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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.

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.

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.

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.

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.

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.

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.

The inheritance of leaf and heading-related traits in Chinese cabbage (Brassica rapa ssp. pekinensis) are poorly understood since only a handful of studies of these traits are available till date. In this study, QTL mapping was done for seven leaf and heading related traits that includes gross weight, number of non-wrapper leaves, head weight, head length, head width, head length/head width ratio, and the number of head-forming leaves in 139 F3 families derived from a cross between two diverse Chinese cabbage lines, ‘501’ with small head and ‘601’ with large head. A total of 27 QTL spread over the nine linkage groups were identified for seven leaf and head-related traits, many of which showed major effects clustered in A2, A4, A6, and A8. The results provided a better understanding of the genetic factors controlling leaf and head-related traits in Chinese cabbage.

Brassica napus (AACC genome) is an important oilseed crop that was formed by the fusion of the diploids B. rapa (AA) and B. oleracea (CC). The complete genomic sequence of the Brassica A genome will be available soon from the B. rapa genome sequencing project, but it is not clear how informative the A genome sequence in B. rapa (A(r)) will be for predicting the structure and function of the A subgenome in the allotetraploid Brassica species B. napus (A(n)). In this paper, we report the results of structural and functional comparative mapping between the A subgenomes of B. napus and B. rapa based on genetic maps that were anchored with bacterial artificial chromosomes (BACs)-sequence of B. rapa. We identified segmental conservation that represented by syntenic blocks in over one third of the A genome; meanwhile, comparative mapping of quantitative trait loci for seed quality traits identified a dozen homologous regions with conserved function in the A genome of the two species. However, several genomic rearrangement events, such as inversions, intra- and inter-chromosomal translocations, were also observed, covering totally at least 5% of the A genome, between allotetraploid B. napus and diploid B. rapa. Based on these results, the A genomes of B. rapa and B. napus are mostly functionally conserved, but caution will be necessary in applying the full sequence data from B. rapa to the B. napus as a result of genomic rearrangements in the A genome between the two species.

Cabbage head splitting can greatly affect both the quality and commercial value of cabbage (Brassica oleracea). To detect the genetic basis of head-splitting resistance, a genetic map was constructed using an F2 population derived by crossing “748” (head-splitting-resistant inbred line) and “747” (head-splitting-susceptible inbred line). The map spans 830.9 cM and comprises 270 markers distributed in nine linkage groups, which correspond to the nine chromosomes of B. oleracea. The average distance between adjacent markers was 3.6 cM. A total of six quantitative trait loci (QTLs) conferring resistance to head splitting were detected in chromosome 2, 4, and 6. Two QTLs, SPL-2-1 and SPL-4-1, on chromosomes 2 and 4, respectively, were detected in the experiments over 2 years, suggesting that these two potential loci were important for governing the head-splitting resistance trait. Markers BRPGM0676 and BRMS137, which were tightly linked with head-splitting resistance, were detected in the conserved QTL SPL-2-1 region using bulked segregant analysis. Synteny analysis showed that SPL-2-1 was anchored to a 3.18-Mb genomic region of the B. oleracea genome, homologous to crucifer ancestral karyotype E block in chromosome 1 of Arabidopsis thaliana. Moreover, using a field emission scanning electron microscope, significant differences were observed between the two parental lines in terms of cell structures. Line “747” had thinner cell wall, lower cell density, larger cell size, and anomalous cell wall structure compared with the resistant line “748.” The different cell structures can provide a cytological base for assessing cabbage head splitting.

Panax ginseng, commonly known as Korean ginseng, is a valued source of herbal medicine in Korea and China. We have developed and characterized 35 microsatellite markers in P. ginseng from available BAC end sequences. Characterization of these 35 SSR primer pairs in 14 cultivars of P. ginseng showed 12 primer pairs to be polymorphic and 19 primer pairs to be monomorphic, while the remaining four primer pairs did not produce any product. The number of alleles amplified ranged from 4 to 8 per primer pair, with an average of six alleles per locus. The expected and observed heterozygosities ranged from 0.7500 to 0.9678 and 0.5645 to 0.7109, respectively. None of these loci deviated from Hardy–Weinberg equilibrium. All of the functional primer pairs of P. ginseng showed cross-species transferability with Panax quinquefolium. The cross-species transferable markers could be used for ginseng cultivar identification, for genomic studies, including mapping of specific trait QTL/genes, and for measuring conservation of ginseng.

Background In view of the immense value of Brassica rapa in the fields of agriculture and molecular biology, the multinational Brassica rapa Genome Sequencing Project (BrGSP) was launched in 2003 by five countries. The developing BrGSP has valuable resources for the community, including a reference genetic map and seed BAC sequences. Although the initial B. rapa linkage map served as a reference for the BrGSP, there was ambiguity in reconciling the linkage groups with the ten chromosomes of B. rapa. Consequently, the BrGSP assigned each of the linkage groups to the project members as chromosome substitutes for sequencing. Results We identified simple sequence repeat (SSR) motifs in the B. rapa genome with the sequences of seed BACs used for the BrGSP. By testing 749 amplicons containing SSR motifs, we identified polymorphisms that enabled the anchoring of 188 BACs onto the B. rapa reference linkage map consisting of 719 loci in the 10 linkage groups with an average distance of 1.6 cM between adjacent loci. The anchored BAC sequences enabled the identification of 30 blocks of conserved synteny, totaling 534.9 cM in length, between the genomes of B. rapa and Arabidopsis thaliana. Most of these were consistent with previously reported duplication and rearrangement events that differentiate these genomes. However, we were able to identify the collinear regions for seven additional previously uncharacterized sections of the A genome. Integration of the linkage map with the B. rapa cytogenetic map was accomplished by FISH with probes representing 20 BAC clones, along with probes for rDNA and centromeric repeat sequences. This integration enabled unambiguous alignment and orientation of the maps representing the 10 B. rapa chromosomes. Conclusion We developed a second generation reference linkage map for B. rapa, which was aligned unambiguously to the B. rapa cytogenetic map. Furthermore, using our data, we confirmed and extended the comparative genome analysis between B. rapa and A. thaliana. This work will serve as a basis for integrating the genetic, physical, and chromosome maps of the BrGSP, as well as for studies on polyploidization, speciation, and genome duplication in the genus Brassica.