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.

Comparative Transcriptome-Based Mining of Senescence-Related MADS, NAC, and WRKY Transcription Factors in the Rapid-Senescence Line DLS-91 of Brassica rapa.

Leaf senescence is a developmental process induced by various molecular and environmental stimuli that may affect crop yield. The dark-induced leaf senescence-91 (DLS-91) plants displayed rapid leaf senescence, dramatically decreased chlorophyll contents, low photochemical efficiencies, and upregulation of the senescence-associated marker gene BrSAG12-1. To understand DLS molecular mechanism, we examined transcriptomic changes in DLS-91 and control line DLS-42 following 0, 1, and 4 days of dark treatment (DDT) stages. We identified 501, 446, and 456 DEGs, of which 16.7%, 17.2%, and 14.4% encoded TFs, in samples from the three stages. qRT-PCR validation of 16 genes, namely, 7 MADS, 6 NAC, and 3 WRKY, suggested that BrAGL8-1, BrAGL15-1, and BrWRKY70-1 contribute to the rapid leaf senescence of DLS-91 before (0 DDT) and after (1 and 4 DDT) dark treatment, whereas BrNAC046-2, BrNAC029-2/BrNAP, and BrNAC092-1/ORE1 TFs may regulate this process at a later stage (4 DDT). In-silico analysis of cis-acting regulatory elements of BrAGL8-1, BrAGL42-1, BrNAC029-2, BrNAC092-1, and BrWRKY70-3 of B. rapa provides insight into the regulation of these genes. Our study has uncovered several AGL-MADS, WRKY, and NAC TFs potentially worthy of further study to understand the underlying mechanism of rapid DLS in DLS-91.

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.

Biochemical Analysis of the Role of Leucine-Rich Repeat Receptor-Like Kinases and the Carboxy-Terminus of Receptor Kinases in Regulating Kinase Activity in Arabidopsis thaliana and Brassica oleracea.

Protein post-translational modification by phosphorylation is essential for the activity and stability of proteins in higher plants and underlies their responses to diverse stimuli. There are more than 300 leucine-rich repeat receptor-like kinases (LRR-RLKs), a major group of receptor-like kinases (RLKs) that plays an important role in growth, development, and biotic stress responses in higher plants. To analyze auto- and transphosphorylation patterns and kinase activities in vitro, 43 full-length complementary DNA (cDNA) sequences were cloned from genes encoding LRR-RLKs. Autophosphorylation activity was found in the cytoplasmic domains (CDs) of 18 LRR-RLKs; 13 of these LRR-RLKs with autophosphorylation activity showed transphosphorylation in Escherichiacoli. BRI1-Associated Receptor Kinase (BAK1), which is critically involved in the brassinosteroid and plant innate immunity signal transduction pathways, showed strong auto- and transphosphorylation with multi-specific kinase activity within 2 h of induction of Brassica oleraceae BAK1-CD (BoBAK1-CD) in E. coli; moreover, the carboxy-terminus of LRR-RLKs regulated phosphorylation and kinase activity in Arabidopsis thaliana and vegetative crops.

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.

Genomic and Post-Translational Modification Analysis of Leucine-Rich-Repeat Receptor-Like Kinases in Brassica rapa.

Among several receptor-like kinases (RLKs), leucine-rich-repeat receptor-like kinases (LRR-RLKs) are a major group of genes that play crucial roles in growth, development and stress responses in plant systems. Given that they have several functional roles, it is important to investigate their roles in Brassica rapa. In the present study, 303 LRR-RLKs were identified in the genome of B. rapa and comparative phylogenetic analysis of 1213 combined LRR-RLKs of B. rapa, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa helped us to categorize the gene family into 15 subfamilies based on their sequence and structural similarities. The chromosome localizations of 293 genes allowed the prediction of duplicates, and motif conservation and intron/exon patterns showed differences among the B. rapa LRR-RLK (BrLRR-RLK) genes. Additionally, computational function annotation and expression analysis was used to predict their possible functional roles in the plant system. Biochemical results for 11 selected genes showed variations in phosphorylation activity. Interestingly, BrBAK1 showed strong auto-phosphorylation and trans-phosphorylation on its tyrosine and threonine residues compared with AtBAK1 in previous studies. The AtBAK1 receptor kinase is involved in plant growth and development, plant innate immunity, and programmed cell death, and our results suggest that BrBAK1 might also be involved in the same functions. Another interesting result was that BrBAK1, BrBRI1, BrPEPR1 and BrPEPR2 showed activity with both anti-phosphotyrosine and anti-phosphothreonine antibodies, indicating that they might have dual-specificity kinase activity. This study provides comprehensive results for the BrLRR-RLKs, revealing expansion of the gene family through gene duplications, structural similarities and variations among the genes, and potential functional roles according to gene ontology, transcriptome profiling and biochemical analysis.