Abstract
Self-incompatibility is the system that inhibits pollen germination and pollen tube growth by self-pollen. This trait is important for the breeding of Brassica and Raphanus species. In these species, self-incompatibility is governed by the S locus, which contains three linked genes (a set called the S haplotype), i.e., S-locus receptor kinase, S-locus cysteine-rich protein/S-locus protein 11, and S-locus glycoprotein. A large number of S haplotypes have been identified in Brassica oleracea, B. rapa, and Raphanus sativus to date, and the nucleotide sequences of their many alleles have also been registered. In this state, it is important to avoid confusion between S haplotypes, i.e., an identical S haplotype with different names and a different S haplotype with an identical S haplotype number. To mitigate this issue, we herein constructed a list of S haplotypes that are easily accessible to the latest nucleotide sequences of S-haplotype genes, together with revisions to and an update of S haplotype information. Furthermore, the histories of the S-haplotype collection in the three species are reviewed, the importance of the collection of S haplotypes as a genetic resource is discussed, and the management of information on S haplotypes is proposed.
Keywords: self-incompatibility, S haplotype, S-locus receptor kinase, S-locus cysteine-rich protein/S-locus protein, S-locus glycoprotein, Brassica, Raphanus
Introduction
Self-incompatibility is the system that inhibits germination and pollen tube growth by self-pollen. It is present in many flowering plants (de Nettancourt 2001) and was investigated and described by Darwin (1876) in his work. Self-incompatibility has been considered to have evolved to prevent inbreeding depression and maintain genetic variation in populations. Many cruciferous species have a self-incompatibility system. The self-incompatibility has been extensively studied, particularly in Brassica and Arabidopsis species and their relatives. In this system, incompatible pollen grains are unable to germinate on stigma papilla cells, which are epidermal cells of the stigma, or incompatible pollen tubes cannot penetrate papilla cells. Genetic studies on this trait were performed in the 1950s and revealed that this response is controlled by a single locus, the S locus, with multiple alleles (Bateman 1955). The self-incompatibility phenotype of pollen and the stigma is sporophytically determined by the diploid S-genotype of the parent plant. Genetic dominance relationships between S alleles have been observed in both the stigma and pollen (Hatakeyama et al. 1998a, Thompson and Taylor 1966). Biochemical and molecular genetic studies revealed three important genes at the S-locus, two of which are key factors for self-recognition in the self-incompatibility response: S-receptor kinase (SRK) as a female determinant (Stein et al. 1991) and S-locus cysteine rich protein/S-locus protein 11 (SCR/SP11) as a male determinant (Schopfer et al. 1999, Suzuki et al. 1999). The third gene is S-locus glycoprotein (SLG), which is highly similar to the extracellular domain (S-domain) of SRK (Nasrallah et al. 1987). These three genes are tightly linked to each other at the S-locus and their alleles are inherited by progeny as one set. A genetic set of the alleles of these genes at the same S locus is designated as ‘S haplotype’ (Nasrallah and Nasrallah 1993) (Fig. 1). The term ‘S haplotype’ is used in this review, because ‘S-allele’ and ‘S-variant’ used in previous studies corresponds to ‘S haplotype’.
Fig. 1.
Linkage of SRK, SCR/SP11 and SLG at the S locus. Three genes, SRK, SCR/SP11 and SLG, located on the S locus and a set of alleles of three genes are called S haplotype.
A large number of S haplotypes have been collected in major Brassica and Raphanus vegetable species, i.e., Brassica oleracea, B. rapa, and Raphanus sativus, with nucleotide sequence information on SRK, SCR/SP11, and/or SLG alleles (Cho and Kim 2022, Oikawa et al. 2011). Based on the extent of sequence similarity shared by the SLG and SRK alleles, S haplotypes are grouped into two major classes, class I and II (Nasrallah et al. 1991). Genome resequencing has recently progressed in many inbred lines and cultivars of these species. These projects allow us to discover new S haplotypes. Furthermore, novel S haplotypes are expected to be found in uninvestigated resources. Actually, we newly identified four S haplotypes in B. rapa. Therefore, the number of identified S haplotypes is expected to further increase. Under this forecasted case, it is important to avoid confusion between S haplotypes, i.e., an identical S haplotype with different names and a different S haplotype with an identical S haplotype number. Therefore, we revised and updated S haplotype information on these three species and constructed a list on the Tohoku University Brassica Seed Bank website, which is easily accessible for the nucleotide sequence information of the latest S-haplotype genes. In this paper, firstly we review the historical processes of the collection and identification of S-haplotypes in these species. Next, we add information on eight novel S haplotypes that we newly identified. The nucleotide sequences of the newly identified alleles of known S haplotypes and information on novel S haplotypes collected from genome resequences have also been added to the list. Moreover, the development of DNA markers to identify or discriminate between S haplotypes is reviewed. Genome resequences provide information on the SP11-methylation-inducing region (SMI) and SMI2, which are factors that induce the epigenetic control of dominance relationships on the pollen side. This information will facilitate investigations on and predictions of dominance relationships between S haplotypes. Therefore, we herein describe discovered SMI and SMI2 sequences. In the end, we discuss the importance of the collection of S haplotypes as a genetic resource and propose the management of information on S haplotypes and S-tester lines.
The alleles of S-locus genes and S haplotypes have been commonly represented by numbers. According to standard nomenclature, alleles are shown with “+” or “–”, e.g., S+1 or S-1 (Meinke and Koornneef 1997, Østergaard and King 2008). To designate alleles of the S-locus and S haplotypes, “–” followed by Arabic numerals is hereafter used. Additionally, letters that abbreviate the species name are prefixed to the S haplotypes and alleles: e.g., S haplotypes of B. rapa are represented by BrS-1.
Collection and unification of S haplotypes
At the beginning of the collection of S haplotypes, the S genotype of a plant was identified based on a pollination assay. Selfed seeds were obtained through bud pollination from a plant, and a family was then produced. The S genotype of each plant in a family was identified by self- and cross-compatibility in intra-family diallel pollinations. Compatibility was determined based on the seed set or pollen tube behavior on the stigma through observations by fluorescence microscopy. Following this step, the S homozygotes produced by each family were subjected to diallel pollinations in order to identify the S genotype, and the S homozygote with the independent S haplotype was then a S-tester line. After the establishment of S-tester lines, the S genotyping of unidentified plants was conducted by test crossing with S-tester lines. This became the standard method for identifying S haplotypes.
To reduce the time and labor associated with the handling of many S-tester lines, an isoelectric focusing analysis of S glycoproteins (=SLG) was developed to identify S-haplotypes (Nishio and Hinata 1978, 1980, Nou et al. 1993a, 1993b). After the cloning of SLG genes, a genomic Southern blot analysis of SLG was used to identify S-haplotypes (Nasrallah et al. 1988, Okazaki et al. 1999). Furthermore, a PCR-RFLP analysis of SLG was developed (Brace et al. 1993, Nishio et al. 1994). This method has been useful for discovering new S haplotypes and discriminating between S haplotypes. Many alleles of SCR, SRK, and SLG have been isolated due to the development of many primer sets, and their nucleotide sequences have been elucidated. Through the methods described above, the collection of S haplotypes in the following species has progressed: B. oleracea, B. rapa, and R. sativus.
B. oleracea
The collection of S alleles (‘S haplotype’ is currently the proper term) was started for B. oleracea in the 1960s by Dr. Thompson’s group (Plant Breeding Institute, Cambridge, England) and Dr. van Hal’s group (Unilever Research, Holland). The S-tester lines of 23 S haplotypes from marrow stem kale and 15 S haplotypes from Brussels sprout were established by the former group (Thompson 1968) and the latter group (van Hal and Verhoven 1968), respectively. By 1973, 47 S-tester lines were produced by Dr. Thomson and transferred to Dr. Ockendon’s group, i.e., the National Vegetable Research Station at Wellsbourne. Since the 47 S-tester lines included redundantly identical S haplotypes, Dr. Ockendon spent several years solving this issue and successfully discriminated between 36 independent S haplotypes using pollination tests and the production of S-tester lines for these S haplotypes. In this collection, the S-haplotype number designated by Dr. Thompson was inherited. The four S haplotypes discovered by van Hal and not found in Thompson’s collection were referred to as S-60 to S-63 (Ockendon 1974). Information on 40 S haplotypes is listed in the study by Ockendon (2000). From 1975 onwards, Ockendon’s group continued to search S haplotypes in B. oleracea crops, such as broccoli, cabbage, and wild forms (Ockendon 1980, 1982, Stern et al. 1982), and newly identified and added 10 S haplotypes to the list, resulting in 50 S haplotypes (Ockendon 2000). These S-tester lines were transferred to Dr. Nishio’s group in Tohoku University, Japan.
Using S-tester lines and F1 cultivars, Dr. Nishio’s group cloned and elucidated the full or partial sequences of the SLG, SRK, and SCR/SP11 alleles of many S haplotypes (summarized in Oikawa et al. 2011) as shown in Table 1, i.e., 41 S haplotypes; however, unidentified alleles remained. Together with information on several S haplotypes identified by other groups, the SLG, SRK, or SCR/SP11 alleles were identified in 45 S haplotypes by 2011 (Oikawa et al. 2011, Sato et al. 2002). In parallel with their identification, the re-production of S-tester lines advanced to solve late flowering in specific S-homozygous lines of Ockendon (2000), i.e., S-3, -8, -9, -12, -22, -23, -29, -46, -58, -60, and -62 (Oikawa et al. 2011). These lines were crossed with the broccoli cultivar ‘Ryokurei’ and respective S-homozygous lines were selected from their offspring. A series of B. oleracea S-tester lines (Table 1) are stored in a laboratory of Plant Breeding and Genetics at Tohoku University in Japan.
Table 1.
S haplotypes of Brassica oleracea
a Sato et al. 2002, b Takuno et al. 2007, c Kusaba et al. 2000, d Delorme et al. 1995, e Cabrillac et al. 1999, f Stein et al. 1991, g Fujimoto et al. 2006, h Tian et al. 2013, i Suzuki et al. 2000, j Kusaba and Nishio 1999, k Fujimoto et al. 2004 (unpublished), l Kumar and Trick 1994, m Kimura et al. 2002, n Oikawa et al. 2011, o Shiba et al. 2002, p Vanoosthuyse et al. 2001, q Schopfer et al. 1999, r Lan and Li 2007 (unpublished), s Sato et al. 2003, t Kusaba et al. 1997, u Nasrallah et al. 1987, v Takuno et al. 2008, w Trick and Flavell 1989. * class-II S haplotype. $ pseudogene.
B. rapa
In the 1970s, Dr. Hinata and colleagues in Tohoku University produced S-homozygous lines from a wild population collected at the Oguni-machi area of Yamagata prefecture in Japan. Thirteen S-homozygous lines, S-1 to S-13, were developed (Kitashiba and Nishio 2013, Nishio and Hinata 1978). These lines were subjected to an analysis of S glycoproteins in the stigma by isoelectric focusing (Nishio and Hinata 1978). After this analysis, three lines, i.e., S-8, S-9, and S-12, were frequently used and played a significant role in the elucidation of the molecular mechanisms underlying self-incompatibility genetically, physiologically, and biochemically (Schopfer et al. 1999, Suzuki et al. 1999, Takayama et al. 2001). After the collection of S haplotypes, Dr. Hinata’s group newly identified 15 S haplotypes from an independent wild population collected at another spot of Oguni-machi and gave a number to each S haplotype, i.e., S-22, S-25, S-26, S-28, S-29, S-34, S-35, S-36, S-38, S-39, S-41, S-44, S-46, S-45, and S-99 (Nou et al. 1993a, 1993b). S-28, S-34, and S-26 were speculated to be identical to S-9, S-5, and S-7, respectively, based on a diallel pollination assay and immunoblot analysis (Nou et al. 1993b). At this time, the renewed S numbers S-43, S-24, and S-42 were given to S-8, S-12, and S-13, respectively. Dr. Hinata’s group also surveyed another population collected at the Balcesme area in Turkey and 16 S haplotypes were identified by a pollination assay and S-locus glycoprotein analysis (Nou et al. 1993a). S-homozygous lines were subjected to a diallel pollination assay together with the 18 S-homozygous lines developed from Oguni-machi, and 29 S haplotypes were ultimately estimated in the examined materials (Nou et al. 1993b). In this experiment, eleven S haplotypes, i.e., S-21, S-23, S-30, S-31, S-32, S-33, S-37, S-40, S-47, S-48, and S-49, which were identified from Balcesme, were newly numbered. In a series of experiments by Nou et al. (1991, 1993a, 1993b), four S haplotypes, i.e., S-22, S-24, S-35, and S-42 (S-13) were common between the Japanese and Turkey populations.
In the 1990s, the identification of the sequences in most SLG alleles and several SRK alleles was advanced by Japanese researchers (Hatakeyama et al. 1998b, Kusaba et al. 1997, Watanabe et al. 1994, Yamakawa et al. 1994, summarized in Oikawa et al. 2011). In 1999, two new S haplotypes, S-52 and S-60, were added by a pollination assay and the identification of SLG alleles (Takasaki et al. 1999). The sequences of the S domain and kinase domain of many SRK alleles were identified in the 2000s (Fujimoto et al. 2006, Okamoto et al. 2007, Sato et al. 2002, Takuno et al. 2007). Regarding SCR/SP11 alleles, after the identification of the SP11 allele of the S-9 haplotype by Suzuki et al. (1999) and the S-8, S-12, and S-52 haplotypes by Takayama et al. (2000), the sequences of the SCR/SP11 alleles for 15 S haplotypes were elucidated (Watanabe et al. 2000), followed by the sequencing of many SCR/SP11 alleles in B. oleracea (Kimura et al. 2002, Sato et al. 2002). In the latter half of the 2000s, Dr. Nishio’s group newly identified 16 S haplotypes from resources stored in the Tohoku Univ. Brassica Seed Bank, Kaneko Seed Company (Japan), and IPK Gene Bank (Gatersleben, Germany) mainly by the sequencing of SCR/SP11 alleles (Fujimoto et al. 2006, Oikawa et al. 2011, Takuno et al. 2007, 2010) and assigned S-53 to S-56 and S-61 to S-72. The SLG sequences of old dead seeds of the original S-tester lines (Nishio and Hinata 1978) were revealed to correspond to reported nucleotide sequences (Kitashiba and Nishio 2013). In addition, S-31 and S-39 were the same as S-29 and S-40, respectively. On the other hand, the S-23 and S-42 lines had been lost and any sequences of SLG, SRK and SCR/SP11 alleles were not elucidated. Either SLG, SRK, and SCR/SP11 alleles have been identified in total of 48 S haplotypes in B. rapa (Table 2).
Table 2.
S haplotypes of Brassica rapa
a Yamakawa et al. 1995, b Okamoto et al. 2007, c Takuno et al. 2007, d Sato et al. 2002, e Hatakeyama et al. 1998a, f Kakizaki et al. 2006, g Hatakeyama et al. 1998b, h Kusaba and Nishio 1999, i Fujimoto et al. 2006, j Fukai et al. 2003, k Schopfer et al. 1999, l Suzuki et al. 1999, m Takayama et al. 2000, n Watanabe et al. 2000, o Wang et al. 2019a, p Takuno et al. 2010, q Sato et al. 2003, r Shiba et al. 2002, s Isokawa et al. 2010, t Dwyer et al. 1991, u Watanabe et al. 1994, v Yamakawa et al. 1994, w Kusaba et al. 1997, x Takasaki et al. 1999, y Takuno et al. 2008, z Chung et al. 2011 (unpublished). * class-II S haplotype. N. A. means “not amplified”.
While the alleles of many S-haplotypes as described above were identified, Dr. Nishio’s group re-developed S-tester lines for some S haplotypes in B. rapa to solve the inbreeding depression of S-homozygous lines. To achieve this, previously developed S-homozygous lines for S-8, S-9, S-12, S-29, S-30, S-34, S-37, S-45, S-47, S-48, and S-49 (Nou et al. 1991, 1993a, 1993b) were crossed with an F1 cultivar ‘Osome’ and the respective S-homozygous lines were selected from their offspring. A series of B. rapa S-tester lines are stored in a laboratory of Plant Breeding and Genetics at Tohoku University in Japan, although several S-tester lines, such as S-63 and S-64, lost the germination ability of seeds.
R. sativus
R. sativus is closely related to B. rapa and B. oleracea, and also has a self-incompatibility system. Niikura and Matsuura (1997) produced R. sativus inbred lines and 17 S haplotypes were designated S-201 to S-217 through pollination tests. Furthermore, they identified an additional 20 S haplotypes by a PCR-RFLP analysis for SLG alleles mainly using Japanese landrace cultivars along with East Asian and European radish cultivars, although their SLG sequences have not been determined (Niikura and Matsuura 1999).
Dr. Nishio’s group also collected S haplotypes in R. sativus. They produced inbred lines from F1 cultivars and performed a Southern blot analysis using SLG probes and a PCR-RFLP analysis (Sakamoto et al. 1998). In experiments by Sakamoto et al. (1998), thirteen class-I and five class-II S haplotypes were identified and S-1 to S-18 were assigned to the inbred lines used. In addition, the SLG alleles of S-1 to S-8 were sequenced (Sakamoto et al. 1998). Using the same materials along with two S-homozygous lines for S-19 and S-21, Okamoto et al. (2004) determined the nucleotide sequences of the second exon of SCR/SP11 encoding the mature protein in ten S haplotypes, i.e., S-1, S-2, S-4, S-6, S-8, S-9, S-14, S-18, S-19, and S-21 as well as those of the S-domain of SRK alleles in nine S haplotypes (S-1, S-2, S-4, S-6, S-8, S-9, S-18, S-19, and S-21). The class-I S haplotypes, S-1 to S-8, S-14, S-18, S-19, and S-21 along with the class-II S haplotypes, S-9, S-11, and S-15 were identified with the sequences of either gene of the S haplotypes. In 2018, further nucleotide sequencing was performed using the same materials along with newly developed S-homozygous lines (Haseyama et al. 2018). This additional analysis resulted in the identification of 24 S haplotypes by 2018, i.e., S-1 to S-9, S-11, S-14, S-15, S-17 to S-19, S-21 to S-23, S-25, S-26, and S-28 to S-31. In which, 23 SCR/SP11 alleles, 19 S-domains, and 14 kinase domains of 19 SRK alleles and 18 SLG alleles were identified. On the other hand, Lim et al. (2002) reported the nucleotide sequences of nine SLG alleles and independently assigned them as S-1 to S-10. However, the SLG sequences of S-6, S-2, and S-7 by Lim et al. (2002) were highly similar to those of S-1, S-2, and S-4, respectively, by Sakamoto et al. (1998), with 100% or more than 99% similarity at the deduced amino acid sequences (Nishio and Sakamoto 2017). Furthermore, Lim’s group at the National Horticultural Research Institute (Korea) deposited eight SLG sequences in a public DNA database in 2004. S-12 and S-18 assigned by the group were found to be identical to S-3 and S-6, respectively, by Sakamoto et al. (1998) (Nishio and Sakamoto 2017). Five SCR-allele sequences were registered in the database with the S-haplotype names of S-1 to S-5 by Kim’s group at Sunchon National University (Korea) in 2003. Similarly, the SCR sequences of both S-4 and S-5 were highly similar to that of S-9 by Okamoto et al. (2004), while the SCR sequences of S-1, S-2, and S-3 were almost identical to those of S-26, S-15, and S-29 by Haseyama et al. (2018). As explained above, the same or highly similar sequences were reported with different S haplotype names, and different sequences were also registered with the same S-haplotype names. In contrast to cases in B. rapa and B. oleracea, the confusion of S-haplotype names occurred in R. sativus. To overcome this issue, the unification of S-haplotype names was performed by comparisons of the sequences between alleles (Haseyama et al. 2018) because Nishio’s group identified most of the SCR/SP11, SRK, and SLG sequences from the majority of the S haplotypes they collected. Following this unification, Wang et al. (2019b) identified 15 S haplotypes in Chinese local varieties, including three new S haplotypes that were named based on the list by Haseyama et al. (2018). In 2022, Cho and Kim (2022) integrated the 27 S haplotypes, which were previously identified in the Korean breeding lines by SLG and SRK nucleotide sequencing (Kim and Kim 2019), into the unified list. In 2022, 40 S haplotypes have so far been identified (Table 3).
Table 3.
S haplotypes of Raphanus sativus
a Okamoto et al. 2004, b Haseyama et al. 2018, c Lim et al. 2002, d Kim and Kim 2019, e Shirasawa et al. 2020, f Lim et al. 2004 (unpublished), g Kim et al. 2016 (unpublished), h Kim and Cho 2021 (unpublished), i Wang et al. 2019b, j Zhou et al. 2009 (unpublished), k Kim et al. 2003 (unpublished), l Sakamoto et al. 1998, m Niikura and Matsuura 1997, n Zhao et al. 2006 (unpublished), o Jeong et al. 2016, p Kitashiba et al. 2014. * class-II S haplotype. N. A. means “not amplified”.
Updated and comprehensive lists of S haplotypes in B. oleracea, B. rapa, and R. sativus
Survey of SRK, SCR, and SLG genes in public databases
To update previous lists of the B. oleracea, B. rapa, and R. sativus S haplotypes as described above, the available nucleotide sequences of SRK, SLG, and SCR of these three species were searched in published studies and public databases. In the designation of S haplotypes and the alleles of S haplotypes in each species, letters that abbreviate the species name are hereafter prefixed to the S haplotypes and alleles: e.g., the S-1 haplotype of B. rapa is represented by BrS-1 and the SLG allele of BrS-1 by BrSLG-1. Comparisons among SCR sequences revealed that the S-x and S-z haplotypes of B. rapa (Isokawa et al. 2010) were identical to BrS-61 and BrS-70 of B. rapa (Takuno et al. 2010), respectively (Table 2). In radish, eight S haplotypes in R. raphanistrum (Rr), which is a close relative to R. sativus, were deposited in the public database (Supplemental Table 1). Two of the R. raphanistrum S haplotypes, namely, RrSCR-6 and RrSCR-8, were highly similar to RsSCR-15 (99%) and RsSCR-26 (95%), respectively.
Identification of novel B. rapa and R. sativus S haplotypes
We recently identified novel S haplotypes from Brassica and Raphanus species. Four nucleotide sequences of SLG were identified from the following materials: B. rapa Fast Plants (In The Woods, Aomori, Japan), B. rapa variety utilis (Watanabe Seed Co., Miyagi, Japan), and the B. rapa C-121 accession (Tohoku University Brassica Seed Bank) (see Supplemental Text 1), and were designated as BrSLG-75, BrSLG-76, BrSLG-77, and BrSLG-78 (Table 2). A BrSCR-77 sequence was also identified (Table 2). In R. sativus, three novel nucleotide sequences of SLG were identified and designated as RsSLG-53, RsSLG-54, and RsSLG-55 following the last number given by Cho and Kim (2022). Moreover, a novel SCR sequence (RsSCR-56) was identified from a R. sativus cultivar stored in the National Agriculture and Food Research Organization Genebank Project (Genebank JP Number 26992 and 26993) (Table 3). Overall, four novel B. rapa S haplotypes and four novel R. sativus S haplotypes were discovered.
New SLG and SCR nucleotide sequences of known S haplotypes were identified. In the BrS-62, BrS-63, BrS-66, BrS-67, BrS-70, BrS-71, and BrS-72 haplotypes, only the SCR/SP11 alleles of the S-haplotype genes were determined. Using the same materials as the S-tester lines by Nishio’s group, partial fragments of SLG alleles containing hyper variable regions (HVR) were amplified and sequenced (see Supplemental Text 1, Table 2). The partial sequence of BrSLG-71 was highly similar (99.2%) to that of S locus protein 6 (SG6) [accession number JF427568] at the nucleotide and protein levels, respectively, suggesting that SG6 corresponded to BrSLG-71. The BrSCR-22 gene had a shorter first intron than other class-I SCR alleles (Wang et al. 2019a). Since BrS-22 and BoS-45 are an interspecific pair (Sato et al. 2002), the BoSCR-45 gene was amplified and sequenced using the BrSCR22-F and BrSCR22-R primers, which were designed based on the BrSCR-22 sequence (see Supplemental Text 1, Table 1). Sequencing revealed that BoSCR-45 had a short first intron, similar to BrSCR-22 (MG708357).
Phylogenetic trees of the protein sequences of SCR, the SRK S domain and SLG, were constructed (Supplemental Figs. 1–3). A phylogenetic analysis of SCR and the SRK S domain showed that seven novel S haplotypes (BrS-75, -76, -77, and -78 and RsS-53, -54, and -55) were class-I S haplotypes, while RsS-56 was a class-II S haplotype.
Identification of SRK, SCR and SLG full-length nucleotide sequences from recent genome information
Next-generation sequencing technology enables the sequencing of many B. rapa and R. sativus varieties and, thus, provides the full-length nucleotide sequences of SRK, SCR, and SLG. A BLAST (https://blast.ncbi.nlm.nih.gov/) analysis with BrSCR as the query sequence revealed that the B. rapa S-60 and S-22 haplotypes were on chromosome A07 of B. rapa subsp. pekinensis, the inbred line Chiifu-401-42 genome (Zhang et al. 2018), and chromosome A07 of the B. rapa FPsc genome (https://phytozome-next.jgi.doe.gov/info/BrapaFPsc_v1_3), respectively. Brara.G02663.1 in the B. rapa FPsc genome was similar to the BrSRK-22 partial sequence, which led to the identification of the full-length nucleotide sequence of BrSRK-22. The B. oleracea S-2b haplotype is on chromosome C06 of the B. oleracea var. capitata 02-12 genome (Liu et al. 2014).
The radish draft genome contains RsS-19 haplotypes (Kitashiba et al. 2014, Mitsui et al. 2015), and the full-length nucleotide sequence of RsSRK-19 (accession number, KX961694) has been reported by Kim and Kim (2018). Our previous analysis showed that the full-length nucleotide sequences of RsSCR-19 and RsSLG-19 were also present in the radish draft genome (Kitashiba et al. 2014, Mitsui et al. 2015). Cho and Kim (2022) reported that the S locus of the RsS-9 haplotype was located in chromosome 7 (Rs7) of the ‘WK10039’ genome (Jeong et al. 2016). We found that the cultivated radish ‘QZ-16’ genome (accession number, PRJEB37015) also contained the RsS-9 haplotype in chromosome 8 (Chr8), which corresponded to Rs7 reported by Kitashiba et al. (2014) and Jeong et al. (2016). In the ‘WK10039’ genome, Rs385140.1 and Rs385170.1 corresponded to RsSLG-9 and the first exon of RsSRK-9, respectively (Supplemental Fig. 4A). Two identical RsSCR-9 genes were found in the ‘WK10039’ genome (Supplemental Fig. 4A). On the other hand, RsSCR-9 was only located in region 23,318,086–23,317,740 of Chr8 in the ‘QZ-16’ genome. RsSRK-9 and RsSLG-9 were located in regions 23,333,706–23,340,153 and 23,369,603–23,370,928, respectively, of Chr8 in the ‘QZ-16’ genome. The coding sequences of RsSRK-9 and RsSLG-9 were predicted based on these genome sequences (Table 3). In addition to the RsS-9 haplotype, RsSCR-30 in the RUS00293_1 scaffold and RsSRK-30 and RsSLG-30 in the RUS00537_1 scaffold were found in the ‘WK10039’ genome (Supplemental Fig. 4B). This result suggested that the S genotype of ‘WK10039’ was the RsS-9/RsS-30 heterozygote. Furthermore, Rs549690.1 and Rs549670.1 corresponded to RsSLG-30 and the first exon of RsSRK-30, respectively (Supplemental Fig. 4B). Two identical RsSCR-30 genes were located in the RUS00293_1 scaffold (Supplemental Fig. 4B). Based on this analysis, the RsSRK-30 and RsSCR-30 coding sequences from the ‘WK10039’ genome were predicted (Table 3).
A BLAST analysis using RsSCR sequences revealed that the ‘Okute-Sakurajima’ R. sativus cultivar genome (Shirasawa et al. 2020) contained the RsS-6 and RsS-23 haplotypes. RSAskr1.0R7g73939 and RSAskr1.0R7g73939 corresponded to the S domain and kinase domain of RsSRK-23, respectively. The full-length nucleotide sequence of RsSCR-23 was found in the region 9,898,692–9,899,212 of Rs7. Consistent with the absence of the amplification of RsSLG-23 fragments by PCR (Haseyama et al. 2018), the SLG sequence was not found in Rs7 of the ‘Okute-Sakurajima’ genome. The S locus of RsS-6 was also present in Rs4 of the ‘Okute-Sakurajima’ genome (RSAskr1.0). Although the RsS-6 locus is assigned at different positions from the RsS-23 locus, this discrepancy may be attributed to errors in genome assembly because the S locus is highly diverse among S haplotypes (Fujimoto et al. 2006). RSAskr1.0R4g52319 and RSAskr1.0R4g52310 are identical to RsSRK-6 and RsSLG-6, respectively. The first exon of RsSCR-6 was located in region 53,308,717–53,308,777 of Rs4 in RSAskr1.0. The two second exons were found in regions 53,313,905–53,314,083 and 53,324,607–53,324,785 of Rs4 in RSAskr1.0 (Table 3).
Re-investigation of RsS-3, RsS-4, RsS-21, and RsS-30 haplotypes
Cho and Kim (2022) reported that the SRK kinase domain sequences of the RsS-21 and RsS-30 haplotypes (Haseyama et al. 2018) showed lower levels of identity (i.e., 94.06 and 85.96%, respectively) than those of the reported S haplotypes, i.e., RsS12 and RsS1, by Kim and Kim (2019). However, these inconsistencies were not observed in the SRK S-domain sequences of the RsS-21 and RsS-30 haplotypes. Re-investigations of the nucleotide sequences of the SRK kinase domain in the same RsS-21 and RsS-30 tester lines as those in Haseyama et al. (2018) revealed that the nucleotide sequence of an amplified kinase domain was highly similar (99.9% identity at the nucleotide level) to the SRK kinase domain of RsS1 and mapped to the nucleotide sequence of RsSRK-30 in the genome of ‘WK10039’ with more than 99.9% identity. A SRK cDNA fragment from RsS-21 was amplified and nucleotide sequences contained the SRK S domain of RsS-21 (accession number AB114854) and the kinase domain of RsS12 (Kim and Kim 2019). Based on these findings, we revised sequence information for RsSRK-21 and RsSRK-30 as listed in Table 3.
We also noted an error in the sequence of the SRK S domain between RsS-3 and RsS-4. We confirmed that the nucleotide sequence of the SRK S domain of R. sativus previously annotated as RsS-4 (Okamoto et al. 2004; accession number AB114848) was actually RsS-3. Therefore, the AB114848 designation was revised to correspond to the nucleotide sequence of the RsSRK-3 S domain (Table 3).
We summarized the reported primers for the amplification of the SCR, SRK, and SLG genes in Supplemental Table 2. Either primer combination will amplify the SCR, SRK, and SLG genes or a partial region of these genes.
Interspecific/intergenic pairs of S haplotypes
As the number of identified S haplotypes increases, S haplotypes with high similarity to those from different species (or genus) in the SLG, SRK, or SCR/SP11 sequences have been identified (Kimura et al. 2002, Kusaba et al. 1997). These pairs are called interspecific/intergenic pairs. Twenty-one interspecific/intergenic pairs have been found by comparisons between S haplotypes from B. rapa, B. oleracea, and R. sativus (Haseyama et al. 2018, Kimura et al. 2002, Kusaba and Nishio 1999, Kusaba et al. 1997, Oikawa et al. 2011, Okamoto et al. 2004, Sato et al. 2003, 2004). These pairs are as follows: BrS-8 (BrS-43)/BoS-32, BrS-9 (BrS-28)/RsS-21, BrS-12 (BrS-24)/BoS-51, BrS-22/BoS-45, BrS-25/BoS-14, BrS-27/BoS-8/Rs-14, BrS-35/BoS-31/RsS-22, BrS-32/BoS-68, BrS-36/BoS-24, BrS-40/BoS-5, BrS-41/BoS-64, BrS-44/BoS-2b, BrS-46/BoS-7, BrS-47/BoS-12, BrS-49/BoS-61, BrS-52/RsS-30, BrS-53/BoS-39, BrS-54/BoS-28, BrS-60/BoS-15, BrS-61/BoS-13, and BrS-69/RsS-25. Additionally, according to Supplemental Figs. 1 and 2, high similarity was observed between SCR/SP11 alleles and between SRK alleles in the BrS-34 and RsS-17, BoS-20 and RsS-19, and BoS-4 and RsS-23 pairs, suggesting that these three pairs are intergenic pairs. As demonstrated and discussed by Kimura et al. (2002), Sato et al. (2003, 2004, 2006), and Okamoto et al. (2004), each pair is considered to have originated from a common ancestral S haplotype. Interspecific/intergenic pairs are useful materials for studies on the domains affecting recognition specificity. Sato et al. (2004) found that the sequences of two regions of SCR/SP11, i.e., regions III and V, were highly conserved between interspecific pairs and were predicted to be necessary for the recognition of self SRK. Similarly, these materials will contribute to further studies, such as those on the evolution and diversification of S haplotypes.
Development of S haplotype identification using DNA polymorphisms
As the number of identified S haplotypes increases, the management of the nucleotide sequences of alleles of S haplotypes is important to avoid confusion with S haplotype names. Several consensus primer sets have been developed to amplify the fragments of SLG, the S-domains and kinase domains of SRK and SCR/SP11 from genomic DNA or cDNA (Supplemental Table 2), and nucleotide sequences have been determined as described above. Many SCR/SP11s have been amplified by several primers designed from the first exon region as a forward primer and the oligo-dT primer as a reverse primer. The second exon of the SCR/SP11 sequences were found to be highly diverse among alleles (average 55%; Sato et al. 2002). High diversity among alleles was also observed in the regions, i.e., HVR_I, II, and III, of the S domains of SRK and SLG. These findings suggest that the sequencing of SCR/SP11 and the S domains of SRK and SLG is an effective approach to identify new S haplotypes. On the other hand, the kinase domain of SRK is the least diverse among alleles. For example, the kinase domains of BrSRK-8 and BrSRK-46 were shown to be almost identical (Kusaba and Nishio 1999). Therefore, if new S haplotypes are examined, the cloning and sequencing of the S-domains of SRK, SLG, and SCR/SP11 are recommended.
Besides sequencing, DNA markers for an analysis of DNA polymorphisms are also very useful for the discrimination or identification of S haplotypes. PCR-RFLP markers have been developed based on polymorphisms in SLG alleles (Brace et al. 1993, Nishio et al. 1996) and the kinase domains of SRK alleles (Nishio et al. 1997, Park et al. 2002). Since this method is very simple and reliable, it is generally used for practical breeding, such as F1 hybrid breeding programs and seed purity tests, as well as for basic research on self-incompatibility. However, the PCR-RFLP method cannot easily identify the S genotypes of heterozygous plants because of complex band patterns. To overcome this issue, Oikawa et al. (2011) developed a method to identify 40 and 33 S haplotypes in B. rapa and B. oleracea, respectively. In this method, allele-specific primer sets and allele-specific oligonucleotide probes for each SCR/SP11 allele were designed from the sequences of each SCR/SP11 allele. Digoxigenin-labeled DNA fragments were amplified from an unknown S-genotype sample using multiplex primer pairs and the products obtained were hybridized with dot-blotted allele-specific probes, resulting in S-allele-specific signals. Distinct signals can be detected, even in heterozygous samples. Additionally, the developed allele-specific primer sets (Oikawa et al. 2011) are useful for S-genotyping. Based on the determined sequences of SLG alleles and the S-domain of SRK alleles, Haseyama et al. (2018) recently developed a dot-blot hybridization method for S-genotyping using SRK allele-specific probes designed from HVR polymorphisms. Similarly, the magnetic bead hybridization method has been developed to target the HVRs of SRK (Tonosaki et al. 2013). These methods will facilitate further research on new S haplotypes in an uninvestigated population and the confirmation of the S genotyping of materials.
Dominance relationships between S haplotypes
Genetic dominance relationships are observed between S haplotypes on the pollen and stigma sides. Thompson and Taylor (1966) and Hatakeyama et al. (1998a) reported that class-I S haplotypes were generally dominant to class-II S haplotypes on the pollen side of B. oleracea and B. rapa, although dominance relationships were also observed between class-I S haplotypes. Shiba et al. (2002) reported that transcripts of the SCR/SP11 allele of the recessive class-II S haplotype were not detected in the anther tapetum of B. rapa heterozygous plants having class-I and class-II S haplotypes. Shiba et al. (2006) subsequently showed that the suppression of recessive class-II SCR/SP11 occurred epigenetically by the de novo methylation of the 5ʹ-promoter region. Furthermore, Tarutani et al. (2010) found inverted genomic sequences similar to the sequence of the 5ʹ-promoter region in class-II SCR/SP11 in the flanking region of SLG of class-I S haplotypes. The inverted repeat sequence, called SMI, produced trans-acting small non-coding RNA (small RNA). The transcript Smi induced the methylation of the promoters of recessive class-II SCR/SP11 alleles, resulting in the suppression of class-II SCR/SP11 transcription.
Four class-II S haplotypes (BrS-44, S-60, S-40, and S-29) and three class-II S haplotypes (BoS-2b, S-5, and S-15) have been reported in B. rapa and B. oleracea, respectively. These S haplotypes show a linear dominance hierarchy on the pollen side in the order of BrS-44 > BrS-60 > BrS-40 > BrS-29 (Kakizaki et al. 2006) and BoS-2b > BoS-5 > BoS-15 (Thompson and Taylor 1966). Plants heterozygous for combinations of these class-II S haplotypes exhibited de novo promoter methylation and the suppression of the transcription of recessive SCR/SP11 alleles in anthers (Shiba et al. 2006). Yasuda et al. (2016) found another inverted repeat sequence, SMI2, on the S-locus in B. rapa, and demonstrated that the linear dominance hierarchy of B. rapa class-II S haplotypes was governed by transcripts, i.e., Smi2.
The dominance relationships between class-I and class-II S haplotypes in R. sativus currently remain unknown. Consistent with the findings obtained for B. rapa, those on the expression of accession ‘WK10039’ in the Radish Genome Database (http://radish-genome.org) showed that class-I RsSCR-30 was expressed in anther tissues (Supplemental Fig. 4B), whereas class-II RsSCR-9 expression was low (Supplemental Fig. 4A). A BLAST analysis against the ‘WK10039’ genome was performed using the stem-loop region of BrS-9_SMI as a query sequence. A BrS-9_SMI-like sequence (Supplemental Fig. 4C), designated as RsS-30_SMI in region 19,931–20,026 of the RUS00537_1 scaffold, was found. This region was located downstream of the 3ʹ untranslated region of RsSLG-30, as reported in B. rapa SMI (Tarutani et al. 2010). Three BrS-9_SMI-like sequences in the respective two S-loci of the ‘Okute-Sakurajima’ genome (Shirasawa et al. 2020) and in the RSA1.0_01586.1 scaffold of the ‘Aokubi’ genome (Kitashiba et al. 2014) were also found. These sequences were designated as RsS-6_SMI, RsS-23_SMI, and RsS-19_SMI, respectively (Supplemental Fig. 4C). Although a homologous sequence of BrS-9_SMI was observed in class-II S haplotypes in B. rapa (Tarutani et al. 2010), BrS-9_SMI-like sequences were not found in the class-II RsS-9 S locus in the ‘WK10039’ or ‘QZ-16’ genome. A highly homologous region to the RsS-30_Smi sequence was found in the RsSCR-9 promoter region (Supplemental Fig. 4D), suggesting that recessive class-II SCR expression was suppressed by Smi produced from the dominant class-I S locus in R. sativus, similar to observations in B. rapa (Tarutani et al. 2010).
On the other hand, a B. rapa SMI2-like sequence was detected in region 23,368,070–23,368,499 of Chr 8 in the ‘QZ-16’ genome. This location corresponds to a region between SRK and SLG, as observed in B. rapa SMI2 (Yasuda et al. 2016). However, the predicted RsS-9_Smi2 precursor had a longer terminal loop than the B. rapa Smi2 precursors (Supplemental Fig. 5). Further expression and functional analyses of RsS-9_Smi2 are needed to establish whether RsS-9_Smi2 suppresses RsSCR expression in recessive S haplotype(s) in a similar manner to that observed for B. rapa (Yasuda et al. 2016).
Regarding the dominance relationships of S haplotypes in the stigma, genetic analyses of B. oleracea and B. rapa were performed by Thompson and Taylor (1966) and Hatakeyama et al. (1998a), respectively. However, the underlying molecular mechanisms remain unclear. Although Hatakeyama et al. (2001) attempted an expression analysis, the recessiveness of S haplotypes in the stigma was not associated with lower SRK expression levels (Hatakeyama et al. 2001), suggesting that dominance relationships are influenced not by differences in the relative expression levels of SRK, but by the features of the SRK protein itself. BrS-8 (BrS-43) was co-dominant to BrS-46 and BrS-54, while BrS-54 was recessive to BrS-46 on the stigma side. The sequence of a kinase domain of BrSRK-8 was identical to that of BrSRK-46 at the amino acid level, and both sequences showed high similarity (98.3%) to that of BrSRK-54 (Takuno et al. 2007). On the other hand, the S domains of these SRKs were more divergent (77.8 to 85.3%). These findings suggest the importance of the S-domain for the dominance relationships of SRK. Furthermore, the possibility that the intensity of affinity between SRKs is involved in dominance relationships was proposed by Naithani et al. (2007). However, these inferences are not sufficient to explain dominance relationships. Further genetic and biochemical studies are needed. The collected SRK sequences and S-tester lines will be helpful for these purposes.
Future prospects
Brassica and Raphanus species diversified from a common ancestral species that had been established after whole-genome triplication ca. 15 million years ago (Liu et al. 2014, Lysak et al. 2005, Yang et al. 2006). In the ancestral species, a large number of S haplotypes were inferred to have evolutionarily arisen and were inherited by each species. The current number of S haplotypes is unknown. Nou et al. (1993a) estimated the number of S haplotypes in B. rapa based on the number of S haplotypes in wild populations in Japan and Turkey, and more than 100 S haplotypes are expected to exist in B. rapa species. Wild populations collected in India, Algeria, Egypt, and other places in Turkey are stored in our laboratory at Tohoku University (Tohoku Univ. Brassica Seed Bank), and S haplotypes have not yet been surveyed in the resources. Even in a Chinese landrace, the presence of novel S haplotypes has been suggested (Takuno et al. 2010), although the sequences of these S haplotypes remain unknown. Ockendon (2000) predicted that the total number of S haplotypes in B. oleracea was approximately 50 based on his long-term study of S-haplotype collections. By expanding the target for the collection of S haplotypes to the same C-genome species as B. oleracea, such as B. incana, B. villosa, and B. cretica, novel S haplotypes are expected to be discovered. To date, information on S-haplotypes in R. sativus has mainly been collected from Asian populations and cultivars in Japan, Korea, and China. World R. sativus populations are genetically classified into major 4 groups, i.e., the European group, South and Southeast Asian group, East Asian group, including China and Korea, and Japanese group (Kobayashi et al. 2020). The S haplotypes of radish populations in the European group and South and Southeast Asian group have not yet been surveyed in detail. Therefore, novel S haplotypes are expected to be identified in these areas. Although three interspecific pairs in the class-II S haplotype between B. rapa and B. oleracea have been identified, no interspecific pairs have been discovered in the class-II S haplotype between R. sativus and these species. This suggests that unidentified class-II S haplotypes are present in each species. In the exhaustive collection of S haplotypes, the development of an efficient method for S haplotype identification using the latest techniques and machines, such as a next-generation sequencer, is required. In addition, more recently, information of pangenome sequences published in B. rapa and B. oleracea species (Belser et al. 2018, Golicz et al. 2016) has been available for identification of alleles of some genes such as resistance gene analog (RGA) classes (Bayer et al. 2019). Although nucleotide sequences of each allele of S haplotypes are highly variable, especially SCR/SP11, the pangenome information will also contribute to further identification of new S haplotypes.
S haplotypes have been established through a long evolutionary history. Therefore, identified and unidentified S haplotypes are important gene resources and must not be ruined. Although it may be difficult to exchange plant samples, i.e., S-tester lines, internationally between countries or institutes due to the Convention on Biological Diversity or the private matters of each research institution, it is necessary to at least manage nucleotide sequence information on S haplotypes. Therefore, we opened a website to provide sequence information on B. rapa, B. oleracea, and R. sativus (URL: https://www.agri.tohoku.ac.jp/pbreed/S-haplotype/resource.html). The lists on the website will be useful for comparing a query sequence with the listed SLG, SRK and SCR/SP11 sequences. Following the discovery of new S haplotypes by researchers, the sharing of their sequences will be very useful. Please let us know the sequences along with deposition in public databases, such as DDBJ, NCBI or GenBank. We will add them to the S-haplotype list with references. The sharing of S-tester lines is vital. The propagation of our S-tester lines of B. rapa, B. oleracea, and R. sativus is in progress and they may be distributed to interested researchers in near future. We also accept deposits of S-homozygote or S-heterozygote samples of B. rapa, B. oleracea, and R. sativus.
The information collected on S haplotypes has contributed to studies on self-incompatibility in Brassicaceae species. However, many questions remain, such as the mechanisms responsible for the intensity and stability of self-incompatibility, dominance relationships between S haplotypes on the stigma side and even on the pollen sides, the signal pathway after self-recognition, and the evolution of S haplotypes. We expect the further collection and sharing of S haplotypes to provide information that will resolve these questions.
Author Contribution Statement
MY and HK designed the research. All authors preformed the research and analyzed the data. MY and HK wrote the manuscript.
Supplementary Material
Acknowledgments
We thank members of the Tohoku University Plant Breeding and Genetics Laboratory, Takii & Co., Ltd., Sakata Seed Corporation, and Kaneko Seeds Co., Ltd. for constructing the S-tester lines of B. rapa, B. oleracea, and R. sativus. We also thank Takeshi Nishio, Professor Emeritus of Tohoku Univ., for his deep review and comments on the history of S-haplotype collection and unification. This work was supported, in part, by the Japan Society for the Promotion of Science KAKENHI (grant numbers 20K05979 to M.Y., and 17K07599 to H.K.) and the research program on the development of innovative technology grants from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN) (grant number JPJ007097 to M.Y.).
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