Molecular markers, such as PCR-based and SNP-based markers, are extremely useful for plant genetics and crop breeding. Marker-assisted selection (MAS) has been widely applied in plant breeding to improve crop yield, quality, and tolerance to biotic and abiotic stresses. To develop gene-based (or -specific) molecular markers, three different approaches have been used in Brassica species: Known-gene-based, RNA seq/Exon-based and RNA seq/Intron-based molecular marker development for several years. Using these techniques, molecular markers have been developed to identify flowering time, anthocyanin accumuation and abiotic stresses in B. rapa and B. oleracea. Markers were distributed in exons as well as introns, and coding sequences and untranslated regions (UTRs). All markers developed have been transformed into SNP marker after HRM confirmation. I will discuss efficiency, accuracy, and potential problems and contribution of these markers for Brassica breeding.

Chinese cabbage is one of most important vegetable crop in Eastern Asian countries including Korea. Because Chinese cabbage is a leafy vegetable, genetic research with respect to the leaf morphology is important. In this research, we have used two inbred lines of Chinese cabbages (Kenshin and RCBr) and generated recombinant lines having various leaf morphology. In F2 population of Kenshin X RCBr, leaf shape showed very dramatic variations with normal distribution in terms of leaf size, petiole length, leaf margin and etc. Microarray with a 135K DNA chip (version 3) integrated 2 sets of total Chinese cabbage genes. Biological process of candidate genes was classified into transcription factor, genes encoding kinase activity protein, protein folding related genes, oxidation-reduction process genes. Putative leaf-morphology-related genes were 142 that are involed in phytohormone pathway genes, cell proliferation & cell elongation related genes and genes controlling leaf morphogenesis etc. These genes are further classified to phytohormone signaling-associated genes (SAUR44, PIN2, CPK6, RDUF2), leaf development regulating genes (DWF4, CUC2, TCP15, BLH4, NGA4), and cell division and cell growth related genes (ILP1, TCTP, EMB1027).

To select genes associated with the high-temperature tolerance from Brassica, two transcriptomic analyses have been used: microarray and RNA Seq. Using two contrasting inbred lines of B. rapa, Chiifu and Kenshin, version 3 microarray (135 K microarray) was conducted to RNA samples extracted from series of 45℃-treated leaves and 29 genes were selected for genomic DNA cloning of cabbage. Of 29 genes, 8 genes contain 40 SNPs, 11 SSRs and 23 In-Del markers that distinguish high-temperature tolerant and susceptible cabbages, BN1 and BN2. These 8 genes include a unknown gene, AP2, SMP, FBD, SKP2B, IAA16, HSP21 and OLI2-2. We also selected 16 cabbage genes from RNA Seq analysis using two inbred lines, BN1 and BN2: 5 genes for BN1-high expression, 5 genes for BN1-specific expression, 5 genes for BN2-specific expression, and BoCaMB. Using RNA sequences, genomic DNAs corresponding to 16 genes have been clones and analyzed to find out molecular markers. Markers were further transformed into PCR-based marker and confirmed with additional cabbage genetic lines. We are currently transforming PCR-makers into SNP markers. To examine function of high-temperature tolerant genes, we also transformed 5 genes into Arabidopsis plants. We will describe detailed methods and results in a poster. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (the Next-Generation Genomics Center No. PJ009085), Rural Development Administration, Republic of Korea]

Shaggy-like kinases (SKs), also known as Glycogen synthase kinase 3 (GSK3) proteins, play many important roles in cellular signaling in animals, fungi and amoebae. In particular, SKs participate in key developmental signaling pathways and also regulate the cytoskeleton. SKs -encoding genes are also present in all land plants and in algae, raising questions about possible ancestral functions in eukaryotes. Unlike in animals and Dictyostelium, land plant SKs are encoded by relatively large multi-gene families whose members share high sequence similarity. Along with the studied 10 ASKs (Arabidopsis shaggy-like kinases) indicate that plant SK proteins are actively implicated in hormonal signalling networks during development as well as in biotic and abiotic stress responses. In this study, 18 BrSKs are identified from Chinese cabbage, and they are classified into four groups according to the classification of Arabidopsis. The characterization, classification, gene structure and phylogenetic construction of BrSK proteins are performed. Distribution mapping shows that BrSKs are absented in A02 and A10 chromosome. 8 orthologous gene pairs are shared by Chinese cabbage and Arabidopsis. The expression patterns of BrSK genes exhibit differences in five tissues based on RNA-seq data in public data base. Specially, BrSKβ-1 and BrSKβ-2 show floral buds specifically expressed, which indicate that BrSKβ may play a key role during flower or pollen development. We deomonatrated that suppresion of Arabdiopsis orthology of BrSKβ impaired the late pollen in Arabidopsis plants. Taken together, our analyses provided insights into the characterization of the BrSK genes in Chinese cabbage, providing foundation of further functional studies of those genes. [This work was supported by a grant from the Next-Generation BioGreen 21 Program (the Next-Generation Genomics Center No. PJ008118), Rural Development Administration, Republic of Korea]

Numerous environmental stresses, such as abiotic and biotic stresses, cause significant yield loss in crops and can significantly affect their development. Un the field conditions, crops are exposed to a variety of concurrent stresses. Combined high temperature and linked diseases can cause considerable damage that eventually leads to crop death. Hence, this study was conducted to characterize the genes encoding the nucleotide-binding site (NBS) motif obtained from transcriptome profiles of two cabbage genotypes with contrasting responses to heat stress. We selected 80 up-regulated genes form a total of 264 loci, among which 17 were confirmed to be complete and incomplete members of the TIR-NBS-LRR (TNL) class families, and another identified as a NFYA-HAP2 family member. Expression analysis using qRT-PCR revealed that 8 genes showed significant responses to heat shock treatment and F. oxysporum infection. Additionally, in the commercial B. oleracea cultivars with resistance to F. oxysporum, Bol007132, Bol016084, and Bol030522 genes showed dramatically higher expression levels in the F. oxysporum resistant line than the intermediate and susceptible lines. The results of this study may facilitate the identification and development of molecular markers based on multiple stress resistance genes related to heat and fungal stress under field conditions in B. oleracea.

Cold stress, which includes chilling (<20℃) and/or freezing (<0℃) temperatures, adversely affects the growth and development of plants and significantly constraints the spatial distribution of plants and agricultural productivity. Cold signal in plants is transmitted to activate C-repeat/drought-responsive elements-binding factor (CBF)-dependent and independent transcriptional pathway. In Arabidopsis, cold-regulated genes have been estimated to constitute ~4% to 20% of the genome. Chinese cabbage (Brassica rapa ssp. pekinensis), like Arabidopsis, is a member of the Cruciferae family. With expectation that Chinese cabbage has similar cold-responding process as Arabidopsis, transcriptome profiles were examined in two comparable DH lines, Chiifu and Kenshin, using 24K microarray and expression of some of genes was analyzed by RT-PCR. Hundreds genes showed over two fold change upon freezing treatment, but only four genes specific for each line. Most of examined CBF-dependent and -independent pathway related genes have a similar expression patterns between Chiifu and Kenshin, except BrICE1 (inducer of CBF expression 1), MYB15, BrRAP2.1 and BrRAP2.6 (ethylene-responsive transcription factor 2.6). The BrICEL (inducer of CBF expression 1 like) showed specifically expression in Kenshin. The expression levels of vernalization related genes (BrRTV1, BrVRN5, BrVIN3L, VIP3 and VIP5) showed no difference between Chiifu and Kenshin and did not response to freezing treatment in our experiment. We will discuss more detailed expression data on poster.

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.

Abiotic stresses such as extreme temperatures frequently limit the plant growth and productivity of major crop species. Two Chinese cabbage DH lines that have different geographic origins, in that Chiifu is from temperate regions, while Kenshin is from subtropical and tropical regions have been expected to show the specific response to high or low temperature. To find the temperature response genes between Chiifu and Kenshin, we analyzed transcriptomic profiling from light-chilling (6h at 4°C) and high temperature (6h at 38°C) treated plants using the KBGP-24K chip. Distribution of genes classified by PI (probe intensity) values showed remarkable difference between Chiifu and Kenshin. The number of genes up- and down-regulated gens by both temperatures were 135 and 79 genes, respectively. These genes may be temperature stress-related genes. Genes involved in the response to stress were changed by light-chilling stress. Chiifu specifically up-regulated genes upon light chilling-stress belong to cold acclimation proteins, calcium binding proteins, cell wall biogenesis proteins and lipoxygenase. On the other hand, Kenshin specifically up-regulated genes by heat-shock treatment include heat-shock proteins, phosphatases, protein folding and phosphorylation-associated ones. Further study on these specific genes function may provide insight to adaptation of Chinese cabbage and clue to develop molecular markers.