Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum)

Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum) Shiori Otsuka, Takashi Hara, Koji Ishiguro, Kenichi Matsushima, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4702901/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Buckwheat ( Fagopyrum esculentum ) is a heterostylous self-incompatible crop that requires outcrossing for seed formation. Pollen parents influence the traits of seeds in many plants, but their influence in buckwheat is unknown. Here, we crossed self-incompatible (SI) and self-compatible (SC) lines with different rutin contents. The average rutin contents of SI leading cultivars were all 0.15–0.21 mg/g, and a SI high rutin content line that we had developed was 0.6 mg/g, although it has wide range SDs (0.12–0.41). On the other hand, the average rutin contents of SC lines were 0.01–0.06 mg/g, with stable SDs (0.02–0.03). In crosses between high- and low-rutin parents, the average rutin content of F 1 seeds was significantly lower than that of the high-rutin parent and higher than that of the low-rutin parent, indicating that the pollen parent influences the rutin content in seeds of buckwheat. RNA-seq analysis confirmed that alleles of several genes encoding enzymes involved in rutin synthesis derived from pollen parents were expressed during seed formation. buckwheat rutin pollen xenia RNA-seq self-incompatibility Figures Figure 1 Figure 2 Figure 3 Introduction Buckwheat is a pseudocereal crop in the Polygonaceae and is widely grown, notably in Russia, China, France, and Japan (FAOSTAT, 2023 ). It is used to make bread, noodles, and ethnic foods in combination with wheat, rice, or maize in many countries (Krkosková and Mrázová, 2005 ). In Japan, buckwheat noodles have been eaten for more than 400 years and are considered a traditional food (Krkosková and Mrázová, 2005 ; Qu et al., 2013 ). Known for their health benefits (Krkosková and Mrázová, 2005 ), buckwheat grains contain abundant starch, vitamins, minerals, an well-balanced amino acid composition, fiber (Huda et al., 2021 ), rutin (Matsui and Walker, 2020 ), a flavonoid with antioxidant, anti-inflammatory, anti-diabetic, anti-cancer, and pro-lipid-metabolism effects (Bhatt et al., 2022 ; Chu et al., 2014 ; Lee et al., 2016 ; Qu et al., 2013 ). Since rutin is not present in other major crops, new buckwheat lines with a high rutin content are desired. However, it is difficult to develop new lines, because buckwheat is an outcrossing plant on account of heterostylous self-incompatibility (Matsui and Yasui, 2020 ). Buckwheat has two flower types, pin and thrum: pin flowers have a long style and short stamens, whereas thrum flowers have a short style and long stamens (Darwin, 1897 ). It is possible to cross only between plants with different flower types, so all resultant seeds are F 1 s with high heterozygosity (Matsui and Yasui, 2020 ). Self-compatible buckwheat lines have been developed from an interspecific cross between F. esculentum and F. homotropicum (Aii et al., 1998 ; Campbell, 1995 ; Matsui et al., 2003 ; Wang et al., 2005 ; Woo et al., 1999 ). We developed the self-compatible line ‘Kyushu PL4’ (Matsui et al., 2008 ), which has been used as a maternal line to introduce self-compatibility into other lines, such as ‘Kyukei SC7’ (Hara et al., 2020 ; Takeshima et al., 2021 , 2022 ). A PL4 genome database recently developed by a research group including ourselves (Fawcett et al., 2023 ) has provided much genetic information. Flavonoids, including rutin, also known as quercetin-glycoside-rhamnoside, are synthesized via the flavonoid biosynthesis pathway in several sequential steps within the phenylpropanoid biosynthesis pathway (Matsui and Walker, 2020 ). Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL) convert phenylalanine into p -coumaroyl-CoA. Chalcone synthase (CHS), chalcone isomerase (CHI), and flavone 3-hydroxylase (F3H) catalyze p -coumaroyl-CoA into dihydrokaempferol. From dihydrokaempferol, flavonoid 3′-hydroxylase (F3′H), flavonoid 3′5′-hydroxylase (F3′5′H), and flavonol synthase (FLS) produce quercetin (Matsui and Walker, 2020 ; Zhang et al., 2017 ). The quercetin is then glycosylated by glycosyltransferases (GTRs) including GT (glucosyltransferase) and RT (rhamnosyltransferase) to produce rutin (Matsui and Walker, 2020 ; Zhang et al., 2017 ) (Fig. 1 ). It is well known that pollen can influence the character of seeds or fruits, a phenomenon called xenia (effect on endosperm and embryos) or metaxenia (effect on surrounding tissues) (Denney, 1992 ). For example, the pollen parent affects the fruit set, size, and mass of grapes and peonies (Sabir, 2015 ; Xie et al., 2017 ); the mass and ripeness of highbush blueberries (Doi et al., 2021 ); the color of the seed coat of Trifolium alexandrinum (Malaviya et al., 2019 ); and the contents of chemical components in peonies, almonds, rapeseed, and Siraitia grosvenorii (Kodad et al., 2009 ; Sánchez-Pérez et al., 2012 ; Wang et al., 2010 ; Xie et al., 2017 ; Yan et al., 2019 ). Although buckwheat requires cross-pollination, little is known about the influence of pollen parents. Here, by measuring the rutin contents of F 1 seeds produced by crosses between lines with high and low rutin contents, we clarified that the pollen parent influences the rutin content of F 1 seeds. RNA-seq analysis of maturing F 1 seeds detected several alleles related to rutin synthesis derived from parental lines. Materials and methods Plant materials We used five self-incompatible (SI) lines and two self-compatible (SC) lines (Table S1 ). Four of the SI lines—‘Kitamitsuki’ (KTM), ‘Kitawasesoba’ (KTW), ‘Reranokaori’ (RRN), and ‘Kitayuki’ (KTY)—are leading cultivars in Hokkaido, Japan (Ohsawa, 2020 ). By recurrent repeated individual selection for rutin content over 12 generations, we bred the SI ‘High Rutin content line No. 8’ (HR8) from ‘Botansoba’, which has high heterogeneity. We bred the SC line ‘Kyushu PL4’ (PL4) from a cross between F. esculentum and F. homotropicum (Matsui et al., 2008 ; Matsui and Yasui, 2020 ). We also selected a low-rutin-content SC line (LoR) from an F 4 segregating line produced from a cross between SC ‘Kyukei SC2’ (Matsui et al., 2003 ) and SC ‘C0408-0RP’, which was bred at Kade Research Ltd. (Hara et al., 2011 ). Production of F 1 seeds in a field for measurement of rutin content Lines were crossed at the Hokkaido Agricultural Research Center (42°88.3′N, 143°05.5′E) in the combinations listed in Table S2. Each cross was performed in a mesh-netted plot of two rows 1 m long and 60 cm between rows, with 10 to 20 plants of one line per row. For SI × SI crosses, pin plants of one line were grown in one row and thrum plants from the other line were grown in the other row. For SI × SC crosses, pin plants of the SI line were planted in one row and plants of the SC line were planted in the other row (Matsui and Yasui, 2020 ). Plants were pollinated by flies. Seeds were harvested at maturity, dried, and stored in a refrigerator. Production of F 1 seeds in a glasshouse with hand-pollination for RNA sequencing To clarify whether genes related to rutin synthesis derived from pollen parents are expressed in F 1 seeds, we crossed HR8 × PL4 and HR8 × HR8. The plants were grown in pots in a glasshouse at the Institute of Crop Science (36°03.0′N, 140°09.9′E). As HR8 is SI, each plant had a different genotype. To detect alleles from the pollen parent, we prepared two HR8 pin plants (HR8-pin-A and HR8-pin-B) as maternal plants; and two HR8 thrum plants (HR8-thrum-C and HR8-thrum-D) and two PL4 long-homostyle plants (PL4-LH-A and PL4-LH-B) as pollen parents. Using different branches, we crossed HR8-pin-A × PL4-LH-A, HR8-pin-A × HR8-thrum-C, HR8-B × HR8-thrum-D, and HR8-B × PL4-LH-B (Fig. S1 ). Each cross was hand pollinated and then bagged to prevent crosses with other plants. Immature seeds were harvested 10–20 days after crossing and frozen quickly in liquid nitrogen. Four seeds derived from each cross combination were used for RNA extraction. Measurement of rutin contents by high-performance liquid chromatography (HPLC) One seed was placed in a 2.0-mL tube with a small bead and crushed (2500 rpm for 60 s) in a Micro Smash MS-100 cell disruptor (Tomy Seiko, Tokyo, Japan). Then 1 mL of 80% ethanol was added, and the samples were incubated at 37°C for 3 h. They were centrifuged at 21 000 × g for 5 min at 4°C and the supernatant was analyzed for rutin content by HPLC (Ishiguro et al., 2016 ). The HPLC system consisted of two pumps (LC-20AD), an autoinjector (SIL-20AC), and a column oven (CTO-20AC, all from Shimadzu, Kyoto, Japan). Into a reversed-phase column (3 µm, 150 mm × 2 mm i.d., Cadenza CD-C18, Imtakt Co., Ltd., Kyoto, Japan) at 40°C was injected 2 µL of extract. The mobile phase was composed of phase A (7.5% v/v acetonitrile containing 0.1% v/v trifluoroacetic acid [TFA]) and phase B (50% v/v acetonitrile containing 0.1% v/v TFA). Samples were eluted with a 35% solution of phase B in phase A at 0.3 mL/min for 18 min. Rutin was identified from the retention time and UV-vis spectra of a standard, and was quantified against an external standard on a calibration curve based on detection at 360 nm. Maternal and pollen effects on rutin content Five plants were selected from each row (Table S2). The rutin content of each of 15 seeds per plant was measured. Means were compared by Fisher’s Least Significant Difference (LSD) test at P = 0.05, 0.01, or 0.001 in BellCurve for Excel software (Social Survey Research Information Co., Ltd. Tokyo, Japan). Maternal and pollen effects on the rutin content were calculated based on the method of Wang et al. ( 2010 ). The rutin content of F 1 seeds, F 1 , was calculated as: F 1 = mP m + (1 – m ) P p where P m and P p are the rutin contents of the maternal and pollen parents, and m is the maternal effect, calculated as m = ( F 1 – P p ) / ( P m – P p ), and therefore the pollen parent effect = 1 – m . The mid-parental value (MP) = ( P m + P p ) / 2. RNA sequencing analysis for identifying alleles derived from pollen parent and calculation of the ratio of alleles derived from pollen parent We investigated the expression of genes related to rutin synthesis in the phenylpropanoid and flavonoid biosynthesis pathways in maturing seeds of HR8 pin plants × PL4 pollen parent and of HR8 pin plants × HR8 thrum plants to confirm that any allelic differences are not caused by natural variation in HR8. Maturing seeds were frozen in liquid nitrogen and homogenized with a mortar and pestle. Total RNA was extracted from the seeds with a Maxwell RSC Plant RNA Kit in a Maxwell RSC Instrument (Promega) according to the manufacturer’s protocol. A Next Generation Sequencing library was constructed with a TruSeq stranded mRNA Library Preparation Kit (Illumina), and RNAs were sequenced on a NovaSeq 6000 sequencer to generate 150-bp paired-end reads. Short reads were cleaned in fastp software (Chen et al., 2018 ), and the first and last 6 bases of each locus were trimmed because the reliability of the ratio of nucleotide in each SNP was low due to low quality of base-call in this region. The cleaned short reads were mapped to the cDNA sequence of PL4 (Fawcett et al., 2023 ) in Bowtie 2 software (Langmead and Salzberg, 2012 ). The mapped data were visualized in Integrative Genomics Viewer (IGV) software (Robinson et al., 2011 ). Genes encoding enzymes involved in rutin synthesis were selected based on the report by Fawcett et al. ( 2023 ). After the RNA-seq data were mapped to the reference sequence, loci of expressed genes were determined and SNPs were searched in IGV. Alleles derived from pollen parents were detected by comparison of the results between HR8 × HR8 and HR8 × PL4. We also estimated the ratio of expression of alleles derived from pollen parent by calculating the ratio of nucleotides derived from pollen parent on each SNP indicated by IGV to determine the relationship between the ratio of expression of alleles derived from pollen parent and rutin content in seed (Fig. S2). Results Rutin contents among materials The average rutin contents of KTM, KTW, KTY and RRN were all 0.15–0.21 mg/g, and that of HR8 was 0.6 mg/g. Because these are all SI and thus heterozygosity is high, SD had a wide range (0.12–0.41; Table 1 ). On the other hand, the average rutin contents of SC lines PL4 and LoR were both 0.01–0.06 mg/g, with stable SDs (0.02–0.03; Table 1 ). From these results, we classified these lines into three groups by rutin content—low ( 0.5 mg/g)—and investigated the effects of the pollen parent with crosses among these groups (Table S1 ). Table 1 Parental lines and seed rutin contents. Parent Average rutin content (mg/g) SD HR8 0.60 0.41 PL4 0.06 0.03 LoR 0.01 0.02 KTM 0.18 0.09 KTW 0.21 0.13 RRN 0.15 0.12 KTY 0.17 0.14 Flower type and self-compatibility of these lines are shown in Table S2. Effect of pollen parent on rutin content Because only HR8 had a high rutin content, we used this line in all crossing combinations. When HR8 as the maternal parent was crossed with KTM, KTW, KTY, and RRN as the pollen parents, the rutin contents of the seeds (0.31–0.40 mg/g; Table 2 ) were significantly lower than that of HR8 (0.60 mg/g; Table 1 ), indicating that the rutin content was influenced by the pollen parent (Figs. 2 a–d). Table 2 Parental effects on the rutin contents of F 1 seeds. Cross combination by rutin contents Cross combination of lines Cross No. 1) Mid-parental value Rutin content (mg/g) SD Significance 2) Maternal effect value Pollen parent effect value P m vs. F 1 P p vs. F 1 High (♀) × medium or low (♂) HR8 × KTM a1 0.39 0.38 0.26 *** *** 0.49 0.51 HR8 × KTW b1 0.40 0.36 0.36 *** ** 0.40 0.60 HR8 × RRN c1 0.37 0.40 0.26 *** *** 0.56 0.44 HR8 × KTY d1 0.38 0.31 0.26 *** ** 0.32 0.68 HR8 × PL4 e 0.33 0.44 0.28 ** *** 0.71 0.29 HR8 × LoR f 0.30 0.45 0.27 ** *** 0.74 0.26 average – – – – – – 0.54 0.46 Medium (♀) × high (♂) KTM × HR8 a2 0.39 0.32 0.28 ** *** 0.65 0.35 KTW × HR8 b2 0.40 0.22 0.19 0.87 *** 0.98 0.02 RRN × HR8 c2 0.37 0.21 0.16 0.16 *** 0.86 0.14 KTY × HR8 d2 0.38 0.28 0.20 * *** 0.75 0.25 average – – – – – – 0.81 0.19 Flower type and self-compatibility of these lines are shown in Table S2. 1) Cross No. with the same letter indicates that they were grown and crossed in the same plot. 2) Means were compared by LSD test. P 1 , maternal parent; P 2 , pollen parent. Significant differences at *0.05, **0.01, and ***0.001 probability levels. Similarly, when KTM and KTY as the maternal parents were crossed with HR8 as the pollen parent, the rutin contents of the seeds (0.32, 0.28 mg/g; Table 2 ) were significantly higher than that of each maternal parent (0.18, 0.17 mg/g; Figs. 2 a, d; Table 1 ). On the other hand, when KTW and RRN as the maternal parents were crossed with HR8 as the pollen parent, the rutin content of the seeds (0.22, 0.21 mg/g; Table 2 ) was only marginally higher than that of the seed parents (0.21, 0.15 mg/g; Figs. 2 b, c; Table 1 ). In the crosses between HR8 and PL4 or LoR, the rutin contents of the seeds (0.44 and 0.45 mg/g) were significantly lower than those of HR8 (0.60 mg/g; Table 2 ) and higher than those of PL4 or LoR (0.06, 0.01 mg/g; Figs. 2 e, f; Table 1 ), indicating that the rutin content was influenced by the pollen parent. Identification of expressed genes derived from pollen parents Genes encoding enzymes in the rutin biosynthesis pathway were expressed in maturing seeds (Table 3 ), indicating the synthesis of rutin during seed development. Approximately 35% of involved genes were expressed (Table 3 ). Allele sequences derived from the pollen parent PL4 were recognized at 14 loci (Fig. 3 ; Table 3 ). Table 3 Gene families encoding enzymes involved in the rutin biosynthesis pathway and the number of genes expressed during seed formation. Enzyme Number of loci Detected in reference Expressed Pollen parent alleles detected Phenylalanine ammonia-lyase (PAL) 4 4 3 Cinnamate-4-hydroxylase (C4H) 6 2 1 4-Coumarate CoA ligase (4CL) 2 2 1 Chalcone synthase (CHS) 13 5 3 Chalcone isomerase (CHI) 3 1 1 Flavanone-3-hydroxylase (F3H) 2 2 1 Flavonoid-3′-hydroxylase (F3′H) 2 2 1 Flavonoid-3′-5′-hydroxylase (F3′5′H) 16 3 3 Flavonol synthase (FLS) 2 1 0 Glycosyltransferase (GTR) 1) 12 (1) (1) Total 1) 62 22 14 1) Gene for GTR was expressed in only one plant. The total does not include GTR. Discussion Paternal and maternal effects on the rutin content of F 1 hybrid seeds Pollen parents have a direct genetic influence on F 1 seeds in some plants (Denney, 1992 ; Kanade et al., 2024 ). They influence sugar content and enzymes for secondary metabolites in palm fruit (Shahsavar and Shahhosseini, 2022 ) and yield and anthocyanin content in highbush blueberry (Doi et al., 2021 ). However, to our knowledge, there are no reports in buckwheat. We clarified the effects of the pollen parent on the rutin content of F 1 seeds through the use of the SC lines PL4 and LoR and of the high-rutin-content line HR8. As most buckwheat cultivars are SI, it is difficult to develop lines in which rutin content is fixed, especially at a high level, because of the absence of genetic information. Using only common cultivars, it would be difficult to determine whether rutin content is influenced by the pollen parent or simply varies within a line. The low rutin contents of PL4 and LoR are fixed. So when they were crossed with HR8, the effect of the pollen parent was clearly detected (Fig. 2 e, f). HR8 was developed by long-term recurrent selection for high rutin content and has a significantly higher content than the other SI lines, although variation is still large. Significant differences in the rutin content between the F 1 seeds and the maternal parent lines were found when HR8 as the maternal parent was crossed with the other SI lines as the pollen parents and when KTM and KTY as the maternal parents were crossed with HR8, but not when KTW and RRN as the maternal parents were crossed with HR8 as the pollen parent. The lack of significant differences in the rutin content between F 1 seeds and the maternal parents in some cross combination would be probably caused by the broad range of both lines. HR8 can help clarify the effect of the pollen parent on the rutin content of seeds, but the development of better-fixed lines would be more useful. In crosses between medium-rutin-content lines KTM, KTW, KTY, and RRN as the maternal parents and HR8 as the pollen parent, the effect value of the pollen parent on rutin content averaged 0.19 (range, 0.02–0.35; Table 2 ), lower than that of the opposite crosses including crosses between HR8 and PL4 or LoR (0.46; range, 0.26–0.68). The reason for the difference is not evident, and the development of an SC line with high rutin content would help. Detection of alleles derived from pollen parents and relationship between the ratio of alleles and rutin contents If rutin contents are influenced by pollen parents, alleles related to rutin synthesis of the pollen parent should be expressed during seed development. We detected the expression of pollen parent alleles in F 1 seeds for several genes encoding enzymes involved in rutin synthesis. Approximately 60% of the alleles derived from the pollen parents were recognized in the expressed loci (Table 3 ). Approximately 40% of the loci, the ratio of pollen alleles was 20–40% (Table 4 ), comparable to the influence of the pollen parent on the rutin content of PL4 × HR8 (0.29; Table 2 ). However, some genes lay outside this range, in which the lowest influence of pollen parent on genes was 9.6% and the highest was 74.2% (Table 4 ). Buckwheat seeds are composed of a diploid embryo, a triploid endosperm, and a diploid testa derived from the maternal parent, with different rutin contents, which are highest in the embryo (Suzuki et al., 2002 ). The expression levels of genes involved in rutin synthesis seem to depend on maturity stages (Penin et al., 2021 ). Thus, a larger embryo size and a larger proportion of embryo in the seed would mean a higher rutin content. The expression of genes related to rutin synthesis may increase as the embryo grows larger, but the embryo’s size in the seed may be regulated by a number of genes. Further study will be needed to clarify the relationships between the ratio of each allele and rutin content. Table 4 SNPs detected in expressed genes related to flavonoid biosynthesis and the ratio of alleles derived from pollen parent. Enzyme Locus Total number of nucleotides HR8-pin-A × PL4-LH-A HR8-pin-B × PL4-LH-B Number of SNPs Ratio of pollen parent allele Number of SNPs Ratio of pollen parent allele Pattern I 1) Pattern II 1) Average ± SE Pattern I 1) Pattern II 1) Average ± SE PAL FesPL4_r1.1_Chr3.g195460.1 2173 22 3 30.3 1.94 15 10 16.9 4.02 FesPL4_r1.1_Chr4.g269240.1 2110 20 18 22.5 9.80 43 5 14.6 5.60 FesPL4_r1.1_Chr8.g155630.1 2113 4 12 44.4 6.63 8 7 47.1 6.55 C4H FesPL4_sc0109.1.g001280.1 1516 39 5 20.7 2.05 22 7 26.7 5.11 4CL FesPL4_r1.1_Chr4.g271010.1 1642 13 1 38.5 - 35 2 18.0 2.00 CHS FesPL4_r1.1_Chr4.g217000.1 1183 3 7 68.0 3.15 3 7 66.1 5.61 FesPL4_r1.1_Chr7.g094080.1 1180 5 0 - - 4 1 74.2 0.00 FesPL4_r1.1_Chr7.g094660.1 1180 5 0 - - 4 1 9.6 0.00 CHI FesPL4_r1.1_Chr3.g000530.1 772 11 0 - - 10 0 - - F3H FesPL4_r1.1_Chr5.g258370.1 1105 10 3 21.4 0.91 13 3 20.0 2.52 F3′H FesPL4_r1.1_Chr8.g248260.1 1588 9 16 54.9 8.55 8 16 53.9 7.66 F3′5′H FesPL4_r1.1_Chr4.g265140.1 1834 13 1 36.4 - 15 0 - - FesPL4_r1.1_Chr4.g265180.1 1876 31 1 11.8 - 18 0 - - FesPL4_r1.1_Chr4.g265190.1 1576 5 0 - - 5 0 - - 1) Two different SNP patterns are recognized because HR8 is SI and keeps heterozygosity in many loci (Fig. S2). Conclusion We confirmed that the rutin content of buckwheat is influenced by the pollen parent in both combinations of crossing a high-rutin line with low- or medium-rutin lines. The pollen parent alleles of several genes encoding enzymes involved in rutin synthesis were expressed. Since the pollen parent affects multiple traits in many species, it is likely that the pollen parent also affects traits other than rutin content in buckwheat. As buckwheat requires outcrossing, this result could be important knowledge for breeding and growing buckwheat. Declarations Competing interests The authors declare that they have no competing interests. Funding This work was supported by NARO and partly supported by the Toyo Suisan Foundation. Author Contribution SO and KM conceived and designed the experiments. SO and KM crossed the plants. SO, TH, and KI measured rutin by HPLC. TH and KIM developed plant materials. SO, YY, and KM performed RNA sequencing analysis. SO and KM wrote the manuscript. 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New Phytologist 159(3):701–708. https://doi.org/10.1046/j.1469-8137.2003.00840.× . Matsui K, Tetsuka T, Kara T, Morishita T (2008) Breeding and characterization of a new self-compatible common buckwheat parental line,” Buckwheat Norin-PL1”. Bull Natl Agric Res Cent Kyushu Okinawa Reg 49:1–17 (Japanese with English summary). Matsui K, Walker A (2020) Biosynthesis and regulation of flavonoids in buckwheat. Breeding Science 70(1):74–84. https://doi.org/10.1270/jsbbs.19041 . Matsui K, Yasui Y (2020) Genetic and genomic research for the development of an efficient breeding system in heterostylous self-incompatible common buckwheat ( Fagopyrum esculentum ). Theoretical and Applied Genetics 133(5):1641–1653. https://doi.org/10.1007/s00122-020-03572-6 . Ohsawa R (2020) Current status and prospects of common buckwheat breeding in Japan. Breeding Science 70(1):3–12. https://doi.org/10.1270/jsbbs.19108 . Penin AA, Kasianov AS, Klepikova AV, Kirov IV, Gerasimov ES, Fesenko AN, Logacheva MD (2021) High-resolution transcriptome atlas and improved genome assembly of common buckwheat, Fagopyrum esculentum . Frontiers in Plant Science 12. https://doi.org/10.3389/fpls.2021.612382 . Qu Y, Yasuda T, Nakajima K, Hiwatashi A, Moroi C, Sanada H, Egashira Y (2013) Effect of rutin in buckwheat noodle on lipid metabolism in rats. Food Science and Technology Research 19(6):1011–1018. https://doi.org/10.3136/fstr.19.1011 . Robinson JT, Thorvaldsdóttir H, Winckler W, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nature Biotechnology, 29(1):24–26. Sabir A (2015) Xenia and Metaxenia in grapes: differences in berry and seed characteristics of maternal grape cv. ‘Narince’ ( Vitis vinifera L.) as influenced by different pollen sources. Plant Biology 17(2):567–573. https://doi.org/10.1111/plb.12266 . Shahsavar AR, Shahhosseini A (2022) The metaxenia effects of different pollen grains on secondary metabolites enzymes and sugars of ‘Piarom’ date palm fruit. Scientific Reports 12(1):10058. https://doi.org/10.1038/s41598-022-14373-w . Suzuki T, Honda Y, Funatsuki W, Nakatsuka K (2002) Purification and characterization of flavonol 3-glucosidase, and its activity during ripening in tartary buckwheat seeds. Plant Science 163(3):417–423. https://doi.org/10.1016/s0168-9452(02)00158-9 . Sánchez-Pérez R, Arrázola G, Martín ML, Grané N, Dicenta F (2012) Influence of the pollinizer in the amygdalin content of almonds. Scientia Horticulturae, 139:62–65. https://doi.org/10.1016/j.scienta.2012.02.028 . Takeshima R, Ogiso-Tanaka E, Yasui Y, Matsui K (2021) Targeted amplicon sequencing plus next-generation sequencing-based bulked segregant analysis identified genetic loci associated with preharvest sprouting tolerance in common buckwheat ( Fagopyrum esculentum ). BMC Plant Biology 22(1):353. https://doi.org/10.1186/s12870-020-02790-w . Takeshima R, Yabe S, Matsui K (2022) Genetic basis of maturity time is independent from that of flowering time and contributes to ecotype differentiation in common buckwheat ( Fagopyrum esculentum Moench). BMC Plant Biology 22(1):353. https://doi.org/10.1186/s12870-022-03722-6 . Wang XF, Liu GH, Yang Q, Hua W, Liu J, Wang HZ (2010) Genetic analysis on oil content in rapeseed ( Brassica napus L.). Euphytica 173(1):17–24. https://doi.org/10.1007/s10681-009-0062-x . Wang YJ, Scarth R, Campbell C (2005) S h and S c -two complementary dominant genes that control self-compatibility in buckwheat. Crop Science, 45(4):1229–1234. https://doi.org/10.2135/cropsci2003.0560 . Woo SH, Adachi T, Jong SK, Campbell CG (1999) Inheritance of self-compatibility and flower morphology in an inter-specific buckwheat hybrid. Canadian Journal of Plant Science 79(4):483–490. https://doi.org/10.4141/p98-117 . Xie LH, Niu LX, Zhang YL, Jin M, Ji D, Zhang XX (2017) Pollen sources influence the traits of seed and seed oil in Paeonia ostii ‘Feng Dan’. Hortscience, 52(5):700–705. https://doi.org/10.21273/hortsci11803-17 . Yan ZG, Xiao D, Xu YL, Ma J, Liu F, Bai LH, Ma XJ (2019) Effects of metaxenia on the carbohydrate and mogroside content and related enzyme activities in Siraitia grosvenorii fruit. Acta Physiologiae Plantarum, 41(7):1–11. https://doi.org/10.1007/s11738-019-2887-9 . Zhang LJ, Li XX, Ma B, Gao Q, Du HL, Han YH, Qiao ZJ et al (2017) The tartary buckwheat genome provides insights into rutin biosynthesis and abiotic stress tolerance. Molecular Plant 10(9):1224–1237. https://doi.org/10.1016/j.molp.2017.08.013 . Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigTablesOtsukabuckwheat.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4702901","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":331791052,"identity":"d15b44cd-da10-4b62-87f0-b7183d26f595","order_by":0,"name":"Shiori Otsuka","email":"","orcid":"","institution":"NARO","correspondingAuthor":false,"prefix":"","firstName":"Shiori","middleName":"","lastName":"Otsuka","suffix":""},{"id":331791056,"identity":"dc05efd9-2d6b-4a40-a6dd-541c4687289d","order_by":1,"name":"Takashi Hara","email":"","orcid":"","institution":"NARO","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Hara","suffix":""},{"id":331791058,"identity":"1f9a7da0-daa8-4958-b6ba-5c33aceef845","order_by":2,"name":"Koji Ishiguro","email":"","orcid":"","institution":"NARO","correspondingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Ishiguro","suffix":""},{"id":331791059,"identity":"0ac27186-aa09-4e91-97ab-a4a7f3963e9a","order_by":3,"name":"Kenichi Matsushima","email":"","orcid":"","institution":"Shinshu University","correspondingAuthor":false,"prefix":"","firstName":"Kenichi","middleName":"","lastName":"Matsushima","suffix":""},{"id":331791060,"identity":"b31843aa-fc41-4482-9241-ea94084ab185","order_by":4,"name":"Yasuo Yasui","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Yasuo","middleName":"","lastName":"Yasui","suffix":""},{"id":331791061,"identity":"00986426-8cf0-4422-98d8-42e600490ae9","order_by":5,"name":"Katsuhiro Matsui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYNACgwQ5KEuOH8pgw6OcGazFGMozlmwgTgtDQmIDmhbcQLf9/OHPPAVp6dvZzz58wFBjIGHefoD5xQcGvjxcWszOJLNJ8xjk5O7sSTc2YDhmICFzJoHNcgYDWzFOLQeS2Zh5DCpyNxxIY5NgYPtTJwEkjXkY2GBOxdRy/jHzZ6CWdIPzz4Ba/hlIENZyI5kB5LAEgxtAWxjbwFqYH+PX8thMco5BmuGGG8+YDRL7gFp4EtsYZxjg8cv5xMcf3vxJljc4n8b44MM3oBb2w4c/fKg4hjPEUEECmGRsk2AwOJZAnBYoYP7AwFBDmpZRMApGwSgYzgAA7glNXpVT6DgAAAAASUVORK5CYII=","orcid":"","institution":"NARO","correspondingAuthor":true,"prefix":"","firstName":"Katsuhiro","middleName":"","lastName":"Matsui","suffix":""}],"badges":[],"createdAt":"2024-07-08 05:56:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4702901/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4702901/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61571670,"identity":"1cf82d7f-917a-4cdc-b639-dd085c2dbb8d","added_by":"auto","created_at":"2024-08-01 11:03:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":752485,"visible":true,"origin":"","legend":"\u003cp\u003eRutin biosynthesis pathway in buckwheat.\u003c/p\u003e\n\u003cp\u003eEnzymes are indicated in upper-case blue letters. Arrows indicate reactions catalyzed by the indicated enzymes. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:Coa ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase; GTR, flavonol glycosyltransferase.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4702901/v1/cb15e453ba05ec26f99de378.png"},{"id":61572276,"identity":"2a6e7d36-6a80-406f-88a9-b012a1576f6c","added_by":"auto","created_at":"2024-08-01 11:11:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":127735,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of rutin contents of F\u003csub\u003e1\u003c/sub\u003e seeds and their parent lines.\u003c/p\u003e\n\u003cp\u003eHR8 was crossed with (a) KTM, (b) KTW, (c) RRN, (d) KTY, (e) PL4, (f) LoR.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4702901/v1/28ffa49b43379444fe6e8016.png"},{"id":61572277,"identity":"131d5530-a0cf-4284-8926-6e35efb15b68","added_by":"auto","created_at":"2024-08-01 11:11:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":534748,"visible":true,"origin":"","legend":"\u003cp\u003eExample of gene expression in maturing seeds of (rows 1 and 3) HR8 × HR8 and (rows 2 and 4) HR8 × PL4.\u003c/p\u003e\n\u003cp\u003eThe reference sequence is PL4, and nucleotides that are the same as in PL4 are shown in gray. Proportions of nucleotides that differ from those in PL4 are shown by color, and the nucleotide is indicated. For example, at 216 bp, the nucleotide in rows 1 and 3 (HR8 × HR8) is C (blue), whereas that in rows 2 and 4 (HR8 × PL4) are C (blue) and G (orange). This difference indicates that the “G” allele of the pollen parent “PL4” was expressed.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4702901/v1/29b2bd4a42d33f1187ee23b3.png"},{"id":69005868,"identity":"1a3c887a-1580-4154-9759-65283331ad83","added_by":"auto","created_at":"2024-11-14 12:39:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2429825,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4702901/v1/54a7a28e-b8dd-4f72-aad8-599881d76bf0.pdf"},{"id":61571671,"identity":"bb0e6548-9384-4e4f-908b-f334b5e41b9e","added_by":"auto","created_at":"2024-08-01 11:03:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":471790,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigTablesOtsukabuckwheat.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4702901/v1/b1cf377a9311103ee0081705.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBuckwheat is a pseudocereal crop in the Polygonaceae and is widely grown, notably in Russia, China, France, and Japan (FAOSTAT, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is used to make bread, noodles, and ethnic foods in combination with wheat, rice, or maize in many countries (Krkoskov\u0026aacute; and Mr\u0026aacute;zov\u0026aacute;, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In Japan, buckwheat noodles have been eaten for more than 400 years and are considered a traditional food (Krkoskov\u0026aacute; and Mr\u0026aacute;zov\u0026aacute;, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Qu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Known for their health benefits (Krkoskov\u0026aacute; and Mr\u0026aacute;zov\u0026aacute;, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), buckwheat grains contain abundant starch, vitamins, minerals, an well-balanced amino acid composition, fiber (Huda et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), rutin (Matsui and Walker, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), a flavonoid with antioxidant, anti-inflammatory, anti-diabetic, anti-cancer, and pro-lipid-metabolism effects (Bhatt et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chu et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Qu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince rutin is not present in other major crops, new buckwheat lines with a high rutin content are desired. However, it is difficult to develop new lines, because buckwheat is an outcrossing plant on account of heterostylous self-incompatibility (Matsui and Yasui, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Buckwheat has two flower types, pin and thrum: pin flowers have a long style and short stamens, whereas thrum flowers have a short style and long stamens (Darwin, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1897\u003c/span\u003e). It is possible to cross only between plants with different flower types, so all resultant seeds are F\u003csub\u003e1\u003c/sub\u003es with high heterozygosity (Matsui and Yasui, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSelf-compatible buckwheat lines have been developed from an interspecific cross between \u003cem\u003eF. esculentum\u003c/em\u003e and \u003cem\u003eF. homotropicum\u003c/em\u003e (Aii et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Campbell, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Matsui et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Woo et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). We developed the self-compatible line \u0026lsquo;Kyushu PL4\u0026rsquo; (Matsui et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), which has been used as a maternal line to introduce self-compatibility into other lines, such as \u0026lsquo;Kyukei SC7\u0026rsquo; (Hara et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Takeshima et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A PL4 genome database recently developed by a research group including ourselves (Fawcett et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) has provided much genetic information.\u003c/p\u003e \u003cp\u003eFlavonoids, including rutin, also known as quercetin-glycoside-rhamnoside, are synthesized via the flavonoid biosynthesis pathway in several sequential steps within the phenylpropanoid biosynthesis pathway (Matsui and Walker, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL) convert phenylalanine into \u003cem\u003ep\u003c/em\u003e-coumaroyl-CoA. Chalcone synthase (CHS), chalcone isomerase (CHI), and flavone 3-hydroxylase (F3H) catalyze \u003cem\u003ep\u003c/em\u003e-coumaroyl-CoA into dihydrokaempferol. From dihydrokaempferol, flavonoid 3\u0026prime;-hydroxylase (F3\u0026prime;H), flavonoid 3\u0026prime;5\u0026prime;-hydroxylase (F3\u0026prime;5\u0026prime;H), and flavonol synthase (FLS) produce quercetin (Matsui and Walker, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The quercetin is then glycosylated by glycosyltransferases (GTRs) including GT (glucosyltransferase) and RT (rhamnosyltransferase) to produce rutin (Matsui and Walker, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is well known that pollen can influence the character of seeds or fruits, a phenomenon called xenia (effect on endosperm and embryos) or metaxenia (effect on surrounding tissues) (Denney, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). For example, the pollen parent affects the fruit set, size, and mass of grapes and peonies (Sabir, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e); the mass and ripeness of highbush blueberries (Doi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e); the color of the seed coat of \u003cem\u003eTrifolium alexandrinum\u003c/em\u003e (Malaviya et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); and the contents of chemical components in peonies, almonds, rapeseed, and \u003cem\u003eSiraitia grosvenorii\u003c/em\u003e (Kodad et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; S\u0026aacute;nchez-P\u0026eacute;rez et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yan et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough buckwheat requires cross-pollination, little is known about the influence of pollen parents. Here, by measuring the rutin contents of F\u003csub\u003e1\u003c/sub\u003e seeds produced by crosses between lines with high and low rutin contents, we clarified that the pollen parent influences the rutin content of F\u003csub\u003e1\u003c/sub\u003e seeds. RNA-seq analysis of maturing F\u003csub\u003e1\u003c/sub\u003e seeds detected several alleles related to rutin synthesis derived from parental lines.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eWe used five self-incompatible (SI) lines and two self-compatible (SC) lines (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Four of the SI lines\u0026mdash;\u0026lsquo;Kitamitsuki\u0026rsquo; (KTM), \u0026lsquo;Kitawasesoba\u0026rsquo; (KTW), \u0026lsquo;Reranokaori\u0026rsquo; (RRN), and \u0026lsquo;Kitayuki\u0026rsquo; (KTY)\u0026mdash;are leading cultivars in Hokkaido, Japan (Ohsawa, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). By recurrent repeated individual selection for rutin content over 12 generations, we bred the SI \u0026lsquo;High Rutin content line No. 8\u0026rsquo; (HR8) from \u0026lsquo;Botansoba\u0026rsquo;, which has high heterogeneity. We bred the SC line \u0026lsquo;Kyushu PL4\u0026rsquo; (PL4) from a cross between \u003cem\u003eF. esculentum\u003c/em\u003e and \u003cem\u003eF. homotropicum\u003c/em\u003e (Matsui et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Matsui and Yasui, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We also selected a low-rutin-content SC line (LoR) from an F\u003csub\u003e4\u003c/sub\u003e segregating line produced from a cross between SC \u0026lsquo;Kyukei SC2\u0026rsquo; (Matsui et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and SC \u0026lsquo;C0408-0RP\u0026rsquo;, which was bred at Kade Research Ltd. (Hara et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eProduction of F\u003csub\u003e1\u003c/sub\u003e seeds in a field for measurement of rutin content\u003c/h2\u003e \u003cp\u003eLines were crossed at the Hokkaido Agricultural Research Center (42\u0026deg;88.3\u0026prime;N, 143\u0026deg;05.5\u0026prime;E) in the combinations listed in Table S2. Each cross was performed in a mesh-netted plot of two rows 1 m long and 60 cm between rows, with 10 to 20 plants of one line per row. For SI \u0026times; SI crosses, pin plants of one line were grown in one row and thrum plants from the other line were grown in the other row. For SI \u0026times; SC crosses, pin plants of the SI line were planted in one row and plants of the SC line were planted in the other row (Matsui and Yasui, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plants were pollinated by flies. Seeds were harvested at maturity, dried, and stored in a refrigerator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProduction of F\u003csub\u003e1\u003c/sub\u003e seeds in a glasshouse with hand-pollination for RNA sequencing\u003c/h2\u003e \u003cp\u003eTo clarify whether genes related to rutin synthesis derived from pollen parents are expressed in F\u003csub\u003e1\u003c/sub\u003e seeds, we crossed HR8 \u0026times; PL4 and HR8 \u0026times; HR8. The plants were grown in pots in a glasshouse at the Institute of Crop Science (36\u0026deg;03.0\u0026prime;N, 140\u0026deg;09.9\u0026prime;E). As HR8 is SI, each plant had a different genotype. To detect alleles from the pollen parent, we prepared two HR8 pin plants (HR8-pin-A and HR8-pin-B) as maternal plants; and two HR8 thrum plants (HR8-thrum-C and HR8-thrum-D) and two PL4 long-homostyle plants (PL4-LH-A and PL4-LH-B) as pollen parents. Using different branches, we crossed HR8-pin-A \u0026times; PL4-LH-A, HR8-pin-A \u0026times; HR8-thrum-C, HR8-B \u0026times; HR8-thrum-D, and HR8-B \u0026times; PL4-LH-B (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Each cross was hand pollinated and then bagged to prevent crosses with other plants. Immature seeds were harvested 10\u0026ndash;20 days after crossing and frozen quickly in liquid nitrogen. Four seeds derived from each cross combination were used for RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of rutin contents by high-performance liquid chromatography (HPLC)\u003c/h2\u003e \u003cp\u003eOne seed was placed in a 2.0-mL tube with a small bead and crushed (2500 rpm for 60 s) in a Micro Smash MS-100 cell disruptor (Tomy Seiko, Tokyo, Japan). Then 1 mL of 80% ethanol was added, and the samples were incubated at 37\u0026deg;C for 3 h. They were centrifuged at 21 000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C and the supernatant was analyzed for rutin content by HPLC (Ishiguro et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The HPLC system consisted of two pumps (LC-20AD), an autoinjector (SIL-20AC), and a column oven (CTO-20AC, all from Shimadzu, Kyoto, Japan). Into a reversed-phase column (3 \u0026micro;m, 150 mm \u0026times; 2 mm i.d., Cadenza CD-C18, Imtakt Co., Ltd., Kyoto, Japan) at 40\u0026deg;C was injected 2 \u0026micro;L of extract. The mobile phase was composed of phase A (7.5% v/v acetonitrile containing 0.1% v/v trifluoroacetic acid [TFA]) and phase B (50% v/v acetonitrile containing 0.1% v/v TFA). Samples were eluted with a 35% solution of phase B in phase A at 0.3 mL/min for 18 min. Rutin was identified from the retention time and UV-vis spectra of a standard, and was quantified against an external standard on a calibration curve based on detection at 360 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMaternal and pollen effects on rutin content\u003c/h2\u003e \u003cp\u003eFive plants were selected from each row (Table S2). The rutin content of each of 15 seeds per plant was measured. Means were compared by Fisher\u0026rsquo;s Least Significant Difference (LSD) test at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05, 0.01, or 0.001 in BellCurve for Excel software (Social Survey Research Information Co., Ltd. Tokyo, Japan).\u003c/p\u003e \u003cp\u003eMaternal and pollen effects on the rutin content were calculated based on the method of Wang et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The rutin content of F\u003csub\u003e1\u003c/sub\u003e seeds, \u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e, was calculated as:\u003c/p\u003e \u003cp\u003e \u003cem\u003eF\u003c/em\u003e \u003csub\u003e \u003cem\u003e1\u003c/em\u003e \u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003emP\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e + (1 \u0026ndash; \u003cem\u003em\u003c/em\u003e)\u003cem\u003eP\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eP\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e are the rutin contents of the maternal and pollen parents, and \u003cem\u003em\u003c/em\u003e is the maternal effect, calculated as \u003cem\u003em\u003c/em\u003e = (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e \u0026ndash; \u003cem\u003eP\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e) / (\u003cem\u003eP\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e \u0026ndash; \u003cem\u003eP\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e), and therefore the pollen parent effect\u0026thinsp;=\u0026thinsp;1 \u0026ndash; \u003cem\u003em\u003c/em\u003e. The mid-parental value (MP) = (\u003cem\u003eP\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e + \u003cem\u003eP\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e) / 2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA sequencing analysis for identifying alleles derived from pollen parent and calculation of the ratio of alleles derived from pollen parent\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe investigated the expression of genes related to rutin synthesis in the phenylpropanoid and flavonoid biosynthesis pathways in maturing seeds of HR8 pin plants \u0026times; PL4 pollen parent and of HR8 pin plants \u0026times; HR8 thrum plants to confirm that any allelic differences are not caused by natural variation in HR8.\u003c/p\u003e \u003cp\u003eMaturing seeds were frozen in liquid nitrogen and homogenized with a mortar and pestle. Total RNA was extracted from the seeds with a Maxwell RSC Plant RNA Kit in a Maxwell RSC Instrument (Promega) according to the manufacturer\u0026rsquo;s protocol. A Next Generation Sequencing library was constructed with a TruSeq stranded mRNA Library Preparation Kit (Illumina), and RNAs were sequenced on a NovaSeq 6000 sequencer to generate 150-bp paired-end reads. Short reads were cleaned in fastp software (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and the first and last 6 bases of each locus were trimmed because the reliability of the ratio of nucleotide in each SNP was low due to low quality of base-call in this region. The cleaned short reads were mapped to the cDNA sequence of PL4 (Fawcett et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) in Bowtie 2 software (Langmead and Salzberg, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The mapped data were visualized in Integrative Genomics Viewer (IGV) software (Robinson et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenes encoding enzymes involved in rutin synthesis were selected based on the report by Fawcett et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). After the RNA-seq data were mapped to the reference sequence, loci of expressed genes were determined and SNPs were searched in IGV. Alleles derived from pollen parents were detected by comparison of the results between HR8 \u0026times; HR8 and HR8 \u0026times; PL4.\u003c/p\u003e \u003cp\u003eWe also estimated the ratio of expression of alleles derived from pollen parent by calculating the ratio of nucleotides derived from pollen parent on each SNP indicated by IGV to determine the relationship between the ratio of expression of alleles derived from pollen parent and rutin content in seed (Fig. S2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRutin contents among materials\u003c/h2\u003e \u003cp\u003eThe average rutin contents of KTM, KTW, KTY and RRN were all 0.15\u0026ndash;0.21 mg/g, and that of HR8 was 0.6 mg/g. Because these are all SI and thus heterozygosity is high, SD had a wide range (0.12\u0026ndash;0.41; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On the other hand, the average rutin contents of SC lines PL4 and LoR were both 0.01\u0026ndash;0.06 mg/g, with stable SDs (0.02\u0026ndash;0.03; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). From these results, we classified these lines into three groups by rutin content\u0026mdash;low (\u0026lt;\u0026thinsp;0.1 mg/g), medium (0.1\u0026ndash;0.5 mg/g), and high (\u0026gt;\u0026thinsp;0.5 mg/g)\u0026mdash;and investigated the effects of the pollen parent with crosses among these groups (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParental lines and seed rutin contents.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAverage rutin content (mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHR8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePL4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLoR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKTM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKTW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRRN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKTY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eFlower type and self-compatibility of these lines are shown in Table S2.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pollen parent on rutin content\u003c/h2\u003e \u003cp\u003eBecause only HR8 had a high rutin content, we used this line in all crossing combinations. When HR8 as the maternal parent was crossed with KTM, KTW, KTY, and RRN as the pollen parents, the rutin contents of the seeds (0.31\u0026ndash;0.40 mg/g; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were significantly lower than that of HR8 (0.60 mg/g; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that the rutin content was influenced by the pollen parent (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;d).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParental effects on the rutin contents of F\u003csub\u003e1\u003c/sub\u003e seeds.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCross combination by rutin contents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCross combination of lines\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCross No. \u0026nbsp;\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMid-parental value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRutin content (mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eSignificance\u0026nbsp;\u003csup\u003e2)\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMaternal effect value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePollen parent effect value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP\u003csub\u003em\u003c/sub\u003e vs. F\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eP\u003csub\u003ep\u003c/sub\u003e vs. F\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eHigh (♀) \u0026times; medium or low (♂)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR8 \u0026times; KTM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR8 \u0026times; KTW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR8 \u0026times; RRN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR8 \u0026times; KTY\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ed1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR8 \u0026times; PL4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ee\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR8 \u0026times; LoR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eaverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eMedium (♀) \u0026times; high (♂)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKTM \u0026times; HR8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ea2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKTW \u0026times; HR8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eb2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRRN \u0026times; HR8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKTY \u0026times; HR8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ed2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eaverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003eFlower type and self-compatibility of these lines are shown in Table S2.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e1) Cross No. with the same letter indicates that they were grown and crossed in the same plot.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e2) Means were compared by LSD test. P\u003csub\u003e1\u003c/sub\u003e, maternal parent; P\u003csub\u003e2\u003c/sub\u003e, pollen parent. Significant differences at *0.05, **0.01, and ***0.001 probability levels.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSimilarly, when KTM and KTY as the maternal parents were crossed with HR8 as the pollen parent, the rutin contents of the seeds (0.32, 0.28 mg/g; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were significantly higher than that of each maternal parent (0.18, 0.17 mg/g; Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, d; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On the other hand, when KTW and RRN as the maternal parents were crossed with HR8 as the pollen parent, the rutin content of the seeds (0.22, 0.21 mg/g; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) was only marginally higher than that of the seed parents (0.21, 0.15 mg/g; Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the crosses between HR8 and PL4 or LoR, the rutin contents of the seeds (0.44 and 0.45 mg/g) were significantly lower than those of HR8 (0.60 mg/g; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and higher than those of PL4 or LoR (0.06, 0.01 mg/g; Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that the rutin content was influenced by the pollen parent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of expressed genes derived from pollen parents\u003c/h2\u003e \u003cp\u003eGenes encoding enzymes in the rutin biosynthesis pathway were expressed in maturing seeds (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating the synthesis of rutin during seed development. Approximately 35% of involved genes were expressed (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Allele sequences derived from the pollen parent PL4 were recognized at 14 loci (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGene families encoding enzymes involved in the rutin biosynthesis pathway and the number of genes expressed during seed formation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEnzyme\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eNumber of loci\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDetected in reference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExpressed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePollen parent alleles detected\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenylalanine ammonia-lyase (PAL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCinnamate-4-hydroxylase (C4H)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-Coumarate CoA ligase (4CL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChalcone synthase (CHS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChalcone isomerase (CHI)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavanone-3-hydroxylase (F3H)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonoid-3\u0026prime;-hydroxylase (F3\u0026prime;H)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonoid-3\u0026prime;-5\u0026prime;-hydroxylase (F3\u0026prime;5\u0026prime;H)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonol synthase (FLS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycosyltransferase (GTR)\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u0026nbsp;\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e1) Gene for GTR was expressed in only one plant. The total does not include GTR.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePaternal and maternal effects on the rutin content of F\u003csub\u003e1\u003c/sub\u003e hybrid seeds\u003c/h2\u003e \u003cp\u003ePollen parents have a direct genetic influence on F\u003csub\u003e1\u003c/sub\u003e seeds in some plants (Denney, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Kanade et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). They influence sugar content and enzymes for secondary metabolites in palm fruit (Shahsavar and Shahhosseini, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and yield and anthocyanin content in highbush blueberry (Doi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, to our knowledge, there are no reports in buckwheat.\u003c/p\u003e \u003cp\u003eWe clarified the effects of the pollen parent on the rutin content of F\u003csub\u003e1\u003c/sub\u003e seeds through the use of the SC lines PL4 and LoR and of the high-rutin-content line HR8. As most buckwheat cultivars are SI, it is difficult to develop lines in which rutin content is fixed, especially at a high level, because of the absence of genetic information. Using only common cultivars, it would be difficult to determine whether rutin content is influenced by the pollen parent or simply varies within a line. The low rutin contents of PL4 and LoR are fixed. So when they were crossed with HR8, the effect of the pollen parent was clearly detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003eHR8 was developed by long-term recurrent selection for high rutin content and has a significantly higher content than the other SI lines, although variation is still large. Significant differences in the rutin content between the F\u003csub\u003e1\u003c/sub\u003e seeds and the maternal parent lines were found when HR8 as the maternal parent was crossed with the other SI lines as the pollen parents and when KTM and KTY as the maternal parents were crossed with HR8, but not when KTW and RRN as the maternal parents were crossed with HR8 as the pollen parent. The lack of significant differences in the rutin content between F\u003csub\u003e1\u003c/sub\u003e seeds and the maternal parents in some cross combination would be probably caused by the broad range of both lines. HR8 can help clarify the effect of the pollen parent on the rutin content of seeds, but the development of better-fixed lines would be more useful.\u003c/p\u003e \u003cp\u003eIn crosses between medium-rutin-content lines KTM, KTW, KTY, and RRN as the maternal parents and HR8 as the pollen parent, the effect value of the pollen parent on rutin content averaged 0.19 (range, 0.02\u0026ndash;0.35; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), lower than that of the opposite crosses including crosses between HR8 and PL4 or LoR (0.46; range, 0.26\u0026ndash;0.68). The reason for the difference is not evident, and the development of an SC line with high rutin content would help.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of alleles derived from pollen parents and relationship between the ratio of alleles and rutin contents\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIf rutin contents are influenced by pollen parents, alleles related to rutin synthesis of the pollen parent should be expressed during seed development. We detected the expression of pollen parent alleles in F\u003csub\u003e1\u003c/sub\u003e seeds for several genes encoding enzymes involved in rutin synthesis. Approximately 60% of the alleles derived from the pollen parents were recognized in the expressed loci (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Approximately 40% of the loci, the ratio of pollen alleles was 20\u0026ndash;40% (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), comparable to the influence of the pollen parent on the rutin content of PL4 \u0026times; HR8 (0.29; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, some genes lay outside this range, in which the lowest influence of pollen parent on genes was 9.6% and the highest was 74.2% (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Buckwheat seeds are composed of a diploid embryo, a triploid endosperm, and a diploid testa derived from the maternal parent, with different rutin contents, which are highest in the embryo (Suzuki et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The expression levels of genes involved in rutin synthesis seem to depend on maturity stages (Penin et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Thus, a larger embryo size and a larger proportion of embryo in the seed would mean a higher rutin content. The expression of genes related to rutin synthesis may increase as the embryo grows larger, but the embryo\u0026rsquo;s size in the seed may be regulated by a number of genes. Further study will be needed to clarify the relationships between the ratio of each allele and rutin content.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSNPs detected in expressed genes related to flavonoid biosynthesis and the ratio of alleles derived from pollen parent.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eEnzyme\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eLocus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTotal number of nucleotides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eHR8-pin-A \u0026times; PL4-LH-A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c12\" namest=\"c9\"\u003e \u003cp\u003eHR8-pin-B \u0026times; PL4-LH-B\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNumber of SNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eRatio of pollen parent allele\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eNumber of SNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eRatio of pollen parent allele\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePattern I\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePattern II\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026plusmn; SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePattern I\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePattern II\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026plusmn; SE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr3.g195460.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e16.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr4.g269240.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e14.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr8.g155630.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e47.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC4H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_sc0109.1.g001280.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e26.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4CL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr4.g271010.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e18.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr4.g217000.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e66.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e5.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr7.g094080.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e74.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr7.g094660.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr3.g000530.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e772\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF3H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr5.g258370.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e20.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF3\u0026prime;H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr8.g248260.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e54.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e53.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e7.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF3\u0026prime;5\u0026prime;H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr4.g265140.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1834\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr4.g265180.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFesPL4_r1.1_Chr4.g265190.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003e1) Two different SNP patterns are recognized because HR8 is SI and keeps heterozygosity in many loci (Fig. S2).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe confirmed that the rutin content of buckwheat is influenced by the pollen parent in both combinations of crossing a high-rutin line with low- or medium-rutin lines. The pollen parent alleles of several genes encoding enzymes involved in rutin synthesis were expressed. Since the pollen parent affects multiple traits in many species, it is likely that the pollen parent also affects traits other than rutin content in buckwheat. As buckwheat requires outcrossing, this result could be important knowledge for breeding and growing buckwheat.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by NARO and partly supported by the Toyo Suisan Foundation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSO and KM conceived and designed the experiments. SO and KM crossed the plants. SO, TH, and KI measured rutin by HPLC. TH and KIM developed plant materials. SO, YY, and KM performed RNA sequencing analysis. SO and KM wrote the manuscript. All authors edited and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Kiku Fujii and Mayumi Hayashida for technical assistance. We also thank Tomohiro Takakura and Kazaha Nagashima for growing the plants.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during this study are available from the corresponding author on reasonable request. The raw RNA-Seq data were submitted to the DDBJ Sequence Read Archive (https://www.ddbj.nig.ac.jp/dra/index-e.html) under accession numbers DRA018730, DRA018757, DRA018758, and DRA018772.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAii J, Nagano M, Penner GA, Campbell CG, Adachi T (1998) Identification of RAPD markers linked to the homostylar (\u003cem\u003eHo\u003c/em\u003e) gene in buckwheat. 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Molecular Plant 10(9):1224\u0026ndash;1237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.molp.2017.08.013\u003c/span\u003e\u003cspan address=\"10.1016/j.molp.2017.08.013\" 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":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"buckwheat, rutin, pollen, xenia, RNA-seq, self-incompatibility","lastPublishedDoi":"10.21203/rs.3.rs-4702901/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4702901/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBuckwheat (\u003cem\u003eFagopyrum esculentum\u003c/em\u003e) is a heterostylous self-incompatible crop that requires outcrossing for seed formation. Pollen parents influence the traits of seeds in many plants, but their influence in buckwheat is unknown. Here, we crossed self-incompatible (SI) and self-compatible (SC) lines with different rutin contents. The average rutin contents of SI leading cultivars were all 0.15\u0026ndash;0.21 mg/g, and a SI high rutin content line that we had developed was 0.6 mg/g, although it has wide range SDs (0.12\u0026ndash;0.41). On the other hand, the average rutin contents of SC lines were 0.01\u0026ndash;0.06 mg/g, with stable SDs (0.02\u0026ndash;0.03). In crosses between high- and low-rutin parents, the average rutin content of F\u003csub\u003e1\u003c/sub\u003e seeds was significantly lower than that of the high-rutin parent and higher than that of the low-rutin parent, indicating that the pollen parent influences the rutin content in seeds of buckwheat. RNA-seq analysis confirmed that alleles of several genes encoding enzymes involved in rutin synthesis derived from pollen parents were expressed during seed formation.\u003c/p\u003e","manuscriptTitle":"Pollen parent affects rutin content of seeds of buckwheat (Fagopyrum esculentum)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-01 11:03:47","doi":"10.21203/rs.3.rs-4702901/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"02fddaf3-5beb-496c-9db1-aeb9a3a34a0b","owner":[],"postedDate":"August 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-14T12:38:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-01 11:03:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4702901","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4702901","identity":"rs-4702901","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}