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 SRKs (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. 4B), 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 F2 individuals. The SC phenotype was detected in F1 and heterozygous F2 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 F1 hybrid breeding. Due to unstable SI phenotypes of some inbred lines, inadvertent self-pollination of maternal lines of F1 hybrids frequently can result in a low genetic purity of F1 cultivars. For this reason, male-sterility has replaced SI systems as a more stable genetic emasculation tool. Although male-sterility is used for F1 hybrid breeding, propagation of inbred parental lines should be performed by self-pollination of parental lines using high concentrations of CO2. However, if the SC phenotype is introgressed to parental lines, such expensive treatment with CO2 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.