An enhanced genome assembly and functional, high-throughput molecular markers enable genomics-assisted breeding of waxy sorghum [Sorghum bicolor (L.) Moench]

An enhanced genome assembly and functional, high-throughput molecular markers enable genomics-assisted breeding of waxy sorghum [Sorghum bicolor (L.) 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Moench] Melinda Yerka, Zhiyuan Liu, Scott Bean, Deepti Nigam, Chad Hayes, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4883126/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Aug, 2025 Read the published version in Journal of Applied Genetics → Version 1 posted 5 You are reading this latest preprint version Abstract Several mutations of the sorghum [ Sorghum bicolor (L.) Moench] GRANULE-BOUND STARCH SYNTHASE ( GBSS ) gene [ Sobic.010G022600 ] result in a low amylose:amylopectin starch ratio in the endosperm and confer a glutinous, “waxy” texture; hence, the wild-type gene is commonly referred to as Waxy ( Wx ). Recessive waxy ( wx ) alleles improve starch digestibility in ethanol production, human foods and beverages, and animal feed. However, breeding waxy sorghum can be time-consuming due to the need for grain to reach physiological maturity before the trait can be phenotyped and ongoing reliance on PCR markers for genotyping, which are not amenable to next-generation sequencing (NGS). Modern genomics-assisted breeding requires conducing high-throughput genotyping and selection in large, segregating populations prior to flowering. This study provides the first published NGS markers for the two mostly commonly used waxy ( wx ) alleles of sorghum and is the first to fully characterize the large insertion that is causal of the wx a allele. An enhanced genome assembly was constructed from the B.Tx623 reference genome (v3.1.1) to include the 5.6 kb la rge r etrotransposon d erivative (LARD) in the wx a allele. This improved read mapping at Sobic.010G022600 in wx a individuals, identified 78 new uniquely mapped reads, and made it possible to distinguish different Waxy genotypes using short-read sequencing data. Functional PACE-PCR markers, suitable for genomic selection, were developed for Wx , wx a , and wx b alleles and validated in three public and private breeding programs. These new molecular breeding resources will improve the efficiency of developing commercial waxy sorghum hybrids. sorghum waxy grain quality molecular markers breeding Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Sorghum [ Sorghum bicolor (L.) Moench] is the fifth-most produced grain crop in the world. It is undergoing major development as a climate-smart species due to its wide genetic diversity and its ability to tolerate hot, dry, low-input environments. Sorghum can be marketed with gluten-free, non-GMO, and ancient grain labels in specialty foods, food ingredients, and alcoholic beverages. As a diploid (2 n = 2 x = 20) with a genome size of ~ 700 Mbp, several published reference genomes (Deschamps et al. 2018 , McCormick et al. 2018 , Cooper et al. 2019 , Tao et al. 2021 ) and pangenomes (Deschamps et al. 2018 , Ruperao et al. 2021 ), publicly available mutant and mapping populations (Kumar et al., Xin et al. 2009 , Yerka et al. 2015 , Brenton et al. 2016 , Boyles et al. 2019 , Mace et al. 2021 , Xin et al. 2023 ), and large whole-genome sequencing data of diversity populations (Mace et al. 2013 , Boatwright et al. 2022 ), it is also an emerging model grass species, particularly for sugarcane [ Saccharum oficinarum L.]. Certain mutations of the sorghum GRANULE-BOUND STARCH SYNTHASE ( GBSS ) gene [ Sobic.010G022600 ] result in a low amylose:amylopectin starch ratio. In seeds, low-amylose endosperms have a sticky, “waxy” texture; hence, the wild-type gene is commonly referred to as Waxy . GBSS is highly conserved in the Poaceae and has been extensively reviewed for its role in cereal starch synthesis (Tetlow and Emes 2017 , Tetlow 2018 , Nakamura 2023 ), including in waxy maize [ Zea mays L.] (Talukder et al. 2023 , Wu et al. 2023 ), glutinous rice [ Oryza sativa L.] (Okpala et al. 2022 , Jin et al. 2023 , Nakamura 2023 ), and sorghum (Kang et al. 2023 ). Among sorghum breeders, the waxy grain phenotype is generally considered to be ~ 95–100% amylopectin. A dosage effect on amylopectin content depends on how many recessive waxy alleles are inherited by the triploid endosperm (Karper 1933 , Yerka et al. 2016 ). In 1933, R.E. Karper first documented the Mendelian inheritance of individual alleles of wild-type ( Wx ) and waxy ( wx ) grain phenotypes in sorghum (Karper 1933 ). Pedersen et al. (Pedersen et al. 2005 ) were first to characterize the waxy a ( wx a ) and waxy b ( wx b ) alleles with respect to the production of GBSS: wx a is a knock-out allele producing no GBSS, whereas wx b is “leaky” and produces low levels of non-functional GBSS. Sattler et al. (Sattler et al. 2009 ) performed the first molecular characterization of wx a and wx b alleles and identified the causal mutations of each one: the wx a allele contains a large insertion in the third exon, whereas the wx b allele contains a C to T transition converting amino acid 268 from glutamine to histidine. Since then, two additional alleles have been reported in East Asia, although their nomenclature is inconsistent across publications (Kawahigashi et al. 2013 , Lu et al. 2013 ). The wx c allele described by Lu et al. ( 2013 ) has a G deletion at the 5' splicing site of the ninth intron, causing a frameshift mutation and early translation termination. The wx d allele described by the same authors contains a mutation at the 3' splicing site of the tenth intron, causing a splicing site shift and deletion of five amino acids (GTGKK) in the predicted translated protein. While sorghum is a key subsistence crop for human food and animal feed in Sub-Saharan African and Southeast Asia, it has historically been bred for grain yield and animal feed in North and South America, Europe, and Australia. Waxy sorghum grain has long been associated with improved whole-grain digestibility by animals (Sherrod et al. 1969 , Walker and Lichtenw.Re 1974, Myer and Gorbet 1985 , Schroeder et al. 1998 ), rapid fermentation into ethanol for biofuel production (Yan et al. 2011 , Wu et al. 2013 ), the production of alcoholic beverages (Hsieh and Pi 1979 , Mugode et al. 2011 , Mezgebe et al. 2018 ), and improved baking properties (Sang et al. 2008 , Elhassan et al. 2015 ). Despite its potential uses, sorghum breeders have historically reported conflicting results with waxy sorghum grain yield and germination (Jampala et al. 2012 ), causing a delay in the adoption of this trait by seed companies. In 2015 the first near-isogenic waxy/wild-type lines and hybrids that overcame these challenges were publicly released, leading to current commercial interest (Yerka et al. 2015 , Yerka et al. 2015 ). Consequently, waxy grain is the first value-added food and beverage quality trait in sorghum to be commercially developed in the U.S. The first generation of commercial waxy hybrids are now in the final stages of development by several seed companies and should be released within the next few years. Nevertheless, most of these efforts are limited to Mendelian approaches that rely on PCR-based molecular markers specific to alleles of the Waxy locus. The genomic relationship between yield, germination, and the waxy trait has not been resolved, and continues to hinder the efficiency of developing high-yielding hybrids. Improved genomics-assisted breeding tools would greatly enhance these efforts. The present study was conducted to develop an updated molecular breeding toolkit for waxy sorghum that can be combined with genomic selection for rapid crop improvement. The full-length wx a allele was integrated into chromosome 10 of the B.Tx623 reference genome (v3.1.1) to improve read mapping in diverse waxy materials at Sobic.010G022600 . Functional competitive allele-specific PCR (PACE-PCR) markers were developed for Wx , wx a , and wx b alleles (the most frequently used alleles in U.S. breeding programs) and validated in numerous pedigrees in public and private breeding programs. This new molecular toolkit should enhance the efficiency of waxy sorghum breeding for the production of climate-smart foods, animal feeds, and biofuels. Materials and Methods Plant Materials . Three sets of sorghum genotypes contributed to this analysis: publicly available materials in the University of Nevada, Reno (UNR; Reno, NV) sorghum breeding program, publicly available materials at the USDA-ARS Plant Stress and Germplasm Development Research Unit (Lubbock, TX), and pre-commercial materials (experimental hybrid parents) at Richardson Seeds (Vega, TX), a subsidiary of Nuseed (Victoria, Australia) (Supplementary Table 1). The UNR materials included B.Tx623 and a selection of previously reported waxy and wild-type near-isogenic lines (referred to hereafter as waxy NILs) (Yerka et al. 2015 a, Yerka et al. 2015 b, Yerka et al. 2016 ) upon which additional phenotypic selection has been imposed for uniformity. The USDA materials included 98 experimental breeding lines in the F 4 generation from various population structures (biparental, multiparental, backcross). The pre-commercial Richardson Seeds materials were coded for privacy and include 19 inbred lines in the F 7 generation or later from biparental populations. All plant materials were grown in Lubbock, TX in 2022. Panicles were mechanically threshed to obtain seeds for analysis. Aliquots (20 g) of bulk seed from 2–3 panicles per entry were evaluated for % amylose content on a whole-grain, dry weight basis (dwb) using published near-infrared spectroscopy (NIRS) protocols at the USDA-ARS Grain Quality and Structure Research Unit in Manhattan, KS (Peiris et al. 2021 ). NIR data quality was assured by only reporting those values with Mahalanobis distances < 4.0 (Whitfield et al. 1987 ). The use of both public and private materials from three independent sorghum breeding programs was a strategy to ensure that new molecular markers are robust and will work across diverse populations and molecular breeding programs. Sequencing the full-length wx a insertion . Full-length sequence of the wx a allele was needed to develop an enhanced assembly inclusive of both sequence and structural variations present at the Waxy locus of chromosome 10. Due to the repetitive nature of the transposon insertion in wx a genotypes, a contiguous assembly of the wx a allele was not possible using PE150 “short” reads from our WGS data, nor in published WGS data from the Sorghum Association Panel (SAP). Instead, a de novo full-length sequence of the wx a allele of ‘Tx2907’ (a common trait donor) was generated using long-range PCR, as follows. Genomic DNA was extracted from immature sorghum leaves using the CTAB method (Porebski et al. 1997 ). A Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the quality and concentration of DNA. Forward (AF: 5′-GGCCTGGATTCAATGTTCTT-3′) and referees (AR: 5′-GCAGCTGGTTGTCCTTGTAG-3′) primers were used with Q5 High-Fidelity 2X Master Mix (NEB, UK) under the following conditions: DNA was denatured at 98°C for 30 sec followed by 33 cycles of 98°C for 10 sec, annealing at 64°C for 30 sec, extension at 72°C for 5 min, and a final extension for 2 min at 72°C. Samples were held at 4°C and stored at -20°C for future use. The amplicon was purified with Monarch PCR & DNA Cleanup Kit (NEB, UK). Cloning was conducted using the Zero Blunt TOPO PCR Cloning Kit. Briefly, the purified PCR product (60–80 ng) was cloned into the TOPO vector and transformed into competent cells of E. coli . After 1-h propagating, the E. coli was streaked onto a 50 µg mL − 1 kanamycin plate and incubated overnight before colony selection and plasmid extraction. Sanger sequencing of the plasmid DNA was conducted by a commercial service provider. The conserved domain of the sequence was annotated at NCBI ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). These efforts were used to enhance the sorghum reference genome (v3.1.1) to contain the full-length wx a allele on chromosome 10 (referred to hereafter as the “enhanced genome assembly”). Specifically, an in-house Biopython program was developed to add the wx a insertion to the reference genome (v3.1.1) by substituting it and its flanking regions for chr10 positions 1862384 to 1862906. Allele-specific PACE marker development . The principle of PACE-PCR is that primers having unique tail sequences target known SNPs and bind to specific alleles (von Maydell 2023 ). Subsequently, during amplification, allele-specific fluorescent tags are added and detected spectrophotometrically so that different alleles can be identified (von Maydell 2023 ). In this study, primers were designed with the tails FAM (5′-GAAGGTGACCAAGTTCATGCT-3′) and HEX (5′-GAAGGTCGGAGTCAACGGATT-3′). The detailed sequences of each marker are shown in Table S1 . Validation of the PACE-PCR markers. The wx a allele was validated using fragment length polymorphisms amplified in PCR and visualized in gel electrophoresis. The primers and PCR conditions were the same as those above for amplifying the full-length wx a insertion, despite differences in fragment lengths. The wx b allele was validated with Sanger sequencing using the forward primer BF (5′-CCTCTGTCATGCTACCTCAAG-3′) and the reverse primer BR (5′-CATTCATTGCATACCCGTCG-3′) in the same PCR conditions as above. Fragments were separated in a 1% agarose gel. Purified PCR products were used for Sanger sequencing validation. PACE-PCR genotyping was conducted on all plant materials following manufacturer instructions (3CR Bioscience, Harlow, United Kingdom) of a CFX96 Touch Deep Well Real-Time PCR Detection System (BIO-RAD, USA). Specifically, 0.15 µL of the assay mix (12 µM for each allele-specific forward primer and 30 µM for a single common reverse primer in the final PCR reaction), 1 µL gDNA (100–200 ng), 3.85 µL ultra-pure distilled water, and 5 µL PACE™ 2.0 Genotyping Master Mix (3CR, Bioscience) were added to a total reaction volume of 10 µL. DNA was denatured at 94°C, then a touch down (65 − 57°C) step was performed for 10 cycles for primer annealing and initial extension. An additional 30 cycles were used for further extension to generate more tail-specific sequences. Fluorescence was visualized and digitalized for automated analysis with Bio-Rad CFX Maestro software. Whole-genome sequencing (WGS) . WGS data was obtained to generate whole-genome coverage at greater depth and coverage than GBS in selected plant materials to enhance our ability to assess read mapping in Wx versus wx a materials using the enhanced genome assembly. These materials included the grain sorghum parent lines ‘KS115’ ( Wx ) and ‘R.Tx2907’ ( wx a ). PE150 libraries were prepared for Illumina sequencing of gDNA (24 Gb/sample, ~ 30x coverage). To evaluate the quality of the raw reads, FastQC ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) (Andrews 2010 ) was utilized. Trimming was conducted by removing nucleotide calls with Phred scores below 20 (Q20) from the 3' ends of the reads. Subsequently, high-quality reads were selected for alignments using Bowtie2 (Langmead and Salzberg 2012 ) with default parameters. Whole-genome sequence of R.Tx2907 was retrieved from publicly available source (Boatwright et al. 2022 ). SAM/BAM alignments obtained were analyzed using the SAMtools (Li et al. 2009 ) and BAMtools (Barnett et al. 2011 ) software suites. The aligned reads mapped to the Waxy locus were visualized and compared using the IGV tool (Thorvaldsdóttir et al. 2013 ). Results The full-length wx a insertion . The causal mutation of the wx a allele is a 5,174 bp insertion containing RNase H (RNH), Reverse Transcriptase (RT), gap-pre-integrase, zf (Zinc knuckle) and Transposase domains; a long terminal repeat (LTR) region, and a GAG (gag-polypeptide) domain (Fig. 1 ). It is a large retrotransposon derivative (LARD) element that belongs to LTR retrotransposons (Kalendar et al. 2004 ). LARD elements are nonautonomous retrotransposons, relying on other autonomous TEs to transpose. LARDs consist of non-coding internal domains and LTR (Havecker et al. 2004 ). Within the LARD, the LTR region is a pair of identical 422 bp sequences at each end of the insertion, which serve as regulatory elements for GAG and POL domains needed for gene expression (Finnegan et al., 2012). RNH protein degrades the 5’ LTR to transcribe RNA and promotes priming of the 3’LTR for RT to synthesize a new cDNA molecule. A target region in the genome is cut by integrase and the newly synthesized cDNA is inserted. The GAG domain contains coding sequences for two structural proteins which are believed to play an important, but as-yet undetermined role (Chaparro et al. 2015 ). PACE-PCR markers . The initial PACE-PCR markers provided excellent discrimination of Wx , wx a , and wx b alleles in homozygous genotypes (Figs. 2 A and 2 C). Primer pairs AF/AR, used to discriminate between B.Tx623 ( Wx ) and R.Tx2907 ( wx a ), amplified bands of 523 bp and 5,702 bp, respectively (Fig. 2 B). Sanger sequencing confirmed the presence of the G-T SNP in wx b genotypes (Fig. 2 D). To validate the performance of the markers in breeding, genotyping was conducted blind (not in the breeders’ labs), without a priori knowledge of pedigrees or Waxy allele(s) in each line. The markers were used to screen a total of 258 genotypes in the UNR, USDA, and Richardson Seeds breeding programs, including individuals that were homozygous and heterozygous for various combinations of Waxy alleles (Fig. 3 ). To test whether the genotyping results matched the amylose contents expected by the breeders, we compared the Waxy alleles of plants (determined from young leaf tissue) to the amylose content of mature grain (Table S2 ). The amylose contents of the UNR and Richardson Seeds programs largely agreed with the genotyping results. Out of 98 USDA-ARS lines genotyped, ten were still segregating for different Waxy alleles (heterozygous) and eliminated from the analysis. Twenty-four out of 26 expected homozygous lines were determined to be homozygous for the correct allele (either wx a or wx b ) and had grain amylose contents below 5%, which is the threshold typically considered to define the waxy phenotype. However, two of those 26 lines were not in fact homozygous and were still segregating for wild-type or heterowaxy ( Wx / wx a or Wx / wx b ) plants that were not genotyped but were harvested and included in the grain quality analysis. Alternatively, some homozygous waxy plants could have been adventitiously cross-pollinated by wild-type pollen despite every attempt to cover them prior to flowering. Nevertheless, the average amylose content of the USDA-ARS waxy lines was 2.12%. Four heterowaxy lines had amylose contents between 10.6% and 17.3%, and 58 of 60 wild-type lines had amylose contents over 15%, consistent with the recessive nature of wx alleles and their dose response in the triploid endosperm (Fig. 4 ). Our results demonstrate the importance of high-throughput molecular markers when breeding with waxy materials, as the phenotype is not visible in the grain and adventitious outcrossing with wild-type pollen carrying dominant alleles can easily contaminate breeding stocks after genotyping is conducted early in the season to raise amylose content through xenia (Karper 1933 , Gorbet and Weibel 1972 ). Overall, the PACE genotypes were consistent with expected grain amylose contents and were able to rapidly identify off-types. They are therefore suitable for detecting adventitious outcrossing (through analysis of harvested grain or leaf tissues in the following generation) as a routine quality control measure leading up to commercialization. As the parental lines are from different breeding programs using different genetic backgrounds, the results indicate that these markers can be broadly used in waxy sorghum breeding. Read-mapping at the Waxy locus . The presence of the large insertion in wx a genotypes results in broken reads when flanking sequences that partially cover both wild-type and insertion sequences fail to align to the wild-type reference genome. This limits the efficiency of local short read mapping during molecular breeding and makes linkage disequilibrium analyses more difficult. The enhanced genome assembly was able to overcome this challenge by providing reference sequences for read mapping across the full-length insertion. When WGS data for the inbred lines KS115 ( Wx ) and R.Tx2907 ( wx a ) were mapped to the wild-type (B.Tx623, v3.1.1) and enhanced reference genome (containing the wx a insertion), the same number of reads were detected between Chr. 10: 1,860,965-1,865,278 for KS115, but an additional 78 uniquely mapped reads covering the insertion were mapped for R.Tx2907 (Fig. 5 ). Discussion Breeding for grain quality traits of sorghum . Historic breeding efforts in U.S. sorghum have focused on grain yield more than quality because phenotyping for quality is typically more time-consuming and expensive; and because producers were paid primarily for yield. However, advances in high-throughput phenotyping and genotyping methods have made it easier than ever to phenotype for quality traits and their interactions with whole-plant (grain + biomass) productivity and per-area yield. There is also a small but rapidly growing world-wide market for grain sorghum with grain quality traits, like low amylose content, so breeding programs are beginning to address them. The global value of the gluten-free products market was estimated at USD 6.3 billion in 2022 and projected to reach USD 11.8 billion by 2030 at a compound annual growth rate of 9.5%, based on a Vantage Market Research report. Highlights of the same report noted that a considerable amount of the increase in value could be attributed to more health-conscious consumers who were interested in methods to extend the shelf life of baked goods, especially breads, without the addition of preservatives – a known property of waxy sorghum flour. Waxy sorghum grain has been studied for many years in the food sciences is a high-value endosperm trait associated with high digestibility by animals (Sherrod et al. 1969 , Walker and Lichtenw.Re 1974, Myer and Gorbet 1985 , Schroeder et al. 1998 ), rapid fermentation into ethanol (Yan et al. 2011 , Wu et al. 2013 ), the production of alcoholic beverages (Hsieh and Pi 1979 , Mugode et al. 2011 , Mezgebe et al. 2018 ), and improved baking properties (Sang et al. 2008 , Elhassan et al. 2015 ) due to high-amylopectin starch content. While the waxy trait has been identified since the 1930s (Karper 1933 ), recent hybrids are available with no yield drag or poor agronomic performance compared to near-isogenic or closely-related wild-type hybrids (Jampala et al. 2012 , Yerka et al. 2016 ). It has also been linked to lower resistant starch content, an important factor in food functionality that must be balanced with diet because, when consumed in excess, it could result in unhealthy gut microbiomes, based on human stool microbiomes and mouse feeding studies (Yang et al. 2023 ). This is because the increased digestibility of waxy starches can raise the glycemic index (Frei et al. 2003 , Fitzgerald et al. 2011 ). Sorghum breeders are interested in combining waxy grain with additional target traits such as larger seed size, improved protein content and digestibility, improved germination, sugarcane aphid resistance, earlier maturity, etc. to improve their commercial value, expand production into additional regions, and help open underexplored markets where value-added, climate-smart crops are urgently needed. In particular, the western U.S. may be ideal for the development of new grain quality traits due to minimal precipitation throughout the growing season and harvest period, which can reduce grain quality in more central and eastern regions of the country. Developing waxy sorghum hybrids with improved trait combinations for deployment in new areas will be most efficiently achieved using advanced molecular breeding techniques. The PACE-PCR markers and updated chromosome 10 assembly presented herein provide a solid foundation for those efforts. Molecular breeding resources for adapting sorghum yield and quality to local environments . Genome-to-phenome (G2P) modeling combines physiological information about plant developmental processes with genomic, agronomic, and environomic data. These methods, which often use artificial intelligence (AI) to optimize the predictive capacity of genetic models for improved gene discovery, have emerged as the most effective method for clarifying relationships between inputs and outputs in cropping systems (Harfouche et al. 2019 , Xu et al. 2022 ). Hence, to secure both the abundance and quality of food systems, machine learning will become increasingly important to bring phenotyping for quality traits up to the speed and efficiency of phenotyping for yield for rapid improvements to the sustainable production of microscale, biochemical traits (Yan and Wang 2023 ), like amylose or amylopectin content in climate-smart species like sorghum. The current study reports functional markers for waxy grain, which is not amenable to high-throughput phenotyping in field environments prior to the time of flowering. However, genotyping young plants with NGS markers in genomic selection is becoming standard in many companies, so the reported markers will provide immediate improvements to breeding efficiency by shortening the commercial breeding cycle. They will also assist with ongoing quality control by identifying progeny that are outcrosses derived from adventitious cross-pollination of maternal plants in the previous generation. The improved read mapping achieved in wx a genotypes using the enhanced genome assembly is consistent with what other researchers have demonstrated when incorporating presence-absence and copy number variation into the reference genomes used for making marker-trait associations (Bayer et al. 2020 , Jayakodi et al. 2020 , Tao et al. 2021 , Li et al. 2022 , Sun et al. 2022 ). Future work in the sorghum grain quality space should focus on creating pangenomes that account for even more quality traits not expressed in existing reference genomes. Breeding for human food and animal feed end uses will depend upon identifying functional markers for micro- and macronutrient contents (e.g. starch, protein, oil, amylose, lysine, beta carotene, zinc, iron, etc.), percent vitreosity, kernel hardness, resistant starch content, fiber, seed size, and the interactions of diverse grain physiochemistry profiles with grain weathering and molding, as no in-field, high-throughput phenotyping platform currently exists for them. The ongoing reliance of most breeding programs on laboratory-based quality testing lengthens the breeding cycle when grain must first be harvested and analyzed before superior genotypes can be identified and advanced to the next generation. In addition, the interaction between functional grain qualities and production environments can be impacted by presence-absence variation that plays a role in other aspects of plant physiology and adaptation, such as loci controlling plant height, maturity, and thermal or photoperiod sensitivity. As reference genomes improve, plant breeders will be increasingly able to develop plant populations with improved combinations of yield and quality traits that express well in target environments. Conclusions The waxy trait is one of the first specialty grain quality traits of U.S. sorghum to be commercially produced. The validated markers in our study will be beneficial for various practical applications, such as germplasm characterization, allele mining, and marker assisted selection during backcrossing and forward breeding programs to improve sorghum food, feed, and fuel applications. We have built upon previous efforts to genetically characterize the Waxy locus by developing an enhanced chromosome 10 assembly for read mapping and functional NGS markers. These new resources provide an updated molecular toolkit for sorghum breeding that was validated in three public and private programs. This toolkit will improve the efficiency of breeding waxy sorghum using genomic selection and genome-to-phenome methodologies for rapid functional trait adaptation to target production environments. Declarations Data Availability All datasets generated during and/or analyzed during the current study are available in the supplementary files associated with this manuscript. STATEMENTS AND DECLARATIONS Funding. Funding for the development and analysis of the UNR breeding lines was provided by a United States Department of Agriculture Multi-State Hatch Award (NEV00763) and a United States Department of Agriculture Award (2019-67014-29174) to MKY. Funding for PACE-PCR assays, genomic analyses, and field studies at Texas Tech University (TTU) was provided by new faculty start-up funding to YJ. ZL was supported by the Graduate Support Stipend fellowship from the TTU Davis college of Agricultural Science and Natural Resources obtained by YJ and GP. Funding for the development of the USDA experimental breeding lines was provided by United States Department of Agriculture, Agricultural Research Service Project Plan 3096-21000-024-000-D. Richardson Seeds funded the development of their own experimental breeding lines and provided in-kind funding to MKY to support field studies conducted in Vega, TX during the development of the UNR breeding lines. Competing Interests. The authors have no relevant financial or non-financial interests to disclose. Author Contributions MY and ZL are co-first authors. MY and YJ are co-corresponding authors who designed the studies and analyzed data. MY, YJ, and GP obtained funding to conduct the work. ZL and DN developed the PACE-PCR markers and analyzed data. MY developed the UNR plant materials and led field studies using the UNR and Richardson Seeds plant materials with the assistance of YJ and ZL in Lubbock, TX. CH developed and conducted field studies with the USDA sorghum materials. DD, GK, SM, and GC developed the pre-commercial materials and analyzed data. SB conducted grain quality analyses. All authors read and approved the final manuscript. References Sorghum bicolor BTx642 v1.1, DOE-JGI, http://phytozome.jgi.doe.gov/ Sorghum bicolor RTx430 v2.1, DOE-JGI, http://phytozome.jgi.doe.gov/ Sorghum bicolor SC187 v1.1, DOE-JGI, http://phytozome-next.jgi.doe.gov/info/SbicolorSC187_v1_1 Sorghum bicolor v5.1 DOE-JGI, https://phytozome.jgi.doe.gov/info/Sbicolor_v5_1 Sorghum bicolor Wray v1.1 DOE-JGI, https://phytozome.jgi.doe.gov/info/SbicolorWray_v1_1 Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. 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Crop Sci 56(1):113–121. https://doi.org/10.2135/cropsci2015.03.0151 Supplementary Files SupplementaryFile1WaxyLocusAssemblywithInsertion.docx SupplementaryTables1and2.xlsx Cite Share Download PDF Status: Published Journal Publication published 11 Aug, 2025 Read the published version in Journal of Applied Genetics → Version 1 posted Editorial decision: Major Revisions Needed 04 Feb, 2025 Reviewers agreed at journal 12 Sep, 2024 Reviewers invited by journal 05 Sep, 2024 Editor assigned by journal 12 Aug, 2024 First submitted to journal 08 Aug, 2024 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. We do this by developing innovative software and high quality services for the global research community. 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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-4883126","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":349861822,"identity":"f915d61f-9697-4aea-a80c-6744411f5925","order_by":0,"name":"Melinda Yerka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYFAC5oYDDGwgBpD4AKLYCWphRGhhnAGimInQwgDTwswDtpaABt32xsbDBWV2if3SxxI/2/zaJs/HzMD44WMObi1mZw42HJ5xLjlxZl/aYencvtuGbcwMzJIzt+HRciOx4TBvG3PihjPsDdK5PbcZgVrYmHnxabn/EKSlPnH/Gfbm35Y9t+0Ja7nBCNJyOHEDD9sxaYYftxMJazkDdBjPuePGM86wpVn2NtxObmNmbMbvl+OHD3/mKauW7e9hM77x489t2/ntzQc/fMSjBQYcG0AkYxuYbCCsHgjsIdQfohSPglEwCkbBCAMA1uxVwy7WfpwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7210-8684","institution":"University of Nevada Reno","correspondingAuthor":true,"prefix":"","firstName":"Melinda","middleName":"","lastName":"Yerka","suffix":""},{"id":349861823,"identity":"1f3f7f05-64f7-49c5-9f29-1b85ab02bcc1","order_by":1,"name":"Zhiyuan Liu","email":"","orcid":"","institution":"Texas Tech University","correspondingAuthor":false,"prefix":"","firstName":"Zhiyuan","middleName":"","lastName":"Liu","suffix":""},{"id":349861824,"identity":"cfaee194-c358-4d4f-8e7c-7b3965d929c8","order_by":2,"name":"Scott Bean","email":"","orcid":"","institution":"USDA-ARS: USDA Agricultural Research Service","correspondingAuthor":false,"prefix":"","firstName":"Scott","middleName":"","lastName":"Bean","suffix":""},{"id":349861825,"identity":"ee899147-a045-4041-b975-715ea10df060","order_by":3,"name":"Deepti Nigam","email":"","orcid":"","institution":"Texas Tech University","correspondingAuthor":false,"prefix":"","firstName":"Deepti","middleName":"","lastName":"Nigam","suffix":""},{"id":349861826,"identity":"3d01a004-20b1-4877-b07d-f60cced88a75","order_by":4,"name":"Chad Hayes","email":"","orcid":"","institution":"USDA ARS: USDA Agricultural Research Service","correspondingAuthor":false,"prefix":"","firstName":"Chad","middleName":"","lastName":"Hayes","suffix":""},{"id":349861827,"identity":"dfd8abdb-40dc-4f35-8457-cbfb34e9280a","order_by":5,"name":"Diego Druetto","email":"","orcid":"","institution":"Richardson Seeds","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Druetto","suffix":""},{"id":349861828,"identity":"2c042a5b-753b-4f09-9d2d-e5f601597423","order_by":6,"name":"Gabriel Krishnamoorthy","email":"","orcid":"","institution":"Richardson Seeds","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Krishnamoorthy","suffix":""},{"id":349861829,"identity":"75526067-065e-470e-842f-16bc9d300080","order_by":7,"name":"Shelley Meiwes","email":"","orcid":"","institution":"Richardson Seeds","correspondingAuthor":false,"prefix":"","firstName":"Shelley","middleName":"","lastName":"Meiwes","suffix":""},{"id":349861830,"identity":"05964860-523b-4284-98c1-358d6b6c3b28","order_by":8,"name":"Gonzalo Cucit","email":"","orcid":"","institution":"NuSeed","correspondingAuthor":false,"prefix":"","firstName":"Gonzalo","middleName":"","lastName":"Cucit","suffix":""},{"id":349861831,"identity":"b63c2da6-5612-40e3-94a2-8d12a37ab35b","order_by":9,"name":"Gunvant B. Patil","email":"","orcid":"","institution":"Texas Tech University","correspondingAuthor":false,"prefix":"","firstName":"Gunvant","middleName":"B.","lastName":"Patil","suffix":""},{"id":349861832,"identity":"a5ed40a2-19ae-4347-a3db-83c48ec149f0","order_by":10,"name":"Yinping Jiao","email":"","orcid":"","institution":"Texas Tech University","correspondingAuthor":false,"prefix":"","firstName":"Yinping","middleName":"","lastName":"Jiao","suffix":""}],"badges":[],"createdAt":"2024-08-08 20:30:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4883126/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4883126/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13353-025-00993-1","type":"published","date":"2025-08-11T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66101688,"identity":"fd42b2e1-24eb-4d11-ae63-67042a0d3b25","added_by":"auto","created_at":"2024-10-07 17:17:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106265,"visible":true,"origin":"","legend":"\u003cp\u003eThe genetic architecture of three alleles of the sorghum \u003cem\u003eWaxy\u003c/em\u003e locus, \u003cem\u003eSobic.010G022600\u003c/em\u003e, including the site of the full-length insertion that is causal of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele and the SNP (G-T) that is causal of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e allele. Exons are black. The primers AF/AR and BF/BR are forward/reverse primers that were used to amplify the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e insertion and the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e SNP, respectively. LTR, Long Terminal Repeats; RT, Reverse Transcriptase; gag_p, GAG-pre-integrase domain; zf, Zinc knuckle; GAG, gag-polypeptide\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4883126/v1/a675b119fad5f3a52ac44fba.png"},{"id":66101460,"identity":"cbac85dc-d3ef-46b6-8a9d-03f1f8e34026","added_by":"auto","created_at":"2024-10-07 17:09:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":314497,"visible":true,"origin":"","legend":"\u003cp\u003eOriginal PACE-PCR marker design for \u003cem\u003eWx\u003c/em\u003e, \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e alleles was based on the inbred lines R.Tx2907 (\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e), B.TxARG1 (\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e), and B.Tx623 (\u003cem\u003eWx\u003c/em\u003e). (A) Genotyping results for the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e marker. Three individuals were tested for each genotype. One no-template control (NTC) and one positive control (\u003cem\u003eWx\u003c/em\u003e) were included. Clustered blue and orange dots represent \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eWx\u003c/em\u003e genotypes, respectively, and the black dot represents NTC. (B) PCR validation of the full-length 5,174 bp \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e insertion. Lanes 1-4 are individual plants of R.Tx2907 (\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e) and lanes 5-6 are individual plants of B.Tx623 (\u003cem\u003eWx\u003c/em\u003e). All fragments were amplified with the primer pair AF1/AR1. The band sizes were 5702 bp and 523 bp for the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eWx\u003c/em\u003e alleles, respectively. (C) Original PACE-PCR marker design for \u003cem\u003eWx\u003c/em\u003e and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e alleles. Three individuals were tested for each genotype. One NTC and one positive control (\u003cem\u003eWx\u003c/em\u003e) were included. Clustered blue and orange dots represent \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eWx\u003c/em\u003e genotypes, respectively, and the black dot is the NTC. (D) The primer pair BF1/BR1 was used to amplify the SNP that is causal of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e allele using two individuals each from B.TxARG1, B.N631, and B.N633. A G-T SNP is causal of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e allele\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4883126/v1/c507845b17204a81297dfcfc.png"},{"id":66100959,"identity":"b838604b-1dc7-4942-89cd-b7cf70fc34df","added_by":"auto","created_at":"2024-10-07 17:01:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120835,"visible":true,"origin":"","legend":"\u003cp\u003ePACE-PCR genotyping results for the three breeding programs, including both homozygotes and heterozygotes. (A) Genotyping results using the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e marker. Clustered blue dots represent \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e genotypes, green dots represent \u003cem\u003eWxwx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e genotypes, and orange dots represent \u003cem\u003eWxWx\u003c/em\u003e genotypes. (B) Genotyping results using the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e marker. Clustered blue dots represent \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e genotypes, green dots represent \u003cem\u003eWxwx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e genotypes, and orange dots represent \u003cem\u003eWxWx\u003c/em\u003e genotypes\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4883126/v1/6ea272c536ea6c8297c7e254.png"},{"id":66100962,"identity":"2ba6cace-048c-4980-9fe9-f1ce5d85c278","added_by":"auto","created_at":"2024-10-07 17:01:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":96981,"visible":true,"origin":"","legend":"\u003cp\u003eAmylose contents (% whole grain, dwb) of individuals from waxy, heterowaxy, and wild-type breeding lines, as reported by the USDA-ARS breeding program. A subsample of these individuals was genotyped with PACE-PCR markers using leaf tissue. The red dotted line represents the phenotypic threshold that is conventionally used to define a line as being waxy (5% amylose). Black dots represent the amylose contents of single plants; more individuals per line were evaluated for amylose content than were genotyped. Each column is the expected \u003cem\u003eWaxy \u003c/em\u003ehaplotype reported by the breeder who contributed seeds, with the diploid \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e genotype first and the diploid \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e genotype second: \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(homozygous \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e), \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(homozygous \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e), \u003cem\u003eWxwx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(heterozygous for \u003cem\u003eWx\u003c/em\u003e and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e), \u003cem\u003eWxwx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(heterozygous for \u003cem\u003eWx\u003c/em\u003e and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e), \u003cem\u003eWxWx \u003c/em\u003e(wild-type). The low (\u0026lt; 5%) and intermediate (10-15%) amylose contents in individuals in the wild-type (\u003cem\u003eWxWxWxWx\u003c/em\u003e) column indicate that the breeding line was still segregating for recessive \u003cem\u003ewaxy\u003c/em\u003e alleles in ungenotyped plants that contributed to grain quality analysis, or xenia from adventitious outcrossing with wild-type materials in the previous generation.\u0026nbsp; The intermediate amylose contents (10-11%) in the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eWxWx\u003c/em\u003e and \u003cem\u003eWxWxWxWx\u003c/em\u003e columns indicate that some plants were heterowaxy (\u003cem\u003eWxwx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eWxWx\u003c/em\u003e, \u003cem\u003eWxwx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eWx\u003c/em\u003ewxb, or \u003cem\u003eWxWxWx wx\u003c/em\u003e), also due to segregation or xenia in ungenotyped individuals. These results demonstrate the importance of a rapid and high-throughput genotyping assay while working with the waxy trait to prevent undesirable \u003cem\u003ewaxy\u003c/em\u003e or wild-type alleles from contributing to consecutive generations\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4883126/v1/71483bf1448e6d1d93ec5e70.png"},{"id":66100964,"identity":"a0aa9194-1bed-417a-bc51-18847c9df032","added_by":"auto","created_at":"2024-10-07 17:01:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":463480,"visible":true,"origin":"","legend":"\u003cp\u003eWGS reads from \u003cem\u003eWx\u003c/em\u003e (KS115) and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e (R.Tx2907) inbred lines mapped to the wild-type (\u003cem\u003eWx\u003c/em\u003e) B.Tx623 reference genome (v3.1.1) and the enhanced genome assembly including the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e insertion. The \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e primer pair AF/AR was used to sequence the full-length insertion containing a LARD retrotransposon. An additional 78 unique reads of R.Tx2907 mapped to the enhanced genome assembly compared to the wild-type genome\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4883126/v1/f666c904ed10641bb1e9d306.png"},{"id":89310650,"identity":"1f05bcc4-6541-4632-b6fc-b25dfae7fc6e","added_by":"auto","created_at":"2025-08-18 16:09:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1763557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4883126/v1/72f822e4-f1cc-4948-8c70-5cb07281e4f1.pdf"},{"id":66100963,"identity":"aa7e0fd3-348f-4371-b647-c8625d429253","added_by":"auto","created_at":"2024-10-07 17:01:29","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16493,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile1WaxyLocusAssemblywithInsertion.docx","url":"https://assets-eu.researchsquare.com/files/rs-4883126/v1/c8371a8369870db1a5d49910.docx"},{"id":66101689,"identity":"7824d688-b47e-4a5f-a6a2-cd3c0b1e4327","added_by":"auto","created_at":"2024-10-07 17:17:29","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":24040,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables1and2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4883126/v1/6756dd46e6306c6d9e943e52.xlsx"}],"financialInterests":"","formattedTitle":"An enhanced genome assembly and functional, high-throughput molecular markers enable genomics-assisted breeding of waxy sorghum [Sorghum bicolor (L.) Moench]","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSorghum [\u003cem\u003eSorghum bicolor\u003c/em\u003e (L.) Moench] is the fifth-most produced grain crop in the world. It is undergoing major development as a climate-smart species due to its wide genetic diversity and its ability to tolerate hot, dry, low-input environments. Sorghum can be marketed with gluten-free, non-GMO, and ancient grain labels in specialty foods, food ingredients, and alcoholic beverages. As a diploid (2\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003ex\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) with a genome size of ~\u0026thinsp;700 Mbp, several published reference genomes (Deschamps et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, McCormick et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Cooper et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Tao et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and pangenomes (Deschamps et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Ruperao et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), publicly available mutant and mapping populations (Kumar et al., Xin et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Yerka et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Brenton et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Boyles et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Mace et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Xin et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and large whole-genome sequencing data of diversity populations (Mace et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Boatwright et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), it is also an emerging model grass species, particularly for sugarcane [\u003cem\u003eSaccharum oficinarum\u003c/em\u003e L.].\u003c/p\u003e \u003cp\u003eCertain mutations of the sorghum \u003cem\u003eGRANULE-BOUND STARCH SYNTHASE\u003c/em\u003e (\u003cem\u003eGBSS\u003c/em\u003e) gene [\u003cem\u003eSobic.010G022600\u003c/em\u003e] result in a low amylose:amylopectin starch ratio. In seeds, low-amylose endosperms have a sticky, \u0026ldquo;waxy\u0026rdquo; texture; hence, the wild-type gene is commonly referred to as \u003cem\u003eWaxy\u003c/em\u003e. GBSS is highly conserved in the Poaceae and has been extensively reviewed for its role in cereal starch synthesis (Tetlow and Emes \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Tetlow \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Nakamura \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), including in waxy maize [\u003cem\u003eZea mays\u003c/em\u003e L.] (Talukder et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), glutinous rice [\u003cem\u003eOryza sativa\u003c/em\u003e L.] (Okpala et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Jin et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Nakamura \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and sorghum (Kang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among sorghum breeders, the waxy grain phenotype is generally considered to be ~\u0026thinsp;95\u0026ndash;100% amylopectin. A dosage effect on amylopectin content depends on how many recessive \u003cem\u003ewaxy\u003c/em\u003e alleles are inherited by the triploid endosperm (Karper \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1933\u003c/span\u003e, Yerka et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In 1933, R.E. Karper first documented the Mendelian inheritance of individual alleles of wild-type (\u003cem\u003eWx\u003c/em\u003e) and waxy (\u003cem\u003ewx\u003c/em\u003e) grain phenotypes in sorghum (Karper \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1933\u003c/span\u003e). Pedersen et al. (Pedersen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) were first to characterize the \u003cem\u003ewaxy a\u003c/em\u003e (\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e) and \u003cem\u003ewaxy b\u003c/em\u003e (\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e) alleles with respect to the production of GBSS: \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e is a knock-out allele producing no GBSS, whereas \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e is \u0026ldquo;leaky\u0026rdquo; and produces low levels of non-functional GBSS. Sattler et al. (Sattler et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) performed the first molecular characterization of \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e alleles and identified the causal mutations of each one: the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele contains a large insertion in the third exon, whereas the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e allele contains a C to T transition converting amino acid 268 from glutamine to histidine. Since then, two additional alleles have been reported in East Asia, although their nomenclature is inconsistent across publications (Kawahigashi et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e allele described by Lu et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) has a G deletion at the 5' splicing site of the ninth intron, causing a frameshift mutation and early translation termination. The \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sup\u003e allele described by the same authors contains a mutation at the 3' splicing site of the tenth intron, causing a splicing site shift and deletion of five amino acids (GTGKK) in the predicted translated protein.\u003c/p\u003e \u003cp\u003eWhile sorghum is a key subsistence crop for human food and animal feed in Sub-Saharan African and Southeast Asia, it has historically been bred for grain yield and animal feed in North and South America, Europe, and Australia. Waxy sorghum grain has long been associated with improved whole-grain digestibility by animals (Sherrod et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1969\u003c/span\u003e, Walker and Lichtenw.Re 1974, Myer and Gorbet \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1985\u003c/span\u003e, Schroeder et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), rapid fermentation into ethanol for biofuel production (Yan et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), the production of alcoholic beverages (Hsieh and Pi \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Mugode et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Mezgebe et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and improved baking properties (Sang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Elhassan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Despite its potential uses, sorghum breeders have historically reported conflicting results with waxy sorghum grain yield and germination (Jampala et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), causing a delay in the adoption of this trait by seed companies. In 2015 the first near-isogenic waxy/wild-type lines and hybrids that overcame these challenges were publicly released, leading to current commercial interest (Yerka et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Yerka et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consequently, waxy grain is the first value-added food and beverage quality trait in sorghum to be commercially developed in the U.S. The first generation of commercial waxy hybrids are now in the final stages of development by several seed companies and should be released within the next few years. Nevertheless, most of these efforts are limited to Mendelian approaches that rely on PCR-based molecular markers specific to alleles of the \u003cem\u003eWaxy\u003c/em\u003e locus. The genomic relationship between yield, germination, and the waxy trait has not been resolved, and continues to hinder the efficiency of developing high-yielding hybrids. Improved genomics-assisted breeding tools would greatly enhance these efforts.\u003c/p\u003e \u003cp\u003eThe present study was conducted to develop an updated molecular breeding toolkit for waxy sorghum that can be combined with genomic selection for rapid crop improvement. The full-length \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele was integrated into chromosome 10 of the B.Tx623 reference genome (v3.1.1) to improve read mapping in diverse waxy materials at \u003cem\u003eSobic.010G022600\u003c/em\u003e. Functional competitive allele-specific PCR (PACE-PCR) markers were developed for \u003cem\u003eWx\u003c/em\u003e, \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e alleles (the most frequently used alleles in U.S. breeding programs) and validated in numerous pedigrees in public and private breeding programs. This new molecular toolkit should enhance the efficiency of waxy sorghum breeding for the production of climate-smart foods, animal feeds, and biofuels.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cem\u003ePlant Materials\u003c/em\u003e. Three sets of sorghum genotypes contributed to this analysis: publicly available materials in the University of Nevada, Reno (UNR; Reno, NV) sorghum breeding program, publicly available materials at the USDA-ARS Plant Stress and Germplasm Development Research Unit (Lubbock, TX), and pre-commercial materials (experimental hybrid parents) at Richardson Seeds (Vega, TX), a subsidiary of Nuseed (Victoria, Australia) (Supplementary Table\u0026nbsp;1). The UNR materials included B.Tx623 and a selection of previously reported waxy and wild-type near-isogenic lines (referred to hereafter as waxy NILs) (Yerka et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003ea, Yerka et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003eb, Yerka et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) upon which additional phenotypic selection has been imposed for uniformity. The USDA materials included 98 experimental breeding lines in the F\u003csub\u003e4\u003c/sub\u003e generation from various population structures (biparental, multiparental, backcross). The pre-commercial Richardson Seeds materials were coded for privacy and include 19 inbred lines in the F\u003csub\u003e7\u003c/sub\u003e generation or later from biparental populations. All plant materials were grown in Lubbock, TX in 2022. Panicles were mechanically threshed to obtain seeds for analysis. Aliquots (20 g) of bulk seed from 2\u0026ndash;3 panicles per entry were evaluated for % amylose content on a whole-grain, dry weight basis (dwb) using published near-infrared spectroscopy (NIRS) protocols at the USDA-ARS Grain Quality and Structure Research Unit in Manhattan, KS (Peiris et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). NIR data quality was assured by only reporting those values with Mahalanobis distances\u0026thinsp;\u0026lt;\u0026thinsp;4.0 (Whitfield et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). The use of both public and private materials from three independent sorghum breeding programs was a strategy to ensure that new molecular markers are robust and will work across diverse populations and molecular breeding programs.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSequencing the full-length\u003c/em\u003e wx\u003csup\u003ea\u003c/sup\u003e \u003cem\u003einsertion\u003c/em\u003e. Full-length sequence of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele was needed to develop an enhanced assembly inclusive of both sequence and structural variations present at the \u003cem\u003eWaxy\u003c/em\u003e locus of chromosome 10. Due to the repetitive nature of the transposon insertion in \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e genotypes, a contiguous assembly of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele was not possible using PE150 \u0026ldquo;short\u0026rdquo; reads from our WGS data, nor in published WGS data from the Sorghum Association Panel (SAP). Instead, a \u003cem\u003ede novo\u003c/em\u003e full-length sequence of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele of \u0026lsquo;Tx2907\u0026rsquo; (a common trait donor) was generated using long-range PCR, as follows. Genomic DNA was extracted from immature sorghum leaves using the CTAB method (Porebski et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). A Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the quality and concentration of DNA. Forward (AF: 5\u0026prime;-GGCCTGGATTCAATGTTCTT-3\u0026prime;) and referees (AR: 5\u0026prime;-GCAGCTGGTTGTCCTTGTAG-3\u0026prime;) primers were used with Q5 High-Fidelity 2X Master Mix (NEB, UK) under the following conditions: DNA was denatured at 98\u0026deg;C for 30 sec followed by 33 cycles of 98\u0026deg;C for 10 sec, annealing at 64\u0026deg;C for 30 sec, extension at 72\u0026deg;C for 5 min, and a final extension for 2 min at 72\u0026deg;C. Samples were held at 4\u0026deg;C and stored at -20\u0026deg;C for future use. The amplicon was purified with Monarch PCR \u0026amp; DNA Cleanup Kit (NEB, UK). Cloning was conducted using the Zero Blunt TOPO PCR Cloning Kit. Briefly, the purified PCR product (60\u0026ndash;80 ng) was cloned into the TOPO vector and transformed into competent cells of \u003cem\u003eE. coli\u003c/em\u003e. After 1-h propagating, the \u003cem\u003eE. coli\u003c/em\u003e was streaked onto a 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin plate and incubated overnight before colony selection and plasmid extraction. Sanger sequencing of the plasmid DNA was conducted by a commercial service provider. The conserved domain of the sequence was annotated at NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These efforts were used to enhance the sorghum reference genome (v3.1.1) to contain the full-length \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele on chromosome 10 (referred to hereafter as the \u0026ldquo;enhanced genome assembly\u0026rdquo;). Specifically, an in-house Biopython program was developed to add the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e insertion to the reference genome (v3.1.1) by substituting it and its flanking regions for chr10 positions 1862384 to 1862906.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAllele-specific PACE marker development\u003c/em\u003e. The principle of PACE-PCR is that primers having unique tail sequences target known SNPs and bind to specific alleles (von Maydell \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Subsequently, during amplification, allele-specific fluorescent tags are added and detected spectrophotometrically so that different alleles can be identified (von Maydell \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, primers were designed with the tails FAM (5\u0026prime;-GAAGGTGACCAAGTTCATGCT-3\u0026prime;) and HEX (5\u0026prime;-GAAGGTCGGAGTCAACGGATT-3\u0026prime;). The detailed sequences of each marker are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eValidation of the PACE-PCR markers.\u003c/em\u003e The \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele was validated using fragment length polymorphisms amplified in PCR and visualized in gel electrophoresis. The primers and PCR conditions were the same as those above for amplifying the full-length \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e insertion, despite differences in fragment lengths. The \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e allele was validated with Sanger sequencing using the forward primer BF (5\u0026prime;-CCTCTGTCATGCTACCTCAAG-3\u0026prime;) and the reverse primer BR (5\u0026prime;-CATTCATTGCATACCCGTCG-3\u0026prime;) in the same PCR conditions as above. Fragments were separated in a 1% agarose gel. Purified PCR products were used for Sanger sequencing validation.\u003c/p\u003e \u003cp\u003ePACE-PCR genotyping was conducted on all plant materials following manufacturer instructions (3CR Bioscience, Harlow, United Kingdom) of a CFX96 Touch Deep Well Real-Time PCR Detection System (BIO-RAD, USA). Specifically, 0.15 \u0026micro;L of the assay mix (12 \u0026micro;M for each allele-specific forward primer and 30 \u0026micro;M for a single common reverse primer in the final PCR reaction), 1 \u0026micro;L gDNA (100\u0026ndash;200 ng), 3.85 \u0026micro;L ultra-pure distilled water, and 5 \u0026micro;L PACE\u0026trade; 2.0 Genotyping Master Mix (3CR, Bioscience) were added to a total reaction volume of 10 \u0026micro;L. DNA was denatured at 94\u0026deg;C, then a touch down (65\u0026thinsp;\u0026minus;\u0026thinsp;57\u0026deg;C) step was performed for 10 cycles for primer annealing and initial extension. An additional 30 cycles were used for further extension to generate more tail-specific sequences. Fluorescence was visualized and digitalized for automated analysis with Bio-Rad CFX Maestro software.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWhole-genome sequencing (WGS)\u003c/em\u003e. WGS data was obtained to generate whole-genome coverage at greater depth and coverage than GBS in selected plant materials to enhance our ability to assess read mapping in \u003cem\u003eWx\u003c/em\u003e versus \u003cem\u003ewx\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e materials using the enhanced genome assembly. These materials included the grain sorghum parent lines \u0026lsquo;KS115\u0026rsquo; (\u003cem\u003eWx\u003c/em\u003e) and \u0026lsquo;R.Tx2907\u0026rsquo; (\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e). PE150 libraries were prepared for Illumina sequencing of gDNA (24 Gb/sample, ~\u0026thinsp;30x coverage). To evaluate the quality of the raw reads, FastQC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Andrews \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) was utilized. Trimming was conducted by removing nucleotide calls with Phred scores below 20 (Q20) from the 3' ends of the reads. Subsequently, high-quality reads were selected for alignments using Bowtie2 (Langmead and Salzberg \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) with default parameters. Whole-genome sequence of R.Tx2907 was retrieved from publicly available source (Boatwright et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). SAM/BAM alignments obtained were analyzed using the SAMtools (Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and BAMtools (Barnett et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) software suites. The aligned reads mapped to the \u003cem\u003eWaxy\u003c/em\u003e locus were visualized and compared using the IGV tool (Thorvaldsd\u0026oacute;ttir et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eThe full-length\u003c/em\u003e wx\u003csup\u003ea\u003c/sup\u003e \u003cem\u003einsertion\u003c/em\u003e. The causal mutation of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele is a 5,174 bp insertion containing RNase H (RNH), Reverse Transcriptase (RT), gap-pre-integrase, zf (Zinc knuckle) and Transposase domains; a long terminal repeat (LTR) region, and a GAG (gag-polypeptide) domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It is a large retrotransposon derivative (LARD) element that belongs to LTR retrotransposons (Kalendar et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). LARD elements are nonautonomous retrotransposons, relying on other autonomous TEs to transpose. LARDs consist of non-coding internal domains and LTR (Havecker et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Within the LARD, the LTR region is a pair of identical 422 bp sequences at each end of the insertion, which serve as regulatory elements for GAG and POL domains needed for gene expression (Finnegan et al., 2012). RNH protein degrades the 5\u0026rsquo; LTR to transcribe RNA and promotes priming of the 3\u0026rsquo;LTR for RT to synthesize a new cDNA molecule. A target region in the genome is cut by integrase and the newly synthesized cDNA is inserted. The GAG domain contains coding sequences for two structural proteins which are believed to play an important, but as-yet undetermined role (Chaparro et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003ePACE-PCR markers\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe initial PACE-PCR markers provided excellent discrimination of \u003cem\u003eWx\u003c/em\u003e, \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e alleles in homozygous genotypes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Primer pairs AF/AR, used to discriminate between B.Tx623 (\u003cem\u003eWx\u003c/em\u003e) and R.Tx2907 (\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e), amplified bands of 523 bp and 5,702 bp, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Sanger sequencing confirmed the presence of the G-T SNP in \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the performance of the markers in breeding, genotyping was conducted blind (not in the breeders\u0026rsquo; labs), without \u003cem\u003ea priori\u003c/em\u003e knowledge of pedigrees or \u003cem\u003eWaxy\u003c/em\u003e allele(s) in each line. The markers were used to screen a total of 258 genotypes in the UNR, USDA, and Richardson Seeds breeding programs, including individuals that were homozygous and heterozygous for various combinations of \u003cem\u003eWaxy\u003c/em\u003e alleles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test whether the genotyping results matched the amylose contents expected by the breeders, we compared the \u003cem\u003eWaxy\u003c/em\u003e alleles of plants (determined from young leaf tissue) to the amylose content of mature grain (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The amylose contents of the UNR and Richardson Seeds programs largely agreed with the genotyping results. Out of 98 USDA-ARS lines genotyped, ten were still segregating for different \u003cem\u003eWaxy\u003c/em\u003e alleles (heterozygous) and eliminated from the analysis. Twenty-four out of 26 expected homozygous lines were determined to be homozygous for the correct allele (either \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e) and had grain amylose contents below 5%, which is the threshold typically considered to define the waxy phenotype. However, two of those 26 lines were not in fact homozygous and were still segregating for wild-type or heterowaxy (\u003cem\u003eWx\u003c/em\u003e/\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eWx\u003c/em\u003e/\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e) plants that were not genotyped but were harvested and included in the grain quality analysis. Alternatively, some homozygous waxy plants could have been adventitiously cross-pollinated by wild-type pollen despite every attempt to cover them prior to flowering. Nevertheless, the average amylose content of the USDA-ARS waxy lines was 2.12%. Four heterowaxy lines had amylose contents between 10.6% and 17.3%, and 58 of 60 wild-type lines had amylose contents over 15%, consistent with the recessive nature of \u003cem\u003ewx\u003c/em\u003e alleles and their dose response in the triploid endosperm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur results demonstrate the importance of high-throughput molecular markers when breeding with waxy materials, as the phenotype is not visible in the grain and adventitious outcrossing with wild-type pollen carrying dominant alleles can easily contaminate breeding stocks after genotyping is conducted early in the season to raise amylose content through xenia (Karper \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1933\u003c/span\u003e, Gorbet and Weibel \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). Overall, the PACE genotypes were consistent with expected grain amylose contents and were able to rapidly identify off-types. They are therefore suitable for detecting adventitious outcrossing (through analysis of harvested grain or leaf tissues in the following generation) as a routine quality control measure leading up to commercialization. As the parental lines are from different breeding programs using different genetic backgrounds, the results indicate that these markers can be broadly used in waxy sorghum breeding.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRead-mapping at the\u003c/em\u003e Waxy \u003cem\u003elocus\u003c/em\u003e. The presence of the large insertion in \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e genotypes results in broken reads when flanking sequences that partially cover both wild-type and insertion sequences fail to align to the wild-type reference genome. This limits the efficiency of local short read mapping during molecular breeding and makes linkage disequilibrium analyses more difficult. The enhanced genome assembly was able to overcome this challenge by providing reference sequences for read mapping across the full-length insertion. When WGS data for the inbred lines KS115 (\u003cem\u003eWx\u003c/em\u003e) and R.Tx2907 (\u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e) were mapped to the wild-type (B.Tx623, v3.1.1) and enhanced reference genome (containing the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e insertion), the same number of reads were detected between Chr. 10: 1,860,965-1,865,278 for KS115, but an additional 78 uniquely mapped reads covering the insertion were mapped for R.Tx2907 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eBreeding for grain quality traits of sorghum\u003c/em\u003e. Historic breeding efforts in U.S. sorghum have focused on grain yield more than quality because phenotyping for quality is typically more time-consuming and expensive; and because producers were paid primarily for yield. However, advances in high-throughput phenotyping and genotyping methods have made it easier than ever to phenotype for quality traits and their interactions with whole-plant (grain\u0026thinsp;+\u0026thinsp;biomass) productivity and per-area yield. There is also a small but rapidly growing world-wide market for grain sorghum with grain quality traits, like low amylose content, so breeding programs are beginning to address them.\u003c/p\u003e \u003cp\u003eThe global value of the gluten-free products market was estimated at USD 6.3\u0026nbsp;billion in 2022 and projected to reach USD 11.8\u0026nbsp;billion by 2030 at a compound annual growth rate of 9.5%, based on a Vantage Market Research report. Highlights of the same report noted that a considerable amount of the increase in value could be attributed to more health-conscious consumers who were interested in methods to extend the shelf life of baked goods, especially breads, without the addition of preservatives \u0026ndash; a known property of waxy sorghum flour. Waxy sorghum grain has been studied for many years in the food sciences is a high-value endosperm trait associated with high digestibility by animals (Sherrod et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1969\u003c/span\u003e, Walker and Lichtenw.Re 1974, Myer and Gorbet \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1985\u003c/span\u003e, Schroeder et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), rapid fermentation into ethanol (Yan et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), the production of alcoholic beverages (Hsieh and Pi \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1979\u003c/span\u003e, Mugode et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Mezgebe et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and improved baking properties (Sang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Elhassan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) due to high-amylopectin starch content. While the waxy trait has been identified since the 1930s (Karper \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1933\u003c/span\u003e), recent hybrids are available with no yield drag or poor agronomic performance compared to near-isogenic or closely-related wild-type hybrids (Jampala et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Yerka et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It has also been linked to lower resistant starch content, an important factor in food functionality that must be balanced with diet because, when consumed in excess, it could result in unhealthy gut microbiomes, based on human stool microbiomes and mouse feeding studies (Yang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This is because the increased digestibility of waxy starches can raise the glycemic index (Frei et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Fitzgerald et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSorghum breeders are interested in combining waxy grain with additional target traits such as larger seed size, improved protein content and digestibility, improved germination, sugarcane aphid resistance, earlier maturity, etc. to improve their commercial value, expand production into additional regions, and help open underexplored markets where value-added, climate-smart crops are urgently needed. In particular, the western U.S. may be ideal for the development of new grain quality traits due to minimal precipitation throughout the growing season and harvest period, which can reduce grain quality in more central and eastern regions of the country. Developing waxy sorghum hybrids with improved trait combinations for deployment in new areas will be most efficiently achieved using advanced molecular breeding techniques. The PACE-PCR markers and updated chromosome 10 assembly presented herein provide a solid foundation for those efforts.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMolecular breeding resources for adapting sorghum yield and quality to local environments\u003c/em\u003e. Genome-to-phenome (G2P) modeling combines physiological information about plant developmental processes with genomic, agronomic, and environomic data. These methods, which often use artificial intelligence (AI) to optimize the predictive capacity of genetic models for improved gene discovery, have emerged as the most effective method for clarifying relationships between inputs and outputs in cropping systems (Harfouche et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Xu et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Hence, to secure both the abundance and quality of food systems, machine learning will become increasingly important to bring phenotyping for quality traits up to the speed and efficiency of phenotyping for yield for rapid improvements to the sustainable production of microscale, biochemical traits (Yan and Wang \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), like amylose or amylopectin content in climate-smart species like sorghum. The current study reports functional markers for waxy grain, which is not amenable to high-throughput phenotyping in field environments prior to the time of flowering. However, genotyping young plants with NGS markers in genomic selection is becoming standard in many companies, so the reported markers will provide immediate improvements to breeding efficiency by shortening the commercial breeding cycle. They will also assist with ongoing quality control by identifying progeny that are outcrosses derived from adventitious cross-pollination of maternal plants in the previous generation.\u003c/p\u003e \u003cp\u003eThe improved read mapping achieved in \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e genotypes using the enhanced genome assembly is consistent with what other researchers have demonstrated when incorporating presence-absence and copy number variation into the reference genomes used for making marker-trait associations (Bayer et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Jayakodi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Tao et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Li et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Sun et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Future work in the sorghum grain quality space should focus on creating pangenomes that account for even more quality traits not expressed in existing reference genomes. Breeding for human food and animal feed end uses will depend upon identifying functional markers for micro- and macronutrient contents (e.g. starch, protein, oil, amylose, lysine, beta carotene, zinc, iron, etc.), percent vitreosity, kernel hardness, resistant starch content, fiber, seed size, and the interactions of diverse grain physiochemistry profiles with grain weathering and molding, as no in-field, high-throughput phenotyping platform currently exists for them. The ongoing reliance of most breeding programs on laboratory-based quality testing lengthens the breeding cycle when grain must first be harvested and analyzed before superior genotypes can be identified and advanced to the next generation. In addition, the interaction between functional grain qualities and production environments can be impacted by presence-absence variation that plays a role in other aspects of plant physiology and adaptation, such as loci controlling plant height, maturity, and thermal or photoperiod sensitivity. As reference genomes improve, plant breeders will be increasingly able to develop plant populations with improved combinations of yield and quality traits that express well in target environments.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe waxy trait is one of the first specialty grain quality traits of U.S. sorghum to be commercially produced. The validated markers in our study will be beneficial for various practical applications, such as germplasm characterization, allele mining, and marker assisted selection during backcrossing and forward breeding programs to improve sorghum food, feed, and fuel applications. We have built upon previous efforts to genetically characterize the \u003cem\u003eWaxy\u003c/em\u003e locus by developing an enhanced chromosome 10 assembly for read mapping and functional NGS markers. These new resources provide an updated molecular toolkit for sorghum breeding that was validated in three public and private programs. This toolkit will improve the efficiency of breeding waxy sorghum using genomic selection and genome-to-phenome methodologies for rapid functional trait adaptation to target production environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets generated during and/or analyzed during the current study are available in the supplementary files associated with this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTATEMENTS AND DECLARATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for the development and analysis of the UNR breeding lines was provided by a United States Department of Agriculture Multi-State Hatch Award (NEV00763) and a United States Department of Agriculture Award (2019-67014-29174) to MKY. Funding for PACE-PCR assays, genomic analyses, and field studies at Texas Tech University (TTU) was provided by new faculty start-up funding to YJ. ZL was supported by the Graduate Support Stipend fellowship from the TTU Davis college of Agricultural Science and Natural Resources obtained by YJ and GP. Funding for the development of the USDA experimental breeding lines was provided by United States Department of Agriculture, Agricultural Research Service Project Plan 3096-21000-024-000-D. Richardson Seeds funded the development of their own experimental breeding lines and provided in-kind funding to MKY to support field studies conducted in Vega, TX during the development of the UNR breeding lines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMY and ZL are co-first authors. MY and YJ are co-corresponding authors who designed the studies and analyzed data. MY, YJ, and GP obtained funding to conduct the work. ZL and DN developed the PACE-PCR markers and analyzed data. MY developed the UNR plant materials and led field studies using the UNR and Richardson Seeds plant materials with the assistance of YJ and ZL in Lubbock, TX. CH developed and conducted field studies with the USDA sorghum materials. DD, GK, SM, and GC developed the pre-commercial materials and analyzed data. SB conducted grain quality analyses. 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Crop Sci 56(1):113\u0026ndash;121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2135/cropsci2015.03.0151\u003c/span\u003e\u003cspan address=\"10.2135/cropsci2015.03.0151\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"sorghum, waxy, grain quality, molecular markers, breeding","lastPublishedDoi":"10.21203/rs.3.rs-4883126/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4883126/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeveral mutations of the sorghum [\u003cem\u003eSorghum bicolor\u003c/em\u003e (L.) Moench] \u003cem\u003eGRANULE-BOUND STARCH SYNTHASE\u003c/em\u003e (\u003cem\u003eGBSS\u003c/em\u003e) gene [\u003cem\u003eSobic.010G022600\u003c/em\u003e] result in a low amylose:amylopectin starch ratio in the endosperm and confer a glutinous, \u0026ldquo;waxy\u0026rdquo; texture; hence, the wild-type gene is commonly referred to as \u003cem\u003eWaxy\u003c/em\u003e (\u003cem\u003eWx\u003c/em\u003e). Recessive \u003cem\u003ewaxy\u003c/em\u003e (\u003cem\u003ewx\u003c/em\u003e) alleles improve starch digestibility in ethanol production, human foods and beverages, and animal feed. However, breeding waxy sorghum can be time-consuming due to the need for grain to reach physiological maturity before the trait can be phenotyped and ongoing reliance on PCR markers for genotyping, which are not amenable to next-generation sequencing (NGS). Modern genomics-assisted breeding requires conducing high-throughput genotyping and selection in large, segregating populations prior to flowering. This study provides the first published NGS markers for the two mostly commonly used \u003cem\u003ewaxy\u003c/em\u003e (\u003cem\u003ewx\u003c/em\u003e) alleles of sorghum and is the first to fully characterize the large insertion that is causal of the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele. An enhanced genome assembly was constructed from the B.Tx623 reference genome (v3.1.1) to include the 5.6 kb \u003cem\u003ela\u003c/em\u003erge \u003cem\u003er\u003c/em\u003eetrotransposon \u003cem\u003ed\u003c/em\u003eerivative (LARD) in the \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e allele. This improved read mapping at \u003cem\u003eSobic.010G022600\u003c/em\u003e in \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e individuals, identified 78 new uniquely mapped reads, and made it possible to distinguish different \u003cem\u003eWaxy\u003c/em\u003e genotypes using short-read sequencing data. Functional PACE-PCR markers, suitable for genomic selection, were developed for \u003cem\u003eWx\u003c/em\u003e, \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ewx\u003c/em\u003e\u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e alleles and validated in three public and private breeding programs. These new molecular breeding resources will improve the efficiency of developing commercial waxy sorghum hybrids.\u003c/p\u003e","manuscriptTitle":"An enhanced genome assembly and functional, high-throughput molecular markers enable genomics-assisted breeding of waxy sorghum [Sorghum bicolor (L.) Moench]","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-07 17:01:24","doi":"10.21203/rs.3.rs-4883126/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-02-05T01:54:17+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-09-12T09:56:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-05T07:42:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-12T07:22:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Genetics","date":"2024-08-08T16:30:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"897728dc-ff2c-4794-afb5-1bb1bfc03e85","owner":[],"postedDate":"October 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-18T16:05:43+00:00","versionOfRecord":{"articleIdentity":"rs-4883126","link":"https://doi.org/10.1007/s13353-025-00993-1","journal":{"identity":"journal-of-applied-genetics","isVorOnly":false,"title":"Journal of Applied Genetics"},"publishedOn":"2025-08-11 15:57:48","publishedOnDateReadable":"August 11th, 2025"},"versionCreatedAt":"2024-10-07 17:01:24","video":"","vorDoi":"10.1007/s13353-025-00993-1","vorDoiUrl":"https://doi.org/10.1007/s13353-025-00993-1","workflowStages":[]},"version":"v1","identity":"rs-4883126","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4883126","identity":"rs-4883126","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}