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Article

Development of S Haplotype-Specific Markers to Identify Genotypes of Self-Incompatibility in Radish (Raphanus sativus L.)

by
Seong-Ho Heo
1,2,
Su-Yeon Kim
1,
Suk-Yeon Mo
1 and
Han-Yong Park
1,*
1
Department of Bioresources Engineering, Sejong University, Seoul 05006, Republic of Korea
2
Institute of Breeding Research, DASAN Co., Ltd., Pyeongtaek 17864, Republic of Korea
*
Author to whom correspondence should be addressed.
Plants 2024, 13(5), 725; https://doi.org/10.3390/plants13050725
Submission received: 30 January 2024 / Revised: 25 February 2024 / Accepted: 1 March 2024 / Published: 4 March 2024
(This article belongs to the Collection Advances in Plant Breeding)
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Abstract

:
Radish (Raphanus sativus L.), a root vegetable belonging to the Brassicaceae family, is considered one of the representative crops displaying sporophytic self-incompatibility (SSI). The utilization of a self-incompatibility system in F1 breeding can improve the efficiency of cross-combinations, leading to a reduction in breeding time and aiding in the development of novel F1 varieties. The successful implementation of this system necessitates the rapid and accurate identification of S haplotypes in parental lines. In this study, we identified a total of nine S haplotypes among 22 elite radish lines through Sanger sequencing. Subsequently, we obtained sequences for showing a 95% similarity to nine S haplotypes, along with sequences identified by other researchers using BLAST. Following this, multiple sequence alignment (MSA) was conducted to identify SRK and SLG sequence similarities, as well as polymorphisms within the class I and II groups. Subsequently, S haplotype-specific marker sets were developed, targeting polymorphic regions of SRK and SLG alleles. These markers successfully amplified each of the nine S haplotypes. These markers will play a crucial role in the rapid and precise identification of parental S haplotypes in the radish F1 breeding process, proving instrumental in the radish F1 purity test.

1. Introduction

Radish (Raphanus sativus L.) is a root vegetable that belongs to the Brassicaceae family and is one of the most globally cultivated crops. The radish root is low in calories and rich in various minerals, particularly secondary metabolites such as polyphenols and glucosinolates, known for their anti-cancer effects [1,2]. Furthermore, radish is well known for exhibiting self-incompatibility and heterosis. Self-incompatibility facilitates outcrossing, enabling the maximal utilization of heterosis observed during radish F1 hybrid breeding.
Self-incompatibility (SI) is a mechanism that prevents self-fertilization and promotes outcrossing to maintain genetic diversity in plants [3]. Self-incompatibility can be classified into sporophytic self-incompatibility (SSI) and gametophytic self-incompatibility (GSI), depending on the timing of S gene expression [4,5,6]. Brassicaceae plants, including radish, exhibit sporophytic self-incompatibility (SSI) determined by the genotype of the pollen parent [7]. SSI is controlled by a single multi-allelic locus, called the S locus after ‘S’ for sterility [6,7,8].
Sporophytic self-incompatibility occurs in the stigma, involving the recognition and discrimination between self-pollen and non-self-pollen. This process selectively inhibits pollen tube development, thereby preventing self-fertilization [6,7,8]. There are three major genes that comprise the S locus: SRK (S locus receptor kinase), SLG (S locus glycoprotein), and SCR/SP11 (S locus cysteine-rich protein/S locus protein 11) [9,10]. S locus receptor kinase (SRK) is specifically expressed in the stigma and acts as a maternal determinant [11]. SRK comprises three domains: the receptor domain (S domain), transmembrane domain, and serine/threonine kinase domain. The S domain is crucial in the process of self-pollen recognition, and its structural characteristics include 12 cysteine residues, potential N-glycosylation sites, and three hypervariable regions. The S domain of SRK and SLG alleles has the same structure features, and the sequence similarity of two genes is over 80% within S alleles [12,13]. S locus glycoprotein (SLG) is also specifically expressed in the stigma, and the function of the SLG gene has been reported to be crucial for stabilizing the self-incompatibility response. However, the exact function or mechanism remains unknown [11,14]. S locus cysteine-rich protein/S locus protein11 (SCR/SP11), specifically expressed in pollen, acts as a ligand in the allele-specific recognition between SRK and SCR/SP11. SCR/SP11 activates a signal cascade mediating the SI response [8].
SRK, SLG, and SCR/SP11, involved in the S locus, are tightly linked to each other and tend to be inherited together in the next generation, forming what is called an S haplotype [15]. S haplotypes can be classified into two groups, class I and class II, based on amino acid sequence similarities of SLG and SRK [16]. The amino acid sequences of the S domain of SRK or SLG alleles show a 72% similarity within the same group and a similarity of 70% or less between different groups [17]. Additionally, SLG and the S domain of SRK alleles within the same S haplotype generally show high sequence similarity, especially in the class II group [18,19,20,21]. Between the two groups, there exists a complex genetic dominance relationship, where class I S haplotypes are generally dominant over class II S haplotypes in the pollen. On the other hand, in the stigma, codominance frequently occurs [20,22].
The SI system has been widely applied to F1 hybrid breeding, encompassing radish, cabbage, and other Brassica species. The utilization of the SI system has increased the efficiency of cross-combinations, shortening breeding time and facilitating the development of novel F1 varieties [23]. However, if parents share the same S haplotype and exhibit cross-incompatibility, it is difficult to produce a large scale of F1 hybrid seeds [24]. Therefore, it is necessary to select cross-combinations by early excluding radish breeding lines that exhibit the same S haplotype through the rapid and accurate identification of radish S haplotypes in the F1 hybrid breeding process.
Early studies on the identification of radish S haplotypes involved compatibility index (CI) analysis through test crosses, pollen tube examination, fluorescence analysis, and isoelectric focusing (IEF) gel examination [10,25,26]. However, these methods were labor-intensive, complex, and low-accuracy. With the subsequent advancement of PCR technology, Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) markers and Sequence-Characterized Amplified Region (SCAR) markers that specifically amplify radish SRK or SLG alleles have been developed and used for the identification of radish S haplotypes [27,28,29]. However, when using PCR-RFLP, the presence of identical restriction enzyme sites among some S haplotypes complicates the accurate identification process. With SCAR markers, the high sequence similarity between certain class I and II S haplotypes often leads to the co-amplification of several different S haplotypes [23,28,29].
Recently, the amplification of S locus genes using a universal primer set for class I and II S haplotypes, followed by Sanger sequencing to determine the nucleotide sequence, has become a common method. Subsequently, S haplotype determination involves comparing the obtained sequence with those already deposited in NCBI databases for similarity [23,24]. Even so, the use of gene nucleotide sequences and BLAST search requires the analysis of the nucleotide sequences of all parental lines, making it highly inefficient and unsuitable for large-scale S haplotype identification [23]. Despite the development of various methods, including molecular markers, there is still the challenge of finding an accurate and rapid method for identification of radish S haplotypes.
Furthermore, each researcher has independently assigned a nomenclature for radish S haplotypes in different countries, making the identification of radish S haplotypes more difficult [30,31]. In Japan, Niikura and Matsuura (1998) and Sakamoto (1998) identified nine SLG genes, naming S haplotypes as S201 and S1 to S8 [32,33]. Subsequently, Okamoto (2004) identified 10 SCR genes and 9 SRK genes, adopting the same nomenclature as Sakamoto (1998) [34]. In Korea, Lim (2002, 2004) identified 17 SLG gene sequences, naming them S1 to S26, while Park (2005) identified 5 SCR genes, naming them S1 to S5 [27,31]. Later, Kim (2016, 2019) named S haplotypes RsS-1 to RsS-31, considering combinations of SRK, SLL2, and SP6 genes [17]. More recently, Ni (2022) eliminated overlapping haplotypes based on BLAST analysis and newly named a total of 52 S haplotypes as NAU-S1 to NAU-S52 [23]. Given the various nomenclatures in current databases, it is essential to establish specific criteria for global S haplotype classification to facilitate the sharing of information on individual S haplotype characteristics [31].
Therefore, in this study, we aimed to overcome the limitations of existing S haplotype markers and develop an accurate and rapid method for radish S haplotype identification. Firstly, we classified and compared S haplotypes named differently by Korean, Chinese, and Japanese researchers in the NCBI database according to specific criteria. Secondly, using 22 radish breeding lines, mainly utilized for radish F1 hybrid breeding in Korea and Japan, among other regions, and stabilized through more than seven generations of self-pollination, we developed radish S haplotype-specific markers based on the nucleotide sequences of SRK and SLG alleles. The S haplotype-specific markers developed in this study are expected to be effectively used for identifying the S haplotypes of parental lines, selecting cross-combinations for F1 hybrid breeding, and performing seed purity tests during the F1 hybrid breeding process.

2. Results

2.1. Amplification of SRK, SLG Alleles Using Universal Primers

A total of three class I SRK and SLG universal primer sets, along with four class II SRK and SLG universal primer sets, were used to amplify the kinase domain of SRK and SLG alleles of 22 radish breeding lines (‘SJ-1~22’).
In 14 radish breeding lines (‘SJ-1~14’), the class I kinase domain of SRK was amplified using the UVSRK-F + UVSRK-R primer combination. Additionally, class I SLG alleles were amplified with the SLG-I-F + SLG-I-R and PS22 + SLG-I-R primer combinations, resulting in a total band size of 1000 to 1500 bp (Figure S1).
In eight radish breeding lines (‘SJ-15~22’), the class II kinase domain of SRK was amplified using the KS2 + KA2 primer combination. Additionally, class II SLG alleles were amplified with the SLG-II-F + SLG-II-R, Rs9 SLG-F + Rs9 SLG-R, and UVSLGII-F + UVSLGII-R primer combinations, resulting in a total band size of 800 to 1500 bp (Figure S2).

2.2. Identification of S Haplotypes Based on BLAST Search

We performed BLAST search using SRK and SLG alleles amplified by universal primer sets from ‘SJ-1~22’ to identify the corresponding S haplotypes. These sequences were compared to previously published S haplotypes in the NCBI database or from other researchers. As a result, it was determined that the 22 radish breeding lines possessed a total of nine S haplotypes (Table 1). Nucleotide sequence information is provided in the supplementary materials (Table S4), and if two or more radish lines had the same S haplotype, only the sequence obtained from one of them was indicated.
A total of 14 radish breeding lines (‘SJ-1~14’) were classified as the class I S haplotype. ‘SJ-1~4’ were identified as S1 (Lim), since the kinase domain of SRK and SLG alleles showed 100% and 99% identities to SRK1(Lim) and SLG1(Lim), respectively. ‘SJ-5~6’ were identified as S8 (Lim) due to a 100% identity in the kinase domain of the SRK allele with SRK8 (Lim), SRK19 (Kim), and SRK-19 (Haseyama), along with a 100% identity in the SLG allele with SLG-19 (Haseyama). S8 (Lim) has been reported as the same S haplotype as S19 (Kim) and RsS-19 (Haseyama) [31]. Additionally, the kinase domain of the ‘SJ-5~6’ SRK allele showed a 97% identity with SRK-18 (Haseyama). ‘SJ-7~9’ were identified as S10 (Lim), since the kinase domain of SRK and SLG alleles showed a 99.9% identity to SRK10 (Lim) and SLG10 (Lim), respectively. There were 11 other SRK sequences showing 98 to 99% identities to the kinase domain of the ‘SJ-7~9’ SRK allele (Table 1, Figure S7). ‘SJ-10~11’ were identified as S16 (Lim) due to a 99.9% identity in the kinase domain of the SRK allele with SRK16 (Lim) and SRK-22 (Haseyama), along with a 100% identity in the SLG allele with SLG-22 (Haseyama). S16 (Lim) has been reported as the same S haplotype as RsS-22 (Haseyama) [31]. ‘SJ-12~14’ were identified as S18 (Lim) due to a 99.9% identity in the kinase domain of the SRK allele with SRK18 (Lim) and SRK-6 (Haseyama), along with a 100% identity in the SLG allele with SLG18 (Lim) and SLG-6 (Sakamoto). S18 (Lim) has been reported as the same S haplotype as RsS-6 (Sakamoto) [31].
A total of eight radish breeding lines (‘SJ-15~22’) were classified as the class II S haplotype. ‘SJ-15~17’ were identified as S4 (Lim) due to 99% and 100% identities in the SLG allele with SLG4 (Lim) and SLG-26 (Lim). S4 (Lim) has been reported as the same S haplotype as RsS-26 (Haseyama) [31]. Additionally, the ‘SJ-15~17’ SLG allele showed a 97% identity with SLG-11 (Haseyama). ‘SJ-18~20’ were identified as S5 (Lim) due to a 99% identity in the kinase domain and S domain of the SRK allele with SRK-5 (Wang). S5 (Lim) has been reported as the same S haplotype as S-5 (Wang) [24]. Also, the kinase domain of the ‘SJ-18~20’ SRK allele showed a 99% identity with SRK-9 (Wang) and SRK6 (R. raphanistrum). Additionally, the S domain of the ‘SJ-18~20’ SRK allele showed 97 to 99% identities with 10 other class II S domains of SRK and SLG alleles (Table 1). ‘SJ-21’ was identified as S21 (Lim) due to a 99% identity in the kinase domain of the SRK allele with SRK-9 (Wang) and a 100% identity in the SLG allele with SLG21 (Lim), SLG-9 (Haseyama), and SLG-9 (Wang). S21 (Lim) has been reported as the same S haplotype as RsS-9 (Haseyama) and S-9 (Wang) [24]. Also, the kinase domain of the ‘SJ-21’ SRK allele showed a 99% identity with SRK-5 (Wang) and SRK6 (R. raphanistrum). Additionally, the ‘SJ-21’ SLG allele showed 94 to 100% identities with nine other class II S domains of SRK and SLG alleles (Table 1). ‘SJ-22’ was identified as S26 (Lim) due to a 100% identity in the SLG allele with SLG26 (Lim) and SLG-29 (Haseyama). S26 (Lim) has been reported as the same S haplotype as RsS-29 (Haseyama) [31].

2.3. Multiple Sequence Alignment (MSA) of SRK, SLG Alleles

Multiple sequence alignment (MSA) of nucleotide and amino acid sequences was performed to develop S haplotype-specific markers targeting polymorphic regions of SRK and SLG alleles. In this alignment, we used all the S haplotype sequences registered by Korean, Chinese, and Japanese researchers. This included 7 kinase domains of SRK and 10 SLG alleles from Lim’s group [27]; 10 kinase domains of SRK alleles from Kim’s group [17], 3 kinase domains and 3 S domains of SRK alleles from Wang’s group [24]; 7 SLG alleles from Sakamoto’s group [33]; along with 8 kinase domains, 3 S domains of SRK alleles, and 4 SLG alleles from Haseyama’s group [31]. Only one sequence of the S haplotype was used when the same S haplotype was designated differently.
A total of 19 class I kinase domains of SRK sequences were used for the alignment of the class I kinase domain of SRK alleles. The similarity between the class I kinase domain of SRK nucleotide sequences ranged from 83.86 to 91.85%. All exon regions (4th to 7th) were conserved relative to intron regions (4th to 7th). In the 6th and 7th exons, some nucleotide polymorphisms, including insertions/deletions (InDels), were detected. In the sixth exon region, a small gap (13 bp) was observed. Additionally, a large gap (41 to 49 bp) and a small gap (19 to 29 bp) were observed in the 4th and 6th intron regions, excluding SRK-KD18 (Haseyama) and SRK-KD1 (Haseyama) (Figure S3).
A total of 15 class I SLG sequences were used for the alignment of class I SLG alleles. The similarity between class I SLG nucleotide sequences ranged from 84 to 88.94%. Many single nucleotide polymorphisms (SNPs) were observed in hypervariable regions I to III (HV-I to III). Especially, a small gap (6 bp) was observed in the downstream region of HV-II, excluding SLG4 (Sakamoto). The deduced amino acid sequences of 14 class I SLG alleles revealed characteristics as previously reported, including 12 conserved cysteine residues, the potential N-glycosylation sites, and three hypervariable regions (Figure 1 and Figure S4).
A total of nine class II kinase domains of SRK sequences were used for the alignment of the class II kinase domain of SRK alleles. The similarity between the class II kinase domain of SRK nucleotide sequences ranged from 90.17 to 99.17%. Notably, the SRK26-KD (Wang) nucleotide sequence showed a high similarity (99.17%) with SRK14-KD (Kim). As observed in the case of the class I kinase domain of SRK alleles, all exon regions were conserved relative to intron regions. On the other hand, all exon regions showed high similarity within the same group of the class II kinase domain of SRK alleles. Some single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were observed in the sixth intron region; particularly, a small gap (14 bp) was only detected in SRK-5 (Wang) and SRK-9 (Wang) (Figure S5).
Due to the high similarity between the class II SLG and the S domain of SRK sequences, a total of 12 sequences including SLG and the S domain of SRK alleles were used for the alignment. The similarity between class II SLG nucleotide sequences was 90 to 97.05%, and when including the S domain of SRK sequences, they showed 87.5 to 99.16%. In the upstream region of HV-I, small gaps (9, 12 bp) were observed. In the deduced amino acid sequences of the class II SLG and the S domain of SRK alleles, 12 conserved cysteine residues were detected at the same positions as those in class I SLG alleles, excluding SLG4 and 21 (Lim) and SRK-5 (Wang). Additionally, the potential N-glycosylation sites and three hypervariable regions were found to be located at the same positions (Figure 2 and Figure S6).
Plants 13 00725 g001
Figure 1. Multiple sequence alignment of the amino acid sequences of the 15 class I S haplotype SLG alleles using Clustal Omega program on EMBL-EBI site; a: S haplotype published by Sakamoto [33]; b: S haplotype published by Haseyama [31]; c: S haplotype published by Lim [27]; red box: hypervariable regions I to III of the SLG; filled circle: N-linked glycosylation site; filled triangle: conserved cysteine residues; asterisk: conserved amino acid residues.
Figure 1. Multiple sequence alignment of the amino acid sequences of the 15 class I S haplotype SLG alleles using Clustal Omega program on EMBL-EBI site; a: S haplotype published by Sakamoto [33]; b: S haplotype published by Haseyama [31]; c: S haplotype published by Lim [27]; red box: hypervariable regions I to III of the SLG; filled circle: N-linked glycosylation site; filled triangle: conserved cysteine residues; asterisk: conserved amino acid residues.
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Figure 2. Multiple sequence alignment of the amino acid sequences of the 12 class II S haplotype SLG alleles and the S domain of SRK alleles using Clustal Omega program on EMBL-EBI site; a: S haplotype published by Haseyama [31]; b: S haplotype published by Lim [27]; c: S haplotype published by Wang [24]; red box: hypervariable regions I to III of the SLG; filled circle: N-linked glycosylation site; filled triangle: conserved cysteine residues; empty triangle: the site where non-synonymous mutation was inferred to have occurred; asterisk: conserved amino acid residues.
Figure 2. Multiple sequence alignment of the amino acid sequences of the 12 class II S haplotype SLG alleles and the S domain of SRK alleles using Clustal Omega program on EMBL-EBI site; a: S haplotype published by Haseyama [31]; b: S haplotype published by Lim [27]; c: S haplotype published by Wang [24]; red box: hypervariable regions I to III of the SLG; filled circle: N-linked glycosylation site; filled triangle: conserved cysteine residues; empty triangle: the site where non-synonymous mutation was inferred to have occurred; asterisk: conserved amino acid residues.
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2.4. Development of S Haplotype-Specific Markers

Based on the results of class I and II SRK and SLG sequence alignment, we identified regions showing single nucleotide polymorphisms (SNPs) or small insertions/deletions (INDELs) within class I and II groups. Targeting these regions, we developed S haplotype-specific markers to selectively amplify the nine S haplotypes present in 22 radish breeding lines (‘SJ-1~22’) (Table 4 and Table S2).
A total of four kinase domains of SRK primer sets were designed, with each primer set amplifying specific regions of SRK1 (Lim), SRK8 (Lim), SRK10 (Lim), and SRK18 (Lim) kinase domains, ranging from the 4th to 7th exon of SRK alleles. The expected PCR product size using these primer sets was 105 to 764 bp, designed to exhibit length polymorphism with each primer set (Table 2)
Additionally, nine SLG primer sets were designed, each amplifying specific regions of SLG1 (Lim), SLG4 (Lim), SLG5 (Lim), SLG8 (Lim), SLG10 (Lim), SLG16 (Lim), SLG18 (Lim), SLG21 (Lim), and SLG26 (Lim), ranging from HV-I to III of SLG alleles. The expected PCR product size using these primer sets was 131 to 529 bp, similar to the kinase domain of SRK alleles, designed to show length polymorphism with each primer set (Table 2).
To verify the specificity of the kinase domain of SRK and SLG primer sets developed in this study, genotyping was conducted on 22 radish breeding lines (‘SJ-1~22’) using the kinase domain of SRK and SLG primer sets. Consequently, all 5 SRK primer sets and 9 SLG primer sets selectively amplified radish breeding lines possessing a distinct S haplotype, and the predicted length of the PCR product from in silico analysis aligned with the observed results (Figure 3 and Figure 4). Furthermore, it was noted that the drawbacks, such as the amplification of nonspecific S haplotypes, which arose from the previously developed SCAR marker, did not occur in the S haplotype-specific markers developed in this study.

3. Discussion and Conclusions

To develop S haplotype-specific markers, it was necessary to determine the sequences of S alleles (SRK, SLG) for a specific S haplotype in radish breeding lines (SJ-1~22). For this purpose, SRK and SLG genes were amplified using three types of class I universal primer sets and four types of class II universal primer sets. Due to the sequence variations in S alleles, amplifying all S alleles with a limited combination of primers posed a challenge. Therefore, we used various primer combinations developed in previous studies [27,33,35,36,37,38,39]. Although PCR amplification was performed using seven class I and II universal primers, it was difficult to identify S haplotypes due to inaccurate amplification of the non-target genes in some cases. For instance, when performing PCR amplification using three universal class II SLG primer sets in ‘SJ-18~20’, it was observed that PCR amplification occurred for the S domain of the SRK allele instead of the SLG allele. These results are consistent with previous reports indicating that the sequences of the S domain of SRK and SLG alleles within the class II group were highly similar [21,24,40]. Additionally, when using the universal class II kinase domain of SRK primer sets in ‘SJ-15~17’ and ‘SJ-22’, other regions with the same primer binding sites were co-amplified, leading to sequencing errors (mixed signal). As a result, the exact kinase domain of SRK sequences of ‘SJ-15~17’ and ‘SJ-22’ could not be identified.
As a result of classifying and comparing class I S haplotypes using BLAST analysis, within the class I S haplotype group, including SRK and SLG alleles of ‘SJ-1~14’, along with those obtained through BLAST search in the NCBI database, there were numerous cases where the kinase domain of SRK alleles exhibited significant similarity to each other. This result corresponds with the observations during the previous identification of class I S haplotypes in the Brassicaceae family, including radishes [24,29]. The kinase domain of SRK alleles has been reported to be the region where recombination could occur, and the observed high similarity within the class I kinase domain of SRK alleles might be attributed to genetic recombination events [41]. In contrast to previous reports suggesting recombination in SLG alleles, we did not observe high similarity within class I SLG alleles [42].
Furthermore, based on Lim’s nomenclature, we compared a total of five class I S haplotypes identified in 14 radish breeding lines (SJ-1~14) with those investigated by other researchers. Through this comparison, we could classify sequences that were similar or identical (Table 1). Consistent with previously reported research, observations revealed that S8 (Lim) corresponds to RsS-19 (Haseyama, Kim), S16 (Lim) corresponds to RsS-22 (Haseyama), and S18 (Lim) corresponds to RsS-6 (Sakamoto, Haseyama) [27,31,33,39]. When BLAST search was conducted using kinase domain sequences of SRK10 (Lim) and SRK16 (Lim) as queries, 11 kinase domains of SRK sequences showing high similarity (98 to 99%) were identified. Subsequent sequence alignment analysis of SRK10 and 16 (Lim) kinase domains with these similar kinase domains revealed nearly identical nucleotide sequences, with only a few single nucleotide polymorphisms (SNPs) (Figure S7). Although the SLG16 (Lim) nucleotide sequence identified in ‘SJ-10~11’ and SLG-7 (Sakamoto) had a 99.9% identity, S16 (Lim) has not been reported to be the same as RsS-7 (Sakamoto). Consequently, a comparison of the total length of two SLG alleles or S domains of SRK alleles was necessary to reclassify the two S haplotypes.
As a result of classifying and comparing class II S haplotypes using BLAST analysis, in the class II S haplotype group, including SRK and SLG alleles of ‘SJ-15~22’, along with those obtained through BLAST search in the NCBI database, the kinase domains of SRK alleles as well as SLG alleles were highly similar to each other, unlike the class I S haplotype group. Previous studies have reported that the kinase domain of SRK and SLG alleles is the region where recombination could occur [41,43]. The class II S haplotype group is inferred to be more recently diverged than the class I S haplotype group, as indicated by the high similarity within the class II S haplotype group [44]. According to the Brassica SI evolutionary model proposed by Uyenoyama, the class II S haplotype group might invade populations at lower rates than the class I S haplotype, leading to a decrease in the occurrence of mutations and divergence within the class II group [45].
Moreover, based on Lim’s nomenclature, we compared a total of four class II S haplotypes identified in eight radish breeding lines (SJ-15~22) with those investigated by other researchers. Through this comparison, we could classify sequences that were similar or identical (Table 1). Consistent with previously reported research, observations have revealed that S4 (Lim) corresponds to RsS-26 (Haseyama), S5 (Lim) corresponds to RsS-5 (Wang), S21 (Lim) corresponds to RsS-9 (Wang, Haseyama), and S26 corresponds to RsS-26 (Haseyama) [24,27,31]. The SLG4 (Lim) sequence identified in ‘SJ-12~14’ showed a 97% similarity with SLG-11 (Haseyama), but S4 (Lim) has not been reported to be the same as RsS-11 (Haseyama). The S domain of the SRK5 (Lim) sequence exhibited a high similarity with SLG21 (Lim) and SLG24 (Lim) at 95% and 96%, respectively. This is presumed to result from the gene duplication of the S domain of SRK alleles, intergenic recombination within SLG alleles, and gene conversion between the S domain of SRK and SLG alleles [18,42,46,47,48]. The kinase domain of SRK5 (Lim) and SRK 21 (Lim) sequences not only showed similarity with each other but also revealed high similarity with the kinase domain of the wild radish SRK allele (99%). It has been reported that class II S haplotypes of cultivated cabbage have been identified in wild species of the cabbage group [49]. This implies that nucleotide sequences more similar or identical to class II S haplotypes of cultivated radish may be discovered in wild radish. High-similarity regions with SLG5 (Lim) and SLG21 (Lim) were searched on radish chromosomes 7 and 8 (OY743213, LR778317). The S locus is a single gene locus located on chromosome 7 [50]. When performing local BLAST with the SLG21 (Lim) sequence as a query on the radish genome ‘QZ-16’ (GCA_902824885.1), including chromosomes 7 (LR778316) and 8 (LR778317), sequences corresponding to SLG21 (Lim) were only found on chromosome 8, with none matching on chromosome 7 (Table S3). Therefore, it is highly probable that the two regions searched on chromosome 8 (LR778317) were the S domain of SRK and SLG alleles rather than S homologs. This issue was inferred to result from errors during the chromosome-scale assembly process, and it could potentially lead to confusion in identifying radish S haplotypes through BLAST search.
In this study, multiple sequence alignment was performed using all the S haplotype sequences identified until now by Korean, Chinese, and Japanese researchers. Through this, variations in SRK and SLG alleles for each class group were detected.
When multiple sequence alignment (MSA) was performed using SRK/SLG sequences of class I S haplotypes, they exhibited a similarity ranging from 83.86% to 91.85%. This closely approximates the similarity range of 80% to 90% reported in previous research [51]. A gap of 19 to 29 bp was observed only in the sixth intron of the SRK6 (Lim) kinase domain and an insertion of 6 bp was observed only in the downstream region of HV-III in SLG-4 (Sakamoto). It was thought that these regions could be used to design candidate S6 (Lim) and S4 (Sakamoto) haplotype-specific markers.
When multiple sequence alignment (MSA) was conducted on the SRK/SLG sequences of class II S haplotypes, they exhibited a similarity ranging from 90% to 99.17%. Additionally, a remarkably high similarity (87.5% to 99.16%) was observed between the SLG gene and the SRK S domain. This observation is consistent with previous research indicating that within the class II group, SRK/SLG sequences show higher similarity compared to the class I group [44,48]. Notably, SLG5 (Lim) and the S domain of SRK21 (Lim) showed the highest similarity (99.16%), although they were different S haplotypes. This result elucidated why the two S haplotypes (S5, S21) were co-amplified when previously developed SCAR markers were used.
Additionally, we observed tyrosine residue in HV-III of SLG4 and 21 (Lim) and the S domain of SRK21 (Lim). It was presumed that non-synonymous mutations might have occurred at that site, substituting the 12th cysteine residue with tyrosine. Based on the observation that mutations have occurred not only in SLG alleles but also in the S domain of SRK alleles, it is considered necessary to further study whether the SNP mutation at that location affects the main function of the S domain of SRK alleles, which perceives and rejects self-pollen in the stigma.
Based on the results of BLAST search and sequence alignment, there was a potential risk of nonspecific amplification of the kinase domain of SRK alleles. For a more accurate identification of the target S haplotype, it might be preferable to prioritize SLG allele-specific primer sets over the kinase domain of SRK primer sets.
When using S haplotype-specific markers developed in this study, it will be possible to overcome the shortcomings of previously developed SCAR markers, such as the amplification of nonspecific S haplotypes. These markers can be used for the more accurate and rapid amplification of target S haplotypes in radish (Figure 3 and Figure 4). These markers are anticipated to aid in identifying the S haplotypes of parental lines and selecting cross-combinations during radish F1 breeding processes. Furthermore, these markers are expected to be effectively applied in the F1 seed purity test.
To identify additional S haplotypes rapidly and accurately, in addition to the nine S haplotypes already identified, it is considered imperative to develop other S allele-specific markers based on the methods applied in this study.

4. Materials and Methods

4.1. Plant Materials

The 22 elite cultivated radish lines (‘SJ-1~22’), serving as parental lines for F1 hybrid breeding in Korea, Japan, and other regions, were used as plant materials in this study [52]. The plant materials were rendered homozygous through self-pollination via bud pollination for more than 7 generations at a plant breeding house in Gyeonggi-do, Yeoju, Yanggui-ri, Republic of Korea (Table 3).

4.2. Amplification and Sequencing of SRK, SLG Alleles

4.2.1. Extraction of Genomic DNA and Amplification of SRK, SLG Alleles

Genomic DNA was extracted from seedling leaves (15 to 20 days after germination) of each radish breeding line using the CTAB method [53]. The concentration and purity of all extracted genomic DNAs were assessed using a nanodrop machine (DeNovix Co., Wilmington, DE, USA).
For the PCR amplification of SRK and SLG nucleotide sequences of ‘SJ-1~22’, 7 class I, II SRK and SLG universal primer sets were used in this study (Table 1 and Table S1). The UVSLGII primer set was designed to amplify the greatest region of the SLG21 (Lim) nucleotide sequence with the multiple sequence alignment of 12 class II SLG alleles and S domains of SRK sequences identified from radish. This primer set targeted the 5’ and 3’ class II SLG-conserved nucleotide sequences. PCR amplification was performed in a 15 μL reaction mixture, consisting of 7.5 μL Dyne Ready 2X-GO (Star Plus Taq with Dye; Dynebio Co., Gyeonggido, Republic of Korea), 1.5 μL 5X Tune-up solution, 1.5 μL forward primer (5 μM), 1.5 μL reverse primer (5 μM), 1.5 μL template DNA (50 ng/μL), and 1.5 μL distilled water. The PCR amplification conditions involved an initial denaturation at 95 °C for 3 min and a final elongation at 72 °C for 5 min, with the optimal annealing temperature applied to each primer (Table 4).

4.2.2. Identification of S Haplotypes Based on BLAST Search

The PCR products obtained by amplifying SRK and SLG alleles of 22 radish breeding lines were separated by electrophoresis on a 2% agarose gel stained with 7.2 μL of EcoDye (Biofact Co., Daejeon, Republic of Korea) in 360 mL of 0.5X TBE buffer at 250 V for 40 min and visualized using an Ultraviolet (UV) Transilluminator. Subsequently, gel extraction was carried out to obtain accurate nucleotide sequence information for SRK and SLG alleles. Slices of the agarose gel containing the amplified target SRK and SLG alleles were cut to approximately 0.2 to 0.3 g, transferred to a 2 mL micro-centrifuge tube, and purified using the LaboPass Gel and PCR Clean-up Kit (Cosmogenetech Co., Seoul, Republic of Korea). Following purification, Sanger sequencing was performed by Cosmogenetech Co. (Seoul, Republic of Korea). To address base noise, sequencing was performed twice, using both forward and reverse primers. Any base with a base quality value (QV) of 20 or less was excluded from the sequencing analysis.
To determine the S haplotypes of the 22 elite breeding lines (‘SJ-1~22’), the sequencing data were compared with other kinase domains and S domains of SRK and SLG alleles registered in the National Center for Biotechnology Information (NCBI) or other database using the Basic Local Alignment Search Tool (BLAST) program.
Since the nomenclature of S haplotype differed for each researcher in each country and was not unified, the S haplotypes were classified based on the nomenclature proposed by Lim [27].

4.3. Multiple Sequence Alignment (MSA)

To develop S haplotype-specific markers, we attempted to identify target regions of SRK and SLG alleles that showed sequence or structural variation. We performed multiple sequence alignment (MSA) for both nucleotide and amino acid sequences. This analysis involved the S haplotypes amplified by PCR from 22 radish breeding lines, with S haplotypes showing more than 94% similarity through a BLAST search, and additional S haplotypes identified by researchers in Korea [17,27], China [24], and Japan [31,33].
Using BLAST, we acquired SRK and SLG nucleotide sequences with more than 94% similarity to SRK and SLG alleles of ‘SJ-1~22’. Subsequently, MSA of both nucleotide and amino acid sequences was performed using EMBL-EBI (European Bioinformatics Institute), MAFFT version 7.0, and MEGA version 11.0 programs. Hypervariable regions 1 to 3 (HV-I to III) of SRK and SLG alleles were identified through the EMBL-EBI sixpack tool.

4.4. Development of S Haplotype-Specific Markers

Based on the results of MSA, S haplotype-specific markers were developed, targeting specific regions that show polymorphism in SRK and SLG alleles of ‘SJ-1~22’. The Primer-BLAST program was used, with the primer length set between 20 and 30 bp, the melting temperature (Tm) value ranging from 52 to 66 °C, and the GC content around 60%. The annealing temperature was calculated using NEB Tm calculator version 1.15.0.
First, candidate S haplotype-specific markers that selectively amplified the kinase domain of SRK and SLG of each ‘SJ-1~22’ in silico were chosen. Primer sets that exhibited overlapping binding sites, low polymorphism, or amplified regions of 100 bp or less were discarded. Subsequently, PCR amplification and gel electrophoresis were performed on the 22 elite breeding lines (‘SJ-1~22’) using the selected S haplotype-specific markers to assess whether they specifically amplified the corresponding S haplotypes.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants13050725/s1. Figure S1. PCR result of 22 radish breeding lines (‘SJ-1~22’) using class I, II kinase domains of SRK (SRK-KD) primer set. (A) PCR amplification of class I SRK-KD using UVSRK-F + UVSRK-R primer set; (B): PCR amplification of class II SRK-KD using KS2 + KA2 primer set; M: 100 bp size marker. Figure S2: PCR result of 22 radish breeding lines (‘SJ-1~22’) using class I, II SLG primer sets. (A) PCR amplification of class I SLG using SLG-I-F + SLG-I-R primer set; (B): PCR amplification of class I SLG using PS22 + SLG-I-R primer set; (C): PCR amplification of class II SLG using SLG-II-F + SLG-II-R primer set; (D): PCR amplification of class II SLG using Rs9 SLG-F + Rs9 SLG-R primer set; M: 100 bp size marker; (E): PCR amplification of class II SLG using UVSLGII-F + UVSLGII-R primer set; M: 100 bp sizer marker. Figure S3. Multiple sequence alignment of the nucleotide sequences of the 19 class I S haplotype kinase domains of SRK alleles; a: S haplotype published by Haseyama (2018); b: S haplotype published by Kim (2016); c: S haplotype published by Lim (2002); red box: 4th to 7th exons of the SRK allele; asterisk: conserved nucleotide. Figure S4. Multiple sequence alignment of the nucleotide sequences of the 15 class I S haplotype SLG alleles; a: S haplotype published by Sakamoto (1998); b: S haplotype published by Haseyama (2018); c: S haplotype published by Lim (2002); red box: hypervariable regions I to III of the SLG allele; asterisk: conserved nucleotide. Figure S5. Multiple sequence alignment of the nucleotide sequences of the 9 class II S haplotype kinase domains of SRK alleles; a: S haplotype published by Kim (2016); b: S haplotype published by Haseyama (2018); c: S haplotype published by Lim (2002); red box: 4th to 7th exons of the SRK allele; asterisk: conserved nucleotide. Figure S6. Multiple sequence alignment of the nucleotide sequences of the 12 class II S haplotype SLG alleles and the S domain of SRK alleles; a: S haplotype published by Haseyama (2018); b: S haplotype published by Lim (2002); c: S haplotype published by Wang (2018); red box: hypervariable regions I to III of SLG allele; asterisk: conserved nucleotide. Figure S7: Multiple sequence alignment of SRK10 (Lim, AY052585), SRK16 (Lim, AY052579), and the 10 other S haplotypes; asterisk: conserved nucleotide; a: S haplotype published by Lim (2002); b: S haplotype published by Haseyama (2018); c: S haplotype published by Kim (2016). Table S1: Information of class I, II S haplotype universal primer used in this study. Table S2: Information of 9 S haplotype-specific primers developed in this study. Table S3: Result of BLASTN 2.14.1+ using SLG21 (Lim) nucleotide sequence to Raphanus sativus genome assembly ‘QZ-16’ (GCA_902824885.1). SLG21 (Lim) sequence is obtained from ‘SJ-21’ radish breeding line; bold letters: putative region of SLG21 (Lim) and SRK21 (Lim) S domain. Table S4: Nucleotide sequences of SRK and SLG alleles of SJ-1~22.

Author Contributions

Conceptualization, S.-H.H. and H.-Y.P.; methodology, S.-H.H., S.-Y.M. and H.-Y.P.; validation, S.-H.H. and S.-Y.K.; formal analysis, S.-H.H. and S.-Y.K.; investigation, S.-H.H., S.-Y.M. and S.-Y.K.; resources, H.-Y.P.; writing—original draft preparation, S.-H.H.; writing—review and editing, S.-H.H., S.-Y.M. and H.-Y.P.; funding acquisition, H.-Y.P. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Digital Breeding Transformation Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (322063-03-3-SB010).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Upon request, the corresponding author can provide access to the research data generated in this study.

Conflicts of Interest

Author S.H.H. was employed by DASAN Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Plants 13 00725 g003
Figure 3. PCR result of 22 radish breeding lines (‘SJ-1~22’) using developed kinase domain of SRK (SRK-KD)-specific primer sets in this study. ‘S1~S26’ are the S haplotypes that each line has. (A): SRK1 (665 bp); (B): SRK8 (271 bp); (C): SRK10 (411 bp); (D): SRK18 (105 bp).
Figure 3. PCR result of 22 radish breeding lines (‘SJ-1~22’) using developed kinase domain of SRK (SRK-KD)-specific primer sets in this study. ‘S1~S26’ are the S haplotypes that each line has. (A): SRK1 (665 bp); (B): SRK8 (271 bp); (C): SRK10 (411 bp); (D): SRK18 (105 bp).
Plants 13 00725 g003
Plants 13 00725 g004
Figure 4. PCR result of class I radish breeding lines (‘SJ-1~22’) using developed SLG-specific primer sets in this study. Each number is the S haplotype that each S homozygous line has. (A): SLG1 (388 bp); (B): SLG4 (529 bp); (C): SLG5 (204 bp); (D): SLG8 (314 bp); (E): SLG10 (369 bp); (F): SLG16 (216 bp); (G): SLG18 (353 bp); (H): SLG21 (445 bp): (I): SLG26 (131 bp).
Figure 4. PCR result of class I radish breeding lines (‘SJ-1~22’) using developed SLG-specific primer sets in this study. Each number is the S haplotype that each S homozygous line has. (A): SLG1 (388 bp); (B): SLG4 (529 bp); (C): SLG5 (204 bp); (D): SLG8 (314 bp); (E): SLG10 (369 bp); (F): SLG16 (216 bp); (G): SLG18 (353 bp); (H): SLG21 (445 bp): (I): SLG26 (131 bp).
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Table 1. Identification and comparison of S haplotypes using BLAST program; Acc.: accession name of radish breeding lines (‘SJ-1~22’) used in this study; NCBI Acc.: accession number of genes registered in NCBI database; bold letters: S haplotype with best hit in result of BLAST search.
Table 1. Identification and comparison of S haplotypes using BLAST program; Acc.: accession name of radish breeding lines (‘SJ-1~22’) used in this study; NCBI Acc.: accession number of genes registered in NCBI database; bold letters: S haplotype with best hit in result of BLAST search.
Acc.S HaplotypeS AllelesToolGeneNCBI Acc.Iden (%)Query Cov
SJ-1~4S1 (Lim)SRK-KD (1063 bp)BLASTnSRK1 (Lim)AY052579100%99%
SLG (1220 bp)BLASTnSLG1 (Lim)AY05257299%99%
SJ-5~6S8 (Lim)SRK-KD (1133 bp)BLASTnSRK8 (Lim)AY052583100%100%
SRK19KX961713100%100%
SRK-19LC341229100%97%
SRK-18LC34122897%97%
SLG (1201 bp)BLASTnSLG-19LC34123899%55%
SJ-7~9S10 (Lim) BLASTnSRK10 (Lim)AY05258599.9%100%
SRK-KD (1051 bp) SRK11 (Lim)AY53453398%100%
SRK16 (Lim)AY53453598%100%
SRK20 (Lim)AY53453798%100%
SRK29 (Lim)AY53454199%100%
SRK-22LC34123198%95%
SRK-23LC34123298%99%
SRK-31LC34123499%96%
SRK7KX96170198%78%
SRK10KX96170498%78%
SRK16KX96171099%78%
SRK17KX96171198%78%
SLG (1202 bp)BLASTnSLG10 (Lim)AY05257699.9%100%
SJ-10~11S16 (Lim)SRK-KD (1071 bp)BLASTnSRK16 (Lim)AY05257999.9%100%
SRK-22LC341231100%94%
SRK10 (Lim)AY05258598%100%
SRK11 (Lim)AY53453398% 100%
SRK20 (Lim)AY53453797%100%
SRK29 (Lim)AY53454198%100%
SRK-23LC34123297%100%
SRK-31LC34123498%94%
SRK7KX96170199% 76%
SRK10KX96170498%76%
SRK16KX96171099%76%
SRK17KX96171199%76%
SLG (1168 bp)BLASTnSLG-22LC341239100%64%
SLG-7AB00968499.9%100%
SJ-12~14S18 (Lim)SRK-KD (1108 bp)BLASTnSRK18 (Lim)AY53453699.9%100%
SRK-6LC34122699.9%95%
SLG (1235 bp)BLASTnSLG18 (Lim)AY527401100%100%
SLG-6AB009682100%100%
SJ-15~17S4 (Lim)SRK-KD
(Not amplified)		-----
SLG (1002 bp)BLASTnSLG4 (Lim)AY05257799%100%
SLG-26LC34124199%99%
SLG-11LC34123697%87%
SJ-18~20S5 (Lim)SRK-KD (1053 bp)BLASTnSRK-5 (Wang)-99%99%
SRK-9 (Wang)-99%99%
SRK6KP11707799%94%
SLG (1004 bp)BLASTnSRK-5 (Wang)-99%94%
SLG5 (Lim)AY05257897%100%
SLG21 (Lim)AY52965095%100%
SLG24 (Lim)AY52965196%100%
SRK1KX96169596%100%
SRK-9AB11485196%98%
SLG-9LC34123595%90%
Rs chr7OY74321395,97%100%
Rs chr8LR77831795,97%100%
SJ-21S21 (Lim)SRK-KD (1015 bp)BLASTnSRK-9 (Wang)-99%99%
SRK-5 (Wang)-99%99%
SRK6KP11707799%96%
SLG (1055 bp)BLASTnSLG21 (Lim)AY529650100%84%
SLG-9LC341235100%77%
Rs chr7OY743213100,94%100%
Rs chr8LR778317100,94%100%
Rs SLG S13-likeXM_056990347100%100%
SLG24 (Lim)AY52965196%84%
SLG5 (Lim)AY05257894%84%
SRK-1KX96169594%100%
SRK-9AB11485194%100%
SJ-22S26 (Lim)SRK-KD
(Not amplified)		-----
SLG (802 bp)BLASTnSLG26 (Lim)AY529652100%100%
SLG-29LC341242100%100%
Table 2. List of 9 S haplotype-specific markers (kinase domain of SRK, SLG) developed in this study.
Table 2. List of 9 S haplotype-specific markers (kinase domain of SRK, SLG) developed in this study.
S haplotypePrimer SetForwardReverseExpected Size
S1 (Lim)SRK1KD1-FKD1-R665 bp
SLG1SLG1-FSLG1-R388 bp
S4 (Lim)SLG4SLG4-FSLG4-R529 bp
S5 (Lim)SLG5SLG5-FSLG5-R409 bp
S8 (Lim)SRK8KD8-FKD8-R271 bp
SLG8SLG8-FSLG8-R314 bp
S10 (Lim)SRK10KD10-FKD10-R411 bp
SLG10SLG10-FSLG10-R369 bp
S16 (Lim)SLG16SLG16-FSLG16-R216 bp
S18 (Lim)SRK18KD18-FKD18-R105 bp
SLG18SLG18-FSLG18-R353 bp
S21 (Lim)SLG21SLG21-FSLG21-R529 bp
S26 (Lim)SLG26SLG26-FSLG26-R131 bp
Table 3. Materials of radish breeding lines used in this study.
Table 3. Materials of radish breeding lines used in this study.
Accession NumberType of Fleshy RootColor of Fleshy RootSource
SJ-1Narrow ellipticGreen and WhiteSouth Chinese
SJ-2AcicularGreen and WhiteSouth Chinese
SJ-3Narrow ellipticGreen and WhiteSouth Chinese
SJ-4OblongGreen and WhiteSouth Chinese
SJ-5Medium ellipticGreen and WhiteNorth Chinese
SJ-6Medium ellipticGreen and WhiteNorth Chinese
SJ-7Bell shapedGreen and WhiteNorth Chinese
SJ-8OblongGreen and WhiteSouth Chinese
SJ-9OblongGreen and WhiteSouth Chinese
SJ-10OblongGreen and WhiteSouth Chinese
SJ-11OblongGreen and WhiteSouth Chinese
SJ-12OvateGreen and WhiteSouth Chinese
SJ-13Medium ellipticGreen and WhiteNorth Chinese
SJ-14Medium ellipticGreen and WhiteNorth Chinese
SJ-15Bell shapedGreen and WhiteNorth Chinese
SJ-16OblongWhiteSouth Chinese
SJ-17OblongWhiteSouth Chinese
SJ-18Bell shapedGreen and WhiteNorth Chinese
SJ-19OblongGreen and WhiteSouth Chinese
SJ-20OblongGreen and WhiteSouth Chinese
SJ-21Bell shapedGreen and WhiteSouth Chinese
SJ-22Narrow ellipticGreen and WhiteNorth Chinese
Table 4. PCR condition for class I, II universal primer sets used in this study.
Table 4. PCR condition for class I, II universal primer sets used in this study.
GeneForwardReverseAnnealing Temperature/Cycle
Class I SRK-KDUVSRK-FUVSRK-R54 °C/34X
Class I SLGSLG-I-FSLG-I-R50 °C/39X
PS22SLG-I-R54 °C/35X
Class II SRK-KDKS2KA261 °C/35X
Class II SLGSLG-II-FSLG-II-R55.5 °C/39X
Rs9SLG-FRs9SLG-R53 °C/35X
UVSLGII-FUVSLGII-R61 °C/30X
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MDPI and ACS Style

Heo, S.-H.; Kim, S.-Y.; Mo, S.-Y.; Park, H.-Y. Development of S Haplotype-Specific Markers to Identify Genotypes of Self-Incompatibility in Radish (Raphanus sativus L.). Plants 2024, 13, 725. https://doi.org/10.3390/plants13050725

AMA Style

Heo S-H, Kim S-Y, Mo S-Y, Park H-Y. Development of S Haplotype-Specific Markers to Identify Genotypes of Self-Incompatibility in Radish (Raphanus sativus L.). Plants. 2024; 13(5):725. https://doi.org/10.3390/plants13050725

Chicago/Turabian Style

Heo, Seong-Ho, Su-Yeon Kim, Suk-Yeon Mo, and Han-Yong Park. 2024. "Development of S Haplotype-Specific Markers to Identify Genotypes of Self-Incompatibility in Radish (Raphanus sativus L.)" Plants 13, no. 5: 725. https://doi.org/10.3390/plants13050725

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