData analysis.\u003c/em\u003e To assess variation in seed shattering, an analysis of variance (ANOVA) was conducted in R version 4.3.1 (R Core Team, 2021) for each of the four phenotyping methods, across four developmental timepoint. Genotype and timepoint were both considered fixed effects. A statistical significance threshold of 0.05 was used in all analyses. Tukey\u0026rsquo;s Honestly Significant Difference (HSD) tests were performed using the \u003cem\u003eAgricolae\u003c/em\u003e package version 1.3.7 (de Mendiburu, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) to identify pairwise differences among groups. To compare phenotyping methods, Pearson\u0026rsquo;s correlation coefficients were calculated with the \u003cem\u003eHmisc\u003c/em\u003e package version 5.2.2 (Harrell, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor reference gene selection, primer efficiency was assessed for each candidate reference gene primer pair by constructing linear regression curves based on the logarithm of the cDNA and the threshold curve cycle (C\u003csub\u003eT\u003c/sub\u003e). Primer efficiency (E) was calculated from the slope (S) of the standard curves generated from serial dilutions, using the equation E= [10\u003csup\u003e(1/\u0026minus;S)\u0026minus;\u003c/sup\u003e1] \u0026times; 100% (Rutledge \u0026amp; Cote, 2003). The coefficients of determination (R\u003csup\u003e2\u003c/sup\u003e) were also computed. Expression stability across tissue types and timepoints were evaluated using RefFinder (Xie et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which also utilizes geNorm (Vandesompele et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Hellemans et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and NormFinder (Andersen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The best candidate reference genes with amplification efficiencies within the optimal range (100% \u0026plusmn; 10%) and minimal variation in expression were selected for assessing gene expression of candidate shattering genes.\u003c/p\u003e\u003cp\u003eGene expression levels of seed shattering-related genes in female and male tissues were quantified using the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e method (Livak \u0026amp; Schmittgen, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). For each sample, threshold cycle (C\u003csub\u003eT\u003c/sub\u003e) values of the target gene were first normalized to the selected reference gene to obtain CT values. Relative expression was then calculated by comparing normalized CT values of each tissue sample to those of the same genes expressed in flag leaf tissue, which was used as the baseline calibrator. The data was transformed using log\u003csub\u003e2\u003c/sub\u003e and presented as relative gene expression. This approach enabled quantification of fold changes in gene expression relative to a consistent non-reproductive tissue control. To evaluate differential expression between the wild type population and the cNWR populations, ANOVA and Tukey\u0026rsquo;s HSD were also performed.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eSeed shattering variability in Northern Wild Rice populations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate genetic and phenotypic variation in seed shattering, we quantified this trait in three \u003cem\u003eZ. palustris\u003c/em\u003e populations using four phenotyping methods at 21, 28, 35, and 42 days after anthesis (DAA). These methods included visual ratings, a drop test, and two biomechanical tension force methods (BTS-P and BTS-B). Statistical analyses (ANOVA, Tukey\u0026rsquo;s HSD; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed visual ratings and the drop test results were significantly influenced by both genotypes and DAA, while BTS-P had significant variation by genotype and BTS-B had no statistical differences (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In general, visual and drop test scores showed increasing seed shattering over time, while BTS-B and BTS-P values, reflecting resistance to detachment, decreased (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Importantly, differences in the shattering phenotype were not due to variation in developmental timing, as all populations reached comparable phenological stages within one to three days of each other (Figure S1).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnalysis of Variance (ANOVA) of four phenotyping methods to characterize seed shattering in Northern Wild Rice (NWR; \u003cem\u003eZizania palustris\u003c/em\u003e) post-anthesis.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSource of Variation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSums of Squares\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMean Squares\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003ep-\u003c/em\u003evalue\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eVisual\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e635.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e211.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e69,705.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34,852.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e896.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e266.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e44.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.37\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e933.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDrop Test\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2,527.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e842.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36,198.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18,099.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e343.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e189.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e47.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,052.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e52.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBTS-P\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18,193.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9,096.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33,073.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16,536.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5,465.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2,732.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36,419.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2,601.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBTS-B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eLegend. BTS-P: Breaking tensile strength by pulling; BTS-B: breaking tensile strength by bending; DAA: days after anthesis; Df: degrees of freedom\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTukey\u0026rsquo;s mean separations analysis for four phenotyping methods to characterize seed shattering in Northern Wild Rice (NWR; Zizania palustris) over time and populations.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"17\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eVisual (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u003cp\u003eDrop Test (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e\u003cp\u003eBTS_Pulling (g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c17\" namest=\"c14\"\u003e\u003cp\u003eBTS_Bending (g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGenotype / DAA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c17\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItasca-C12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.33 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.25 cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e23.23 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e206.78 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e168.67 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e142.93 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.014 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.023 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c17\"\u003e\u003cp\u003e0.01 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFY-C20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.67 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.33 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e10.42 cd\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e35.27 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e185.67 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e139.67 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e44.20 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.027 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.014 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c17\"\u003e\u003cp\u003e0.005 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWild type\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e86.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98.33 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e91.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e98.33 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e100 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e100 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e91.33 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c17\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"17\" nameend=\"c17\" namest=\"c1\"\u003e\u003cp\u003eLegend: Days after anthesis, DAA; not applicable, NA; grams, g; Same letters indicate no statistically significant difference (P\u0026thinsp;=\u0026thinsp;0.05)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"17\" nameend=\"c17\" namest=\"c1\"\u003e\u003cp\u003eMethods: A visual rating, 0-100% shattering; a drop test, 0-100% shattering; breaking tensile strength by pulling, BTS-P, and breaking tensile strength by bending (BTS_B) were measured in force (g)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor visual ratings, the wild type population consistently exhibited greater seed shattering than both cNWR populations, with no significant differences found between Itasca-C12 and FY-C20. Shattering in the wild type increased from 86.67% at 21 DAA to 100% by 35 DAA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). FY-C20 began shattering at 35 DAA, reaching 13.3% by 42 DAA, while Itasca-C12 showed delayed onset, with 8.3% shattering by 42 DAA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The drop test also effectively differentiated the wild type from both cultivated populations across all time points (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The visual and drop test methods were highly correlated with one another (r\u0026thinsp;=\u0026thinsp;0.99, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.001) across all time points (Supplementary Table\u0026nbsp;3).\u003c/p\u003e\u003cp\u003eFor BTS-P, only one time point could be assessed for the wild type, which yielded 91.3 g of force at 28 DAA, compared to 185.7 g for FY-C20 and 206.8 g for Itasca-C12 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). By 42 DAA, Itasca-C12 required greater force to detach seeds than FY-C20 (144 g vs. 44 g; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although BTS-P differences among populations were not statistically significant, Itasca-C12 exhibited higher BTS-P values compared to FY-C20 across all time points. BTS-P was negatively and significantly correlated with both visual and drop test results across time points, except with the visual method at 35 DAA (Supplementary Table\u0026nbsp;3). BTS-B displayed non-significant negative correlations with the visual and drop test methods across timepoints. BTS-B was also non-significantly positively correlated with BTS-P results across timepoints, with one significant correlation between BTS-B and BTS-P at 35 DAA (Supplementary Table\u0026nbsp;3).\u003c/p\u003e\u003cp\u003e\u003cem\u003eHistological analysis of the seed-pedicel abscission layer.\u003c/em\u003e We examined the anatomical structure of the abscission layer in a wild type population, characterized by high levels of seed shattering, and two cultivated populations, Itasca-C12 at 28 DAA and FY-C20 at 21 DAA, which exhibit reduced seed shattering. All three populations developed a distinct abscission layer at the seed-pedicel junction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the wild type population (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), the abscission layer was well-defined, consisting of densely packed and structurally organized parenchyma cells. In contrast, the cultivated populations appear to have more loosely arranged cells and larger intercellular spaces disrupting the continuity of the layer. Among the cultivated populations, FY-C20 showed a more compact and organized abscission layer than Itasca-C12, which had a more diffuse structure and well-defined vascular bundle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ePutative relationships of seed shattering-related genes in Oryzeae.\u003c/em\u003e Protein sequences of five \u003cem\u003eO. sativa\u003c/em\u003e genes involved in seed abscission layer development, \u003cem\u003eSH1\u003c/em\u003e (\u003cem\u003eOsSH1\u003c/em\u003e), \u003cem\u003eqSH1\u003c/em\u003e (\u003cem\u003eOsqSH1\u003c/em\u003e), \u003cem\u003eSh5\u003c/em\u003e (\u003cem\u003eOsSh5\u003c/em\u003e), \u003cem\u003esh4\u003c/em\u003e (\u003cem\u003eOssh4\u003c/em\u003e), and \u003cem\u003eSHAT1\u003c/em\u003e (\u003cem\u003eOsSHAT1\u003c/em\u003e), were queried against the \u003cem\u003eZ. palustris\u003c/em\u003e genome in NCBI GenBank using the BlastP tool (Supplementary Table\u0026nbsp;4). Multiple orthologs were identified in NWR, consistent with a whole-genome duplication (WGD) event in the \u003cem\u003eZizania\u003c/em\u003e lineage following its divergence from \u003cem\u003eOryza\u003c/em\u003e approximately 26 to 30\u0026nbsp;million years ago (Guo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yan et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previously reported candidate orthologs (Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) were confirmed as tops hits based on percent sequence similarity and conserved domain structure, except for \u003cem\u003eOsSHAT1\u003c/em\u003e, where ZPchr0004g38673 (\u003cem\u003eZpSHAT1a\u003c/em\u003e) and ZPchr0458g2256 (\u003cem\u003eZpSHAT1b\u003c/em\u003e) were stronger candidates than the previously reported ZPchr0013g34051 (Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, ZPchr0458g22823 (\u003cem\u003eZpsh4c\u003c/em\u003e) was identified as a novel candidate of \u003cem\u003eOssh4\u003c/em\u003e, based on high protein sequence similarity to \u003cem\u003eZpSh4a, ZpSh4b\u003c/em\u003e, and \u003cem\u003eOssh4\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A percent protein identity (PI) matrix was generated to evaluate the sequence similarity among \u003cem\u003eO. sativa\u003c/em\u003e, \u003cem\u003eZ. palustris\u003c/em\u003e, and \u003cem\u003eZ. latifolia\u003c/em\u003e seed shattering potential orthologs (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The highest PI between \u003cem\u003eO. sativa\u003c/em\u003e and \u003cem\u003eZ. palustris\u003c/em\u003e was observed for \u003cem\u003eSH1\u003c/em\u003e (89.34%), while the lowest PI was for \u003cem\u003eSh5a\u003c/em\u003e (71.23%; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The PI comparisons between NWR and \u003cem\u003eZ. latifolia\u003c/em\u003e ranged from 70.00% for \u003cem\u003eSh5a\u003c/em\u003e to 98.84% for \u003cem\u003eSH1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePercent protein identity (PI) of seed shattering-related genes in \u003cem\u003eOryza sativa\u003c/em\u003e (\u003cem\u003eOs\u003c/em\u003e) and putative \u003cem\u003eZizania palustris\u003c/em\u003e (\u003cem\u003eZp\u003c/em\u003e) and \u003cem\u003eZizania latifolia\u003c/em\u003e (\u003cem\u003eZl\u003c/em\u003e) orthologs.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eO. sativa (Os)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZ. palustris (Zp)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e\u003cem\u003eZ. latifolia (Zl)\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003ePercent Identify Comparisons between Species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGene description\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGene\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eGenBank ID\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eCandidate Gene\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eGenBank ID\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eCandidate genes\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eGenBank IDs (a and b, respectively)\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eZp vs. Os\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eZp\u003c/b\u003e \u003cb\u003evs.\u003c/b\u003e \u003cb\u003eZl a/b\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eOs vs. Zl a/b\u003c/b\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eOsSH1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNP_001389121.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpSH1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0003g18426\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eZlSH1a / ZlSH1b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eABZP36_005173 / ABZP36_018673\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e89.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e91.98 / 98.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e91.94 / 91.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eYABBY transcription factor (Lin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cem\u003eOsSh5\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eXP_015637721.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpSh5a\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0001g31104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eZlSh5a\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eABZP36_013876\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e71.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e78.85 / 70.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e89.56 / 83.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eBel1-type homeobox (Yoon et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpSh5b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0005g15825\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e84.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e90.05 / 95.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpSh5c\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0010g10516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eZlSh5b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eABZP36_030161\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e87.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e95.64 / 89.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpSh5d\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0010g7757\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e85.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e95.64 / 89.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eOsqSH1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eXP_015641948.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpqSH1a\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0001g31318\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eZlqSH1a\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eABZP36_025045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e83.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e85.44 / 91.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e85.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBel1-type homeobox (Konishi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpqSH1b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0007g5872\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eZlqSH1b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eABZP36_016879\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e83.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e90.86 / 83.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e85.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eOsSHAT1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eXP_015636848.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpSHAT1a\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0004g38673\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eZlSHAT1a\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eABZP36_003055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e83.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e90.32 / 88.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e85.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAP2 transcription factor (Zhou et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpSHAT1b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0458g22566\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eZlSHAT1b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eABZP36_007933\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e84.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e90.30 / 96.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e84.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eOssh4\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eXP_015635337.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpsh4a\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0004g38781\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eZlsh4a / Zlsh4b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eABZP36_008339 / ABZP36_004261\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e78.05 / 80.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e76.47 / 80.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eTrihelix transcription factor (Li et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpsh4b\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0458g22499\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e79.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e87.41 / 92.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eZpsh4c\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZPchr0458g22823\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e80.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e86.36 / 84.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e\u003cp\u003eLegend: Zl a and b genes identified in Yan et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zl a and b genes GenBank ID in the \u0026lsquo;Jaiobai\u0026rsquo; genome (GCA_0483537475.1; Guo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); See Supplementary Table\u0026nbsp;4 for protein sequences\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eA cluster analysis of \u003cem\u003eO. sativa\u003c/em\u003e shattering genes and putative \u003cem\u003eZ. palustris\u003c/em\u003e and \u003cem\u003eZ. latifolia\u003c/em\u003e orthologs identified two main clusters in the cladogram, each with moderate bootstrap support of 75%. The first main cluster consisted of two subclusters separating \u003cem\u003eSH1\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;98%) from \u003cem\u003eqSH1\u003c/em\u003e and \u003cem\u003eSH5\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;75%). The second cluster (BS\u0026thinsp;=\u0026thinsp;75%) consisted of \u003cem\u003esh4\u003c/em\u003e and \u003cem\u003eSHAT1\u003c/em\u003e subclusters, supported by BS values of 92% and 93%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Within the \u003cem\u003eSH1\u003c/em\u003e subcluster, \u003cem\u003eOsSH1\u003c/em\u003e formed a distinct branch from \u003cem\u003eZizania\u003c/em\u003e sequences (BS\u0026thinsp;=\u0026thinsp;98%), while the \u003cem\u003eZlSH1b\u003c/em\u003e sequence was the closest ortholog to \u003cem\u003eZpSH1\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;65%; 98.84% PI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the \u003cem\u003eqSH1\u003c/em\u003e subcluster (BS\u0026thinsp;=\u0026thinsp;75%), \u003cem\u003eZpqSH1a\u003c/em\u003e and \u003cem\u003eZlqSH1b\u003c/em\u003e grouped together (BS\u0026thinsp;=\u0026thinsp;98%; 91.43% PI). \u003cem\u003eOsqSH1\u003c/em\u003e, albeit with a weak bootstrap (52%), grouped more closely with \u003cem\u003eZpqSH1b\u003c/em\u003e and \u003cem\u003eZlqSH1a\u003c/em\u003e, who were 90.86% identical and in a clade with a BS of 98%. Within the \u003cem\u003esh5\u003c/em\u003e subcluster (BS\u0026thinsp;=\u0026thinsp;66%), \u003cem\u003eOsSh5\u003c/em\u003e separated from \u003cem\u003eZizania\u003c/em\u003e genes, which grouped with a BS of 82%. \u003cem\u003eZpSH5b\u003c/em\u003e clustered closely with \u003cem\u003eZlSH5b\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;100%; 95.11% PI), while \u003cem\u003eZpSh5a\u003c/em\u003e, \u003cem\u003eZpSh5c\u003c/em\u003e, and \u003cem\u003eZpSh5d\u003c/em\u003e clustered with \u003cem\u003eZlSh5a\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;91%; and PI ranging from 78.85 to 95.64%), although \u003cem\u003eZlSh5a\u003c/em\u003e formed an independent branch separated from the ZpSH5 orthologs (BS\u0026thinsp;=\u0026thinsp;71%). The variation in PI among the \u003cem\u003eZpSh5\u003c/em\u003e orthologs and across species was largely attributed to \u003cem\u003eZpSh5a\u003c/em\u003e. Within the second main cluster, \u003cem\u003eSHAT1\u003c/em\u003e subcluster was well supported (BS\u0026thinsp;=\u0026thinsp;93%), integrated by low supported groups (BS\u0026thinsp;=\u0026thinsp;39%) containing \u003cem\u003eOsSHAT1\u003c/em\u003e, \u003cem\u003eZlSHATb\u003c/em\u003e, and \u003cem\u003eZpSHAT1b\u003c/em\u003e, and another (BS\u0026thinsp;=\u0026thinsp;58%; 90.32% PI) of \u003cem\u003eZlSHAT1a\u003c/em\u003e and \u003cem\u003eZpSHAT1a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The \u003cem\u003esh4\u003c/em\u003e subcluster (92% BS) exhibited a stepwise branching pattern, starting with \u003cem\u003eZlsh4b\u003c/em\u003e, followed by \u003cem\u003eZpsh4a\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;13%), \u003cem\u003eOssh4\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;37%), and \u003cem\u003eZlsh4a\u003c/em\u003e (BS\u0026thinsp;=\u0026thinsp;92%), with \u003cem\u003eZpsh4b\u003c/em\u003e and \u003cem\u003eZpsh4c\u003c/em\u003e forming a well-supported terminal cluster (BS\u0026thinsp;=\u0026thinsp;99% BS). Among the five genes evaluated, \u003cem\u003eZizania\u003c/em\u003e orthologs of \u003cem\u003eOssh4\u003c/em\u003e showed some of the most divergent protein sequences, with PI percentages ranging from 76.47\u0026ndash;80.17% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOrthogroup and Pfam analyses provided additional support for several conserved shattering-related orthologs (Supplementary Table\u0026nbsp;5 and Supplementary Table\u0026nbsp;6). \u003cem\u003eZpSH1\u003c/em\u003e was identified as an ortholog of \u003cem\u003eOsSH1\u003c/em\u003e and assigned to orthogroup OG0010120, with similar sequences in \u003cem\u003eZ. latifolia\u003c/em\u003e (Supplementary Table\u0026nbsp;5). All orthologs contained a YABBY protein domain (Supplementary Table\u0026nbsp;6). For \u003cem\u003eSh5\u003c/em\u003e, OrthoFinder analysis indicated that \u003cem\u003eZpSh5b\u003c/em\u003e was the strongest orthogonal candidate for \u003cem\u003eOsSh5\u003c/em\u003e within orthogroup OG0013611, which also contained \u003cem\u003eZlSh5a\u003c/em\u003e and \u003cem\u003eZlSh5b\u003c/em\u003e (Supplementary Table\u0026nbsp;5). Two closely related sequences \u003cem\u003eZpSh5c\u003c/em\u003e and \u003cem\u003eZpSh5d\u003c/em\u003e grouped within orthogroup OGOO26538, with no clear orthologs in \u003cem\u003eO. sativa\u003c/em\u003e or \u003cem\u003eZ. latifolia\u003c/em\u003e. \u003cem\u003eZpSh5a\u003c/em\u003e could not be assigned to an orthogroup. Pfam analysis found that all \u003cem\u003eSh5\u003c/em\u003e orthologs contain a homeobox domain and a POX domain, except for \u003cem\u003eZpSh5a\u003c/em\u003e (Supplementary Table\u0026nbsp;6). MSA revealed that \u003cem\u003eZpSh5a\u003c/em\u003e exhibited a truncated protein sequence compared to those of other orthologs (Supplementary Fig.\u0026nbsp;2). The alignment of sequences also revealed that \u003cem\u003eOsSh5\u003c/em\u003e and all \u003cem\u003eZpSh5\u003c/em\u003e genes had 100% protein identities for their homeobox domains but differed in their POX domains, within which \u003cem\u003eOsSh5\u003c/em\u003e differed from \u003cem\u003eZpSh5b\u003c/em\u003e and \u003cem\u003eZpSh5c\u003c/em\u003e by four and eight amino acids, respectively (Supplementary Table\u0026nbsp;7). Comparison of \u003cem\u003eOsqSH1\u003c/em\u003e identified \u003cem\u003eZpqSH1b\u003c/em\u003e as a likely ortholog and it was placed in orthogroup OG0008858, along with \u003cem\u003eZlqSH1a\u003c/em\u003e and \u003cem\u003eZlqSH1b\u003c/em\u003e (Supplementary Table\u0026nbsp;5), while \u003cem\u003eZpqSH1a\u003c/em\u003e was not assigned an orthogroup. However, all \u003cem\u003eZ. palustris\u003c/em\u003e orthologs in this group, including \u003cem\u003eZpqSH1a\u003c/em\u003e, have homeobox and POX domains (Supplementary Table\u0026nbsp;6). For \u003cem\u003eOsSHAT1\u003c/em\u003e, \u003cem\u003eZpSHAT1b\u003c/em\u003e was the strongest orthogonal candidate and grouped in orthogroup OG0003844 with \u003cem\u003eZlSHAT1a\u003c/em\u003e and \u003cem\u003eZlSHAT1b\u003c/em\u003e (Supplementary Table\u0026nbsp;5), while \u003cem\u003eZpSHAT1a\u003c/em\u003e was not assigned to an orthogroup. Pfam searches indicated that all \u003cem\u003eSHAT1\u003c/em\u003e ortholog sequences contained two tandem AP2/ERF domains (Supplementary Table\u0026nbsp;6). Lastly, \u003cem\u003eOssh4\u003c/em\u003e along with \u003cem\u003eZpsh4a, Zpsh4b, and Zpsh4c\u003c/em\u003e genes were grouped within the orthogroup OG0004851 (Supplementary Table\u0026nbsp;5). All \u003cem\u003eZ. palustris\u003c/em\u003e ortholog sequences for \u003cem\u003esh4\u003c/em\u003e contained a Myb/SANT-like DNA-binding domain 4 (Supplementary Table\u0026nbsp;6).\u003c/p\u003e\u003cp\u003e\u003cem\u003eReference gene selection for RT-qPCR experiments.\u003c/em\u003e Five commonly used reference genes in plant gene expression studies, ACT1, ACT4, EIF-5, UBI, and GAPDH, were evaluated across leaf, female floret, and male floret tissues to identify those with the most stable expression for normalizing RT-qPCR expression data (Supplementary Table\u0026nbsp;1). Primer efficiencies ranged from 106\u0026ndash;113% with R\u003csup\u003e2\u003c/sup\u003e values between 0.997 and 0.998 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Stability rankings using RefFinder consistently identified \u003cem\u003eACT1\u003c/em\u003e as the most stable and \u003cem\u003eACT4\u003c/em\u003e as the least stable expressed gene (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Based on these results, ACT1 was selected as the internal reference gene for downstream expression analyses.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer efficiency and stability of reference gene candidates for gene expression analyses in male and female floret tissues of Northern Wild Rice (NWR; \u003cem\u003eZizania palustris\u003c/em\u003e) based on RefFinder, which includes geNorm and NormFinder analyses.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZ. palustris gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChr.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePosition (bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eR2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e%E\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eRefFinder\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003egeNorm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNormFinder\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eACT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZPchr0012g19383\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23,232,269\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eActin 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e110\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eACT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZPchr0010g7718\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55,029,365\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eactin-related protein 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e113\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e8.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEIF-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZPchr0006g44672\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52,111,321\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eeukaryotic translation initiation factor 5-like\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e107\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGADPH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZPchr0008g11968\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9,259,023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.74\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUBIQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eZPchr0007g6160\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,416,073\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eubiquitin-conjugating enzyme E2-17 kDa isoform X1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e\u003cp\u003eLegend: primer efficiency, %E; RefFinder (Xie et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2023\u003c/span\u003e); geNorm (Vandesompele et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Hellemans et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e); NormFinder (Andersen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e); lower values indicate higher stability and rank\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eExpression of SH1 and Sh5 orthologs in Z. palustris female florets.\u003c/em\u003e We analyzed the relative expression of NWR candidate orthologs of \u003cem\u003eOsSH1\u003c/em\u003e and \u003cem\u003eOsSh5\u003c/em\u003e, \u003cem\u003eZpSH1\u003c/em\u003e and \u003cem\u003eZpSh5a, ZpSh5b\u003c/em\u003e, and \u003cem\u003eZpSh5c\u003c/em\u003e in NWR female floret tissues (including the seed-pedicel junction) across a time course following anthesis (Supplementary Table\u0026nbsp;2). \u003cem\u003eZpSH1\u003c/em\u003e was selected due to its high expression in female panicle tissue (Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and its conserved role in seed shattering across grass species (Maity et al., 2021). Among four \u003cem\u003eZpSh5\u003c/em\u003e putative paralogs, \u003cem\u003eZpSh5a\u003c/em\u003e, \u003cem\u003eZpSh5b\u003c/em\u003e, and \u003cem\u003eZpSh5c\u003c/em\u003e were included based on their relatively high expression levels (Haas, et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and to explore their potential roles in the regulatory pathways of seed shattering, considering their putative diversification following the WGD. \u003cem\u003eZpSh5d\u003c/em\u003e was omitted from RT-qPCR assays due to its low expression in 8 NWR tissues (Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo characterize candidate gene expression, female florets were collected at 1, 7, 18, 28, and 35 DAA for RT-qPCR analysis. Data from the wild type population were unavailable at 28 and 35 DAA due to near-complete seed shattering (98%) by 28 DAA (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The RT-qPCR gene expression time course profiles of female florets are summarized across genes in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and by population in Supplementary Fig.\u0026nbsp;3. \u003cem\u003eZpSH1\u003c/em\u003e was upregulated compared to leaf tissue, except for FY-C20 at 35 DAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Across DAA, the expression of \u003cem\u003eZpSH1\u003c/em\u003e in the wild type was high on 1 DAA, peaked at 7 DAA, and declined at the last timepoint of the population\u0026rsquo;s collection, 18 DAA. FY-C20\u0026rsquo;s expression was lower than the wild type from 1 to 18 DAA but with peaks at 7 and at 28 DAA and was slightly downregulated by 35 DAA. Itasca-C12 had higher expression than the wild type from 1 to 18 DAA, peaking at 1 DAA and then declining through the rest of the timepoints. For \u003cem\u003eZpSh5a\u003c/em\u003e, expression in female floret tissues was downregulated at 1 and 7 DAA for all populations compared to leaf tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Expression of the wild type was slightly upregulated at 18 DAA, its final timepoint. The expression of \u003cem\u003eZpSh5a\u003c/em\u003e in FY-C20 after 7 DAA was upregulated with a peak at 28 DAA, and undetectable at 35 DAA. In Itasca-C12, the gene was downregulated from 1 DAA through 18 DAA, undetectable at 28 DAA, and upregulated at 35 DAA. The expression of \u003cem\u003eZpSh5b\u003c/em\u003e in female floret tissue was downregulated compared with leaf tissue for all populations and timepoints except for the wild type at 1 DAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The expression of \u003cem\u003eZpSh5c\u003c/em\u003e in female floret tissue was upregulated for all populations and timepoints compared with leaf tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The expression in the wild type was highest at 1 DAA, then declined through 18 DAA. Expression in FY-C20 and Itasca-C12 was higher at 1 DAA than 7 and 18 DAA, after which expression peaked at 28 DAA for both populations and then declined at 35 DAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). However, the expression of FY-C20 throughout the time course was higher relative to Itasca-C12.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eANOVA conducted on a per-gene basis revealed that genotype was not significant for any of the genes (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Expression of \u003cem\u003eZpSH1\u003c/em\u003e and \u003cem\u003eZpSh5a\u003c/em\u003e was influenced by DAA; \u003cem\u003eZpSH1\u003c/em\u003e expression at 7 DAA was different from 35 DAA and \u003cem\u003eZpSh5c\u003c/em\u003e expression at 1 and 7 DAA was different from 28 DAA (Supplementary Table\u0026nbsp;8). No significant differences between populations or time points were observed for candidate genes \u003cem\u003eZpSh5b\u003c/em\u003e and \u003cem\u003eZpSh5c\u003c/em\u003e. ANOVA conducted on a per population basis revealed significant differences between gene expression for all populations, where \u003cem\u003eZpSH1\u003c/em\u003e and \u003cem\u003eZpSh5c\u003c/em\u003e had higher expression than \u003cem\u003eZpSh5a\u003c/em\u003e and \u003cem\u003eZpSh5b\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Supplementary Table\u0026nbsp;8). For FY-C20, there were also significant differences between timepoints; 28 DAA was significantly different from 1 DAA and 35 DAA. For Itasca-C12, the interaction between gene and DAA was significant. Overall, \u003cem\u003eZpSH1\u003c/em\u003e had higher expression, with 1 and 7 DAA being significantly higher than \u003cem\u003eZpSh5b\u003c/em\u003e at 7, 18, and 28 DAA and also, \u003cem\u003eZpSh5a\u003c/em\u003e at 1 and 7 DAA. The expression for all timepoints of \u003cem\u003eZpSh5c\u003c/em\u003e was intermediate and did not differ significantly from any other genes (Supplementary Table\u0026nbsp;8).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAnalysis of Variance (ANOVA) analyses of the relative gene expression of four Northern Wild Rice (NWR; \u003cem\u003eZizania palustris\u003c/em\u003e) candidate orthologs of \u003cem\u003eOryza sativa\u003c/em\u003e genes related to seed shattering in three NWR populations.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSource of Variation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSum of Squares\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMean Squares\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eZpSH1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e83.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.044\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.424\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReplication\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.237\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.766\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e181.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eZpSh5a\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.054\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReplication\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.275\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e84.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eZpSh5b\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.777\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.415\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReplication\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eZpSh5c\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.806\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.221\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReplication\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.062\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genotype\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.641\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e109.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGenotype\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eSource of Variation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eDf\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eSum of Squares\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eMean Squares\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eF-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eP-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eWild type\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.118\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e201.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e67.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e16.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRep\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.882\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.286\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e92.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eFY-C20\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e85.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.018\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e121.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e40.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRep\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.088\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.859\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e189.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eItasca-C12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.892\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e245.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e81.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRep\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.021\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDAA:Genes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e117.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResiduals\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e149.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e*Degrees of freedom, df; days after anthesis, DAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eExpression of SH1 and Sh5 orthologs in male florets.\u003c/em\u003e To investigate potential overlap in the regulatory mechanisms between male and female floret shattering, we analyzed expression in male florets at 7 DAA. Similar to the expression patterns in female florets, \u003cem\u003eZpSh1\u003c/em\u003e and \u003cem\u003eZpSh5c\u003c/em\u003e were upregulated in all populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Expression was highest in Itasca-C12 for \u003cem\u003eZpSH1\u003c/em\u003e and in the wild type for \u003cem\u003eZpSh5c\u003c/em\u003e. At 7 DAA, the expression of \u003cem\u003eZpSh5a\u003c/em\u003e in male florets was downregulated for all populations, consistent with the early DAA in female florets. For \u003cem\u003eZpSh5b\u003c/em\u003e, wild type male florets were upregulated while the cultivated populations were downregulated, consistent with the expression patterns of female florets at 1 DAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When averaged across genes and populations the male floret expression at 7 DAA was significantly correlated with 1 (0.82), 7 (0.80), and 18 (0.38) DAA in the female floret tissue (Supplementary Table\u0026nbsp;9).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cb\u003eSelection-driven changes in cultivated Northern Wild Rice seed retention\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeed shattering is a key adaptive trait in wild grasses, enabling seed dispersal, but it presents a major obstacle in grain domestication. In cNWR, strong selection pressure for seed retention has modified the timing and structure of seed abscission, despite a short domestication history. Our results show that cultivated populations exhibited a 90% reduction and 2-week delay in shattering relative to the wild type (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), possibly due to alteration of the abscission zone. Histological observations support this, where cultivated populations displayed less organized and compact abscission layers, consistent with delayed or disrupted cell separation processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These changes parallel those seen in domesticated \u003cem\u003eOryza\u003c/em\u003e species (Jin and Inouye, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Oba et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Ji et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Thurber et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and suggest that similar cellular mechanisms are being targeted in NWR. The observed changes in tensile strength and shattering progression, particularly in the higher seed-retaining Itasca-C12 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggest population-specific modifications to abscission layer timing or robustness, reflecting the ongoing selection for this trait. While this study identified a potential relationship between seed shattering and the development of cNWR\u0026rsquo;s abscission layer, further investigation into the timing of abscission layer development and degradation is needed, as histological assessments were limited to a single time point per population.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene duplication and divergence underlie shattering variation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGene duplication and divergence are central to trait evolution following WGD events. Our comparative genomic analyses revealed that NWR harbors multiple orthologs, similar to \u003cem\u003eZ. latifolia\u003c/em\u003e (Yan et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), of known \u003cem\u003eO. sativa\u003c/em\u003e seed shattering genes, including \u003cem\u003esh4\u003c/em\u003e, \u003cem\u003eSHAT1\u003c/em\u003e, \u003cem\u003eqSH1\u003c/em\u003e, and \u003cem\u003eSh5\u003c/em\u003e. This redundancy, arising from a WGD and subsequent gene duplications in NWR (Haas et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), suggests that regulatory divergence rather than gene presence/absence may drive variation in NWR seed shattering. For example, \u003cem\u003eSHAT1\u003c/em\u003e orthologs showed evidence of divergence in NWR. \u003cem\u003eZpSHAT1b\u003c/em\u003e clustered with known functional orthologs, while \u003cem\u003eZpSHAT1a\u003c/em\u003e did not cluster with any known orthogroup, possibly reflecting neofunctionalization or regulatory repurposing (Wang et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The exception was a single functional ortholog of \u003cem\u003eOsSH1\u003c/em\u003e, \u003cem\u003eZpSh1\u003c/em\u003e, which retained a YABBY transcription factor and consistently clustered with orthologs from \u003cem\u003eO. sativa\u003c/em\u003e and \u003cem\u003eZ. latifolia\u003c/em\u003e (Supplementary Table\u0026nbsp;5), indicating its conserved role in abscission regulation.\u003c/p\u003e\u003cp\u003eIn particular, \u003cem\u003eZpSh5\u003c/em\u003e, a Bel1-homeobox domain, exhibited paralog divergence among four strong \u003cem\u003eSh5\u003c/em\u003e candidates. \u003cem\u003eZpSh5a\u003c/em\u003e\u0026rsquo;s loss of key homeobox and POX domains, for example, suggests it may be transitioning toward pseudogenization (Panchy et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The retention of multiple \u003cem\u003eSh5-\u003c/em\u003elike genes, with varying degrees of structural and functional integrity, reflects the history of WGD and subsequent segmental or transposon-mediated duplications (Fedoroff, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Leister, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The differential expression of \u003cem\u003eZpSh5\u003c/em\u003e paralogs also supports a role for subfunctionalization or regulatory partitioning in NWR genes following duplication. While some copy number differences between \u003cem\u003eZ. palustris\u003c/em\u003e and \u003cem\u003eZ. latifolia\u003c/em\u003e could be due to assembly artifacts, others likely reflect true genomic expansion, consistent with the larger genome size of \u003cem\u003eZ. palustris\u003c/em\u003e. This complexity highlights the challenge of linking genotype to phenotype in recently duplicated genomes and the need for functional assays to dissect roles of individual paralogs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDevelopmental timing of gene expression differentiates shattering phenotypes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDevelopmental regulation of gene expression is a key determinant of phenotypic variation in domestication traits such as seed shattering. In this study, we identified a general trend of higher expression of \u003cem\u003eZpSH1\u003c/em\u003e and \u003cem\u003eZpSh5c\u003c/em\u003e relative to the other tested genes. \u003cem\u003eZpSH1\u003c/em\u003e exhibited higher expression in the early DAA compared to later DAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), consistent with \u003cem\u003eSH1\u003c/em\u003e\u0026rsquo;s known role in promoting abscission layer formation in grasses (Lin et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and supports the hypothesis that \u003cem\u003eZpSH1\u003c/em\u003e functions as a conserved regulator of shattering in NWR. The expression of \u003cem\u003eZpSh5c\u003c/em\u003e, by contrast, was reduced in cultivated populations compared to the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The delay in peak expression of FY-C20 (28 DAA) aligned with FY-C20\u0026rsquo;s shattering time course, consistent with \u003cem\u003eSh5\u003c/em\u003e\u0026rsquo;s known role in regulating abscission zone differentiation and downstream enzymes (Yoon et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and suggests that altered expression timing could contribute to extended seed retention in NWR. A similar pattern of delay has been observed in U.S. weedy rice (\u003cem\u003eO. sativa\u003c/em\u003e), which has re-evolved its shattering ability, compared to its wild progenitor, \u003cem\u003eOryza rufipogon\u003c/em\u003e (Thurber et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These findings support a model in which selection for seed retention in NWR has acted on regulatory changes, such as the timing and magnitude of gene expression, and that population-specific responses to artificial selection vary in cNWR.\u003c/p\u003e\u003cp\u003eWhile sample size was limited, strong correlations between male and female floret expression in the early DAA also suggest that these tissues may have similar shattering mechanisms. In NWR, male floret shattering occurs prior to female floret shattering and has been used as an early, indirect selection tool for improving seed retention (Kennard et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These results reinforce the utility of this selection method in breeding programs.\u003c/p\u003e\u003cp\u003eOverall, the developmental and regulatory changes identified in this study resemble patterns observed in \u003cem\u003eOryza\u003c/em\u003e, where domestication involved sequential, quantitative shifts in expression across multiple shattering-related loci (Ishikawa et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e. We cannot rule out the unknown interactions between these genes and others not included in this study that can modulate seed shattering in NWR and should be subjects of future research to understand mechanisms of seed shattering in this native North American species.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eDespite the short breeding history of cNWR, the reduction of seed shattering is already evident at the anatomical, physiological, and gene expression levels. Delayed abscission layer development, increased tensile strength, and altered expression of key regulatory genes collectively support the hypothesis that selection has reshaped the developmental and genetic control of shattering in cNWR. Comparative genomic analyses revealed duplicated orthologs of \u003cem\u003eO. sativa\u003c/em\u003e shattering genes, consistent with the WGD history and genomic complexity of \u003cem\u003eZ. palustris\u003c/em\u003e. Among these, \u003cem\u003eZpSH1\u003c/em\u003e and \u003cem\u003eZpSh5c\u003c/em\u003e showed biologically relevant expression variation across populations, suggesting roles in modulating abscission zone formation and seed retention. \u003cem\u003eZpSh5c\u003c/em\u003e\u0026rsquo;s delayed expression in one of the cultivated populations aligns with its delayed shattering phenotype and may reflect a target of selection. Together, these findings highlight the potential of cNWR as a model for investigating the early stages of domestication. Its phylogenetic proximity to \u003cem\u003eOryza\u003c/em\u003e and ongoing artificial selection offer a rare opportunity to study the molecular evolution of domestication traits in real time. Further integrative studies across broader germplasm, incorporating functional assays and regulatory network analysis, will be key to resolving the complex genetic basis of seed retention in this North American cereal.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eanalysis of variance, ANOVA\u003c/p\u003e\u003cp\u003ebreaking tensile strength, BTS\u003c/p\u003e\u003cp\u003ebreaking tensile strength by pulling, BTS-P\u003c/p\u003e\u003cp\u003ebreaking tensile strength by bending, BTS-B\u003c/p\u003e\u003cp\u003echromosome, ch\u003c/p\u003e\u003cp\u003ecomplementary DNA, cDNA\u003c/p\u003e\u003cp\u003ecultivated Northern Wild Rice, cNWR\u003c/p\u003e\u003cp\u003edays after anthesis, DAA\u003c/p\u003e\u003cp\u003eNorthern Wild Rice, NWR\u003c/p\u003e\u003cp\u003e\u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eOs\u003c/em\u003e\u003c/p\u003e\u003cp\u003eprincipal phenological stage, PPS\u003c/p\u003e\u003cp\u003equantitative trait loci, QTL\u003c/p\u003e\u003cp\u003ereverse transcription-quantitative polymerase chain reaction, RT-qPCR\u003c/p\u003e\u003cp\u003eUniversity of Minnesota, UMN\u003c/p\u003e\u003cp\u003ewhole genome duplication, WGD\u003c/p\u003e\u003cp\u003e\u003cem\u003eZizania latifolia\u003c/em\u003e, \u003cem\u003eZp\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eZizania palustris\u003c/em\u003e, \u003cem\u003eZl\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMost data is provided within the manuscript or supplementary information files. Other raw data files associated with this project have also been submitted to the Data Repository for the University of Minnesota (DRUM) and can be accessed via doi XXXX.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the State of Minnesota, Agricultural Research, Education, Extension and Technology Transfer program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR. M.: data generation, expression data generation, figure generation, writing; A. M.: histology data generation; M. B.: data generation, writing, editing; L. M.: figure generation, writing, editing; C. C. M.: data analysis and interpretation; editing; J. 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Genetic control of seed shattering in rice by the APETALA2 transcription factor \u003cem\u003eShattering Abortion1\u003c/em\u003e. \u003cem\u003eThe Plant Cell, 24\u003c/em\u003e(3), 1034–1048.https://doi.org/10.1105/tpc.111.094383\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Zizania palustris, seed retention, abscission layer, Oryza sativa, gene expression, multiple sequence alignment, domestication","lastPublishedDoi":"10.21203/rs.3.rs-7032638/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7032638/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNorthern Wild Rice (NWR; \u003cem\u003eZizania palustris\u003c/em\u003e L.) is an aquatic grain endemic to North America and a member of the Oryzeae tribe. As an outcrossing crop with a short breeding history, domestication progress in cultivated NWR (cNWR) is ongoing and seed shattering remains a major barrier to yield stability. In this study, we investigated the developmental and genetic mechanisms underlying seed retention by integrating phenotypic, anatomical, and molecular analyses across wild and cultivated populations. Time-course phenotyping using four methods revealed a\u0026thinsp;~\u0026thinsp;90% reduction and two-week delay in shattering in cNWR relative to wild populations. Histological analysis indicated anatomical reorganization of the abscission layer in cNWR, consistent with selection for seed retention. Comparative genomic analyses identified multiple NWR orthologs of key \u003cem\u003eOryza sativa\u003c/em\u003e shattering genes, revealing lineage-specific gene duplication, pseudogenization, and divergence from \u003cem\u003eZ. latifolia\u003c/em\u003e. Expression profiling of candidate genes via RT-qPCR across floret developmental stages suggested a regulatory role for \u003cem\u003eZpSh5c\u003c/em\u003e, a putative \u003cem\u003eOsSh5\u003c/em\u003e ortholog, in modulating seed shattering. In cNWR, delayed and reduced expression of \u003cem\u003eZpSh5c\u003c/em\u003e mirrored observed differences in shattering timing, highlighting its potential involvement in abscission zone development. Together, these findings provide new insights into the anatomical and molecular basis of seed shattering in NWR and demonstrate the utility of comparative frameworks for accelerating trait improvement in emerging, non-model crops.\u003c/p\u003e","manuscriptTitle":"Seed Shattering in a North American Oryzeae grain: Developmental and Genomic Signatures of Early Domestication","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 10:31:45","doi":"10.21203/rs.3.rs-7032638/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8fa3d9b-e7fb-4410-adc6-b5228c457ef5","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-29T04:24:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 10:31:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7032638","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7032638","identity":"rs-7032638","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}