Efficient infiltration of water through cell membranes is arbitrated by a family of transmembrane water channels called aquaporins (AQPs). Aquaporin belongs to a highly conserved group of membrane proteins called major intrinsic proteins that facilitate the transport of water and a variety of low molecular weight solutes across biological membranes,which is essential for plants to survive in stress conditions. This study identified 59 BrAQP genes from B. rapa database and Br135K microarray dataset, which was formed by applying low-temperature stresses to contrasting Chinese cabbage two inbreed lines, Chiifu and Kenshin. Based on phylogenetic analyses of BrAQPs revealed four distinct subfamilies, such as plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP) with aquaporin of Tomato and Arabidopsis thaliana. All BrAQP genes were firstly examined through homology study with existing biotic and abiotic stress resistance-related aquaporin genes of other plant species and found a high degree of homology. We selected PIP subfamily genes for expression analysis based on microarray data with high and differential transcript abundance levels and homology study with stress related aquaporin genes of other plant species. In our study, we characterized all B. rapa aquaporin genes and understanding the BrPIP subfamily gene function in plants under various environmental stimuli, the expressions of BrPIP genes under various abiotic stress conditions including cold, drought, salinity, water logging, ABA treatment and Fusarium oxysporum f. sp. Conglutinans infection were investigated by a quantitative real-time reverse transcription-PCR analysis. In our expression analysis, 4 BrPIP genes showed responsive expression against F. oxysporum f. sp. Conglutinans infection. The selected genes showed an organ-specific expression, and 12 out of 22 BrPIP genes were differentially expressed in Chiifu compared to Kenshin under cold stresses. Only 7 genes showed up regulation under drought stress and incase of salt stress 17 BrPIP genes were more responsiveness. Additionally, 18 BrPIP genes were up regulated by ABA treatment and all BrPIP genes showed down regulation under water logging stress. Together with expression and bioinformatic analyses, our results provides novel basis to allocate the stress-related biological function to each PIP gene.

Anthocyanins are responsible for vivid colors of flowers, fruits and vegetative tissues and biosynthesis of it is primarily controlled by several structural and regulatory genes. The regulatory mechanism of this pathway is still unknown. This study identified 19 transcription factors of Brassica rapa and investigated their regulatory function in anthocyanin biosynthesis pathway genes and cold and/or freezing tolerance in B. rapa. Expression analysis of these genes in the pigmented and non-pigmented portion of leaves of different lines of B. rapa revealed that BrMYB2-2 and BrTT8 showed responses contrasting with anthocyanin accumulation and cold stress. Sequences of these genes were analyzed and compared with similar gene sequences from other species and a high degree of homology with their respective functions was found. Co-regulated cis -elements were found in promoters of BrPAL1, BrCHS, BrF3H1, BrF3’H1, BrFLS, BrBAN, BrDFR8, BrANS1, and BrMYB2-2 and BrTT8 had binding sites of the promoters of those structural genes. Thus, the above results suggest the association of BrMYB2-2 and BrTT8 with regulation of anthocyanin biosynthesis pathway genes and cold and freezing stress tolerance and might be useful resources for development of cold resistant Brassica crops with desirable colors as well.

The TIFY family is composed of a plant-specific group of genes with diversity of functions. This family represents four subfamily of proteins viz. ZML, TIFY, PPD and JASMONATE ZIM-domain (JAZ) proteins. TIFY proteins especially, JAZ proteins have been reported to perform different biological processes, such as developmental and stresses and hormone responses in Arabidopsis and rice. However, there is no information about this family genes in Brassicaceae. This study identifies 36 TIFY genes in Brassica rapa, an economically important crop species from this family. An extensive in silico analysis through phylogenetic grouping, protein motif organization and intron-exon distribution also confirmed 4 subfamilies of BrTIFY proteins. Out of 35 BrTIFY genes, we identified 21 under JAZ subfamily besides 7 TIFY, 6 ZML and 2 PPD. An extensive expression profiling of 21 BrTIFY JAZs both in tissues and organs of B. rapa revealed differential expression patterns. Almost all the BrTIFY JAZs predominantly expressed in leaves and flower buds. Besides, in a flower stage specific expression analysis we observed 14 BrTIFY JAZs with constitutive expression patterns. This indicates BrTIFY proteins have a strong involvement in the development of B. rapa flowers. Our protein interaction study also reveals the strong association of these proteins with the fertility and defense processes of B. rapa. To elucidate the stress responsiveness of BrTIFY genes, we analyzed the low temperature-treated whole-genome microarray data set and found almost all the BrTIFY JAZs were having variable transcript abundance in two contrasting inbred lines of B. rapa. Subsequently, all 21 BrTIFY JAZs were validated in response to cold stress in the same two lines via qPCR, where 9 genes were found to show up- regulation. And, a high and differential qPCR expression pattern of all the BrTIFY JAZs was also recorded against JA. Additionally, BrTIFY JAZs were tested against salt, drought, Fusarium, ABA and SA treatments and a considerable number of genes were found to be induced. The extensive annotation and transcriptome profiling reported in this study will be useful for understanding the involvement of TIFY genes in stress resistance and different developmental functions, which ultimately provides the basis for functional characterization and exploitation of the candidate genes for genetic engineering of B. rapa.

Blackleg disease caused by Leptosphaeria maculans, is the most devastating disease of Brassica germplam worldwide that causes million tonnes of crop losses per year throughout the world. To date, a total of 12 race-specific resistance genes of Brassica napus to L. maculans have been reported but linkage mapping analysis reveals that all of those loci are located in A genome i.e., in B. rapa chromosomes. B. oleracea has high ancestral synteny with B. rapa through their evolution. We believe that presence of qualitative resistance is possible in B. oleracea germplasm. The present study was therefore planned to find out any race-specific qualitative resistance gene present in C genome of B. oleracea. A total of 16 microsatellite markers were used which are linked to seven different Rlm and Lep genes of B. napus to screen 32 inbred lines of cabbage. Primers were designed based on homology assessment in corresponding nucleotide sequence available in Bolbase (a B. oleracea genome database, http://www.ocri-genomics.org/bolbase/index.html), located in B. oleracea scaffolds/chromosomes. Out of 16 SSR markers, 13 were found polymorphic which indicates possible existence of resistant genes in cabbage lines. The inbred lines are then assessed against two L. maculans stains with known avirulent genes. Some inbred lines were hypersensitive against gene-specific virulent strains of L. maculans that confirmed existence of Rlm1, Rlm2, Rlm4, LepR3 and LepR4 in the cabbage lines. In this way we were able to select out resistant and susceptible lines against each resistant gene. The gene-specific polymorphic SSR marker regions were cloned and sequenced and candidate SNPs were identified for confirmation of their functionality.

Heterosis is very important for breeding hybrid cultivars and is intensively used to increase the productivity of crop plants. But the molecular basis of heterosis is still unrevealed to the scientists. This study selected 51 heterosis associated genes of Arabidopsis of different family on the basis of their high differential expression in a hybrid compared to its midparent value and identified their orthologues in Brassica oleracea. Then the selected B. oleracea genes were characterized based on their structural properties, recognized functions and expression patterns in a cabbage hybrid progeny (Cabbage-36) of crosses between Cabbage-34 and Cabbage-35 accessions. Among these genes, a good number were found to express highly in the hybrid then the midparent value and better parent in some cases. Moreover, these highly expressed genes are mostly related to the yield contributing characters. Cotyledon and young leaf sizes of these three genotypes also well correlated with gene expression. Thus, it can be said that, the identified genes might be associated with the mechanism of heterosis of B. oleracea hybrid and provide a foundation for the exploration of gene regulatory networks associated with the specification of the phenomenon heterosis in the plant life cycle. Subsequently, these genes would be useful resources for molecular hybrid breeding in Brassica crops as well.

Flavonoids are divided into several structural classes, including anthocyanins, which provide flower and leaf colors and other derivatives with diverse roles in plant development and interactions with the environment. This study characterized four Anthocyanidin Synthase (ANS) genes of Brassica rapa, a structural gene of anthocyanin biosynthetic pathway, and investigated their association with cold and freezing tolerance in B. rapa. Sequences of these genes were analyzed and compared with similar types of gene sequences of other species and found a high degree of homology with their respective functions. In the organ specific expression analysis, these genes showed expression only in the colored portion of leaves of different lines of B. rapa. On the other hand, BrANS genes also showed differential expression with certain time course of cold stress treatment in B. rapa. Thus, the above results suggest probable association of these genes with anthocyanin biosynthesis and cold and freezing tolerance and might be useful resources for developing cold resistant Brassica crops with desirable colors as well. The present work may help explore the molecular mechanism that regulates anthocyanin biosynthesis and its response to abiotic stress at the transcriptional level in plants.

Mitochondria are essential organelles of eukaryotic cells and plant cells contain varying numbers of mitochondrial genome sequences. Sizes and shapes of mitochondria differ within a tissue or in the same cells. Previously sequenced complete mitochondrial genome (NC_016118) of Brassica oleracea size was 360,271 bp, where segmental duplication (repeat block) was 141,800 bp. In this study, we resequenced this whole mitochondrial genome by using WGS (whole genome sequencing) and assembled organelles genome method (unpublished). Newly sequenced mitochondrial genome length was 219,975 bp and circle form. A new sequence segment of approximately 4,800 bp was obtained compared to the previous genome sequence without any large repeat block. Newly obtained mitochondria genome sequence was compared with recently reported mitochondria genome sequences of various species (B. oleracea, B. juncea, B. rapa, B. napus and B. carinata) and subspecies (cabbage, cauliflower, brussels sprouts, kohlrabi, broccoli and kale) by PCR using primers specifying different region of genome sequences. PCR analysis results have also confirmed the variation between previous and newly sequenced mitochondrial genome circles form. Thus, the results suggest new B. oleracea mitotype, including evolutionary events such as inheritance, rearrangement, genome compaction, and diversity

MADS-box transcription factor (TF), primarily involved in the floral organ specification with other several aspects of plant growth and development. Whole genome survey of B. rapa revealed 167 MADS-box genes and categorized into MIKCc, MIKC*, Mα, Mβ and Mγ groups based on phylogeny, protein motif structure and exon-intron organizations. MIKCc group belongs 89 genes, which is the highest in number than in any other crops till date. The MIKCc group has further classified into 13 sub-families. In case of chromosomal localization, remarkably 57 MIKCc type MADS-box genes were found in the duplicated segments of B. rapa genome, whereas only 4 M-type genes have resulted from tandem duplications. Besides floral and vegetative tissue expression we also identified MADS-box genes with their male and female gametophyte specific expression in different stages of flower bud development. Furthermore, from a low temperature treated whole genome microarray data set 19 BrMADS genes were found to show variable transcript abundance in two contrasting double haploid lines of B. rapa. Subsequently, the responsive genes were investigated under three abiotic stresses where they showed differential and corresponsive expression patterns. An extensive annotation and transcriptome profiling undertaken in this study might be useful for understanding the involvement of MADS-box genes in stress resistance besides their growth and developmental functions, which ultimately will provide the basis for functional characterization and exploitation of the candidate genes in the genetic engineering study of B. rapa

Flavonoids including anthocyanins provide flower and leaf colors and other derivatives that play diverse roles in plant development and interactions with the environment and dihydroflavonol 4-reductase (DFR) is part of an important step in the flavonoid biosynthesis pathway of anthocyanins. This study characterized 12 DFR genes of Brassica rapa and investigated their association with anthocyanin coloration, cold and freezing tolerance in several genotypes of B. rapa. Sequences of these genes were analyzed and compared with DFR gene sequences from other species and a high degree of homology was found. Constitutive expression of them in several pigmented and non-pigmented lines of B. rapa showed a correlation with anthocyanin accumulation only for BrDFR8 and 9. Conversely, BrDFR genes also showed responses to cold and freezing stress treatment in B. rapa. BrDFRs were also shown to be regulated by two transcription factors, BrMYB2-2 and BrTT8, contrasting with anthocyanin accumulation and cold and freezing stress. Thus, the above results suggest the association of these genes with anthocyanin biosynthesis and cold and freezing stress tolerance and might be useful resources for development cold and/or freezing resistant Brassica crops with desirable colors as well. The findings presented here may also help explore the molecular mechanism that regulates anthocyanin biosynthesis and its response to abiotic stress at the transcriptional level in plants.

Calcium-binding proteins, like calcineurin B-like (CBL) proteins, represent important roles in plant calcium signaling. Calcium signals mediate a multitude of plant responses to external stimuli and regulate a wide range of physiological processes including pathogens, abiotic stresses and hormones. These proteins form a complex network with their target kinases being the CBL-interacting protein kinases (CIPKs). CBL genes play vital roles in multiple abiotic stress response pathways whereas some of these are more specifically involved in mediating ABA signaling. In this study, we collected 17 CBL genes designated as B. rapa CBL (BrCBL) from the Brassica database and analyzed the sequences. In comparison analysis, these genes showed high homology with published CBL genes of other species. An organ specific expression of these genes was observed in different organs of chinese cabbage plants. In addition, six BrCBL genes showed responsive expression after cold and drought stress treatments at certain time courses. All these data revealed that these CBL genes might be useful resources in developing abiotic stresses resistance Brassica.