Search

Background The nucleotide-binding site–leucine-rich repeat (NBS-LRR) genes are important for plant development and disease resistance. Although genome-wide studies of NBS-encoding genes have been performed in several species, the evolution, structure, expression, and function of these genes remain unknown in radish (Raphanus sativus L.). A recently released draft R. sativus L. reference genome has facilitated the genome-wide identification and characterization of NBS-encoding genes in radish. Results A total of 225 NBS-encoding genes were identified in the radish genome based on the essential NB-ARC domain through HMM search and Pfam database, with 202 mapped onto nine chromosomes and the remaining 23 localized on different scaffolds. According to a gene structure analysis, we identified 99 NBS-LRR-type genes and 126 partial NBS-encoding genes. Additionally, 80 and 19 genes respectively encoded an N-terminal Toll/interleukin-like domain and a coiled-coil domain. Furthermore, 72% of the 202 NBS-encoding genes were grouped in 48 clusters distributed in 24 crucifer blocks on chromosomes. The U block on chromosomes R02, R04, and R08 had the most NBS-encoding genes (48), followed by the R (24), D (23), E (23), and F (17) blocks. These clusters were mostly homogeneous, containing NBS-encoding genes derived from a recent common ancestor. Tandem (15 events) and segmental (20 events) duplications were revealed in the NBS family. Comparative evolutionary analyses of orthologous genes among Arabidopsis thaliana, Brassica rapa, and Brassica oleracea reflected the importance of the NBS-LRR gene family during evolution. Moreover, examinations of cis-elements identified 70 major elements involved in responses to methyl jasmonate, abscisic acid, auxin, and salicylic acid. According to RNA-seq expression analyses, 75 NBS-encoding genes contributed to the resistance of radish to Fusarium wilt. A quantitative real-time PCR analysis revealed that RsTNL03 (Rs093020) and RsTNL09 (Rs042580) expression positively regulates radish resistance to Fusarium oxysporum, in contrast to the negative regulatory role for RsTNL06 (Rs053740). Conclusions The NBS-encoding gene structures, tandem and segmental duplications, synteny, and expression profiles in radish were elucidated for the first time and compared with those of other Brassicaceae family members (A. thaliana, B. oleracea, and B. rapa) to clarify the evolution of the NBS gene family. These results may be useful for functionally characterizing NBS-encoding genes in radish.

Traditional breeding methods usually involve field tests carried out by experienced breeders. However, such methods are costly and time-consuming. Recently, with the development of Next-Generation Sequencing (NGS) technology, molecular markers are being utilized for selection processes in breeding. To implement a high-throughput system using molecular markers in Chinese cabbage (Brassica rapa subsp. pekinensis) breeding, we developed single nucleotide polymorphism (SNP) marker sets for background selection and testing F1 purity using Fluidigm genotyping assays. SNPs were generated using NGS technology on 209 varieties of Chinese cabbage collected from around the world. Those with minor allele frequency ≥ 5% and polymorphism information content ≥ 0.3 were screened, and then based on the physical distribution among the 10 chromosomes, 177 SNPs were selected and synthesized for testing. To obtain marker sets with high selection efficiency, we tested 192 SNPs on 45 types of inbred lines and 29 types of F1 hybrids. Among the 192 SNPs, we selected 96 markers sets for background selection and 24 marker sets for F1 purity testing according to the following criteria; the genotype of the parents was homozygous, and the F1 follows the parents’ genotypes. These SNP sets are suitable for high-throughput systems using the 96.96 and 192.24 integrated fluidic circuit platforms of Fluidigm genotyping assays. These SNP marker sets are not only efficient for selecting of early fixed lines as background selection but are also useful for testing the purity of F1 hybrids.

Clubroot is one of the major diseases adversely affecting Chinese cabbage (Brassica rapa) yield and quality. To precisely characterize the Plasmodiophora brassicae infection of Chinese cabbage, we developed a dual fluorescent staining method for simultaneously examining the pathogen, cell structures, and starch grains. The number of starch (amylopectin) grains increased in B. rapa roots infected by P. brassicae, especially from 14 to 21 days after inoculation. Therefore, the expression levels of 38 core starch metabolism genes were investigated by quantitative real-time PCR. Most genes related to starch synthesis were up-regulated at 7 days after P. brassicae inoculation, whereas the expression levels of starch degradation-related genes were increased at 14 days after inoculation. Then, genes encoding the core enzymes involved in starch metabolism were investigated by assessing their chromosomal distributions, structures, duplication events, and synteny among Brassica species. Genome comparisons indicated that 38 non-redundant genes belonging to six core gene families related to starch metabolism are highly conserved among Arabidopsis thaliana, B. rapa, Brassica nigra, and Brassica oleracea. Previous genome sequencing projects have revealed that P. brassicae obtained host nutrients by manipulating plant metabolism. Starch may serve as a carbon source for P. brassicae colonization, as indicated by histological observations and transcriptomic analysis. Results of this study may elucidate the evolution and expression of core starch metabolism genes and provide researchers with novel insights into the pathogenesis of clubroot in B. rapa.

Background Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed testing for purity was done with a grow-out test (GOT) in the field, but these tests are time consuming and costly. Seed testing with molecular markers was introduced as a replacement for GOT early in the last decade. Recently, Kompetitive allele specific PCR (KASP) markers are promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and could be used with only a small number of accessions or varieties due to the limited genetic information and reference genomes available. Results Here, we identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. Furthermore, the total 2,925 SNPs were selected as accession-specific SNPs, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by NGS analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguish individuals from the mixed population including 50 target accessions from B. rapa core collection and outgroup. Conclusions This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).

Background Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed purity has been assessed with a grow-out test (GOT) in the field, a time consuming and costly venture. Early in the last decade, seed testing with molecular markers was introduced as a replacement for GOT, and Kompetitive allele specific PCR (KASP) markers were recognized as promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and applicable to only a small number of accessions or varieties due to the limited genetic information and reference genomes available. Results We identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. From these, we identified 2,925 SNPs as accession-specific, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by the Next Generation Sequencing (NGS) analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguised individuals from the mixed population including 50 target accessions from B. rapa core collection and the outgroup. Additionally, the marker set we developed here discriminated F1 hybrids and their parental lines with distinct clusters. Conclusions This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl2, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 μg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.

SuccessfulAgrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant’s recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stableAgrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regeneration media. Four-day-oldin vitroderived cotyledon explants were infected withA. tumefaciensstrain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to anAgrobacteriumsuspension (OD600of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO3combination exhibited the highest β-glucuronidase (GUS) expression (94%) and explant regeneration efficiency (76%). After 3 days, the cotyledon explants were subjected to three selection cycles with gradually increasing hygromycin B concentrations (10 to 12 mg/L). The incorporation and expression ofhptIIin T0transformed plants were verified by polymerase chain reaction and Southern blot analyses. These transgenic plants (T0) were fertile and morphologically normal. Using the present protocol, a successful transformation efficiency of 14% was achieved, and this protocol can be applied for genome editing and functional studies to improve Chinese cabbage traits.

The B-box zinc-finger transcription factors are important for plant growth, development, and various physiological processes such as photomorphogenesis, light signaling, and flowering, as well as for several biotic and abiotic stress responses. However, there is relatively little information available regarding Brassica B-box genes and their expression. In this study, we identified 51, 52, and 101 non-redundant genes encoding B-box proteins in Brassica rapa (BrBBX genes), B. oleracea (BoBBX genes), and B. napus (BnBBX genes), respectively. A whole-genome identification, characterization, and evolutionary analysis (synteny and orthology) of the B-box gene families in the diploid species B. rapa (A genome) and B. oleracea (C genome) and in the allotetraploid species B. napus (AC genome) revealed segmental duplications were the major contributors to the expansion of the BrassicaBBX gene families. The BrassicaBBX genes were classified into five subgroups according to phylogenetic relationships, gene structures, and conserved domains. Light-responsive cis-regulatory elements were detected in many of the BBX gene promoters. Additionally, BrBBX expression profiles in different tissues and in response to various abiotic stresses (heat, cold, salt, and drought) or hormones (abscisic acid, methyl jasmonate, and gibberellic acid) were analyzed by qRT-PCR. The data indicated that many B-box genes (e.g., BrBBX13, BrBBX15, and BrBBX17) may contribute to plant development and growth as well as abiotic stress tolerance. Overall, the identified BBX genes may be useful as functional genetic markers for multiple stress responses and plant developmental processes.

Clubroot caused by Plasmodiophora brassicae is a severe disease of cruciferous crops that decreases crop quality and productivity. Several clubroot resistance-related quantitative trait loci and candidate genes have been identified. However, the underlying regulatory mechanism, the interrelationships among genes, and how genes are regulated remain unexplored. MicroRNAs (miRNAs) are attracting attention as regulators of gene expression, including during biotic stress responses. The main objective of this study was to understand how miRNAs regulate clubroot resistance-related genes in P. brassicae-infected Brassica rapa. Two Brassica miRNAs, Bra-miR1885a and Bra-miR1885b, were revealed to target TIR-NBS genes. In non-infected plants, both miRNAs were expressed at low levels to maintain the balance between plant development and basal immunity. However, their expression levels increased in P. brassicae-infected plants. Both miRNAs down-regulated the expression of the TIR-NBS genes Bra019412 and Bra019410, which are located at a clubroot resistance-related quantitative trait locus. The Bra-miR1885-mediated down-regulation of both genes was detected for up to 15 days post-inoculation in the clubroot-resistant line CR Shinki and in the clubroot-susceptible line 94SK. A qRT-PCR analysis revealed Bra019412 expression was negatively regulated by miR1885. Both Bra019412 and Bra019410 were more highly expressed in CR Shinki than in 94SK, the same expression pattern was detected in multiple clubroot-resistant and clubroot-susceptible inbred lines. A 5′ rapid amplification of cDNA ends analysis confirmed the cleavage of Bra019412 by Bra-miR1885b. Thus, miR1885s potentially regulate TIR-NBS gene expression during P. brassicae infections of B. rapa.

A major radish QTL (Fwr1) for fusarium wilt resistance was fine-mapped. Sequence and expression analyses suggest that a gene encoding a serine/arginine-rich protein kinase is a candidate gene for Fwr1. Fusarium wilt resistance locus 1 (Fwr1) is a major quantitative trait locus (QTL) mediating the resistance of radish inbred line ‘B2’ to Fusarium oxysporum, which is responsible for fusarium wilt. We previously detected Fwr1 on radish linkage group 3 (i.e., chromosome 5). In this study, a high-resolution genetic map of the Fwr1 locus was constructed by analyzing 354 recombinant F2 plants derived from a cross between ‘B2’ and ‘835’, the latter of which is susceptible to fusarium wilt. The Fwr1 QTL was fine-mapped to a 139.8-kb region between markers FM82 and FM87 in the middle part of chromosome 5. Fifteen candidate genes were predicted in this region based on a sequence comparison with the ‘WK10039’ radish reference genome. Additionally, we examined the time-course expression patterns of these predicted genes following an infection by the fusarium wilt pathogen. The ORF4 expression level was significantly higher in the resistant ‘B2’ plants than in the susceptible ‘835’ plants. The ORF4 sequence was predicted to encode a serine/arginine-rich protein kinase and includes SNPs that result in nonsynonymous mutations, which may have important functional consequences. This study reveals a novel gene responsible for fusarium wilt resistance in radish. Further analyses of this gene may elucidate the molecular mechanisms underlying the fusarium wilt resistance of plants.

Infectious clones of Korean turnip mosaic virus (TuMV) isolates KIH1 and HJY1 share 88.1% genomic nucleotides and 96.4% polyprotein amino acid identity, and they induce systemic necrosis or mild mosaic, respectively, in Nicotiana benthamiana. Chimeric constructs between these isolates exchanged the 5′, central, and 3′ domains of KIH1 (K) and HJY1 (H), where the order of the letters indicates the origin of these domains. KIH1 and chimeras KHH and KKH induced systemic necrosis, whereas HJY1 and chimeras HHK, HKK, and HKH induced mild symptoms, indicating the determinant of necrosis to be within the 5′ 3.9 kb of KIH1; amino acid identities of the included P1, Helper component protease, P3, 6K1, and cylindrical inclusion N-terminal domain were 90.06, 98.91, 93.80, 100, and 100%, respectively. Expression of P1 or P3 from a potato virus X vector yielded symptom differences only between P3 of KIH1 and HJY1, implicating a role for P3 in necrosis in N. benthamiana. Chimera KKH infected Brassica rapa var. pekinensis ‘Norang’, which was resistant to both KIH1 and HJY1, indicating that two separate TuMV determinants are required to overcome the resistance. Ability of diverse TuMV isolates, chimeras, and recombinants to overcome resistance in breeding lines may allow identification of novel resistance genes.

Infectious clones were generated from 17 new Korean radish isolates of Turnip mosaic virus (TuMV). Phylogenetic analysis indicated that all new isolates, and three previously characterized Korean radish isolates, belong to the basal-BR group (indicating that the pathotype can infect both Brassica and Raphanus spp.). Pairwise analysis revealed genomic nucleotide and polyprotein amino acid identities of >87.9 and >95.7%, respectively. Five clones (HJY1, HJY2, KIH2, BE, and prior isolate R007) had lower sequence identities than other isolates and produced mild symptoms in Nicotiana benthamiana. These isolates formed three distinct sequence classes (HJY1/HJY2/R007, KIH2, and BE), and several differential amino acid residues (in P1, P3, 6K2, and VPg) were present only in mild isolates HJY1, HJY2, and R007. The remaining isolates all induced systemic necrosis in N. benthamiana. Four mild isolates formed a phylogenetic subclade separate from another subclade including all of the necrosis-inducing isolates plus mild isolate KIH2. Symptom severity in radish and Chinese cabbage genotypes was not correlated with pathogenicity in N. benthamiana; indeed, Chinese cabbage cultivar Norang was not infected by any isolate, whereas Chinese cabbage cultivar Chusarang was uniformly susceptible. Four isolates were unable to infect radish cultivar Iljin, but no specific amino acid residues were correlated with avirulence. These results may lead to the identification of new resistance genes against TuMV.

The Brassica genus consists of economically important oil and leafy vegetable crops, which are cultivated worldwide. The Brassica species represent as the “U's triangle” (Nagahara, 1935), which includes the three basic diploid species Brassica rapa (A genome), Brassica nigra (B genome), and Brassica oleracea (C genome), as well as the three amphidiploid species Brassica juncea (A and B genomes), Brassica napus (A and C genomes), and Brassica carinata (B and C genomes). Brassica rapa (2n = 2x = 20) has been cultivated for specific phenotypic characteristics such as heading (i.e., ssp. pekinensis; Chinese cabbage) and non-heading (i.e., ssp. chinesis; pak choi) leafy vegetables, tuberized hypocotyl/roots (i.e., ssp. rapifera; turnip), and oil rich seedpods (i.e., ssp. trilocularis; yellow sarson) in the oil crops. Although the genomes of its subspecies are very similar (Lin et al., 2014), B. rapa demonstrates extreme morphological diversity. Because of the economical value and scientific interest in phenotypic diversity, the genome sequence of mesopolyploid B. rapa ssp. pekinensis Chiifu, a Chinese cabbage, was the first published B. rapa reference genome, and revealed that B. rapa evolved via a two-step whole-genome triplication and, as a result, has three syntenic subgenomes (Wang et al., 2011). The polyploidization is hypothesized to have facilitated the diversification of genes as well as gene fractionation, and as a consequence led to the evolution of different morphotypes within and between related Brassica species. The study of intraspecific diversity in B. rapa offers opportunities to advance our understanding of plant growth, development, and phenotypic evolution (Paterson et al., 2001). Over the last few years, the genome assembly of B. rapa (version 3.0) has been improved using single-molecule sequencing, optical mapping, and chromosome conformation capture technologies (Hi-C), resulting in an approximately 30-fold improvement with a contig N50 size of 1.45 Mb compared with that of previous references (Zhang et al., 2018). The assembly refined the syntenic relationship of genome blocks and centromere locations in the genome, and identified a greater number of annotated transposable elements (TEs) than in previous assemblies. In addition to the whole-genome sequence of Chinese cabbage, chromosome-level genome sequences of other B. rapa subspecies, including yellow sarson (Belser et al., 2018) and pak choi (Li P. et al., 2020; Li Y. et al., 2020), have been recently reported. The studies provide insight to the understanding of genetic drivers underlying the morphological variation among B. rapa subspecies. Moreover, a pangenome from Chinese cabbage, rapid-cycling Brassica, and Japanese vegetable turnip was constructed using Illumina short-read data (Lin et al., 2014), identifying genomic determinants of morphological variation, especially copy number differences in peroxidases associated with the phenylpropanoid biosynthetic pathway in turnip. Turnip (B. rapa L. ssp. rapifera) represent one of the morphotypes in B. rapa that forms tubers (hypocotyl/taproot tubers), produces lobed leaves with long petioles, and can be used to study the genetics underlying storage organ formation (Zhang et al., 2014). Brassica species are susceptible to clubroot disease, caused by Plasmodiophora brassicae (Schwelm et al., 2015), and forms galls (clubs) on infected root tissues with abnormal proliferation, preventing water and nutrient uptake and retarding the normal growth and development of plants, resulting in significantly reduced yield and quality. Sources of known clubroot resistance genes are derived from European turnip, carrying strong resistance to this disease (Matsumoto et al., 1998; Hirani et al., 2018). The resistance genes of European turnip have been introduced into other Brassica crops including vegetables and oilseed rape. In particular, European clubroot differential (ECD) turnips, that exhibited high levels of resistance to clubroot and that consist of four accessions (ECD1–ECD4), were developed and have been helpful for discovering dominant loci conferring clubroot resistance genes including CRa, CRb, and CRc for marker-assisted selection in canola and other Brassica species (Hirani et al., 2018). Here, we report the draft genome assembly of a European turnip, ECD4, which has strong clubroot resistance, that was generated by PacBio single-molecule long-read sequencing technology. This draft genome assembly coupled with transcriptome data derived from various leaf and root tissues will support the discovery of disease resistance genes (R genes) especially clubroot resistance genes, enabling the development of allele-specific markers for marker-assisted selection in Brassica breeding. Moreover, these data provide a valuable resource for studying the morphological diversity and evolution of turnips. For the genome assembly of European turnip (ECD4), a total of 70.63 Gb of PacBio long reads (an average sequencing coverage of 136.36x) and 72.16 Gb of Illumina short reads (139.3x) were generated (Supplementary Table 1). Based on k-mer analysis (k = 21), the estimated genome size of European turnip is approximately 518 Mbp (Figure 1A). The genome assembly resulted in a 315.8 Mb draft genome with 655 contigs (contig N50 length of 1.45 Mb; the longest contig length was 21.92 Mb) (Table 1). Our assembly data covered about 61% of the genome of European turnip, being predicted as mostly euchromatins. The assembly coverage is slightly low compared with those reported in Chinese cabbage (353 Mb) (Zhang et al., 2018), pak choi (370 Mb) (Li P. et al., 2020; Li Y. et al., 2020), and yellow sarson (402 Mb) (Belser et al., 2018). Whole-genome sequences of Chinese cabbage, pak choi, and yellow sarson were assembled at the chromosome-level by using chromosome conformation capture (Hi-C) technology [Chinese cabbage (reference genome version 3.0) and pak choi] and/or BioNano...

The indigenous communities across the globe especially in the rural areas consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many of the traditional food plants are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements and other nutrients. Excessive reliance on the mainstream staple crops has its own disadvantages. TFPs are nowadays considered important crops of the future and can act as supplementary foods for the burgeoning global population. They can also act as emergency foods in times of pandemics and other situations like COVID-19. The current situation necessitates locally available alternative nutritious TFPs for sustainable food production. To increase the cultivation or improve the traits in TFPs, it is essential to understand the molecular basis of the genes that regulate some important traits such as nutritional components and resilience to biotic and abiotic stresses. The integrated use of modern omics and gene editing technologies provide great opportunities to better understand the genetic and molecular basis of superior nutrient content, climate-resilient traits and adaptation to local agroclimatic zones. Recently, realising the importance and benefits of TFPs, scientists have shown interest in the prospection and sequencing of traditional food plants for their improvements, further cultivation and mainstreaming. Integrated omics such as genomics, transcriptomics, proteomics, metabolomics and ionomics are successfully used in plants and have provided a comprehensive understanding of gene-protein-metabolite networks. Combined use of omics and editing tools has led to successful editing of beneficial traits in few TFPs. This suggests that there is ample scope of integrated use of modern omics and editing tools/techniques for improvement of TFPs and their use for sustainable food production. In this article, we highlight the importance, scope and progress towards improvement of TFPs for valuable traits by integrated use of omics and gene editing techniques.

Fusarium wilt is an important disease of radish, leading to severe decrease in yield and quality. In this study, we used 180 F2 populations derived from a cross between radish inbred lines ‘YR4’ and ‘YR18’ to construct linkage group for the detecting quantitative trait locus (QTLs) related to Fusarium oxysporum resistance. Four QTLs related to Fusarium oxysporum resistance were detected on two linkage groups. Of the two major loci for FW resistance, FoRsR7.1FOR59 was delimited to the 2.18-Mb physical interval on chromosome R07, with a high LOD value (5.17–12.84). Additionally, it explained 9.34%-27.97% of the phenotypic variation in three inoculation tests. The other major QTL, FoRsR9.3FOR59, was also detected in all inoculation tests, but it had a relatively low value (3.38–4.52) and explained 6.24%-8.82% of the phenotypic variation. On the basis of the re-sequencing data for the parental lines, we identified 5 R-related genes and 13 unknown genes with sequence variations at the gene and protein levels. A semi-quantitative RT-PCR analysis revealed Rs382940 (TIR-NBS type) and Rs382200 (RLK type) were expressed only in ‘YR4’ from 0 to 6 days after the inoculation. Moreover, Rs382950 (TIR-NBS-LRR type) was more highly expressed in ‘YR4’ from 3 and 6 days after the inoculation. Therefore, these three genes are likely important for the FW resistance of radish. We identified several markers based on the potential candidate genes. These gene set might be useful for breeding system to introduce the Fusarium wilt resistance loci from ‘YR4’ to improve increasing tolerance to Fusarium oxysporum.

Two major QTL associated with resistance to Fusarium wilt (FW) were identified using whole-genome resequencing. Sequence variations and gene expression level differences suggest that TIR-NBS and LRR-RLK are candidate genes associated with FW-resistance. Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. raphani is an important disease in radish, leading to severe decrease in yield and quality. YR4 as a novel genetic source to resistant to FW was confirmed through screening with five pathogen isolates. We have generated F2 and F2:3 populations segregated with FW resistance using YR4 and YR18 inbred lines. The disease symptom was evaluated in F2:3 population (n = 180) in three independent studies over two years. We identified 4 QTL including the two major QTL (FoRsR7.159A and FoRsR9.359A). FoRsR7.159A and FoRsR9.359A were detected in three replicated experiments. FoRsR7.159A was delimited to the 2.18-Mb physical interval on chromosome R07, with a high LOD value (5.17–12.84) and explained phenotypic variation (9.34%–27.97%). The FoRsR9.359A represented relatively low LOD value (3.38–4.52) and explained phenotypic variation (6.24%–8.82%). On the basis of the re-sequencing data for the parental lines, we identified five putative resistance-related genes and 13 unknown genes with sequence variations at the gene and protein levels. A semi-quantitative RT-PCR analysis revealed that Rs382940 (TIR-NBS) and Rs382200 (RLK) were expressed only in ‘YR4’ from 0 to 6 days after the inoculation. Moreover, Rs382950 (TIR-NBS-LRR) was more highly expressed in ‘YR4’ from 3 to 6 days after the inoculation. These three genes might be important for FW-resistance in radish. We identified several markers based on these potential candidate genes. The marker set should be useful for breeding system to introduce the FW resistance loci from ‘YR4’ to improve tolerance to FW.

Clubroot resistance is an economically important trait in Brassicaceae crops. Although many quantitative trait loci (QTLs) for clubroot resistance have been identified in Brassica, disease-related damage continues to occur owing to differences in host variety and constant pathogen variation. Here, we investigated the inheritance of clubroot resistance in a double haploid population developed by crossing clubroot resistant and susceptible lines &ldquo, 09CR500&rdquo, and &ldquo, 09CR501&rdquo, respectively. The resistance of &ldquo, to Plasmodiophora brassicae pathotype &ldquo, Banglim&rdquo, was controlled as a single dominant gene, with the segregation of resistance and susceptibility being nearly 1:1. PbBrA08Banglim was identified as having a logarithm of odds value of 7.9&ndash, 74.8, and a phenotypic variance of 26.0&ndash, 97.1% with flanking marker &ldquo, 09CR.11390652&rdquo, in A08. After aligning QTL regions to the B. rapa reference genome, 11 genes were selected as candidates. PbBrA08Banglim was located near Crr1, CRs, and Rcr9 loci, but differences were validated by marker analysis, gene structural variations, and gene expression levels, as well as phenotypic responses to the pathotype. Genotyping using the &ldquo, marker accurately distinguished the Banglim-resistance phenotypes in the double haploid population. Thus, the developed marker will be useful in Brassica breeding programs, marker-assisted selection, and gene pyramiding to identify and develop resistant cultivars.

Clubroot resistance is an economically important trait in Brassicaceae crops. Although many quantitative trait loci (QTLs) for clubroot resistance have been identified in Brassica, disease-related damage continues to occur owing to differences in host variety and constant pathogen variation. Here, we investigated the inheritance of clubroot resistance in a double haploid population developed by crossing clubroot resistant and susceptible lines “09CR500” and “09CR501”, respectively. The resistance of “09CR500” to Plasmodiophora brassicae pathotype “Banglim” was controlled as a single dominant gene, with the segregation of resistance and susceptibility being nearly 1:1. PbBrA08Banglim was identified as having a logarithm of odds value of 7.9–74.8, and a phenotypic variance of 26.0–97.1% with flanking marker “09CR.11390652” in A08. After aligning QTL regions to the B. rapa reference genome, 11 genes were selected as candidates. PbBrA08Banglim was located near Crr1, CRs, and Rcr9 loci, but differences were validated by marker analysis, gene structural variations, and gene expression levels, as well as phenotypic responses to the pathotype. Genotyping using the “09CR.11390652” marker accurately distinguished the Banglim-resistance phenotypes in the double haploid population. Thus, the developed marker will be useful in Brassica breeding programs, marker-assisted selection, and gene pyramiding to identify and develop resistant cultivars.

Reddish purple Chinese cabbage (RPCC) is a popular variety of Brassica rapa (AA = 20). It is rich in anthocyanins, which have many health benefits. We detected novel anthocyanins including cyanidin 3-(feruloyl) diglucoside-5-(malonoyl) glucoside and pelargonidin 3-(caffeoyl) diglucoside-5-(malonoyl) glucoside in RPCC. Analyses of transcriptome data revealed 32,395 genes including 3345 differentially expressed genes (DEGs) between 3-week-old RPCC and green Chinese cabbage (GCC). The DEGs included 218 transcription factor (TF) genes and some functionally uncharacterized genes. Sixty DEGs identified from the transcriptome data were analyzed in 3-, 6- and 9-week old seedlings by RT-qPCR, and 35 of them had higher transcript levels in RPCC than in GCC. We detected cis-regulatory motifs of MYB, bHLH, WRKY, bZIP and AP2/ERF TFs in anthocyanin biosynthetic gene promoters. A network analysis revealed that MYB75, MYB90, and MYBL2 strongly interact with anthocyanin biosynthetic genes. Our results show that the late biosynthesis genes BrDFR, BrLDOX, BrUF3GT, BrUGT75c1-1, Br5MAT, BrAT-1, BrAT-2, BrTT19-1, and BrTT19-2 and the regulatory MYB genes BrMYB90, BrMYB75, and BrMYBL2-1 are highly expressed in RPCC, indicative of their important roles in anthocyanin biosynthesis, modification, and accumulation. Finally, we propose a model anthocyanin biosynthesis pathway that includes the unique anthocyanin pigments and genes specific to RPCC.

The CCCH-type zinc finger proteins are characterized by their signature motif of three cysteine and one histidine residues. These proteins are RNA binding proteins that function in plant growth, developmental processes, and responses to various environmental stress conditions. In this study, a comprehensive analysis using computational methods allowed the identification of 63 functionally important CCCH genes in the Brassica rapa genome. Several analyses were carried out on the identified genes to understand their roles in this plant. Comparative phylogenetic analysis classified CCCH genes into six clusters, while motif and structural analyses revealed four unique CCCH motifs including different functional motifs and intron/exon variations unique to plants compared to those in other species. Real-time qRT-PCR analysis of 10 randomly selected genes indicated they function at the early stages rather than the later stages of cold and salt stress. This study provides a basic understanding of potential candidate CCCH genes in B. rapa, their structural variation, expression patterns, and their roles under different stress conditions.

The F-box genes form one of the largest functionally important, rapidly evolving plant gene families. The encoded proteins mainly function as part of the Skp1–Cullin–F-box complex involved in ubiquitinating and degrading proteins. The F-box proteins also regulate diverse functions, including embryogenesis, organ development, floral organ identity, self-incompatibility, senescence, homeostasis, signaling, and responses to biotic and abiotic stresses. We identified 571 Brassica rapa F-box genes (BrFBX) and mapped approximately 560 genes onto 10 chromosomes. We also classified the duplicated genes. A phylogenetic tree consisting of the B. rapa F-box genes and an analysis of conserved motif sequences enabled us to categorize the identified genes into 11 subgroups based on node support. Additionally, we determined the intron–exon structural characteristics, which helped detect differences among the BrFBX genes. Gene ontology predictions enabled the classification of 431 genes related to biological processes, 63 genes involved in molecular functions, and 40 genes associated with cellular localization. The 69 genes differentially expressed under abiotic stress conditions (i.e., cold, drought, and salt stresses) and 446 genes expressed in specific tissues (i.e., calli, roots, leaves, stems, flowers, and siliques) were further categorized into three groups based on their expression levels. These genes exhibited various spatiotemporal expression patterns during stress treatments and in specific tissues. Based on motif and expression analyses, we selected approximately 30 BrFBX genes for quantitative reverse transcription polymerase chain reaction analysis, which detected differences in expression among eight tissues during B. rapa (Chiifu) growth. The genome-wide study results reveal the relationships among the BrFBX genes regarding evolution, structural variability, potential functions, and these data can be further used as a resource for the gene characterization in relation to growth, development, and during different stress conditions for the development of Brassica rapa.

A major QTL conferring tolerance to radish (Raphanus sativus) root cracking was mapped for the first time and two calcium regulatory genes were identified that positively associated with the cracking phenomenon. Root cracking is a severe physiological disorder that significantly decreases the yield and commercial value of radish. The genetic and physiological mechanisms underlying this root cracking disorder have not been characterized. In this study, quantitative trait loci (QTLs) putatively associated with radish root cracking were mapped. Ten QTLs were distributed in six linkage groups, among these QTLs, ‘RCr1’ in LG1 was detected over 3 consecutive years and was considered to be a major QTL for root cracking. The QTL ‘RCr1’ was responsible for 4.47–18.11% of variance in the root cracking phenotype. We subsequently identified two candidate genes, RsANNAT and RsCDPK. Both genes encode proteins involved in calcium binding, ion transport, and Ca2+ signal transduction, which are important for regulating plant development and adaptations to the environment. These genes were co-localized to the major QTL region. Additionally, we analyzed physiological changes (i.e., root firmness, cell wall content, and cell-wall-bound calcium content) in two parental lines during different developmental stages. Moreover, we observed that the RsANNAT and RsCDPK expression levels are positively correlated with Ca2+ contents in the roots of the cracking-tolerant ‘835’ cultivar. Thus, these genes may influence root cracking. The data provided herein may support the useful information to understand root cracking behavior in radish and may enable breeders to develop new cultivars exhibiting increased tolerance to root and fruit cracking.

Infectious clones of Korean turnip mosaic virus (TuMV) isolates KIH1 and HJY1 share 88.1% genomic nucleotides and 96.4% polyprotein amino acid identity, and they induce systemic necrosis or mild mosaic, respectively, in

Indigenous communities across the globe, especially in rural areas, consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many TFPs are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements and other nutrients. Excessive reliance on the mainstream staple crops has its own disadvantages. Traditional food plants are nowadays considered important crops of the future and can act as supplementary foods for the burgeoning global population. They can also act as emergency foods in situations such as COVID-19 and in times of other pandemics. The current situation necessitates locally available alternative nutritious TFPs for sustainable food production. To increase the cultivation or improve the traits in TFPs, it is essential to understand the molecular basis of the genes that regulate some important traits such as nutritional components and resilience to biotic and abiotic stresses. The integrated use of modern omics and gene editing technologies provide great opportunities to better understand the genetic and molecular basis of superior nutrient content, climate-resilient traits and adaptation to local agroclimatic zones. Recently, realizing the importance and benefits of TFPs, scientists have shown interest in the prospection and sequencing of TFPs for their improvements, cultivation and mainstreaming. Integrated omics such as genomics, transcriptomics, proteomics, metabolomics and ionomics are successfully used in plants and have provided a comprehensive understanding of gene-protein-metabolite networks. Combined use of omics and editing tools has led to successful editing of beneficial traits in several TFPs. This suggests that there is ample scope for improvement of TFPs for sustainable food production. In this article, we highlight the importance, scope and progress towards improvement of TFPs for valuable traits by integrated use of omics and gene editing techniques.

Long non-coding RNAs (LncRNAs) are a large, diverse class of RNA molecules that has garnered attention for their potential to regulate gene expression and been identified in various organisms. Here we report the first prediction of lncRNAs in Brassica rapa (B. rapa) genome using computational method. We subjected the publicly available full length cDNA sequences and identified 2237 candidate lncRNAs, characterized their functions based on insilico methods. Housekeeping and small regulatory non-coding RNAs (ncRNAs) were removed from the pool. Although 14–15 % of the total sequences were predicted to be non-coding initially, on filtering only 4.6 % of the total sequences were predicted as lncRNAs carrying a large number of simple repeats. The lncRNAs had an average length of 497 bp and were mapped on each chromosome of B. rapa. They were classified to 4 groups based on their origin. Thirty six motifs involving transcription related activities, signaling mechanism and stress response were identified in the lncRNAs. Repeat elements and neighboring genes of the lncRNAs were analyzed since they were associated in function and regulating the expression of these long non-coding RNAs. We believe that this study would be an initial and reference for any further studies regarding long non-coding RNAs in B. rapa and other Brassica crops.

Infectious clones were generated from 17 new Korean radish isolates of

The resistance gene analog (RGA)-based marker strategy is an effective supplement for current marker reservoir of radish disease-resistance breeding. In this study, we identified RGAs based on the conserved nucleotide-binding site (NBS) and S-receptor-like kinase (SRLK) domains. A total of 68 NBS-RGAs and 46 SRLK-RGAs were isolated from two FW-resistant radish inbred lines, B2 and YR31, and one susceptible line, YR15. A BLASTx search revealed that the NBS-RGAs contained six conserved motifs (i.e., P loop, RNBS-A, Kinase-2, RNBS-B, RNBS-C, and GLPL) and the SRLK-RGAs, contained two conserved motifs (i.e., G-type lectin and PAN-AP). A phylogenetic analysis indicated that the NBS-RGAs could be separated into two classes (i.e., toll/interleukin receptor and coiled-coil types), with six subgroups, and the SRLK-RGAs were divided into three subgroups. Moreover, we designed RGA-specific markers from data-mining approach in radish databases. Based on marker analysis, 24 radish inbred lines were clustered into five main groups with a similarity index of 0.44 and showing genetic diversity with resistance variation in those radish inbred lines. The development of RGA-specific primers would be valuable for marker-assisted selection during the breeding of disease-resistant radish cultivars. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-018-1279-y) contains supplementary material, which is available to authorized users.

This study investigated optimum light conditions for enhancing phenylpropanoid biosynthesis and the distribution of phenylpropanoids in organs of Chinese cabbage (Brassica rapa ssp. pekinensis). Blue light caused a high accumulation of most phenolic compounds, including p-hydroxybenzoic acid, ferulic acid, quercetin, and kaempferol, at 12 days after irradiation (DAI). This increase was coincident with a noticeable increase in expression levels of BrF3H, BrF3'H, BrFLS, and BrDFR. Red light led to the highest ferulic acid content at 12 DAI and to elevated expression of the corresponding genes during the early stages of irradiation. White light induced the highest accumulation of kaempferol and increased expression of BrPAL and BrDFR at 9 DAI. The phenylpropanoid content analysis in different organs revealed organ-specific accumulation of p-hydroxybenzoic acid, quercetin, and kaempferol. These results demonstrate that blue light is effective at increasing phenylpropanoid biosynthesis in Chinese cabbage, with leaves and flowers representing the most suitable organs for the production of specific phenylpropanoids.

The resistance to Diamondback moth insect in cabbage is governed by many minor loci in quantitative nature, and at least four genetic loci should be incorporated in marker-assisted breeding program for developing partially resistant DBM cabbage cultivars. The Diamondback moth (DBM), Plutella xylostella (L.), is the most destructive insect infesting cruciferous plants worldwide. Earlier studies have reported that the glossy leaves of cabbage are associated with resistance to this insect. However, until now, genetics of DBM resistance has not been studied in detail, and no QTL/gene mapping for this trait has been reported. In this paper, we report quantitative trait loci (QTL) mapping of DBM-resistant trait using 188 randomly selected segregating F 3 population derived from crossing a partially DBM-resistant glossy leaf cabbage (748) with a susceptible smooth cabbage line (747). Quantitative trait loci mapping using phenotypic data of four consecutive years (2008, 2009, 2010, and 2011) on DBM insect infestation detected a total of eight QTL on five linkage groups suggesting that DBM resistance is a quantitative in nature. Of these QTL, four QTL, i.e., qDbm 1 on LG1, qDbm5 and qDbm6 on LG7, and qDbm8 on LG9, were detected in different tests and years. The QTL, qDbm6 on LG7, was consecutively detected over 3 years. Tightly linked molecular markers have been developed for qDbm8 QTL on LG9 which could be used in marker-assisted breeding program. Our research demonstrated that for desired DBM resistance cultivar breeding, those four genetic loci have to be taken into consideration. Furthermore, the comparative study revealed that DBM resistance QTL is conserved between close relative model plant Arabidopsis thaliana and Brassica oleracea genome.

To better understand carotenoid biosynthesis in Brassica rapa, a genome-wide investigation was performed using the EST/mRNA sequences and the draft genome of B. rapa. We have identified 46 full-length coding sequences in the B. rapa genome by comparing with the Arabidopsis thaliana genes that were involved in carotenoid biosynthesis. The identified genes were categorized to belong to 11 protein families encoding various enzymes involving the biosynthesis. A detailed domain structural analysis of the enzymes showed that these were highly conserved and overlapping. The genes were localized to all 10 chromosomes of the plant, six of which were single-copy genes; whereas others had more than one paralogue. Phylogenetic analysis of A. thaliana and B. rapa sequences revealed sequence identities ranging from 70–90%, indicating the conservation of genes among the species. Approximately 185 single nucleotide polymorphisms (SNPs) were annotated at the exonic and intronic regions of these genes, while comparing the resequenced parental lines RcBr and Kenshin against Chiifu as reference genome. Expression profiling was done for all the identified genes with the available microarray data of inbred lines, Chiifu and RcBr. Finally, a knowledge-based carotenoid biosynthesis pathway was proposed for B. rapa using details retrieved from the comparative study.

Twelve carotenoids were identified in Korean leafy vegetables and paprikas. by high-performance liquid chromatography, Carotenoid contents varied greatly, with red paprika having a higher antheraxanthin and capsanthin contents than other paprikas. Orange paprika had higher levels of zeaxanthin, β-cryptoxanthin, lutein, and α-carotene compared to those of other paprikas. The results of Pearson’s correlation analysis using quantitative data of carotenoids revealed that significant positive relationships were apparent between capsanthin and antheraxanthin (r=0.9870, p

The use of clubroot resistance (CR) genes is an effective and economical approach for controlling Plasmodiophora brassicae, the causal agent of clubroot disease in Chinese cabbage (Brassica rapa) and other Brassica crops. In a previous study, we identified and mapped the CRb locus on chromosome A03 of B. rapa in the doubled-haploid (DH) line ‘CR Shinki DH line’ of Chinese cabbage. In this study, CRb, a dominant gene conferring resistance to pathotype 4 of P. brassicae, was finely mapped in combination with bulked segregant analysis and bioinformatics analysis (BIA). Using 1,486 highly susceptible individuals and 2,896 individuals from two separate F2 populations of ‘702-5’ (B. rapa ssp. chinensis) × ‘CR Shinki DH line,’ the CRb locus was narrowed to a region of approximately 0.14 cM between two flanking markers, TCR79 and TCR108. The sequences of seven newly developed markers linked to CRb were landed on bacterial artificial chromosome (BAC) of the reference B. rapa ‘Chiifu-401-42’ by BIA, and a physical map consisting of three BAC clones was constructed. The CRb locus was defined as an interval of approximately 83.5 kb on a BAC clone (KBrB085J21). The target interval contained one Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR–NBS–LRR) gene, one NBS–LRR gene, and several putative regulatory genes in the B. rapa genome. The CRb gene was tightly linked to two other CR genes, CRa and CRb Kato . These results provide useful information for isolation of the CRb gene and tightly linked molecular markers for breeding CR in B. rapa.

The involvement of MADS-box transcription factors in the development of seeds, flowers, and fruit is well known. Large numbers of MADS-box genes have been characterized and reported in major plants such as Arabidopsis thaliana (107), Oryza sativa (75), Zea mays (75), and Cucumis sativus (43). However, there is little information about MADS-box genes in the economically and morphologically important genus Brassica. Thus, we performed a series of computational analyses on the recently published Brassica rapa genome and identified 167 potential MADS-box genes. Chromosomal localization of these genes revealed many duplicate genes and 10 tandem repeats from B. rapa chromosomes, though none on A04, A08, and A10. Neighbor-joining phylogenetic analysis was performed between B. rapa, A. thaliana, O. sativa, Z. mays, and C. sativus genes, and type-I and type-II groups were differentiated by their gene clades. Based on their phylogeny and functional characters, type-I group was divided into three clades and type-II group into 14 clades. Prediction and comparison of intron and exon patterns of MADS-box genes authenticates the major difference between the groups. Semi-quantitative RT-PCR analysis was carried out for 25 functionally categorized BrMADS-box genes and comparatively studied among buds, sepals, petals, stamens, carpels, and siliques of the parental line RcBr. The structural and functional annotation, as well as expressional profiling of BrMADS in floral organs in this study will be the basis of further functional validation and molecular breeding of B. rapa crops.

무의 가공용 품종의 가공 수율이 현재 약 70%로 버리는 부분이 많아 뿌리의 형태가 가공에 유리하도록 상부와 하부의 굵기가 유사한 품종을 육성코자 하였으며 웅성불임을 이용하여 채종함으로 종자의 순도도 높이고자 본 연구를 실시하였다. 가공용 품종은 특히 순도가 높아야 하므로 계통 육성 과정에서 순계로 고정되었는지 여부를 확인하기 위해 표현형 뿐 아니라 분자표지를 활용하여 증식된 종자의 순도를 검정하였다. 우수 품종 육성을 위해 A친은 (‘관동여름’ x ‘계룡봄무’)의 후대에서 위황병에 저항성이며 추대성이 늦고 고온기 재배에서 근비대성이 타 계통보다 양호한 담록수 계통을 고정하였고, B친은 (‘태상왕’ x ‘서울봄무’)의 후대에서 위황병, 근류병에 저항성이며 잎색은 진한 녹색으로 청수색이 강하고 근비대성, 육질치밀성인 계통을 선발 고정하였다. 화분친(C친)은 개체선발법으로 세대를 진전하였으며 분리세대 순도검정을 위해 각 세대별로 종자 48점을 ACMP-490, cnu-316프라이머를 이용하여 분석한 결과 분리 1 세대 ACMP-490은 64.6%, cnu-316은 66.7%의 고정율을 보였으며, 분리2 세대 ACMP-490은 68.8%, cnu-316은 70.8%의 고정율을 보였고, 분리 3 세대 ACMP-490은 93.8%, cnu-316은 100%의 고정율을 나타냈으며, 분리 4 세대 ACMP-490은 93.8%, cnu-316은 100%의 고정율을 보였고. 분리 5 세대 ACMP-490은 95.8%, cnu-316은 100%의 고정율을 보였고, 분리 6 세대 ACMP-490은 100%, cnu-316은 100%의 고정율을 보여원원종을 확보하였다. 고정이 확인된 계통을 이용하여 조합을 작성한 결과 뿌리의 형태가 가공에 적합하고 순도가 우수하여 이를 ‘YR 오래(YR ORE)’로 2011년에 품종보호 출원하여 2013년 신품종으로 등록 되었다(품종보호 제 4550호, 2013.06.10.).

The diamondback moth, Plutella xylostella (L.), is a major pest responsible for destroying cabbage and other Brassica vegetable crops. A diamondback moth-resistant cabbage line was studied by comparing its metabolite profiles with those of a susceptible cabbage. Fourier transform infrared spectroscopy analysis revealed that carbohydrates, aromatic compounds, and amides were the major factors that distinguished the resistant and susceptible genotypes. Gas chromatography-time-of-flight mass spectrometry profiled 46 metabolites, including 19 amino acids, 15 organic acids, 8 sugars, 3 sugar alcohols, and 1 amine in two genotypes and F1 hybrid cabbages. The levels of glycolic acid, quinic acid, inositol, fumaric acid, glyceric acid, trehalose, shikimic acid, and aspartic acid were found to be very significantly different between the resistant and susceptible genotypes with a P value of

Since the early 1980s, the National Institute of Horticultural & Herbal Sciences has been breeding and collecting diverse radish breeds to select those samples with better horticultural characteristics, to ultimately expand and develop as good radish produce. Genetic diversity is a crucial factor in crop improvement and therefore it is very important to obtain various variations through sample collection. The collected samples were compared with one another in order to assess the level of diversity among the collections, and this procedure allowed for increased application of the gathered resources and aided in determining the direction to secure further samples. Towards this end, this experiment was conducted in order to examine whether the SSR markers derived from Chinese cabbage samples could be transferred to the radish samples. Among the radish breeding lines and introduced resources, 44 lines were used as materials to analyze the genotype using 22 SSR markers selected. As a result, the analysis showed that among all the selected markers, 'cnu_m139' and 'cnu_m289' were the most useful markers for diversity evaluation. The genetic relationship of the radish genetic resources showed that the geographic origins affected the diversity. Furthermore, the different types of radish groups were also determined by the year they were bred. This result demonstrated that there are differences between the older radish breeds and the more recently developed radish breeds. Even though a relatively small number of markers were used in the analysis, it was possible to distinguish whether the radish was bred 30 years ago or in the 2000s, and that the similar physical shapes comprised a particular group, showed that the SSR markers can indeed be successfully applied to to study the diversity within radish breeding lines. Through the results of this study, it can be concluded that the SSR marker developed for the Chinese cabbage can be applied to examine the genetic diversity and analyze the relationship (genetic resource determination) of radish.

Radish is susceptible to all kinds of diseases, and to breed cultivars with high levels of diseases resistance is important for reducing chemical sprays in order to avoid effect on environment. In this chapter, a summary is given on genetic studies of resistance genes in radish. First, we provide an overview of fungi, virus, bacteria, and nematode disease-related QTL studies which were published in recent years and also discuss about the candidate genes/orthologous that were found by comparative QTL/gene mapping on disease resistance loci of Brassicaceae family. Finally, we suggest a few strategies to identify and utilize the resistance genes in radish breeding.

QTLs and candidate gene markers associated with leaf morphological and color traits were identified in two immortalized populations of Brassica rapa, which will provide genetic information for marker-assisted breeding. Brassica rapa is an important leafy vegetable consumed worldwide and morphology is a key character for its breeding. To enhance genetic control, quantitative trait loci (QTLs) for leaf color and plant architecture were identified using two immortalized populations with replications of 2 and 4 years. Overall, 158 and 80 QTLs associated with 23 and 14 traits were detected in the DH and RIL populations, respectively. Among them, 23 common robust-QTLs belonging to 12 traits were detected in common loci over the replications. Through comparative analysis, five crucifer genetic blocks corresponding to morphology trait (R, JU, F and E) and color trait (F, E) were identified in three major linkage groups (A2, A3 and A7). These might be key conserved genomic regions involved with the respective traits. Through synteny analysis with Arabidopsis, 64 candidate genes involved in chlorophyll biosynthesis, cell proliferation and elongation were co-localized within QTL intervals. Among them, SCO3, ABI3, FLU, HCF153, HEMB1, CAB3 were mapped within QTLs for leaf color; and CYCD3;1, CYCB2;4, AN3, ULT1 and ANT were co-localized in QTL regions for leaf size. These robust QTLs and their candidate genes provide useful information for further research into leaf architecture with crop breeding.

Auxins are the key players in plant growth development involving leaf formation, phototropism, root, fruit and embryo development. Auxin/Indole-3-Acetic Acid (Aux/IAA) are early auxin response genes noted as transcriptional repressors in plant auxin signaling. However, many studies focus on Aux/ARF gene families and much less is known about the Aux/IAA gene family in Brassica rapa (B. rapa). Here we performed a comprehensive genome-wide analysis and identified 55 Aux/IAA genes in B. rapa using four conserved motifs of Aux/IAA family (PF02309). Chromosomal mapping of the B. rapa Aux/IAA (BrIAA) genes facilitated understanding cluster rearrangement of the crucifer building blocks in the genome. Phylogenetic analysis of BrIAA with Arabidopsis thaliana, Oryza sativa and Zea mays identified 51 sister pairs including 15 same species (BrIAA-BrIAA) and 36 cross species (BrIAA-AtIAA) IAA genes. Among the 55 BrIAA genes, expression of 43 and 45 genes were verified using Gene-bank B. rapa ESTs and in home developed microarray data from mature leaves of Chiifu and RcBr lines. Despite their huge morphological difference, tissue specific expression analysis of BrIAA genes between the parental lines Chiifu and RcBr showed that the genes followed a similar pattern of expression during leaf development and a different pattern during bud, flower and siliqua development stages. The response of the BrIAA genes to abiotic and auxin stress at different time intervals revealed their involvement in stress response. Single Nucleotide Polymorphisms between IAA genes of reference genome Chiifu and RcBr were focused and identified. Our study examines the scope of conservation and divergence of Aux/IAA genes and their structures in B. rapa. Analyzing the expression and structural variation between two parental lines will significantly contribute to functional genomics of Brassica crops and we belive our study would provide a foundation in understanding the Aux/IAA genes in B. rapa.

Identification of quantitative trait loci (QTLs) governing morphologically important traits enables to comprehend their potential genetic mechanisms in the genetic breeding program. In this study, we used 210 F2 populations derived from a cross between two radish inbred lines (Raphanus sativus) “835” and “B2,” including 258 SSR markers were used to detect QTLs for 11 morphological traits that related to whole plant, leaf, and root yield in 3 years of replicated field test. Total 55 QTLs were detected which were distributed on each linkage group of the Raphanus genome. Individual QTLs accounted for 2.69–12.6 of the LOD value, and 0.82–16.25% of phenotypic variation. Several genomic regions have multiple traits that clustered together, suggested the existence of pleiotropy linkage. Synteny analysis of the QTL regions with A. thaliana genome selected orthologous genes in radish. InDels and SNPs in the parental lines were detected in those regions by Illumina genome sequence. Five identified candidate gene-based markers were validated by co-mapping with underlying QTLs affecting different traits. Semi-quantitative reverse transcriptase PCR analysis showed the different expression levels of these five genes in parental lines. In addition, comparative QTL analysis with B. rapa revealed six common QTL regions and four key major evolutionarily conserved crucifer blocks (J, U, R, and W) harboring QTL for morphological traits. The QTL positions identified in this study will provide a valuable resource for identifying more functional genes when whole radish genome sequence is released. Candidate genes identified in this study that co-localized in QTL regions are expected to facilitate in radish breeding programs.

Brassica rapa L. (AA, 2n = 20), an A genome diploid species of Brassica genus is of researchers interest recent days since enormous amount of data is available about the genome. Since EST analysis is a powerful tool in gene discovery we compared different existing methods and developed a new pipeline for EST computational analysis to analyze the available data. A total of 1,438 expressed sequence tags sizing from 83 to 2,023 base pairs were generated and subjected to various types of analysis. Cluster analysis of these ESTs identified 969 unique sequences called unigenes, with 162 contigs and 807 singlets. Similarity search produced 704 significant hits with E-value ≥ 10−5. The functions of the best hits were annotated by gene ontology (GO) analysis. Additionally, we classified 293 and 541 unigenes based on their functions, using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein structural domain databases, respectively. We also identified and categorized 171 microsatellites into di-, tri-, tetra-, and penta nucleotide repeats, and designed primers. Possible open reading frames (ORFs) were predicted for 960 unigenes, by the comparison with a primary protein sequence database. In silico mapping of partial unigenes were done in bacterial artificial chromosome (BAC) sequences, downloaded from the Brassica genome project website. We determined 149 single nucleotide polymorphisms (SNPs) and 3 indels from the coding region of 27 unigenes of B. rapa and similar Brassica napus ESTs clusters. All the generated EST sequences were submitted to the GenBank EST database (dbEST) as accessions from CO749247 to CO750425.