Recent Transposition of an LTR-Retrotransposon in the Gene Coding for S Receptor Kinase is Responsible for a Novel Self-Compatible Phenotype of Radish (Raphanus Sativus L.)

Recent Transposition of an LTR-Retrotransposon in the Gene Coding for S Receptor Kinase is Responsible for a Novel Self-Compatible Phenotype of Radish (Raphanus Sativus L.) | 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 Recent Transposition of an LTR-Retrotransposon in the Gene Coding for S Receptor Kinase is Responsible for a Novel Self-Compatible Phenotype of Radish ( Raphanus Sativus L.) So-Hyeon Bong, Ganghee Cho, Dong-Seon Kim, Sunggil Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-884506/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 Self-incompatibility (SI) responses of radish ( Raphanus sativus L.) are determined by two tightly linked genes encoding an S receptor kinase (SRK) and an S -locus cysteine-rich protein/ S locus protein 11 ( SCR/SP11 ), respectively. A radish showing an almost self-compatible (SC) phenotype was identified in this study. Inheritance patterns showed that this SC phenotype was dominant over an SI phenotype. In addition, this SC phenotype co-segregated with an S haplotype in an F 2 population. This SC radish contained an RsS-26 haplotype in which duplicate SRK -like genes were previously identified. Full-length sequences of two SRK -like genes of 18,133-bp and 6,200-bp in length were obtained from radish with the RsS-26 haplotype (designated as RsSRK-26-1 and RsSRK-26-2 , respectively). Duplicate SCR/SP11 -like genes were also identified in the radish with the RsS-26 haplotype. Phylogenetic analyses indicated that both duplicate SRK -like and SCR/SP11 -like genes were closely related to other known SRK and SCR/SP11 genes, respectively. No critical mutation was found in the coding region of SRK -like or SCR/SP11 -like gene. However, a 4,146-bp intact LTR-retrotransposon was identified in the third intron of RsSRK-26-1 of the SC radish. Interestingly, this LTR-retrotransposon was not detected in three other breeding lines containing the same RsS-26 haplotype. Except for this LTR-retrotransposon, only two single nucleotide polymorphisms (SNPs) were identified in intronic regions between normal and mutant RsSRK-26-1 alleles. While normal transcription was observed for radish showing RsSRK-26-1 and SI phenotypes in these three breeding lines, no transcript of RsSRK-26-1 was detected in the SC radish, suggesting that recent transposition of an LTR-retrotransposon in the RsSRK-26-1 gene might be responsible for the SC phenotype of radish. Horticulture Molecular Genetics Agronomy Radish (Raphanus sativus L.) Self-incompatibility S haplotype self-compatible mutant Duplicate SRK genes Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Radish ( Raphanus sativus L.) is one of the most important root vegetables in East Asian countries such as China, Japan, and Korea. As a cross-pollinated crop, radish shows hybrid vigor. Its F 1 hybrid varieties that harness such hybrid vigor have been developed (Curtis 2011 ). Self-incompatibility (SI) is defined as inhibition of pollen tube growth in pistils during self-pollination. It has been commercially used for the production of F 1 hybrid seeds in radish (Singh et al. 2001 ). SI systems are widespread in plants. They can promote outcrossing and increase genetic diversity. More than 100 families and almost 40% of angiosperms have adopted SI systems (Igic et al. 2008 ). Generally, a single locus initially named as S (Sterility) is involved in SI responses. Two tightly linked genes consisting of male and female determinants of SI are positioned in the S locus. Nucleotide sequences of these two SI determinants are highly polymorphic and multi-allelic. Since at least two genes are present in the S locus, combinations of these genes can lead to S haplotypes (Stone and Goring 2001 ; Watanabe et al. 2012 ). Types of SI systems are largely categorized into heteromorphic and homomorphic SI. In the case of heteromorphic SI, cross-pollination is possible between different floral morphs. Candidate genes responsible for distyly have been reported in Primula vulgaris (Li et al. 2016 ) and Fagopyrum esculentum (Yasui et al. 2012 ). Homomorphic SI systems are generally classified into gametophytic SI (GSI) and sporophytic SI (SSI) depending on inheritance patterns of SI phenotypes. Such inheritance patterns are determined by genotypes of haploid pollen in GSI and diploid pollen parents in SSI (Silva and Goring 2001 ; Muñoz-Sanz et al. 2020 ). GSI systems based on RNase and S-locus F-box proteins have been extensively studied in Solanaceae, Plantaginaceae, and Rosaceae families (Franceschi et al. 2012 ). The Papaver system based on programmed cell death has been mainly studied in poppy (Wheeler et al. 2009 ). Meanwhile, SSI has been extensively studied in Brassicaceae including radish (Sobotka et al. 2000 ; Takayama and Isogai 2003 ; Tantikanjana et al. 2010 ; Watanabe et al. 2012 ). Two tightly linked genes encoding S receptor kinase (SRK) and S -locus cysteine-rich protein (SCR)/ S locus protein 11(SP11) or SP11/SCR have been revealed as female and male determinants of SSI, respectively (Schopfer et al. 1999 ; Takasaki et al. 2000 ; Takayama et al. 2000 ). Another gene encoding S locus glycoprotein (SLG) has been identified in the S locus, although its precise role remains uncertain (Nasrallah et al. 1985 ; Watanabe et al. 2012 ). Sequences of SLG and S domain of SRK genes are highly homologous within the same S haplotypes (Sato et al. 2002 ; Lim et al. 2002 ; Okamoto et al. 2004 ). Based on sequence diversity of SLG and SRK genes, S haplotypes are classified into class I and class II (Nasrallah et al. 1991 ; Sato et al. 2002 ). Generally, S haplotypes of class I are dominant over those of class II and small noncoding RNAs and DNA methylation are involved in such dominance relationships (Tarutani et al. 2010 ; Yasuda et al. 2016 ). SI was first used in production of F 1 hybrid seeds of cabbage in 1940s by Japanese seed companies. Since then, SI has been exploited for economical production of F 1 hybrid seeds of radish and Brassica crops (Muñoz-Sanz et al. 2020 ). To implement SI systems in radish F 1 hybrid breeding programs, specific S haplotypes of parental lines should be identified to avoid cross-incompatibility between parental lines. At least 35 S haplotypes have been identified in radish (Haseyama et al. 2018 ). More than 50 and 100 S haplotypes have been identified in Chinese cabbage (Nou et al. 1993 ) and cabbage (Ockendon 2000 ), respectively. For efficient identification of S haplotypes, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods have been developed for radish and Brassica crops (Brace et al. 1993 , 1994 ; Nishio et al. 1994 , 1996 , 1997 ; Sakamoto et al. 2000 ; Lim et al. 2002 ). Although PCR-RFLP is more efficient than laborious pollination tests, it has pitfalls such as complicated band patterns of digested PCR products and difficulty in specific PCR amplification of SLG or SRK genes due to the presence of multiple homologous SRK -like genes (Boyes et al. 1991 ; Kim and Kim 2018 ). To overcome these problems, a new S haplotyping system has been developed based on SLL2 and SP6 genes positioned at borders of S core regions and 31 S haplotypes have been identified from diverse breeding lines in our previous studies (Kim et al. 2016 ; Kim and Kim 2019 ). Self-compatible (SC) mutants have been found naturally or induced by irradiation in crops normally showing SI phenotypes (Muñoz-Sanz et al. 2020 ). In some fruit tree species, such SC phenotypes are sometimes advantageous for better fruit set and yield (Claessen et al. 2019 ). A radish breeding line showing a SC phenotype was identified and the critical mutation responsible for the SC phenotype was revealed in this study. In addition, efficient application of this SC phenotype in radish breeding programs is discussed. Materials And Methods Plant materials A radish breeding line (JNUR1537) showing an SC phenotype was introduced from a seed company (Farm Hannong Co., Ltd., Republic of Korea). Detail pedigree of this breeding line is unknown. JNUR1537 was crossed with JNUR1123 to produce F 1 hybrids by hand-pollination. An F 2 population was produced by self-pollination of F 1 hybrids. Three breeding lines (DBRL294-2, DBR2085 and DBR2086) containing the RsS3 haplotype were used to compare SI phenotypes with JNUR1537. S haplotypes of these three breeding lines have been identified in a previous study (Kim and Kim 2019 ). Evaluation of SI phenotypes were performed in greenhouses. Single plants were covered with mesh cages to prevent cross-pollination. No insect pollinator was introduced to mesh cages. PCR amplification, genome walking, and sequencing of PCR products Total genomic DNAs were extracted from leaf tissues using a cetyl trimethylammonium bromide (CTAB) method (Doyle and Doyle 1987 ). PCR amplification and sequencing of PCR products of SLL2 , SP6 , and SRK genes of parental lines of the F 2 population were carried out following methods described in a previous study (Kim and Kim 2019 ). The detail protocol for high-resolution melting (HRM) analysis has been described in a previous study (Seo et al. 2020 ). SYTO®9 green fluorescent nucleic acid stain (Thermo Fisher Scientific, Waltham, MA, USA) was used as a dye. HRM peaks were obtained using a LightCycler® 96 system (Roche Molecular Systems, Pleasanton, CA, USA). Primer sequences of HRM markers are shown in Supplementary Table 1. To obtain full-length genomic DNA sequences of duplicated SRK homologs, long PCR and genome walking were performed. Long PCR reactions were carried out in 50 µL reaction mixtures containing 0.25 µg DNA template, 5 µL 10× PCR buffer, 1.0 µL forward primer (10 µM), 1.0 µL reverse primer (10 µM), 8.0 µL dNTPs (10 mM each), and 0.5 µL Taq polymerase (TaKaRa LA PCR™ Kit Ver. 2.1; Takara Bio, Shiga, Japan). Long PCR amplification was performed with 40 cycles of 98°C for 10 s and 68°C for 15 min. Genome walking was performed using a Universal GenomeWalker kit (Takara Bio) according to the manufacturer’s instructions. Total genomic DNAs used to construct genome walking libraries were isolated from young seedlings using a DNeasy Plant Mini Kit (QIAGEN, Valencia, CA, USA). All PCR products were visualized on 1.5% agarose gels after ethidium bromide staining. Subsequently, PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN). Sequencing was performed by a specialized company (Macrogen, Seoul, Republic of Korea). RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) Total RNAs were extracted from floral buds using a RNeasy Plant Mini Kit (QIAGEN). cDNAs were synthesized using a cDNA synthesis kit (SuperScript™ III first-strand synthesis system for RT-PCR, Invitrogen, Carlsbad, CA, USA). RT-PCR amplification was performed with an initial denaturation step at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 2 min. It was finished with a final 10-min extension step at 72°C. Primer sequences used in RT-PCR are listed in Supplementary Table 1. Radish tubulin sequence (Rs395780) identified from a draft genome sequence (Jeong et al. 2016 ) was used as a control. Construction of phylogenetic trees Genomic DNA, cDNA, or deduced amino acid sequences of SLL2 , SP6 , SRK , and SCR/SP11 genes were aligned using a BioEdit software (Hall 1999 ). Large gaps in alignments were removed using Gblocks program (Castresana 2000 ). Phylogenetic trees were produced using MEGA version X (Kumar et al. 2018 ) with a neighbor-joining method. Node support of the phylogenetic tree was assessed using 1,000 bootstrap replicates. Results Discovery of an SC radish breeding line and inheritance patterns of the SC phenotype Radishes derived from JNUR1537 showed an almost SC phenotype. Compared with an SI radish accession (Fig. 1 A), a large number of pods were formed in the SC radish without any insect pollinators (Fig. 1 B). This SC plant was cross-pollinated with a SI breeding line (JNUR1123). F 1 hybrids showed the SC phenotype, indicating that the SC phenotype was dominant over the SI phenotype. Subsequently, an F 2 population was produced from self-pollination of F 1 hybrids. Ten individuals of the F 2 population were grown in a greenhouse. Each individual plant was separately covered with mech cages. SI phenotypes clearly segregated among F 2 plants (Table 1 ). Table 1 Number of seeds produced from 10 randomly selected F 2 individuals grown in isolated greenhouses Plant code Number of seeds Genotype of S haplotypes F2-1 540 RsS3 / RsS3 F2-2 693 RsS3 / RsS32 F2-3 74 RsS32 / RsS32 F2-4 2,330 RsS3 / RsS32 F2-5 1,483 RsS3 / RsS32 F2-6 174 RsS3 / RsS3 F2-7 1,012 RsS3 / RsS32 F2-8 576 RsS3 / RsS3 F2-9 86 RsS32 / RsS32 F2-10 1,086 RsS3 / RsS32 To test whether the SC phenotype was related to S haplotypes, specific S haplotypes of both SC and SI parental lines were identified using an S haplotyping system developed in a previous study (Kim and Kim 2019 ). From the SI parent, novel sequences of SLL2 , SP6 , and SRK genes closely related to RsSLL2-25 , RsSP6-8 , and RsSRK9 , respectively (Supplementary Figs. 1, 2, 3), were isolated. These novel allele sequences were designated as RsSLL2-31 , RsSP6-26 , and RsSRK21 , respectively. Their nucleotide sequences were deposited into GenBank under accession numbers of MZ383797, MZ383798, and MZ383799, respectively. Since RsSRK21 was clustered with class II SRK genes (Supplementary Fig. 3), RsSK21 was considered as a member of class II SRK genes. The new class II S haplotype consisting of three novel sequences was designated as RsS32 . Nucleotide sequences of both SLL2 and SP6 genes in the SC parent were identical to those of the class II RsS3 haplotype. As expected, a mixture of duplicate SRK -like genes was amplified. In a previous study (Kim and Kim 2019 ), closely related duplicate SRK -like genes have also been detected in the RsS3 haplotype. For efficient genotyping of S haplotypes of F 2 individuals, two HRM markers were developed based on polymorphic sequences of SLL2 and SP6 alleles of parental lines, respectively (Supplementary Fig. 4). After genotyping F 2 seedlings, five individuals of each genotype were separately grown in cages. While SC phenotypes were observed in both homozygous RsS3 and heterozygous individuals, few pods and seeds were formed in homozygous RsS32 individuals (Table 2 ). In addition, SI phenotypes of 10 F 2 individuals previously grown in isolated greenhouse were matched with genotypes of S haplotypes (Table 1 ). These results implied that the SC phenotype was probably related to S core genes such as SRK and SCR/SP11 . Table 2 Numbers of pods and seeds produced from F 2 individuals representing three genotypes of S haplotypes Genotype Plant code Number of pods Number of seeds RsS3 / RsS3 A1 328 844 A2 115 136 A3 232 257 A4 396 761 A5 124 245 A6 76 136 RsS3 / RsS32 H1 280 705 H2 36 67 H3 24 19 H4 192 610 H5 59 161 H6 48 122 RsS32 / RsS32 B1 9 10 B2 12 7 B3 16 13 B4 0 0 B5 0 0 B6 8 7 Assembly of full-length genomic DNA sequences of S core genes in the SC radish To identify any defects in SRK and SCR/SP11 genes in the SC radish, full-length sequences of SRK and SCR/SP11 genes were analyzed. To identify the authentic SRK among duplicate SRK -like genes, full-length genomic DNA sequences of closely related SRK -like genes were obtained in this study. Using primers designed based on conserved regions among radish and three Brassica class II SRK genes (Supplementary Fig. 5), 5’ S domain sequences of SRK were isolated. After obtaining sequences of the last exon7 using genome walking, a full-length SRK sequence was assembled by connecting 5’ S and 3’ kinase domains through long PCR amplifications (Fig. 2 ). In addition, approximately 2-kb 5’ sequences containing a putative promoter was obtained by genome walking, resulting in a 18,133-bp full-length sequence. Meanwhile, a 6,200-bp full-length sequence of another putative SRK gene was obtained using genome walking PCRs with primers designed based on dissimilar sequences between two putative SRK genes (Fig. 2 ). Since partial S domain sequences of the larger putative SRK gene containing a large-sized 11,865-bp intron 3 were identical to those of the SRK-26 deposited in the GenBank (LC341218), the S haplotype of the SC radish might be identical to the RsS-26 haplotype designated by Haseyama et al. ( 2018 ). Indeed, nucleotide sequences of SLG of the SC radish were identical to those of SLG-26 (LC341241) of the RsS-26 haplotype. In addition, partial sequences identical to those of RsSCR-26 (LC325812) were amplified in the SC radish. However, additional 552-bp full-length SCR/SP11 gene sharing 91.0% sequence identities with RsSCR-26 was obtained by genome walking in the SC radish, implying that both SRK and SCR/SP11 genes might be duplicated in the RsS-26 haplotype. The novel SCR/SP11 gene was closely related to other known SCR/SP11 genes (Supplementary Fig. 6). This novel gene was designated as RsSCR-26-2 . Its sequence was deposited into GenBank under the accession number of MZ383800. The full-length RsSCR-26-2 contained intact exons. Following the unified nomenclature suggested by Haseyama et al. ( 2018 ), the RsS3 haplotype was renamed as RsS-26 to avoid confusion. Coding sequences of two putative SRK genes were also intact without any premature stop codons. They shared 91.7% nucleotide sequence identities with each other. Since S core gene sequences were identical to those of the RsS-26 haplotype, the large and small putative SRK genes were designated as RsSRK-26-1 and RsSRK-26-2 , respectively. Full-length sequences of both SRK s were deposited into GenBank under accession numbers of MZ383801 and MT241389, respectively. The phylogenetic tree of radish SRK genes showed that RsSRK-26-1 and RsSRK-26-2 were closely related to each other (Fig. 3 ). Similar to other class II SRK genes, the RsSRK-26-1 gene contained a large-sized intron 3. However, a relatively small intron 3 was identified in the RsSRK-26-2 gene (Supplementary Fig. 7), suggesting that the RsSRK-26-1 gene might be the genuine SRK in the RsS-26 haplotype. Identification of a critical mutation responsible for the radish SC phenotype Interestingly, a transposable element-like sequence was found in the large-sized intron 3 of RsSRK-26-1 (Fig. 2 ). A 4,146-bp intact open reading frame (ORF) was identified in the transposable element-like sequence. Five typical domains of long terminal repeat (LTR)-retrotransposon were identified in the polyprotein region (Fig. 2 ). Since the integrase (INT) domain was positioned upstream of the reverse transcriptase (RT) domain, this LTR-retrotransposon belonged to Copia superfamily (Wicker et al. 2007 ). Target site duplication (TSD) of 4-bp (‘GGAC’) was found at flanking regions of this element. LTR sequences of 284-bp in length positioned at both ends were perfectly identical to each other. This novel LTR-retrotransposon was designated as RsCopia1 . Since the ORF encoding a polyprotein was intact and both LTR sequences were identical to each other, RsCopia1 was assumed to be recently transposed into the RsSRK-26-1 gene. To investigate whether all RsS-26 haplotypes contained RsCopia1 in the SRK gene, three breeding lines (DBRL294-2, DBR2085 and DBR2086) found to contain RsS-26 haplotypes in the previous study (Kim and Kim 2019 ) were analyzed. Results of PCR amplification and sequencing showed no RsCopia1 insertion in any of these three breeding lines (Fig. 4 A). Except for the RsCopia1 insertion, only two single nucleotide polymorphisms (SNPs) were identified in the intron 3 between normal and mutant RsSRK-26-1 alleles. Nucleotide sequence of the normal allele was deposited into GenBank with accession number of MT241388. Unlike SC JNUR1537 containing the mutant SRK allele, three breeding lines harboring normal SRK alleles showed SI phenotypes (Fig. 4 C). Significantly reduced numbers of pods and seeds were produced in these three SI breeding lines (Fig. 4 C, Supplementary Fig. 8). Transcripts of the mutant RsSKR-26-1 were not detected in the SC radish compared with the normal RsSRK-26-1 in three SI breeding lines (Fig. 4 B). There was only a single SNP in putative promoter regions between normal and mutant RsSRK-26-1 alleles. These results suggest that transposition of RsCopia1 in the RsSRK-26-1 gene might be responsible for the inactivation of SRK and the resulting SC phenotype. Discussion Identification of duplicate SRK -like genes in the radish RsS-26 haplotype Full-length genomic DNA sequences of duplicated putative SRK and SCR/SP11 genes were obtained from the radish RsS-26 haplotype in this study (Fig. 2 ). Duplication of S core genes including SCR/SP11 and SRK and its effects on SI responses have been reported in Brassica rapa (Takada et al. 2005 , 2017 ) and Leavenworthia alabamica (Chantha et al. 2013 ), a member of Brassicaceae family. Takada et al. ( 2017 ) have demonstrated that duplicated SUI1 and PUI1 genes corresponding to SRK and SCR/SP11 , respectively, control intraspecific unilateral incompatibility in B. rapa . In the case of Leavenworthia , a novel SI system might have evolved from paralogs ( LaLal2 and LaSCRL ) of SRK and SCR/SP11 genes after loss of the original S locus, which is common in Arabidopsis , Brassica , and Leavenworthia (Chantha et al. 2013 ). The phylogenetic tree indicated that duplication of SRK homologs in the radish RsS-26 haplotype occurred more recently than that in SUI1 and LaLal2 genes (Fig. 3 ). However, it was unlikely that duplication was very recent since significant sequence and length polymorphisms existed between duplicated SRK s (Fig. 2 ). Since duplicate SCR/SP11 genes were also identified, the entire S core region might be duplicated in the RsS-26 haplotype. Alternatively, two separate S core regions might have been merged by homologous recombination-mediated translocation. Further studies are needed to elucidate the exact duplication event and effects of S core region duplication. Isolation of full-length S core regions of the RsS-26 haplotype might provide a clue to resolve these issues. As shown in B. rapa (Takada et al. 2005 , 2017 ) and Leavenworthia (Chantha et al. 2013 ), SI responses in the Brassicaceae family are complex processes. However, they are flexible enough to adopt duplicate paralogous SRK and SCR/SP11 pairs and restore SI systems after losing the original S locus under sufficient selection pressure. Multigene family of SRK homologs in Brassicaceae (Cock et al. 1995 ; Suzuki et al. 1997 ; Pastuglia et al. 1997 ; Kai et al. 2001 ) represents a potential source of such plastic evolution of SI systems. In radish, a total of 61 SRK homologs have been identified from two draft genome sequences (Kim and Kim 2018 ). Further studies such as functional characterization of duplicate SRK and SCR/SP11 genes and analysis of their effect on the strength of SI responses are needed to determine implications of S core region duplication in the evolution of SI systems in Brassicaceae family. The radish RsS-26 haplotype harboring duplicate S core genes represent a valuable material for such studies in the future. Identification of a radish breeding line showing a SC phenotype and its application in radish breeding A radish showing a SC phenotype was identified in this study. To the best of our knowledge, this is the first study to report an SC radish. Among Brassica species, several SC mutants have been previously reported. In Brassica rapa , SC mutants have been identified from two cultivars, Yellow Sarson (Fujimoto et al. 2006a ) and Dahuangyoucai (Zhang et al. 2013 ), containing similar mutant S haplotypes. Another SC phenotype induced by gene conversion from SLG to SRK has been reported (Fujimoto et al. 2006b ). In addition, an SC B. rapa has been artificially developed by silencing of SCR/SP11 using RNAi (Jung et al. 2012 ). In the case of B. oleracea , deletion of exon1 and 2 of SRK is responsible for an SC phenotype (Nasrallah et al. 1994 ). Recently, eight quantitative trait loci (QTLs) controlling a SC phenotype have been identified in an inbred line of B. oleracea (Xiao et al. 2019 ). Duplicate SRK -like genes were identified from the SC radish in this study. Some evidences indicated that the large-sized RsSRK-26-1 might be a genuine SRK . First of all, transcription of RsSRK-26-1 was inactivated in the SC radish in contrast to three other SI breeding lines containing normal RsSRK-26-1 . These results showed a direct relationship between SI phenotypes and RsSRK-26-1 . In addition, the RsSRK-26-1 gene contained a large-sized intron 3 as shown in other Brassica class II SRK genes (Supplementary Fig. 7). Further functional studies are needed to clarify exact roles of both duplicate SRK genes. Since the RsSRK-26-2 gene contained intact exons and its transcripts were more abundant than those of RsSRK-26-1 (Fig. 4 B), further functional characterization of RsSRK-26-2 might be an intriguing topic. An intact LTR-retrotransposon was identified in the large-sized intron 3 of RsSRK-26-1 of the SC radish in this study. Since this element was transposed into an intronic region, this insertion might not have any effect on transcription of SRK. Transcripts of RsSRK-26-1 were not detected in the mutant allele. Because there was only one single SNP in approximately 2.0 kb putative promoter regions between mutant and normal RsSRK-26-1 alleles, insertion of RsCopia1 might be responsible for blockage of transcription. DNA methylation of promoter regions of RsSRK-26-1 is assumed to be induced by transposition of RsCopia1 , although further functional analyses are required. DNA methylation is known to be involved in silencing of transposable elements in plants (Bartels et al. 2018 ). Similarly, transcripts of the SRK gene are not detected in SC Yellow Sarson probably due to insertion of an LTR-retrotransposon in the intron 1, although there is no critical mutation in their promoter regions (Fujimoto et al. 2006a ). In another case, reduced expression of the FLC gene is caused by insertion of an LTR-retrotransposon in the first intron (Michaels et al. 2003 ). The SC phenotype observed in this study was apparent compared with phenotypes of SI breeding lines and F 2 individuals. The SC phenotype was detected in F 1 and heterozygous F 2 individuals, suggesting that SC was dominant over SI in this population. This result indicates that the class II RsS-26 haplotype is dominant over a novel class II RsS32 haplotype. Despite a conspicuous SC phenotype, there were significant variations of pod and seed numbers produced by SC plants in this study. In addition to effects of minor modifying genes, environmental effects might play a significant role in the expression of SC phenotypes. High temperature is known to cause breakdown of SI phenotypes in Brassicaceae (Yamamoto et al. 2019 ). However, variations observed in this study might be largely derived from growth conditions where single plants were covered with mesh cages in the greenhouse. Large variations might result from such inferior growth conditions. When SC plants were grown in open field conditions, seed settings were significantly improved (Supplementary Fig. 9). The SC radish identified in this study would be a valuable material for radish F 1 hybrid breeding. Due to unstable SI phenotypes of some inbred lines, inadvertent self-pollination of maternal lines of F 1 hybrids frequently can result in a low genetic purity of F 1 cultivars. For this reason, male-sterility has replaced SI systems as a more stable genetic emasculation tool. Although male-sterility is used for F 1 hybrid breeding, propagation of inbred parental lines should be performed by self-pollination of parental lines using high concentrations of CO 2 . However, if the SC phenotype is introgressed to parental lines, such expensive treatment with CO 2 might be unnecessary. Regarding fixed varieties, the SC phenotype might greatly improve seed yields. Taken together, the SC radish identified in this study will become an important material for radish breeding programs. Declarations Acknowledgments The authors thank Ji-hwa Heo, Jeong-An Yoo, and Su-jeong Kim for their dedicated technical assistance. Authors’ contribution So-Hyeon Bong and Ganghee Cho performed experiments and drafted the manuscript. Dong-Seon Kim performed phenotypic analyses. Sunggil Kim organized and coordinated this research project and edited the final manuscript. Funding This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through a Golden Seed Project (Center for Horticultural Seed Development, No 213007-05-5-SBB10) and a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ011034). Ethics approval All experiments were performed in compliance with current laws of the Republic of Korea. Consent for publication Not applicable. Competing interests The authors declare no competing interest. 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Nature 459:992-995 Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O, Paux E, SanMiguel P, Schulman AH (2007) A unified classification system for eukaryotic transposable elements. Nat Rev Genet 8:973-982 Xiao Z, Han F, Hu Y, Xue Y, Fang Z, Yang L, Zhang Y, Liu Y, Li Z, Wang Y, Zhuang M, Lv H (2019) Overcoming cabbage crossing incompatibility by the development and application of self-compatibility-QTL- specific markers and genome-wide background analysis. Front Plant Sci 1:189 Yamamoto M, Nishimura K, Kitashiba H, Sakamoto W, Nishio T (2019) High temperature causes breakdown of S haplotype-dependent stigmatic self-incompatibility in self-incompatible Arabidopsis thaliana . J Exp Bot 70:5745-5751 Yasuda S, Wada Y, Kakizaki T, Tarutani Y, Miura-Uno E, Murase K, Fujii S, Hioki T, Shimoda T, Takada Y, Shiba H, Takasaki-Yasuda T, Suzuki G, Watanabe M, Takayama S (2016) A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets. Nat Plants 3:16206 Yasui Y, Mori M, Aii J, Abe T, Matsumoto D, Sato S, Hayashi Y, Ohnishi O, Ota T (2012) S-LOCUS EARLY FLOWERING 3 is exclusively present in the genomes of short-styled buckwheat plants that exhibit heteromorphic self-incompatibility. PLoS One 7:e31264 Zhang X, Ma C, Yin D, Zhu W, Gao C, Zhang J, Fu T (2013) Characterization of S haplotype in a new self-compatible Brassica rapa cultivar Dahuangyoucai. Czech J Genet Plant Breed 49:57-163 Supplementary Files SupplementaryFig.1.SLL2tree.tif Supplementary Fig. 1. Phylogenetic relationship between novel and other previously reported radish SLL2 alleles. The tree was constructed with nucleotide sequences. The novel SLL2 allele identified in this study is shown in boldface. Detailed information about other SLL2 alleles can be found in a previous study (Kim and Kim 2019). SupplementaryFig.2.SP6tree.tif Supplementary Fig. 2. Phylogenetic relationship between novel and other radish SP6 alleles. The tree was constructed with nucleotide sequences. The novel SP6 allele identified in this study is shown in boldface. Detailed information about other SP6 alleles can be found in a previous study (Kim and Kim 2019). SupplementaryFig.3.SRKtree.tif Supplementary Fig. 3. Phylogenetic relationship between novel and other radish SRK alleles. The tree was constructed with genomic DNA sequences. The novel SRK allele identified in this study is shown in boldface. Detailed information about other SRK alleles can be found in a previous study (Kim and Kim 2019). SupplementaryFig.4.HRMmarkers.tif Supplementary Fig. 4. Development of HRM markers used to genotype SLL2 and SP6 genes in an F2 population. A. Polymorphic sequences used to develop HRM markers. Polymorphic sequences are shown as red-colored letters on inverted triangles. Arrow-shaped boxes indicate the 5’-to’3’ direction. Empty and gray boxes indicate exons and introns, respectively. Horizontal arrows indicate primer-binding sites. Primer sequences of HRM markers are shown in Supplementary Table 1. B. Normalized melting peak patterns of HRM markers for SLL2 (Left) and SP6 (Right) genes. SupplementaryFig.5.SRKalignment.tif Supplementary Fig. 5. Alignment of partial nucleotide sequences of full-length class II SRK genes of radish and Brassica species for designing primers to amplify the 5’ end of RsSRK-26-1. Start codons are enclosed in a rectangular box. Horizontal arrows indicate primer-binding sites. Primer sequences are shown in Supplementary Table 1. GenBank accession numbers of RsSRK1, BoSRK-15, BrSRK-60, and BnSRK-6 are KX961695, AB180903, AB097116, and AB270772, respectively. SupplementaryFig.6.SCRtree.tif Supplementary Fig. 6. Phylogenetic relationship between novel and other previously reported radish and Brassica SCR/SP11 genes. The tree was constructed with cDNA sequences. The novel SCR/SP11 gene identified in this study is shown in boldface. GenBank accession numbers are shown in parentheses. SupplementaryFig.7.FulllengthSRKs.tif Supplementary Fig. 7. Phylogenetic relationship between full-length SRK genes of radish and Brassica species, with organization of exon and introns. The tree was constructed with deduced amino acid sequences. GenBank accession numbers: BnSRK-1 (AB270771), BnSRK-6 (AB270772), BoSRK-7 (AB180898), BoSRK-12 (AB180901), BoSRK-3 (X79432), BoSRK-15 (AB180903), BrSRK-8 (AB257127), BrSRK-46 (AB257128), BrSRK-47 (AB180899), BrSRK-54 (AB298592), BrSRK-60 (AB097116), BrSRK-f2 (AB190354), RsSRK19 (KX961694), RsSRK1 (KX961695). Empty and blue-colored boxes indicate exons and introns, respectively. Arrow-shaped boxes indicate the 5′-to-3′ direction. Third introns of class II SRK are shown as green-colored boxes. SupplementaryFig.8.SIandSCphenotypes.tif Supplementary Fig. 8. Comparison of self-compatible and self-incompatible phenotypes of breeding lines containing RsS3 (RsS-26) haplotypes. A. Self-compatible JNUR1537, B. Self-incompatible DBRL294-2, C. Self-incompatible DBR2085, D. Self-incompatible DBR2086. SupplementaryFig.9.SCradishinthefield.tif Supplementary Fig. 9. Self-compatible phenotype of JNUR1537 grown in a field condition. SupplementaryTable1.Primersequences.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-884506","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":53416145,"identity":"a24e1422-328a-4a39-bab4-09291f1a76b1","order_by":0,"name":"So-Hyeon Bong","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"So-Hyeon","middleName":"","lastName":"Bong","suffix":""},{"id":53416146,"identity":"993c204f-3a2d-4897-8a54-50afed91ab58","order_by":1,"name":"Ganghee Cho","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ganghee","middleName":"","lastName":"Cho","suffix":""},{"id":53416147,"identity":"21738d69-f280-4ef4-9568-8d72bd638536","order_by":2,"name":"Dong-Seon Kim","email":"","orcid":"","institution":"FarmHannong Co Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dong-Seon","middleName":"","lastName":"Kim","suffix":""},{"id":53416148,"identity":"10de98bd-b28f-4fe8-96d4-a6ae779a29bb","order_by":3,"name":"Sunggil Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYDCCA0CcwMAgx9gA4RsQrcWYRC1AkAjVQYQWvtuHjz14UHEnvXl2jwHDjxoGY/MGAlokz6WlGySceZbbOOeMAWPPMQYzmQMEtBic4TGTSGw7nNs4I8eAgbeBwUaCkMNgWtIZgVoY/5KiJQGkhRloixlBLZJn2NIkEs4cNmyckVZwWOaYhDFBLXxnmI9J/qg4LG84I3njwzc1NoYzCGmBA8MGcBwRtAMJyJOgdhSMglEwCkYYAABdAj0QwGXJ7AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8555-2995","institution":"Chonnam National University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sunggil","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2021-09-07 20:11:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-884506/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-884506/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":13930246,"identity":"e7972a67-e857-4eaf-b5df-04070a60ce44","added_by":"auto","created_at":"2021-09-23 22:47:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1703719,"visible":true,"origin":"","legend":"Phenotypes of self-incompatible and self-compatible radish breeding lines at the stage of seed harvest. A. A self-incompatible breeding line, JNUR1123, B. A self-compatible breeding line, JNUR1537. ","description":"","filename":"Fig.1.Selfcompatibleradish.png","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/f74ebf306e127d74e2cb27df.png"},{"id":13930706,"identity":"95424597-00f4-45c3-8cf9-0ebec4bee7d3","added_by":"auto","created_at":"2021-09-23 22:50:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20006,"visible":true,"origin":"","legend":"Genomic DNA structures of duplicated putative SRK genes in the radish RsS3 (RsS-26) haplotype. Empty and blue-colored boxes indicate exons and introns, respectively. Arrow-shaped boxes indicate 5′-to-3′ direction. A rectangular box above the RsSRK-26-1 indicates a transposed LTR-retrotransposon. Arrow-shaped box in the rectangular box indicates an ORF encoding a polyprotein and the 5’-to-3’ direction. LTR: long terminal repeat, RH: RNase H, RT: Reverse transcriptase, INT: Integrase, AP: Aspartic proteinase, GAG: Capsid protein. Horizontal arrows indicate positions of primer binding sites.","description":"","filename":"Fig.2.SRKstructure.png","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/9506f96c0c2ca2a6a360aa8e.png"},{"id":13930707,"identity":"98ef6e1a-4636-4c73-9671-b22508f70d36","added_by":"auto","created_at":"2021-09-23 22:50:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27596,"visible":true,"origin":"","legend":"Phylogenetic relationship of S domain sequences of radish SRK genes. The tree was constructed with nucleotide sequences. Duplicate putative SRK genes positioned in the RsS3 (RsS-26) haplotype are shown in boldface. The RsSL1 is a radish SRK-homologous gene, which is most closely related to SRK genes (Kim and Kim 2018). SUI1 (GenBank accession: LC088714) and LaLal2 (GenBank accession: KC981242) are duplicated SRK genes identified from Brassica rapa (Takada et al. 2005, 2017) and Leavenworthia alabamica (Chantha et al. 2013), respectively.","description":"","filename":"Fig.3.SRKtreewithduplicateSRKs.png","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/ccb0351a8d157117dae61cf1.png"},{"id":13930243,"identity":"e179e0ac-8fa0-4644-a547-0d5dc6b9df7e","added_by":"auto","created_at":"2021-09-23 22:47:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95897,"visible":true,"origin":"","legend":"Comparison of breeding lines containing mutant and normal RsSRK-26-1 alleles. A. PCR products of RsSRK-26-1 amplified using a primer pair flanking the LTR-retrotransposon. B. RT-PCR products of RsSRK-26-1 and RsSRK26-2 amplified from cDNAs of floral tissues. C. Comparison of pod and seed numbers produced from four breeding lines. Averages of four plants are shown in graphs. 1: JNUR1537, 2: DBRL294-2, 3: DBR2085, 4: DBR2086.","description":"","filename":"Fig.4.ComparionofSCandSI.png","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/7f9c7ddea7ceb262a46d9ab7.png"},{"id":15675619,"identity":"ec34cf5a-9df1-4b4d-bee2-8b244c45d14e","added_by":"auto","created_at":"2021-11-18 14:29:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1857627,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/8afe00fd-84b7-4848-ba6e-bd3e292ce714.pdf"},{"id":13930244,"identity":"78dc6fc9-89dd-4de1-adbc-094d870f8644","added_by":"auto","created_at":"2021-09-23 22:47:43","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":84008,"visible":true,"origin":"","legend":"Supplementary Fig. 1. Phylogenetic relationship between novel and other previously reported radish SLL2 alleles. The tree was constructed with nucleotide sequences. The novel SLL2 allele identified in this study is shown in boldface. Detailed information about other SLL2 alleles can be found in a previous study (Kim and Kim 2019).","description":"","filename":"SupplementaryFig.1.SLL2tree.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/7f6320875748424950dd999c.tif"},{"id":13930976,"identity":"78806c4c-e70f-4dc8-be63-d577b2bf7673","added_by":"auto","created_at":"2021-09-23 22:53:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":72382,"visible":true,"origin":"","legend":"Supplementary Fig. 2. Phylogenetic relationship between novel and other radish SP6 alleles. The tree was constructed with nucleotide sequences. The novel SP6 allele identified in this study is shown in boldface. Detailed information about other SP6 alleles can be found in a previous study (Kim and Kim 2019).","description":"","filename":"SupplementaryFig.2.SP6tree.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/5018e981c74fbbdf47af38bd.tif"},{"id":13930247,"identity":"c4c032a8-ca66-4bf6-a86d-0a37802fad1d","added_by":"auto","created_at":"2021-09-23 22:47:43","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":107044,"visible":true,"origin":"","legend":"Supplementary Fig. 3. Phylogenetic relationship between novel and other radish SRK alleles. The tree was constructed with genomic DNA sequences. The novel SRK allele identified in this study is shown in boldface. Detailed information about other SRK alleles can be found in a previous study (Kim and Kim 2019).","description":"","filename":"SupplementaryFig.3.SRKtree.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/2015fad58c3c98f58068b464.tif"},{"id":13930709,"identity":"52e1fc0a-b5e1-4bfc-82bb-0eb94cd60768","added_by":"auto","created_at":"2021-09-23 22:50:43","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":284228,"visible":true,"origin":"","legend":"Supplementary Fig. 4. Development of HRM markers used to genotype SLL2 and SP6 genes in an F2 population. A. Polymorphic sequences used to develop HRM markers. Polymorphic sequences are shown as red-colored letters on inverted triangles. Arrow-shaped boxes indicate the 5’-to’3’ direction. Empty and gray boxes indicate exons and introns, respectively. Horizontal arrows indicate primer-binding sites. Primer sequences of HRM markers are shown in Supplementary Table 1. B. Normalized melting peak patterns of HRM markers for SLL2 (Left) and SP6 (Right) genes.","description":"","filename":"SupplementaryFig.4.HRMmarkers.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/7b8d8dcad1ef601587793064.tif"},{"id":13930255,"identity":"88a69124-8516-422e-943f-61429b764d00","added_by":"auto","created_at":"2021-09-23 22:47:43","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":205874,"visible":true,"origin":"","legend":"Supplementary Fig. 5. Alignment of partial nucleotide sequences of full-length class II SRK genes of radish and Brassica species for designing primers to amplify the 5’ end of RsSRK-26-1. Start codons are enclosed in a rectangular box. Horizontal arrows indicate primer-binding sites. Primer sequences are shown in Supplementary Table 1. GenBank accession numbers of RsSRK1, BoSRK-15, BrSRK-60, and BnSRK-6 are KX961695, AB180903, AB097116, and AB270772, respectively.","description":"","filename":"SupplementaryFig.5.SRKalignment.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/8d54a5719ff81fb8d18b9dcc.tif"},{"id":13930249,"identity":"3b0bf0bc-8939-48c4-9e3d-31b0d8fe0c1f","added_by":"auto","created_at":"2021-09-23 22:47:43","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":72322,"visible":true,"origin":"","legend":"Supplementary Fig. 6. Phylogenetic relationship between novel and other previously reported radish and Brassica SCR/SP11 genes. The tree was constructed with cDNA sequences. The novel SCR/SP11 gene identified in this study is shown in boldface. GenBank accession numbers are shown in parentheses.","description":"","filename":"SupplementaryFig.6.SCRtree.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/90fc5ebb07bde233554e7fb8.tif"},{"id":13930252,"identity":"26a4f3bd-4432-4668-a242-b71c2b4f8da8","added_by":"auto","created_at":"2021-09-23 22:47:43","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":353706,"visible":true,"origin":"","legend":"Supplementary Fig. 7. Phylogenetic relationship between full-length SRK genes of radish and Brassica species, with organization of exon and introns. The tree was constructed with deduced amino acid sequences. GenBank accession numbers: BnSRK-1 (AB270771), BnSRK-6 (AB270772), BoSRK-7 (AB180898), BoSRK-12 (AB180901), BoSRK-3 (X79432), BoSRK-15 (AB180903), BrSRK-8 (AB257127), BrSRK-46 (AB257128), BrSRK-47 (AB180899), BrSRK-54 (AB298592), BrSRK-60 (AB097116), BrSRK-f2 (AB190354), RsSRK19 (KX961694), RsSRK1 (KX961695). Empty and blue-colored boxes indicate exons and introns, respectively. Arrow-shaped boxes indicate the 5′-to-3′ direction. Third introns of class II SRK are shown as green-colored boxes.","description":"","filename":"SupplementaryFig.7.FulllengthSRKs.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/7c2696992e9048557e1f8d25.tif"},{"id":13930254,"identity":"390bbcd0-d560-4758-ab02-3cada941bf44","added_by":"auto","created_at":"2021-09-23 22:47:43","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":4861524,"visible":true,"origin":"","legend":"Supplementary Fig. 8. Comparison of self-compatible and self-incompatible phenotypes of breeding lines containing RsS3 (RsS-26) haplotypes. A. Self-compatible JNUR1537, B. Self-incompatible DBRL294-2, C. Self-incompatible DBR2085, D. Self-incompatible DBR2086.","description":"","filename":"SupplementaryFig.8.SIandSCphenotypes.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/fd4794ed439d8cc806215412.tif"},{"id":13930711,"identity":"851b2d56-f69d-446f-9078-1a3f93d3faae","added_by":"auto","created_at":"2021-09-23 22:50:43","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":2216618,"visible":true,"origin":"","legend":"Supplementary Fig. 9. Self-compatible phenotype of JNUR1537 grown in a field condition.","description":"","filename":"SupplementaryFig.9.SCradishinthefield.tif","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/9f66f20619ccc058f0956223.tif"},{"id":13931271,"identity":"e689a9ec-64ba-4fec-b4c4-4a17f637d4e8","added_by":"auto","created_at":"2021-09-23 22:56:43","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":11681,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.Primersequences.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-884506/v1/794a6d716b496909976619da.xlsx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eRecent Transposition of an LTR-Retrotransposon in the Gene Coding for \u003cem\u003eS\u003c/em\u003e Receptor Kinase is Responsible for a Novel Self-Compatible Phenotype of Radish (\u003cem\u003eRaphanus Sativus\u003c/em\u003e L.)\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRadish (\u003cem\u003eRaphanus sativus\u003c/em\u003e L.) is one of the most important root vegetables in East Asian countries such as China, Japan, and Korea. As a cross-pollinated crop, radish shows hybrid vigor. Its F\u003csub\u003e1\u003c/sub\u003e hybrid varieties that harness such hybrid vigor have been developed (Curtis \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Self-incompatibility (SI) is defined as inhibition of pollen tube growth in pistils during self-pollination. It has been commercially used for the production of F\u003csub\u003e1\u003c/sub\u003e hybrid seeds in radish (Singh et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). SI systems are widespread in plants. They can promote outcrossing and increase genetic diversity. More than 100 families and almost 40% of angiosperms have adopted SI systems (Igic et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenerally, a single locus initially named as \u003cem\u003eS\u003c/em\u003e (Sterility) is involved in SI responses. Two tightly linked genes consisting of male and female determinants of SI are positioned in the \u003cem\u003eS\u003c/em\u003e locus. Nucleotide sequences of these two SI determinants are highly polymorphic and multi-allelic. Since at least two genes are present in the \u003cem\u003eS\u003c/em\u003e locus, combinations of these genes can lead to \u003cem\u003eS\u003c/em\u003e haplotypes (Stone and Goring \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Types of SI systems are largely categorized into heteromorphic and homomorphic SI. In the case of heteromorphic SI, cross-pollination is possible between different floral morphs. Candidate genes responsible for distyly have been reported in \u003cem\u003ePrimula vulgaris\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and \u003cem\u003eFagopyrum esculentum\u003c/em\u003e (Yasui et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHomomorphic SI systems are generally classified into gametophytic SI (GSI) and sporophytic SI (SSI) depending on inheritance patterns of SI phenotypes. Such inheritance patterns are determined by genotypes of haploid pollen in GSI and diploid pollen parents in SSI (Silva and Goring \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Mu\u0026ntilde;oz-Sanz et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). GSI systems based on RNase and S-locus F-box proteins have been extensively studied in Solanaceae, Plantaginaceae, and Rosaceae families (Franceschi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The \u003cem\u003ePapaver\u003c/em\u003e system based on programmed cell death has been mainly studied in poppy (Wheeler et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Meanwhile, SSI has been extensively studied in Brassicaceae including radish (Sobotka et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Takayama and Isogai \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Tantikanjana et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwo tightly linked genes encoding \u003cem\u003eS\u003c/em\u003e receptor kinase (SRK) and \u003cem\u003eS\u003c/em\u003e-locus cysteine-rich protein (SCR)/\u003cem\u003eS\u003c/em\u003e locus protein 11(SP11) or SP11/SCR have been revealed as female and male determinants of SSI, respectively (Schopfer et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Takasaki et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Takayama et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Another gene encoding \u003cem\u003eS\u003c/em\u003e locus glycoprotein (SLG) has been identified in the \u003cem\u003eS\u003c/em\u003e locus, although its precise role remains uncertain (Nasrallah et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Sequences of \u003cem\u003eSLG\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e domain of \u003cem\u003eSRK\u003c/em\u003e genes are highly homologous within the same \u003cem\u003eS\u003c/em\u003e haplotypes (Sato et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Okamoto et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Based on sequence diversity of \u003cem\u003eSLG\u003c/em\u003e and \u003cem\u003eSRK\u003c/em\u003e genes, \u003cem\u003eS\u003c/em\u003e haplotypes are classified into class I and class II (Nasrallah et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Sato et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Generally, \u003cem\u003eS\u003c/em\u003e haplotypes of class I are dominant over those of class II and small noncoding RNAs and DNA methylation are involved in such dominance relationships (Tarutani et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yasuda et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSI was first used in production of F\u003csub\u003e1\u003c/sub\u003e hybrid seeds of cabbage in 1940s by Japanese seed companies. Since then, SI has been exploited for economical production of F\u003csub\u003e1\u003c/sub\u003e hybrid seeds of radish and \u003cem\u003eBrassica\u003c/em\u003e crops (Mu\u0026ntilde;oz-Sanz et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To implement SI systems in radish F\u003csub\u003e1\u003c/sub\u003e hybrid breeding programs, specific \u003cem\u003eS\u003c/em\u003e haplotypes of parental lines should be identified to avoid cross-incompatibility between parental lines. At least 35 \u003cem\u003eS\u003c/em\u003e haplotypes have been identified in radish (Haseyama et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). More than 50 and 100 \u003cem\u003eS\u003c/em\u003e haplotypes have been identified in Chinese cabbage (Nou et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and cabbage (Ockendon \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), respectively.\u003c/p\u003e \u003cp\u003eFor efficient identification of \u003cem\u003eS\u003c/em\u003e haplotypes, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods have been developed for radish and \u003cem\u003eBrassica\u003c/em\u003e crops (Brace et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Nishio et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Sakamoto et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Lim et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Although PCR-RFLP is more efficient than laborious pollination tests, it has pitfalls such as complicated band patterns of digested PCR products and difficulty in specific PCR amplification of \u003cem\u003eSLG\u003c/em\u003e or \u003cem\u003eSRK\u003c/em\u003e genes due to the presence of multiple homologous \u003cem\u003eSRK\u003c/em\u003e-like genes (Boyes et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Kim and Kim \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To overcome these problems, a new \u003cem\u003eS\u003c/em\u003e haplotyping system has been developed based on \u003cem\u003eSLL2\u003c/em\u003e and \u003cem\u003eSP6\u003c/em\u003e genes positioned at borders of \u003cem\u003eS\u003c/em\u003e core regions and 31 \u003cem\u003eS\u003c/em\u003e haplotypes have been identified from diverse breeding lines in our previous studies (Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kim and Kim \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSelf-compatible (SC) mutants have been found naturally or induced by irradiation in crops normally showing SI phenotypes (Mu\u0026ntilde;oz-Sanz et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In some fruit tree species, such SC phenotypes are sometimes advantageous for better fruit set and yield (Claessen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A radish breeding line showing a SC phenotype was identified and the critical mutation responsible for the SC phenotype was revealed in this study. In addition, efficient application of this SC phenotype in radish breeding programs is discussed.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eA radish breeding line (JNUR1537) showing an SC phenotype was introduced from a seed company (Farm Hannong Co., Ltd., Republic of Korea). Detail pedigree of this breeding line is unknown. JNUR1537 was crossed with JNUR1123 to produce F\u003csub\u003e1\u003c/sub\u003e hybrids by hand-pollination. An F\u003csub\u003e2\u003c/sub\u003e population was produced by self-pollination of F\u003csub\u003e1\u003c/sub\u003e hybrids. Three breeding lines (DBRL294-2, DBR2085 and DBR2086) containing the \u003cem\u003eRsS3\u003c/em\u003e haplotype were used to compare SI phenotypes with JNUR1537. \u003cem\u003eS\u003c/em\u003e haplotypes of these three breeding lines have been identified in a previous study (Kim and Kim \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Evaluation of SI phenotypes were performed in greenhouses. Single plants were covered with mesh cages to prevent cross-pollination. No insect pollinator was introduced to mesh cages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePCR amplification, genome walking, and sequencing of PCR products\u003c/h2\u003e \u003cp\u003eTotal genomic DNAs were extracted from leaf tissues using a cetyl trimethylammonium bromide (CTAB) method (Doyle and Doyle \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). PCR amplification and sequencing of PCR products of \u003cem\u003eSLL2\u003c/em\u003e, \u003cem\u003eSP6\u003c/em\u003e, and \u003cem\u003eSRK\u003c/em\u003e genes of parental lines of the F\u003csub\u003e2\u003c/sub\u003e population were carried out following methods described in a previous study (Kim and Kim \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The detail protocol for high-resolution melting (HRM) analysis has been described in a previous study (Seo et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). SYTO\u0026reg;9 green fluorescent nucleic acid stain (Thermo Fisher Scientific, Waltham, MA, USA) was used as a dye. HRM peaks were obtained using a LightCycler\u0026reg; 96 system (Roche Molecular Systems, Pleasanton, CA, USA). Primer sequences of HRM markers are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003eTo obtain full-length genomic DNA sequences of duplicated \u003cem\u003eSRK\u003c/em\u003e homologs, long PCR and genome walking were performed. Long PCR reactions were carried out in 50 \u0026micro;L reaction mixtures containing 0.25 \u0026micro;g DNA template, 5 \u0026micro;L 10\u0026times; PCR buffer, 1.0 \u0026micro;L forward primer (10 \u0026micro;M), 1.0 \u0026micro;L reverse primer (10 \u0026micro;M), 8.0 \u0026micro;L dNTPs (10 mM each), and 0.5 \u0026micro;L Taq polymerase (TaKaRa LA PCR\u0026trade; Kit Ver. 2.1; Takara Bio, Shiga, Japan). Long PCR amplification was performed with 40 cycles of 98\u0026deg;C for 10 s and 68\u0026deg;C for 15 min. Genome walking was performed using a Universal GenomeWalker kit (Takara Bio) according to the manufacturer\u0026rsquo;s instructions. Total genomic DNAs used to construct genome walking libraries were isolated from young seedlings using a DNeasy Plant Mini Kit (QIAGEN, Valencia, CA, USA). All PCR products were visualized on 1.5% agarose gels after ethidium bromide staining. Subsequently, PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN). Sequencing was performed by a specialized company (Macrogen, Seoul, Republic of Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and reverse transcription polymerase chain reaction (RT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNAs were extracted from floral buds using a RNeasy Plant Mini Kit (QIAGEN). cDNAs were synthesized using a cDNA synthesis kit (SuperScript\u0026trade; III first-strand synthesis system for RT-PCR, Invitrogen, Carlsbad, CA, USA). RT-PCR amplification was performed with an initial denaturation step at 94\u0026deg;C for 3 min, followed by 35 cycles of 94\u0026deg;C for 30 s, 68\u0026deg;C for 30 s, and 72\u0026deg;C for 2 min. It was finished with a final 10-min extension step at 72\u0026deg;C. Primer sequences used in RT-PCR are listed in Supplementary Table\u0026nbsp;1. Radish tubulin sequence (Rs395780) identified from a draft genome sequence (Jeong et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) was used as a control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of phylogenetic trees\u003c/h2\u003e \u003cp\u003eGenomic DNA, cDNA, or deduced amino acid sequences of \u003cem\u003eSLL2\u003c/em\u003e, \u003cem\u003eSP6\u003c/em\u003e, \u003cem\u003eSRK\u003c/em\u003e, and \u003cem\u003eSCR/SP11\u003c/em\u003e genes were aligned using a BioEdit software (Hall \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Large gaps in alignments were removed using Gblocks program (Castresana \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Phylogenetic trees were produced using MEGA version X (Kumar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with a neighbor-joining method. Node support of the phylogenetic tree was assessed using 1,000 bootstrap replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eDiscovery of an SC radish breeding line and inheritance patterns of the SC phenotype\u003c/h2\u003e\n \u003cp\u003eRadishes derived from JNUR1537 showed an almost SC phenotype. Compared with an SI radish accession (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), a large number of pods were formed in the SC radish without any insect pollinators (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). This SC plant was cross-pollinated with a SI breeding line (JNUR1123). F\u003csub\u003e1\u003c/sub\u003e hybrids showed the SC phenotype, indicating that the SC phenotype was dominant over the SI phenotype. Subsequently, an F\u003csub\u003e2\u003c/sub\u003e population was produced from self-pollination of F\u003csub\u003e1\u003c/sub\u003e hybrids. Ten individuals of the F\u003csub\u003e2\u003c/sub\u003e population were grown in a greenhouse. Each individual plant was separately covered with mech cages. SI phenotypes clearly segregated among F\u003csub\u003e2\u003c/sub\u003e plants (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber of seeds produced from 10 randomly selected F\u003csub\u003e2\u003c/sub\u003e individuals grown in isolated greenhouses\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlant code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of seeds\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenotype of \u003cem\u003eS\u003c/em\u003e haplotypes\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e693\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS32 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2,330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1,483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1,012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e576\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS32 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF2-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1,086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eTo test whether the SC phenotype was related to \u003cem\u003eS\u003c/em\u003e haplotypes, specific \u003cem\u003eS\u003c/em\u003e haplotypes of both SC and SI parental lines were identified using an \u003cem\u003eS\u003c/em\u003e haplotyping system developed in a previous study (Kim and Kim \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). From the SI parent, novel sequences of \u003cem\u003eSLL2\u003c/em\u003e, \u003cem\u003eSP6\u003c/em\u003e, and \u003cem\u003eSRK\u003c/em\u003e genes closely related to \u003cem\u003eRsSLL2-25\u003c/em\u003e, \u003cem\u003eRsSP6-8\u003c/em\u003e, and \u003cem\u003eRsSRK9\u003c/em\u003e, respectively (Supplementary Figs. 1, 2, 3), were isolated. These novel allele sequences were designated as \u003cem\u003eRsSLL2-31\u003c/em\u003e, \u003cem\u003eRsSP6-26\u003c/em\u003e, and \u003cem\u003eRsSRK21\u003c/em\u003e, respectively. Their nucleotide sequences were deposited into GenBank under accession numbers of MZ383797, MZ383798, and MZ383799, respectively. Since \u003cem\u003eRsSRK21\u003c/em\u003e was clustered with class II \u003cem\u003eSRK\u003c/em\u003e genes (Supplementary Fig. 3), \u003cem\u003eRsSK21\u003c/em\u003e was considered as a member of class II \u003cem\u003eSRK\u003c/em\u003e genes. The new class II \u003cem\u003eS\u003c/em\u003e haplotype consisting of three novel sequences was designated as \u003cem\u003eRsS32\u003c/em\u003e. Nucleotide sequences of both \u003cem\u003eSLL2\u003c/em\u003e and \u003cem\u003eSP6\u003c/em\u003e genes in the SC parent were identical to those of the class II \u003cem\u003eRsS3\u003c/em\u003e haplotype. As expected, a mixture of duplicate \u003cem\u003eSRK\u003c/em\u003e-like genes was amplified. In a previous study (Kim and Kim \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), closely related duplicate \u003cem\u003eSRK\u003c/em\u003e-like genes have also been detected in the \u003cem\u003eRsS3\u003c/em\u003e haplotype.\u003c/p\u003e\n \u003cp\u003eFor efficient genotyping of \u003cem\u003eS\u003c/em\u003e haplotypes of F\u003csub\u003e2\u003c/sub\u003e individuals, two HRM markers were developed based on polymorphic sequences of \u003cem\u003eSLL2\u003c/em\u003e and \u003cem\u003eSP6\u003c/em\u003e alleles of parental lines, respectively (Supplementary Fig. 4). After genotyping F\u003csub\u003e2\u003c/sub\u003e seedlings, five individuals of each genotype were separately grown in cages. While SC phenotypes were observed in both homozygous \u003cem\u003eRsS3\u003c/em\u003e and heterozygous individuals, few pods and seeds were formed in homozygous \u003cem\u003eRsS32\u003c/em\u003e individuals (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, SI phenotypes of 10 F\u003csub\u003e2\u003c/sub\u003e individuals previously grown in isolated greenhouse were matched with genotypes of \u003cem\u003eS\u003c/em\u003e haplotypes (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These results implied that the SC phenotype was probably related to \u003cem\u003eS\u003c/em\u003e core genes such as \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumbers of pods and seeds produced from F\u003csub\u003e2\u003c/sub\u003e individuals representing three genotypes of \u003cem\u003eS\u003c/em\u003e haplotypes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGenotype\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePlant code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of pods\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of seeds\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e844\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e136\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e257\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e396\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e761\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e245\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e136\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cem\u003eRsS3 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e705\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e192\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e610\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e161\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e122\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cem\u003eRsS32 / RsS32\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eB6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003ch2\u003eAssembly of full-length genomic DNA sequences of \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eS\u003c/span\u003e core genes in the SC radish\u003c/h2\u003e\n \u003cp\u003eTo identify any defects in \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e genes in the SC radish, full-length sequences of \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e genes were analyzed. To identify the authentic \u003cem\u003eSRK\u003c/em\u003e among duplicate \u003cem\u003eSRK\u003c/em\u003e-like genes, full-length genomic DNA sequences of closely related \u003cem\u003eSRK\u003c/em\u003e-like genes were obtained in this study. Using primers designed based on conserved regions among radish and three \u003cem\u003eBrassica\u003c/em\u003e class II \u003cem\u003eSRK\u003c/em\u003e genes (Supplementary Fig. 5), 5\u0026rsquo; \u003cem\u003eS\u003c/em\u003e domain sequences of \u003cem\u003eSRK\u003c/em\u003e were isolated. After obtaining sequences of the last exon7 using genome walking, a full-length \u003cem\u003eSRK\u003c/em\u003e sequence was assembled by connecting 5\u0026rsquo; \u003cem\u003eS\u003c/em\u003e and 3\u0026rsquo; kinase domains through long PCR amplifications (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, approximately 2-kb 5\u0026rsquo; sequences containing a putative promoter was obtained by genome walking, resulting in a 18,133-bp full-length sequence. Meanwhile, a 6,200-bp full-length sequence of another putative \u003cem\u003eSRK\u003c/em\u003e gene was obtained using genome walking PCRs with primers designed based on dissimilar sequences between two putative \u003cem\u003eSRK\u003c/em\u003e genes (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eSince partial \u003cem\u003eS\u003c/em\u003e domain sequences of the larger putative \u003cem\u003eSRK\u003c/em\u003e gene containing a large-sized 11,865-bp intron 3 were identical to those of the \u003cem\u003eSRK-26\u003c/em\u003e deposited in the GenBank (LC341218), the \u003cem\u003eS\u003c/em\u003e haplotype of the SC radish might be identical to the \u003cem\u003eRsS-26\u003c/em\u003e haplotype designated by Haseyama et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Indeed, nucleotide sequences of \u003cem\u003eSLG\u003c/em\u003e of the SC radish were identical to those of \u003cem\u003eSLG-26\u003c/em\u003e (LC341241) of the \u003cem\u003eRsS-26\u003c/em\u003e haplotype. In addition, partial sequences identical to those of \u003cem\u003eRsSCR-26\u003c/em\u003e (LC325812) were amplified in the SC radish. However, additional 552-bp full-length \u003cem\u003eSCR/SP11\u003c/em\u003e gene sharing 91.0% sequence identities with \u003cem\u003eRsSCR-26\u003c/em\u003e was obtained by genome walking in the SC radish, implying that both \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e genes might be duplicated in the \u003cem\u003eRsS-26\u003c/em\u003e haplotype. The novel \u003cem\u003eSCR/SP11\u003c/em\u003e gene was closely related to other known \u003cem\u003eSCR/SP11\u003c/em\u003e genes (Supplementary Fig. 6). This novel gene was designated as \u003cem\u003eRsSCR-26-2\u003c/em\u003e. Its sequence was deposited into GenBank under the accession number of MZ383800. The full-length \u003cem\u003eRsSCR-26-2\u003c/em\u003e contained intact exons. Following the unified nomenclature suggested by Haseyama et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e), the \u003cem\u003eRsS3\u003c/em\u003e haplotype was renamed as \u003cem\u003eRsS-26\u003c/em\u003e to avoid confusion.\u003c/p\u003e\n \u003cp\u003eCoding sequences of two putative \u003cem\u003eSRK\u003c/em\u003e genes were also intact without any premature stop codons. They shared 91.7% nucleotide sequence identities with each other. Since \u003cem\u003eS\u003c/em\u003e core gene sequences were identical to those of the \u003cem\u003eRsS-26\u003c/em\u003e haplotype, the large and small putative \u003cem\u003eSRK\u003c/em\u003e genes were designated as \u003cem\u003eRsSRK-26-1\u003c/em\u003e and \u003cem\u003eRsSRK-26-2\u003c/em\u003e, respectively. Full-length sequences of both \u003cem\u003eSRK\u003c/em\u003es were deposited into GenBank under accession numbers of MZ383801 and MT241389, respectively. The phylogenetic tree of radish \u003cem\u003eSRK\u003c/em\u003e genes showed that \u003cem\u003eRsSRK-26-1\u003c/em\u003e and \u003cem\u003eRsSRK-26-2\u003c/em\u003e were closely related to each other (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Similar to other class II \u003cem\u003eSRK\u003c/em\u003e genes, the \u003cem\u003eRsSRK-26-1\u003c/em\u003e gene contained a large-sized intron 3. However, a relatively small intron 3 was identified in the \u003cem\u003eRsSRK-26-2\u003c/em\u003e gene (Supplementary Fig. 7), suggesting that the \u003cem\u003eRsSRK-26-1\u003c/em\u003e gene might be the genuine \u003cem\u003eSRK\u003c/em\u003e in the \u003cem\u003eRsS-26\u003c/em\u003e haplotype.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003eIdentification of a critical mutation responsible for the radish SC phenotype\u003c/h2\u003e\n \u003cp\u003eInterestingly, a transposable element-like sequence was found in the large-sized intron 3 of \u003cem\u003eRsSRK-26-1\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). A 4,146-bp intact open reading frame (ORF) was identified in the transposable element-like sequence. Five typical domains of long terminal repeat (LTR)-retrotransposon were identified in the polyprotein region (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Since the integrase (INT) domain was positioned upstream of the reverse transcriptase (RT) domain, this LTR-retrotransposon belonged to \u003cem\u003eCopia\u003c/em\u003e superfamily (Wicker et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). Target site duplication (TSD) of 4-bp (\u0026lsquo;GGAC\u0026rsquo;) was found at flanking regions of this element. LTR sequences of 284-bp in length positioned at both ends were perfectly identical to each other. This novel LTR-retrotransposon was designated as \u003cem\u003eRsCopia1\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eSince the ORF encoding a polyprotein was intact and both LTR sequences were identical to each other, \u003cem\u003eRsCopia1\u003c/em\u003e was assumed to be recently transposed into the \u003cem\u003eRsSRK-26-1\u003c/em\u003e gene. To investigate whether all \u003cem\u003eRsS-26\u003c/em\u003e haplotypes contained \u003cem\u003eRsCopia1\u003c/em\u003e in the \u003cem\u003eSRK\u003c/em\u003e gene, three breeding lines (DBRL294-2, DBR2085 and DBR2086) found to contain \u003cem\u003eRsS-26\u003c/em\u003e haplotypes in the previous study (Kim and Kim \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) were analyzed. Results of PCR amplification and sequencing showed no \u003cem\u003eRsCopia1\u003c/em\u003e insertion in any of these three breeding lines (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Except for the \u003cem\u003eRsCopia1\u003c/em\u003e insertion, only two single nucleotide polymorphisms (SNPs) were identified in the intron 3 between normal and mutant \u003cem\u003eRsSRK-26-1\u003c/em\u003e alleles. Nucleotide sequence of the normal allele was deposited into GenBank with accession number of MT241388.\u003c/p\u003e\n \u003cp\u003eUnlike SC JNUR1537 containing the mutant \u003cem\u003eSRK\u003c/em\u003e allele, three breeding lines harboring normal \u003cem\u003eSRK\u003c/em\u003e alleles showed SI phenotypes (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Significantly reduced numbers of pods and seeds were produced in these three SI breeding lines (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, Supplementary Fig. 8). Transcripts of the mutant \u003cem\u003eRsSKR-26-1\u003c/em\u003e were not detected in the SC radish compared with the normal \u003cem\u003eRsSRK-26-1\u003c/em\u003e in three SI breeding lines (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). There was only a single SNP in putative promoter regions between normal and mutant \u003cem\u003eRsSRK-26-1\u003c/em\u003e alleles. These results suggest that transposition of \u003cem\u003eRsCopia1\u003c/em\u003e in the \u003cem\u003eRsSRK-26-1\u003c/em\u003e gene might be responsible for the inactivation of \u003cem\u003eSRK\u003c/em\u003e and the resulting SC phenotype.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003ch2\u003eIdentification of duplicate \u003cem\u003eSRK\u003c/em\u003e-like genes in the radish \u003cem\u003eRsS-26\u003c/em\u003e haplotype\u003c/h2\u003e\n\u003cp\u003eFull-length genomic DNA sequences of duplicated putative \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e genes were obtained from the radish \u003cem\u003eRsS-26\u003c/em\u003e haplotype in this study (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Duplication of \u003cem\u003eS\u003c/em\u003e core genes including \u003cem\u003eSCR/SP11\u003c/em\u003e and \u003cem\u003eSRK\u003c/em\u003e and its effects on SI responses have been reported in \u003cem\u003eBrassica rapa\u003c/em\u003e (Takada et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) and \u003cem\u003eLeavenworthia alabamica\u003c/em\u003e (Chantha et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), a member of Brassicaceae family. Takada et al. (\u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) have demonstrated that duplicated \u003cem\u003eSUI1\u003c/em\u003e and \u003cem\u003ePUI1\u003c/em\u003e genes corresponding to \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e, respectively, control intraspecific unilateral incompatibility in \u003cem\u003eB. rapa\u003c/em\u003e. In the case of \u003cem\u003eLeavenworthia\u003c/em\u003e, a novel SI system might have evolved from paralogs (\u003cem\u003eLaLal2\u003c/em\u003e and \u003cem\u003eLaSCRL\u003c/em\u003e) of \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e genes after loss of the original \u003cem\u003eS\u003c/em\u003e locus, which is common in \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eBrassica\u003c/em\u003e, and \u003cem\u003eLeavenworthia\u003c/em\u003e (Chantha et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). The phylogenetic tree indicated that duplication of \u003cem\u003eSRK\u003c/em\u003e homologs in the radish \u003cem\u003eRsS-26\u003c/em\u003e haplotype occurred more recently than that in \u003cem\u003eSUI1\u003c/em\u003e and \u003cem\u003eLaLal2\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eHowever, it was unlikely that duplication was very recent since significant sequence and length polymorphisms existed between duplicated \u003cem\u003eSRK\u003c/em\u003es (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Since duplicate \u003cem\u003eSCR/SP11\u003c/em\u003e genes were also identified, the entire \u003cem\u003eS\u003c/em\u003e core region might be duplicated in the \u003cem\u003eRsS-26\u003c/em\u003e haplotype. Alternatively, two separate \u003cem\u003eS\u003c/em\u003e core regions might have been merged by homologous recombination-mediated translocation. Further studies are needed to elucidate the exact duplication event and effects of \u003cem\u003eS\u003c/em\u003e core region duplication. Isolation of full-length \u003cem\u003eS\u003c/em\u003e core regions of the \u003cem\u003eRsS-26\u003c/em\u003e haplotype might provide a clue to resolve these issues.\u003c/p\u003e\n\u003cp\u003eAs shown in \u003cem\u003eB. rapa\u003c/em\u003e (Takada et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) and \u003cem\u003eLeavenworthia\u003c/em\u003e (Chantha et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), SI responses in the Brassicaceae family are complex processes. However, they are flexible enough to adopt duplicate paralogous \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e pairs and restore SI systems after losing the original \u003cem\u003eS\u003c/em\u003e locus under sufficient selection pressure. Multigene family of \u003cem\u003eSRK\u003c/em\u003e homologs in Brassicaceae (Cock et al. \u003cspan class=\"CitationRef\"\u003e1995\u003c/span\u003e; Suzuki et al. \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e; Pastuglia et al. \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e; Kai et al. \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e) represents a potential source of such plastic evolution of SI systems. In radish, a total of 61 \u003cem\u003eSRK\u003c/em\u003e homologs have been identified from two draft genome sequences (Kim and Kim \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Further studies such as functional characterization of duplicate \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e genes and analysis of their effect on the strength of SI responses are needed to determine implications of \u003cem\u003eS\u003c/em\u003e core region duplication in the evolution of SI systems in Brassicaceae family. The radish \u003cem\u003eRsS-26\u003c/em\u003e haplotype harboring duplicate \u003cem\u003eS\u003c/em\u003e core genes represent a valuable material for such studies in the future.\u003c/p\u003e\n\u003ch2\u003eIdentification of a radish breeding line showing a SC phenotype and its application in radish breeding\u003c/h2\u003e\n\u003cp\u003eA radish showing a SC phenotype was identified in this study. To the best of our knowledge, this is the first study to report an SC radish. Among \u003cem\u003eBrassica\u003c/em\u003e species, several SC mutants have been previously reported. In \u003cem\u003eBrassica rapa\u003c/em\u003e, SC mutants have been identified from two cultivars, Yellow Sarson (Fujimoto et al. \u003cspan class=\"CitationRef\"\u003e2006a\u003c/span\u003e) and Dahuangyoucai (Zhang et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), containing similar mutant \u003cem\u003eS\u003c/em\u003e haplotypes. Another SC phenotype induced by gene conversion from \u003cem\u003eSLG\u003c/em\u003e to \u003cem\u003eSRK\u003c/em\u003e has been reported (Fujimoto et al. \u003cspan class=\"CitationRef\"\u003e2006b\u003c/span\u003e). In addition, an SC \u003cem\u003eB. rapa\u003c/em\u003e has been artificially developed by silencing of \u003cem\u003eSCR/SP11\u003c/em\u003e using RNAi (Jung et al. \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). In the case of \u003cem\u003eB. oleracea\u003c/em\u003e, deletion of exon1 and 2 of \u003cem\u003eSRK\u003c/em\u003e is responsible for an SC phenotype (Nasrallah et al. \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e). Recently, eight quantitative trait loci (QTLs) controlling a SC phenotype have been identified in an inbred line of \u003cem\u003eB. oleracea\u003c/em\u003e (Xiao et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eDuplicate \u003cem\u003eSRK\u003c/em\u003e-like genes were identified from the SC radish in this study. Some evidences indicated that the large-sized \u003cem\u003eRsSRK-26-1\u003c/em\u003e might be a genuine \u003cem\u003eSRK\u003c/em\u003e. First of all, transcription of \u003cem\u003eRsSRK-26-1\u003c/em\u003e was inactivated in the SC radish in contrast to three other SI breeding lines containing normal \u003cem\u003eRsSRK-26-1\u003c/em\u003e. These results showed a direct relationship between SI phenotypes and \u003cem\u003eRsSRK-26-1\u003c/em\u003e. In addition, the \u003cem\u003eRsSRK-26-1\u003c/em\u003e gene contained a large-sized intron 3 as shown in other \u003cem\u003eBrassica\u003c/em\u003e class II \u003cem\u003eSRK\u003c/em\u003e genes (Supplementary Fig.\u0026nbsp;7). Further functional studies are needed to clarify exact roles of both duplicate \u003cem\u003eSRK\u003c/em\u003e genes. Since the \u003cem\u003eRsSRK-26-2\u003c/em\u003e gene contained intact exons and its transcripts were more abundant than those of \u003cem\u003eRsSRK-26-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), further functional characterization of \u003cem\u003eRsSRK-26-2\u003c/em\u003e might be an intriguing topic.\u003c/p\u003e\n\u003cp\u003eAn intact LTR-retrotransposon was identified in the large-sized intron 3 of \u003cem\u003eRsSRK-26-1\u003c/em\u003e of the SC radish in this study. Since this element was transposed into an intronic region, this insertion might not have any effect on transcription of \u003cem\u003eSRK.\u003c/em\u003e Transcripts of \u003cem\u003eRsSRK-26-1\u003c/em\u003e were not detected in the mutant allele. Because there was only one single SNP in approximately 2.0 kb putative promoter regions between mutant and normal \u003cem\u003eRsSRK-26-1\u003c/em\u003e alleles, insertion of \u003cem\u003eRsCopia1\u003c/em\u003e might be responsible for blockage of transcription. DNA methylation of promoter regions of \u003cem\u003eRsSRK-26-1\u003c/em\u003e is assumed to be induced by transposition of \u003cem\u003eRsCopia1\u003c/em\u003e, although further functional analyses are required. DNA methylation is known to be involved in silencing of transposable elements in plants (Bartels et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, transcripts of the \u003cem\u003eSRK\u003c/em\u003e gene are not detected in SC Yellow Sarson probably due to insertion of an LTR-retrotransposon in the intron 1, although there is no critical mutation in their promoter regions (Fujimoto et al. \u003cspan class=\"CitationRef\"\u003e2006a\u003c/span\u003e). In another case, reduced expression of the \u003cem\u003eFLC\u003c/em\u003e gene is caused by insertion of an LTR-retrotransposon in the first intron (Michaels et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe SC phenotype observed in this study was apparent compared with phenotypes of SI breeding lines and F\u003csub\u003e2\u003c/sub\u003e individuals. The SC phenotype was detected in F\u003csub\u003e1\u003c/sub\u003e and heterozygous F\u003csub\u003e2\u003c/sub\u003e individuals, suggesting that SC was dominant over SI in this population. This result indicates that the class II \u003cem\u003eRsS-26\u003c/em\u003e haplotype is dominant over a novel class II \u003cem\u003eRsS32\u003c/em\u003e haplotype. Despite a conspicuous SC phenotype, there were significant variations of pod and seed numbers produced by SC plants in this study. In addition to effects of minor modifying genes, environmental effects might play a significant role in the expression of SC phenotypes. High temperature is known to cause breakdown of SI phenotypes in Brassicaceae (Yamamoto et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, variations observed in this study might be largely derived from growth conditions where single plants were covered with mesh cages in the greenhouse. Large variations might result from such inferior growth conditions. When SC plants were grown in open field conditions, seed settings were significantly improved (Supplementary Fig.\u0026nbsp;9).\u003c/p\u003e\n\u003cp\u003eThe SC radish identified in this study would be a valuable material for radish F\u003csub\u003e1\u003c/sub\u003e hybrid breeding. Due to unstable SI phenotypes of some inbred lines, inadvertent self-pollination of maternal lines of F\u003csub\u003e1\u003c/sub\u003e hybrids frequently can result in a low genetic purity of F\u003csub\u003e1\u003c/sub\u003e cultivars. For this reason, male-sterility has replaced SI systems as a more stable genetic emasculation tool. Although male-sterility is used for F\u003csub\u003e1\u003c/sub\u003e hybrid breeding, propagation of inbred parental lines should be performed by self-pollination of parental lines using high concentrations of CO\u003csub\u003e2\u003c/sub\u003e. However, if the SC phenotype is introgressed to parental lines, such expensive treatment with CO\u003csub\u003e2\u003c/sub\u003e might be unnecessary. Regarding fixed varieties, the SC phenotype might greatly improve seed yields. Taken together, the SC radish identified in this study will become an important material for radish breeding programs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Ji-hwa Heo, Jeong-An Yoo, and Su-jeong Kim for their dedicated technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSo-Hyeon Bong and Ganghee Cho performed experiments and drafted the manuscript. Dong-Seon Kim performed phenotypic analyses. Sunggil Kim organized and coordinated this research project and edited the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through a Golden Seed Project (Center for Horticultural Seed Development, No 213007-05-5-SBB10) and a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ011034).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in compliance with current laws of the Republic of Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBartels A, Han Q, Nair P, Stacey L, Gaynier H, Mosley M, Huang QQ, Pearson JK, Hsieh T, An YC, Xiao W (2018) Dynamic DNA methylation in plant growth and development. 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PLoS One 7:e31264\u003c/li\u003e\n \u003cli\u003eZhang X, Ma C, Yin D, Zhu W, Gao C, Zhang J, Fu T (2013) Characterization of \u003cem\u003eS\u003c/em\u003e haplotype in a new self-compatible \u003cem\u003eBrassica rapa\u003c/em\u003e cultivar Dahuangyoucai. Czech J Genet Plant Breed 49:57-163\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Radish (Raphanus sativus L.), Self-incompatibility, S haplotype, self-compatible mutant, Duplicate SRK genes","lastPublishedDoi":"10.21203/rs.3.rs-884506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-884506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSelf-incompatibility (SI) responses of radish (\u003cem\u003eRaphanus sativus\u003c/em\u003e L.) are determined by two tightly linked genes encoding an \u003cem\u003eS\u003c/em\u003e receptor kinase (SRK) and an \u003cem\u003eS\u003c/em\u003e-locus cysteine-rich protein/\u003cem\u003eS\u003c/em\u003e locus protein 11 (\u003cem\u003eSCR/SP11\u003c/em\u003e), respectively. A radish showing an almost self-compatible (SC) phenotype was identified in this study. Inheritance patterns showed that this SC phenotype was dominant over an SI phenotype. In addition, this SC phenotype co-segregated with an \u003cem\u003eS\u003c/em\u003e haplotype in an F\u003csub\u003e2\u003c/sub\u003e population. This SC radish contained an \u003cem\u003eRsS-26\u003c/em\u003e haplotype in which duplicate \u003cem\u003eSRK\u003c/em\u003e-like genes were previously identified. Full-length sequences of two \u003cem\u003eSRK\u003c/em\u003e-like genes of 18,133-bp and 6,200-bp in length were obtained from radish with the \u003cem\u003eRsS-26\u003c/em\u003e haplotype (designated as \u003cem\u003eRsSRK-26-1\u003c/em\u003e and \u003cem\u003eRsSRK-26-2\u003c/em\u003e, respectively). Duplicate \u003cem\u003eSCR/SP11\u003c/em\u003e-like genes were also identified in the radish with the \u003cem\u003eRsS-26\u003c/em\u003e haplotype. Phylogenetic analyses indicated that both duplicate \u003cem\u003eSRK\u003c/em\u003e-like and \u003cem\u003eSCR/SP11\u003c/em\u003e-like genes were closely related to other known \u003cem\u003eSRK\u003c/em\u003e and \u003cem\u003eSCR/SP11\u003c/em\u003e genes, respectively. No critical mutation was found in the coding region of \u003cem\u003eSRK\u003c/em\u003e-like or \u003cem\u003eSCR/SP11\u003c/em\u003e-like gene. However, a 4,146-bp intact LTR-retrotransposon was identified in the third intron of \u003cem\u003eRsSRK-26-1 \u003c/em\u003eof the SC radish. Interestingly, this LTR-retrotransposon was not detected in three other breeding lines containing the same \u003cem\u003eRsS-26\u003c/em\u003e haplotype. Except for this LTR-retrotransposon, only two single nucleotide polymorphisms (SNPs) were identified in intronic regions between normal and mutant \u003cem\u003eRsSRK-26-1\u003c/em\u003e alleles. While normal transcription was observed for radish showing \u003cem\u003eRsSRK-26-1\u003c/em\u003e and SI phenotypes in these three breeding lines, no transcript of \u003cem\u003eRsSRK-26-1\u003c/em\u003e was detected in the SC radish, suggesting that recent transposition of an LTR-retrotransposon in the \u003cem\u003eRsSRK-26-1\u003c/em\u003e gene might be responsible for the SC phenotype of radish.\u003c/p\u003e","manuscriptTitle":"Recent Transposition of an LTR-Retrotransposon in the Gene Coding for S Receptor Kinase is Responsible for a Novel Self-Compatible Phenotype of Radish (Raphanus Sativus L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-09-23 22:47:41","doi":"10.21203/rs.3.rs-884506/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":"683cd838-8f3c-44e1-9b94-1f2a29ebdb2f","owner":[],"postedDate":"September 23rd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":7384701,"name":"Horticulture"},{"id":7384702,"name":"Molecular Genetics"},{"id":7384703,"name":"Agronomy"}],"tags":[],"updatedAt":"2021-11-12T13:36:36+00:00","versionOfRecord":[],"versionCreatedAt":"2021-09-23 22:47:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-884506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-884506","identity":"rs-884506","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}