A GBS-based genetic linkage map and quantitative trait loci (QTL) associated with resistance to Xanthomonas campestris pv. campestris race 1 identified in Brassica oleracea.

The production of Brassica oleracea, an important vegetable crop, is severely affected by black rot disease caused by the bacterial pathogen Xanthomonas campestris pv. campestris. Resistance to race 1, the most virulent and widespread race in B. oleracea, is under quantitative control; therefore, identifying the genes and genetic markers associated with resistance is crucial for developing resistant cultivars. Quantitative trait locus (QTL) analysis of resistance in the F2 population developed by crossing the resistant parent BR155 with the susceptible parent SC31 was performed. Sequence GBS approach was used to develop a genetic linkage map. The map contained 7,940 single nucleotide polymorphism markers consisting of nine linkage groups spanning 675.64 cM with an average marker distance of 0.66 cM. The F2:3 population (N = 126) was evaluated for resistance to black rot disease in summer (2020), fall (2020), and spring (2021). QTL analysis, using a genetic map and phenotyping data, identified seven QTLs with LOD values between 2.10 and 4.27. The major QTL, qCaBR1, was an area of overlap between the two QTLs identified in the 2nd and 3rd trials located at C06. Among the genes located in the major QTL interval, 96 genes had annotation results, and eight were found to respond to biotic stimuli. We compared the expression patterns of eight candidate genes in susceptible (SC31) and resistant (BR155) lines using qRT-PCR and observed their early and transient increases or suppression in response to Xanthomonas campestris pv. campestris inoculation. These results support the involvement of the eight candidate genes in black rot resistance. The findings of this study will contribute towards marker-assisted selection, additionally the functional analysis of candidate genes may elucidate the molecular mechanisms underlying black rot resistance in B. oleracea.

Identification of accession-specific variants and development of KASP markers for assessing the genetic makeup of Brassica rapa seeds.

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).

Starch content changes and metabolism-related gene regulation of Chinese cabbage synergistically induced by Plasmodiophora brassicae infection.

Clubroot is one of the major diseases adversely affecting Chinese cabbage (Brassica rapa) yield and quality. To precisely characterize the Plasmodiophora brassicae infection on 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 seven days after the P. brassicae inoculation, whereas the expression levels of the starch degradation-related genes increased at 14 days after the 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. 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 the histological observation 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.

Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese Cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector.

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.

L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis).

Successful Agrobacterium-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 stable Agrobacterium 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-old in vitro derived cotyledon explants were infected with A. tumefaciens strain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to an Agrobacterium suspension (OD600 of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO3 combination exhibited the highest ß-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 of hptII in T0 transformed 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.

Genome-Wide Identification, Evolution, and Comparative Analysis of B-Box Genes in Brassica rapa, B. oleracea, and B. napus and Their Expression Profiling in B. rapa in Response to Multiple Hormones and Abiotic Stresses.

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.

MiR1885 Regulates Disease Tolerance Genes in Brassica rapa during Early Infection with Plasmodiophora brassicae.

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.

QTL mapping for Fusarium wilt resistance based on the whole-genome resequencing and their association with functional genes in Raphanus sativus.

KEY MESSAGE: 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.

Integrating Omics and Gene Editing Tools for Rapid Improvement of Traditional Food Plants for Diversified and Sustainable Food Security.

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.

Genome-wide identification and characterization of NBS-encoding genes in Raphanus sativus L. and their roles related to Fusarium oxysporum resistance.

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.

Quantitative Trait Locus Mapping of Clubroot Resistance and Plasmodiophora brassicae Pathotype Banglim-Specific Marker Development in Brassica rapa.

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.

Red Chinese Cabbage Transcriptome Analysis Reveals Structural Genes and Multiple Transcription Factors Regulating Reddish Purple Color.

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.

Fine-mapping of a major QTL (Fwr1) for fusarium wilt resistance in radish.

KEY MESSAGE: 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.

Genetic and physiological analyses of root cracking in radish (Raphanus sativus L.).

KEY MESSAGE: 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.

A Turnip Mosaic Virus Determinant of Systemic Necrosis in Nicotiana benthamiana and a Novel Resistance-Breaking Determinant in Chinese Cabbage Identified from Chimeric Infectious Clones.

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.

Sequence Variations Among 17 New Radish Isolates of Turnip mosaic virus Showing Differential Pathogenicity and Infectivity in Nicotiana benthamiana, Brassica rapa, and Raphanus sativus.

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.

Isolation and characterization of fusarium wilt resistance gene analogs in radish.

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.

Integrated analysis of leaf morphological and color traits in different populations of Chinese cabbage (Brassica rapa ssp. pekinensis).

KEY MESSAGE: 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, J&U, 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.

Genome-Wide Analysis and Characterization of Aux/IAA Family Genes in Brassica rapa.

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 Genebank 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.

Quantitative Trait Loci for Morphological Traits and their Association with Functional Genes in Raphanus sativus.

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.