Vignesh Dhandapani

Genic microsatellite markers in Brassica rapa: development, characterization, mapping, and their utility in other cultivated and wild Brassica relatives.

Genic microsatellite markers, also known as functional markers, are preferred over anonymous markers as they reveal the variation in transcribed genes among individuals. In this study, we developed a total of 707 expressed sequence tag-derived simple sequence repeat markers (EST-SSRs) and used for development of a high-density integrated map using four individual mapping populations of B. rapa. This map contains a total of 1426 markers, consisting of 306 EST-SSRs, 153 intron polymorphic markers, 395 bacterial artificial chromosome-derived SSRs (BAC-SSRs), and 572 public SSRs and other markers covering a total distance of 1245.9 cM of the B. rapa genome. Analysis of allelic diversity in 24 B. rapa germplasm using 234 mapped EST-SSR markers showed amplification of 2 alleles by majority of EST-SSRs, although amplification of alleles ranging from 2 to 8 was found. Transferability analysis of 167 EST-SSRs in 35 species belonging to cultivated and wild brassica relatives showed 42.51% (Sysimprium leteum) to 100% (B. carinata, B. juncea, and B. napus) amplification. Our newly developed EST-SSRs and high-density linkage map based on highly transferable genic markers would facilitate the molecular mapping of quantitative trait loci and the positional cloning of specific genes, in addition to marker-assisted selection and comparative genomic studies of B. rapa with other related species.

The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases

Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes.

Quantitative Trait Loci Mapping in Brassica rapa Revealed the Structural and Functional Conservation of Genetic Loci Governing Morphological and Yield Component Traits in the A, B, and C Subgenomes of Brassica Species

Brassica rapa is an important crop species that produces vegetables, oilseed, and fodder. Although many studies reported quantitative trait loci (QTL) mapping, the genes governing most of its economically important traits are still unknown. In this study, we report QTL mapping for morphological and yield component traits in B. rapa and comparative map alignment between B. rapa, B. napus, B. juncea, and Arabidopsis thaliana to identify candidate genes and conserved QTL blocks between them. A total of 95 QTL were identified in different crucifer blocks of the B. rapa genome. Through synteny analysis with A. thaliana, B. rapa candidate genes and intronic and exonic single nucleotide polymorphisms in the parental lines were detected from whole genome resequenced data, a few of which were validated by mapping them to the QTL regions. Semi-quantitative reverse transcriptase PCR analysis showed differences in the expression levels of a few genes in parental lines. Comparative mapping identified five key major evolutionarily conserved crucifer blocks (R, J, F, E, and W) harbouring QTL for morphological and yield components traits between the A, B, and C subgenomes of B. rapa, B. juncea, and B. napus. The information of the identified candidate genes could be used for breeding B. rapa and other related Brassica species.

Identification of Potential microRNAs and Their Targets in Brassica rapa L.

MicroRNAs (miRNAs) are recently discovered, noncoding, small regulatory RNA molecules that negatively regulate gene expression. Although many miRNAs are identified and validated in many plant species, they remain largely unknown in Brassica rapa (AA 2n =, 20). B. rapa is an important Brassica crop with wide genetic and morphological diversity resulting in several subspecies that are largely grown for vegetables, oilseeds, and fodder crop production. In this study, we identified 186 miRNAs belonging to 55 families in B. rapa by using comparative genomics. The lengths of identified mature and pre-miRNAs ranged from 18 to 22 and 66 to 305 nucleotides, respectively. Comparison of 4 nucleotides revealed that uracil is the predominant base in the first position of B. rapa miRNA, suggesting that it plays an important role in miRNA-mediated gene regulation. Overall, adenine and guanine were predominant in mature miRNAs, while adenine and uracil were predominant in pre-miRNA sequences. One DNA sequence producing both sense and antisense mature miRNAs belonging to the BrMiR 399 family, which differs by 1 nucleotide at the, 20th position, was identified. In silico analyses, using previously established methods, predicted 66 miRNA target mRNAs for 33 miRNA families. The majority of the target genes were transcription factors that regulate plant growth and development, followed by a few target genes that are involved in fatty acid metabolism, glycolysis, biotic and abiotic stresses, and other cellular processes. Northern blot and qRT-PCR analyses of RNA samples prepared from different B. rapa tissues for 17 miRNA families revealed that miRNAs are differentially expressed both quantitatively and qualitatively in different tissues of B. rapa.

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.

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.

Identification of the BrRHP1 locus that confers resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis) and development of linked molecular markers.

Inheritance of resistance to downy mildew (Hyaloperonospora parasitica) in Chinese cabbage (Brassica rapa ssp. pekinensis) was studied using inbred parental lines RS1 and SS1 that display strong resistance and severe susceptibility, respectively. F1, F2, and BC1F1 populations were evaluated for their responses to downy mildew infection. Resistance to downy mildew was conditioned by a single dominant locus designated BrRHP1. A random amplified polymorphic DNA (RAPD) marker linked to BrRHP1 was identified using bulked segregant analysis and two molecular markers designated BrPERK15A and BrPERK15B were developed. BrPERK15B was polymorphic between the parental lines used to construct the reference linkage map of B. rapa, allowing the mapping of the BrRHP1 locus to the A1 linkage group. Using bacterial artificial chromosome clone sequences anchored to the A1 linkage group, six simple polymerase chain reaction (PCR) markers were developed for use in marker-assisted breeding of downy mildew resistance in Chinese cabbage. Four simple PCR markers flanking the BrRHP1 locus were shown to be collinear with the long-arm region of Arabidopsis chromosome 3. The two closely linked flanking markers delimit the BrRHP1 locus within a 2.2-Mb interval of this Arabidopsis syntenic region.

Development of EST database and transcriptome analysis in the leaves of Brassica rapa using a newly developed pipeline

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.Graphical abstract

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.

Development of a leafy Brassica rapa fixed line collection for genetic diversity and population structure analysis

Brassica rapa is an economically important crop with a wide range of morphologies. Developing a set of fixed lines and understanding their diversity has been challenging, but facilitates resource conservation. We investigated the genetic diversity and population structure of 238 fixed lines of leafy B. rapa with 45 new simple sequence repeat markers and 109 new NGS (next-generation sequencing)-generated single nucleotide polymorphism markers evenly distributed throughout the B. rapa genome. Phylogenetic analysis classified the vegetable fixed lines into four subgroups, with the three oil types forming a separate and relatively distant cluster. A model-based population structure analysis identified four subpopulations corresponding to geographical origins and morphological traits, and revealed extensive allelic admixture. In particular, the Chinese cabbage cluster was subdivided into three groups and showed considerable correlation with leaf- and heading-related traits (leaf and heading shape). The vegetable B. rapa fixed lines successfully developed in our study could be valuable materials for establishing a multinational Brassica rapa diversity resource. Understanding the genetic diversity and population structure could be useful for utilization of the representative genetic variation and further genomic analysis.