Self-incompatibility phenotypes of SRK mutants can be predicted with high accuracy.

Only very limited information is available on why some non-synonymous variants severely alter gene function while others have no effect. To identify the characteristic features of mutations that strongly influence gene function, this study focused on S-locus receptor kinase, SRK, which encodes a highly polymorphic receptor kinase expressed in stigma papillary cells that underlies a female determinant of self-incompatibility in Brassicaceae. A set of 299 Arabidopsis thaliana transformants expressing mutated SRKb from A. lyrata was constructed and analyzed to determine the genotype and self-incompatibility phenotype of each transformant. Almost all the transformants showing the self-incompatibility defect contained mutations in AlSRKb that altered localization to the plasma membrane. The observed mutations occurred in amino acid residues that were highly conserved across S haplotypes and whose predicted locations were in the interior of the protein. These mutations were likely to underlie the self-incompatibility defect as they caused significant changes to amino acid properties. Such findings suggested that mutations causing the self-incompatibility defect were more likely to result from changes to AlSRKb biosynthesis than from loss of function. In addition, this study showed the RandomForest and Extreme Gradient Boosting methods could predict self-incompatibility phenotypes of SRK mutants with high accuracy.

S haplotype collection in Brassicaceae crops-an updated list of S haplotypes.

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.

Generation of Arabidopsis thaliana transformants showing the self-recognition activity of Brassica rapa.

Self-incompatibility in the Brassicaceae family is governed by SRK and SCR, which are two highly polymorphic genes located at the S-locus. Previously, the Arabidopsis lyrata SRK and SCR genes were introduced into Arabidopsis thaliana to generate self-incompatible lines. However, there are no reports showing that Brassica SRK and SCR genes confer self-incompatibility in A. thaliana. Doing so would further advance the mechanistic understanding of self-incompatibility in Brassicaceae. Therefore, we attempted to generate A. thaliana transformants showing the self-recognition activity of Brassica rapa by introducing BrSCR along with a chimeric BrSRK (BrSRK chimera, in which the kinase domain of BrSRK was replaced with that of AlSKR-b). We found that the BrSRK chimera and BrSCR of B. rapa S-9 and S-46 haplotypes, but not those of S-29, S-44, and S-60 haplotypes, conferred self-recognition activity in A. thaliana. Analyses of A. thaliana transformants expressing mutant variants of the BrSRK-9 chimera and BrSCR-9 revealed that mutations at the amino acid residues involved in BrSRK9-BrSCR9 interaction caused defects in the self-incompatibility response. The method developed in this study for generating self-incompatible A. thaliana transformants showing B. rapa self-recognition activity will be useful for analysis of self-recognition mechanisms in Brassicaceae.

Loss-of-function of Nicotiana tabacum L. eukaryotic translation initiation factors eIF4E1-S and eIF(iso)4E-T synergistically confers high-level resistance to both Potato virus Y (PVY) and resistance-breaking PVY.

The plant eukaryotic translation-initiation factors eIF4E and eIF(iso)4E play key roles in infection by plant RNA viruses, especially potyviruses. Mutations in the genes that encode these factors reduce susceptibility to the viruses. In the amphidiploid plant tobacco (Nicotiana tabacum L.), eIF4E1-S deletion mutants resist Potato virus Y (PVY), but resistance-breaking strains (RB-PVY) have appeared. In an earlier study, we demonstrated that the loss-of-function of eIF(iso)4E-T reduces susceptibility to RB-PVY. Here, we show that simultaneous inhibition of eIF4E1-S and eIF(iso)4E-T synergistically confers enhanced resistance to both PVY and RB-PVY without host growth or development defects. PVY symptoms and accumulation in a tobacco line lacking eIF4E1-S were detected at 14 days post-inoculation (dpi) and RB-PVY symptoms in lines without functional eIF(iso)4E-T were observed at 24 dpi. RB-PVY emerged in a PVY-infected tobacco line lacking eIF4E1-S. In contrast, lines without functional eIF4E1-S and eIF(iso)4E-T were nearly immune to PVY and RB-PVY, and little accumulation of either virus was detected even at 56 dpi. Thus, the lines will be promising for PVY-resistance breeding. This study provides a novel strategy to develop tobacco highly resistant to PVY and RB-PVY, and insights into the mechanisms responsible for high-level resistance.

Characterization of PcLEA14, a Group 5 Late Embryogenesis Abundant Protein Gene from Pear (Pyrus communis).

Fruit trees need to overcome harsh winter climates to ensure perennially; therefore, they are strongly influenced by environmental stress. In the present study, we focused on the pear homolog PcLEA14 belonging to the unique 5C late embryogenesis abundant (LEA) protein group for which information is limited on fruit trees. PcLEA14 was confirmed to belong to this protein group using phylogenetic tree analysis, and its expression was induced by low-temperature stress. The seasonal fluctuation in its expression was considered to be related to its role in enduring overwinter temperatures, which is particularly important in perennially. Moreover, the function of PcLEA14 in low-temperature stress tolerance was revealed in transgenic Arabidopsis. Subsequently, the pear homolog of dehydration-responsive element-binding protein/C-repeat binding factor1 (DREB1), which is an important transcription factor in low-temperature stress tolerance and is uncharacterized in pear, was analyzed after bioinformatics analysis revealed the presence of DREB cis-regulatory elements in PcLEA14 and the dormancy-related gene, both of which are also expressed during low temperatures. Among the five PcDREBs, PcDREB1A and PcDREB1C exhibited similar expression patterns to PcLEA14 whereas the other PcDREBs were not expressed in winter, suggesting their different physiological roles. Our findings suggest that the low-temperature tolerance mechanism in overwintering trees is associated with group 5C LEA proteins and DREB1.

Reduced susceptibility to a tobacco bushy top virus Malawi isolate by loss of function in host eIF(iso)4E genes.

Tobacco bushy top disease (TBTD) is a viral disease of tobacco (Nicotiana tabacum L.) caused by mixed infection of Tobacco bushy top virus or Ethiopian tobacco bushy top virus and a helper virus. Despite its damage to tobacco, practical genetic resources for disease resistance have not been found. Here, we report that a mutation of tobacco eIF(iso)4E genes (eIF(iso)4E-S and eIF(iso)4E-T), which encode eukaryotic translation initiation factors, confers resistance (reduced susceptibility) to TBTD caused by a virus from Malawi (designated as tobacco bushy top virus Malawi isolate, TBTV-MW). RNAi lines in which eIF(iso)4E genes were silenced showed reduced susceptibility to TBTV-MW. We also tested chemically-induced single (eIF(iso)4E-S or eIF(iso)4E-T) and double (eIF(iso)4E-S and eIF(iso)4E-T) nonsense mutants for resistance to TBTV-MW. Suppression of eIF(iso)4E-S showed reduced susceptibility, and the resistance of the double mutant tended to be even stronger. eIF(iso)4E mutants also showed reduced susceptibility to TBTV-MW transmitted by aphids. To the best of our knowledge, the eIF(iso)4E-S mutant is the first genetic resource for TBTD resistance breeding in tobacco.

Genome sequence and analysis of a Japanese radish (Raphanus sativus) cultivar named 'Sakurajima Daikon' possessing giant root.

AIM: The complex genome of a Japanese radish (Raphanus sativus) cultivar named 'Okute-Sakurajima' with an extremely large edible round root was analysed to explore its genomic characteristics. METHODS AND RESULTS: Single-molecule real-time technology was used to obtain long sequence reads to cover 60× of the genome. De novo assembly generated 504.5 Mb contigs consisting of 1,437 sequences with the N50 value of 1.2 Mb and included 94.1% of the core eukaryotic genes. Nine pseudomolecules, comprising 69.3% of the assembled contigs, were generated along with a high-density SNP genetic map. The sequence data thus established revealed the presence of structural variations and rearrangements in the Brassicaceae genomes. CONCLUSION AND PERSPECTIVE: A total of 89,915 genes were identified in the 'Okute-Sakurajima' genome, 30,033 of which were newly found in this study. The genome information reported here will not only contribute to the establishment of a new resource for the radish genomics but also provide insights into the molecular mechanisms underlying formation of the giant root.

Identification of genome-wide single-nucleotide polymorphisms among geographically diverse radish accessions.

Radish (Raphanus sativus L.) is cultivated around the world as a vegetable crop and exhibits diverse morphological and physiological features. DNA polymorphisms are responsible for differences in traits among cultivars. In this study, we determined genome-wide single-nucleotide polymorphisms (SNPs) among geographically diverse radish accessions using the double-digest restriction site-associated DNA sequencing (ddRAD-Seq) method. A total of 52,559 SNPs was identified in a collection of over 500 radish accessions (cultivated and wild) from East Asia, South and Southeast Asia, and the Occident and Near East. In addition, 2,624 SNP sites without missing data (referred to as common SNP sites) were identified among 510 accessions. Genetic diversity analyses, based on the common SNP sites, divided the cultivated radish accessions into four main groups, each derived from four geographical areas (Japan, East Asia, South and Southeast Asia, and the Occident and Near East). Furthermore, we discuss the origin of cultivated radish and its migration from the West to East Asia. SNP data generated in this work will facilitate further genetic studies on the radish breeding and production of DNA markers.

High temperature causes breakdown of S haplotype-dependent stigmatic self-incompatibility in self-incompatible Arabidopsis thaliana.

Commercial seeds of Brassicaceae vegetable crops are mostly F1 hybrids, the production of which depends on self-incompatibility during pollination. Self-incompatibility is known to be weakened by exposure to elevated temperatures, which may compromise future breeding and seed production. In the Brassicaceae, self-incompatibility is controlled by two genes, SRK and SCR, which function as female and male determinants of recognition specificity, respectively. However, the molecular mechanisms underlying the breakdown of self-incompatibility under high temperature are poorly understood. In this study, we examined the self-incompatibility phenotypes of self-incompatible Arabidopsis thaliana SRK-SCR transformants under normal (23 °C) and elevated (29 °C) temperatures. Exposure to elevated temperature caused defects in the stigmatic, but not the pollen, self-incompatibility response. In addition, differences in the response to elevated temperature were observed among different S haplotypes. Subcellular localization revealed that high temperature disrupted the targeting of SRK to the plasma membrane. SRK localization in plants transformed with different S haplotypes corresponded to their self-incompatibility phenotypes, further indicating that defects in SRK localization were responsible for the breakdown in the self-incompatibility response at high temperature. Our results provide new insights into the causes of instability in self-incompatibility phenotypes.

SCR-22 of pollen-dominant S haplotype class is recessive to SCR-44 of pollen-recessive S haplotype class in Brassica rapa.

SCR/SP11 encodes the male determinant of recognition specificity of self-incompatibility (SI) in Brassica species and is sporophytically expressed in the anther tapetum. Based on dominance relationships in pollen and nucleotide sequence similarity, the S haplotypes in Brassica have been classified as class I or class II, with class-I S haplotypes being dominant over class-II S haplotypes. Here, we revealed that S-22 in B. rapa belonging to class I is recessive to class-II S-44 and class-I S-36 in pollen, whereas it is dominant over S-60, S-40, and S-29 based on pollination tests. SCR/SP11 of S-22 (SCR-22) was sequenced, revealing that the deduced amino-acid sequence of SCR-22 has the longest C-terminal domain among the SCR/SP11 sequences. The expression of SCR-22 was found to be suppressed in S-22/S-44 and S-22/S-36 heterozygotes. Normal transcription of SCR-44 was considered to be due to the transcription suppression of Smi sRNA of the S-22 haplotype and a very low methylation state of the SCR-44 promoter region in the tapetum of S-22/S-44 heterozygotes. In SCR-22, only the cytosine residue located at the -37 bp position of the promoter region was hypermethylated in the tapetum of S-22/S-44 heterozygotes, and few methylated cytosines were detected in the promoter and coding regions of SCR-22 in S-22/S-36 heterozygotes. SCR-22 was also expressed in microspores in S-22 homozygotes but not in S-22/S-44 and S-22/S-36 heterozygotes. These results suggest that a mechanism different from class-II SCR/SP11 suppression may operate for the suppression of recessive class-I SCR-22 in S heterozygotes.

Sensitive mutant detection by concentrating mutant DNA with allele-specific capture and its application to analysis of contaminated grains in rice.

KEY MESSAGE: We developed a method for detection of mutants in a large number of plants, and found this method to be applicable to detection of a mutant allele at a concentration of 1/1000. Many techniques for SNP analysis have been developed, but most of these techniques are not so sensitive to be used for detection of mutants in a large number of plants. Although some highly sensitive methods of SNP analysis have been reported, they are costly. In the present study, a method for concentrating mutant DNA was examined for sensitive detection of an SNP allele in a bulked DNA sample. PCR products of mutant alleles were captured by biotin-labeled oligonucleotide conjugated with streptavidin-coated magnetic beads. By repeated captures of each strand and combining both strands, mutant alleles with a concentration of 1/1000 in wild-type alleles were detectable by CAPS or dCAPS analysis. Indirect capture of a mutant allele was possible, but efficiency was slightly lower than that of the direct capture. The developed method was applied to detection of contamination of rice grains by grains of a different cultivar. Possible applications of this method are discussed.

A 2-Oxoglutarate-Dependent Dioxygenase Mediates the Biosynthesis of Glucoraphasatin in Radish.

Glucosinolates (GSLs) are secondary metabolites whose degradation products confer intrinsic flavors and aromas to Brassicaceae vegetables. Several structures of GSLs are known in the Brassicaceae, and the biosynthetic pathway and regulatory networks have been elucidated in Arabidopsis (Arabidopsis thaliana). GSLs are precursors of chemical defense substances against herbivorous pests. Specific GSLs can act as feeding blockers or stimulants, depending on the pest species. Natural selection has led to diversity in the GSL composition even within individual species. However, in radish (Raphanus sativus), glucoraphasatin (4-methylthio-3-butenyl glucosinolate) accounts for more than 90% of the total GSLs, and little compositional variation is observed. Because glucoraphasatin is not contained in other members of the Brassicaceae, like Arabidopsis and cabbage (Brassica oleracea), the biosynthetic pathways for glucoraphasatin remain unclear. In this report, we identified and characterized a gene encoding GLUCORAPHASATIN SYNTHASE 1 (GRS1) by genetic mapping using a mutant that genetically lacks glucoraphasatin. Transgenic Arabidopsis, which overexpressed GRS1 cDNA, accumulated glucoraphasatin in the leaves. GRS1 encodes a 2-oxoglutarate-dependent dioxygenase, and it is abundantly expressed in the leaf. To further investigate the biosynthesis and transportation of GSLs in radish, we grafted a grs1 plant onto a wild-type plant. The grafting experiment revealed a leaf-to-root long-distance glucoraphasatin transport system in radish and showed that the composition of GSLs differed among the organs. Based on these observations, we propose a characteristic biosynthesis pathway for glucoraphasatin in radish. Our results should be useful in metabolite engineering for breeding of high-value vegetables.

Identification of loci associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis.

KEY MESSAGE: We identified three physical positions associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. We also confirmed their genetic effects on the embryo yield. Isolated microspore culture is well utilized for the production of haploid or doubled-haploid plants in Brassica crops. Brassica rapa cv. 'Ho Mei' is one of the most excellent cultivars in embryo yield of microspore culture. To identify the loci associated with microspore embryogenesis, segregation analysis of 154 DNA markers anchored to B. rapa chromosomes (A01-A10) was performed using a population of microspore-derived embryos obtained from an F1 hybrid between 'CR-Seiga', a low yield cultivar in microspore-derived embryos, and 'Ho Mei'. Three regions showing significant segregation distortion with increasing 'Ho Mei' alleles were detected on A05, A08 and A09, although these regions showed the expected Mendelian segregation ratio in an F2 population. The additive effect of alleles in these regions on embryo yield was confirmed in a BC3F1 population. One region on A08 containing Br071-5c had a higher effect than the other regions. Polymorphism of nucleotide sequences around the Br071-5c locus was investigated to find the gene possibly responsible for efficient embryogenesis from microspores.

Identification of a gene controlling variation in the salt tolerance of rapeseed (Brassica napus L.).

MAIN CONCLUSION: By genome-wide association study, QTLs for salt tolerance in rapeseed were detected, and a TSN1 ortholog was identified as a candidate gene responsible for genetic variation in cultivars. Dissecting the genomic regions governing abiotic stress tolerance is necessary for marker-assisted breeding to produce elite breeding lines. In this study, a world-wide collection of rapeseed was evaluated for salt tolerance. These rapeseed accessions showed a large variation for salt tolerance index ranging from 0.311 to 0.999. Although no significant correlation between salt tolerance and Na(+) content was observed, there was a significant negative correlation between shoot biomass production under a control condition and salt tolerance. These rapeseed accessions were genotyped by DArTseq for a total of 51,109 genetic markers, which were aligned with 'pseudomolecules' representative of the genome of rapeseed to locate their hypothetical order for association mapping. A total of 62 QTLs for salt tolerance, shoot biomass, and ion-homeostasis-related traits were identified by association mapping using both the P and Q+K models. Candidate genes located within the QTL regions were also shortlisted. Sequence analysis showed many polymorphisms for BnaaTSN1. Three of them in the coding region resulting in a premature stop codon or frameshift were found in most of the sensitive lines. Loss-of-function mutations showed a significant association with salt tolerance in B. napus.

Map-based cloning of a candidate gene conferring Fusarium yellows resistance in Brassica oleracea.

KEY MESSAGE: We identified the candidate gene conferring yellow wilt resistance (YR) in B. oleracea . This work will facilitate YR breeding programs for B. oleracea and its closely related species. Yellow wilt disease is one of the most serious diseases of cabbage worldwide. Type A resistance to the disease is controlled by a single dominant gene that is used in cabbage breeding. Our previous QTL study identified the FocBo1 locus controlling type A resistance. In this study, the FocBo1 locus was fine-mapped by using 139 recombinant F2 plants derived from resistant cabbage (AnjuP01) and susceptible broccoli (GCP04) DH lines. As a result, we successfully delimited the location of FocBo1 within 1.00 cM between markers, BoInd 2 and BoInd 11. Analysis of BAC and cosmid sequences corresponding to the FocBo1 locus identified an orthologous gene of Bra012688 that was recently identified as an candidate gene that confers yellows resistance in Chinese cabbage. The candidate gene-specific DNA markers and phenotypes in F1 cabbage cultivars and their selfed F2 populations showed a perfect correlation. Our identification of the candidate gene for FocBo1 will assist introduction of fusarium resistance into B. oleracea cultivars and contribute further understanding of interaction between Brassica plants and fusarium.

Comparative transcriptome analysis of leaves and roots in response to sudden increase in salinity in Brassica napus by RNA-seq.

Amphidiploid species in the Brassicaceae family, such as Brassica napus, are more tolerant to environmental stress than their diploid ancestors.A relatively salt tolerant B. napus line, N119, identified in our previous study, was used. N119 maintained lower Na(+) content, and Na(+)/K(+) and Na(+)/Ca(2+) ratios in the leaves than a susceptible line. The transcriptome profiles of both the leaves and the roots 1 h and 12 h after stress were investigated. De novo assembly of individual transcriptome followed by sequence clustering yielded 161,537 nonredundant sequences. A total of 14,719 transcripts were differentially expressed in either organs at either time points. GO and KO enrichment analyses indicated that the same 49 GO terms and seven KO terms were, respectively, overrepresented in upregulated transcripts in both organs at 1 h after stress. Certain overrepresented GO term of genes upregulated at 1 h after stress in the leaves became overrepresented in genes downregulated at 12 h. A total of 582 transcription factors and 438 transporter genes were differentially regulated in both organs in response to salt shock. The transcriptome depicting gene network in the leaves and the roots regulated by salt shock provides valuable information on salt resistance genes for future application to crop improvement.

Self-incompatibility in Brassicaceae crops: lessons for interspecific incompatibility.

Most wild plants and some crops of the Brassicaceae express self-incompatibility, which is a mechanism that allows stigmas to recognize and discriminate against "self" pollen, thus preventing self-fertilization and inbreeding. Self-incompatibility in this family is controlled by a single S locus containing two multiallelic genes that encode the stigma-expressed S-locus receptor kinase and its pollen coat-localized ligand, the S-locus cysteine-rich protein. Physical interaction between receptor and ligand encoded in the same S locus activates the receptor and triggers a signaling cascade that results in inhibition of "self" pollen. Sequence information for many S-locus haplotypes in Brassica species has spurred studies of dominance relationships between S haplotypes and of S-locus structure, as well as the development of methods for S genotyping. Furthermore, molecular genetic studies have begun to identify genes that encode putative components of the self-incompatibility signaling pathway. In parallel, standard genetic analysis and QTL analysis of the poorly understood interspecific incompatibility phenomenon have been initiated to identify genes responsible for the inhibition of pollen from other species by the stigma. Herewith, we review recent studies of self-incompatibility and interspecific incompatibility, and we propose a model in which a universal pollen-inhibition pathway is shared by these two incompatibility systems.

Draft sequences of the radish (Raphanus sativus L.) genome.

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥ 300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.

Nucleotide sequence variation of GLABRA1 contributing to phenotypic variation of leaf hairiness in Brassicaceae vegetables.

GLABRA1 (GL1) belongs to the group of R2R3-MYB transcription factors and is known to be essential for trichome initiation in Arabidopsis. In our previous study, we identified a GL1 ortholog in Brassica rapa as a candidate for the gene controlling leaf hairiness by QTL analysis and suggested that a 5-bp deletion (B-allele) and a 2-bp deletion (D-allele) in the exon 3 of BrGL1 and a non-synonymous SNP (C-allele) in the second nucleotide of exon 3 possibly cause leaf hairlessness. In this study, we transformed a B. rapa line having the B-allele with the A-allele (wild type) or the C-allele of BrGL1 under the control of the CaMV 35S promoter. The transgenic plants with the A-allele showed dense coverage of seedling tissues including stems, young leaves and hypocotyls with trichomes, whereas the phenotypes of those with the C-allele were unchanged. In order to obtain more information about allelic variation of GL1 in different plant lineages and its correlation with leaf hairiness, two GL1 homologs, i.e., RsGL1a and RsGL1b, in Raphanus sativus were analyzed. Allelic variation of RsGL1a between a hairless line and a hairy line was completely associated with hairiness in their BC1F1 population. Comparison of the full-length of RsGL1a in the hairless and hairy lines showed great variation of nucleotides in the 3' end, which might be essential for its function and expression.

QTL analysis using SNP markers developed by next-generation sequencing for identification of candidate genes controlling 4-methylthio-3-butenyl glucosinolate contents in roots of radish, Raphanus sativus L.

SNP markers for QTL analysis of 4-MTB-GSL contents in radish roots were developed by determining nucleotide sequences of bulked PCR products using a next-generation sequencer. DNA fragments were amplified from two radish lines by multiplex PCR with six primer pairs, and those amplified by 2,880 primer pairs were mixed and sequenced. By assembling sequence data, 1,953 SNPs in 750 DNA fragments, 437 of which have been previously mapped in a linkage map, were identified. A linkage map of nine linkage groups was constructed with 188 markers, and five QTLs were detected in two F(2) populations, three of them accounting for more than 50% of the total phenotypic variance being repeatedly detected. In the identified QTL regions, nine SNP markers were newly produced. By synteny analysis of the QTLs regions with Arabidopsis thaliana and Brassica rapa genome sequences, three candidate genes were selected, i.e., RsMAM3 for production of aliphatic glucosinolates linked to GSL-QTL-4, RsIPMDH1 for leucine biosynthesis showing strong co-expression with glucosinolate biosynthesis genes linked to GSL-QTL-2, and RsBCAT4 for branched-chain amino acid aminotransferase linked to GSL-QTL-1. Nucleotide sequences and expression of these genes suggested their possible function in 4MTB-GSL biosynthesis in radish roots.