Yul-Kyun Ahn

OsWRKY30 is a transcription activator that enhances rice resistance to the Xanthomonas oryzae pathovar oryzae

The WRKY family proteins are transcription factors involved in the regulation of plant defense response pathways. In our present study, we characterized a rice WRKY gene, OsWRKY30 that had been previously isolated through systematic expression analysis of OsWRKYs during defense responses. The endogenous expression of OsWRKY30 was found to be abolished in salicylic acid (SA)-nonaccumulating NahG transgenic rice. OsWRKY30 activates transcription in maize protoplasts and its N-terminus retains transcription activation ability in yeast. OsWRKY30-GFP fusion proteins were found to localize in the nuclei of maize protoplasts. OsWRKY30 binds to the consensus W-box motif in yeast. Overexpression of OsWRKY30 in rice upregulates an SA-responsive gene OsWRKY45, and enhances disease resistance to the Xanthomonas oryzae pathovar oryzae. These results suggest that OsWRKY30 functions as a positive regulator of rice disease resistance via an SA signaling pathway.

Resequencing of Capsicum annuum parental lines (YCM334 and Taean) for the genetic analysis of bacterial wilt resistance

BackgroundBacterial wilt (BW) is a widespread plant disease that affects a broad range of dicot and monocot hosts and is particularly harmful for solanaceous plants, such as pepper, tomato, and eggplant. The pathogen responsible for BW is the soil-borne bacterium, Ralstonia solanacearum, which can adapt to diverse temperature conditions and is found in climates ranging from tropical to temperate. Resistance to BW has been detected in some pepper plant lines; however, the genomic loci and alleles that mediate this are poorly studied in this species.ResultsWe resequenced the pepper cultivars YCM344 and Taean, which are parental recombinant inbred lines (RIL) that display differential resistance phenotypes against BW, with YCM344 being highly resistant to infection with this pathogen. We identified novel single nucleotide polymorphisms (SNPs) and insertions/deletions (Indels) that are only present in both parental lines, as compared to the reference genome and further determined variations that distinguish these two cultivars from one another. We then identified potentially informative SNPs that were found in genes related to those that have been previously associated with disease resistance, such as the R genes and stress response genes. Moreover, via comparative analysis, we identified SNPs located in genomic regions that have homology to known resistance genes in the tomato genomes.ConclusionsFrom our SNP profiling in both parental lines, we could identify SNPs that are potentially responsible for BW resistance, and practically, these may be used as markers for assisted breeding schemes using these populations. We predict that our analyses will be valuable for both better understanding the YCM334/Taean-derived populations, as well as for enhancing our knowledge of critical SNPs present in the pepper genome.

Development of an Efficient Agrobacterium- Mediated Transformation System and Production of Herbicide-Resistant Transgenic Plants in Garlic (Allium sativum L.)

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.

Construction of a draft reference transcripts of onion (Allium cepa) using long-read sequencing

To obtain intact and full-length RNA transcripts of onion (Allium cepa), long-read sequencing technology was first applied. Total RNAs extracted from four tissues; flowers, leaves, bulbs and roots, of red–purple and yellow-colored onions (A. cepa) were sequenced using long-read sequencing (RSII platform, P4-C2 chemistry). The 99,247 polished high-quality isoforms were produced by sequence correction processes of consensus calling, quality filtering, orientation verification, misread-nucleotide correction and dot-matrix view. The dot-matrix view was subsequently used to remove artificial inverted repeats (IRs), and resultantly 421 IRs were removed. The remaining 98,826 isoforms were condensed to 35,505 through the removal process of redundant isoforms. To assess the completeness of the 35,505 isoforms, the ratio of full-length isoforms, short-read mapping to the isoforms, and differentially expressed genes among the four tissues were analyzed along with the gene ontology across the tissues. As a result, the 35,505 isoforms were verified as a collection of isoforms with high completeness, and designated as draft reference transcripts (DRTs, ver 1.0) constructed by long-read sequencing.

Plant growth and fruit quality as affected by canopy locations in ‘Shiranuhi’ mandarin

Fruit quality (soluble solids and acidity) and growth properties of fruiting branches in different canopy positions (upper, middle, and lower) of ‘Shiranuhi’ mandarin [(C. unshiu × C. sinensis) × C. reticulate] trees were determined during fruit maturation. Length, diameter, and number of leaves per fruiting branch increased in order from the upper, through the middle, to the lower parts of the canopy. Fruit diameter, fruit weight, pulp weight and pulp thickness were also highest in the upper part. Transverse sections of stem end protrusions in fruit from the upper, middle, and lower parts of the tree canopy were 37.3 ± 2.3 mm, 33.2 ± 0.2 mm and 32.1 ± 0.4 mm, respectively. Soluble solid content increased during fruit maturation at all locations within the canopy and was highest in the upper part. Likewise, acid content decreased with fruit maturity and was lowest in the lower part. These results suggested that harvesting be staggered starting with fruit from the upper part of the canopy as part of crop management in ‘Shiranuhi’ mandarin for direct effect on fruit quality.

Development of a high-throughput SNP marker set by transcriptome sequencing to accelerate genetic background selection in Brassica rapa

Among the molecular markers used today, single nucleotide polymorphisms (SNP) are the most common type used in genetic diversity analysis due to their abundance. To develop a high-throughput SNP marker set to accelerate genetic background selection in Brassica rapa breeding, we sequenced the transcriptomes of 20 Chinese cabbage accessions representing diversity in traits such as head type, maturity, inner leaf color, and disease resistance. We identified 13,976 SSRs and 380,198 SNPs by aligning their contigs. We chose 189 SNPs that covered the entire B. rapa genome through a filtering process based on criteria such as depth, level of polymorphism, segregation ratio, lack of adjacent SNPs, copy number, and PIC value. To validate the SNP marker set, we genotyped 23 Chinese cabbage accessions and constructed a phylogenetic tree. The results showed that the SNP genotyping data could distinguish the Chinese cabbage accessions according to their phenotypic variations. The 23 accessions were classified into two groups that were characterized by phenotypic traits, especially head type and maturity. In conclusion, the selected SNP marker set is a reliable breeding tool for distribution analysis or selection of different Chinese cabbage accessions and may be applicable for rapid genetic background selection of Chinese cabbages for breeding.

Comparative analysis of disease-linked single nucleotide polymorphic markers from Brassica rapa for their applicability to Brassica oleracea.

Numerous studies using single nucleotide polymorphisms (SNPs) have been conducted in humans, and other animals, and in major crops, including rice, soybean, and Chinese cabbage. However, the number of SNP studies in cabbage is limited. In this present study, we evaluated whether 7,645 SNPs previously identified as molecular markers linked to disease resistance in the Brassica rapa genome could be applied to B. oleracea. In a BLAST analysis using the SNP sequences of B. rapa and B. oleracea genomic sequence data registered in the NCBI database, 256 genes for which SNPs had been identified in B. rapa were found in B. oleracea. These genes were classified into three functional groups: molecular function (64 genes), biological process (96 genes), and cellular component (96 genes). A total of 693 SNP markers, including 145 SNP markers [BRH—developed from the B. rapa genome for high-resolution melt (HRM) analysis], 425 SNP markers (BRP—based on the B. rapa genome that could be applied to B. oleracea), and 123 new SNP markers (BRS—derived from BRP and designed for HRM analysis), were investigated for their ability to amplify sequences from cabbage genomic DNA. In total, 425 of the SNP markers (BRP-based on B. rapa genome), selected from 7,645 SNPs, were successfully applied to B. oleracea. Using PCR, 108 of 145 BRH (74.5%), 415 of 425 BRP (97.6%), and 118 of 123 BRS (95.9%) showed amplification, suggesting that it is possible to apply SNP markers developed based on the B. rapa genome to B. oleracea. These results provide valuable information that can be utilized in cabbage genetics and breeding programs using molecular markers derived from other Brassica species.

Watermelon (Citrullus lanatus) late-embryogenesis abundant group 3 protein, ClLEA3-1, responds to diverse abiotic stresses

Plants are exposed to various environmental stresses such as drought, cold, and high salinity during their sessile lifetime. The Late-Embryogenesis Abundant (LEA) proteins are involved in protection against different environmental stresses. LEA proteins have been classified into 7 groups based on sequence similarity and conserved domains. Among these groups, the functions of only group 1, 2 and 3 genes in regulating abiotic stress tolerance have been investigated. The productivity of watermelon (Citrullus lanatus), the most economically important cucurbitaceous crop, is affected by environmental stresses, but the functions of environmental stress-related genes in watermelon have not been reported yet. Therefore, abiotic and biotic stress-related research on watermelon is necessary. In this study, we selected Cl017745 (ClLEA3-1) as a representative group 3 LEA protein based on the watermelon database (http://www.icugi.org) and reported its expression patterns in response to diverse stresses and ABA treatment. Tissue-specific expression pattern of ClLEA3-1 was induced by dehydration, low-temperature, high-salinity, and abscisic acid (ABA) treatment, but expression of ClLEA3-1 was not increased by inoculation with a pathogen. Subcellular localization analysis showed that ClLEA3-1 was a cytosolic protein. Therefore, we recommend that ClLEA3-1 (Cl017745) can be used as an abiotic stress marker gene in watermelon.

Molecular marker information from de novo assembled transcriptomes of chilli pepper ( Capsicum annuum L.) varieties based on next-generation sequencing technology

Next-generation sequencing technique has been known as a useful tool for de novo transcriptome assembly, functional annotation of genes and identification of molecular markers. This study was carried out to mine molecular markers from de novo assembled transcriptomes of four chilli pepper varieties, the highly pungent ‘Saengryeg 211’ and non-pungent ‘Saengryeg 213’ and variably pigmented ‘Mandarin’ and ‘Blackcluster’. Pyrosequencing of the complementary DNA library resulted in 361,671, 274,269, 279,221, and 316,357 raw reads, which were assembled in 23,607, 19,894, 18,340 and 20,357 contigs, for the four varieties, respectively. Detailed sequence variant analysis identified numerous potential single-nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) for all the varieties for which the primers were designed. The transcriptome information and SNP/SSR markers generated in this study provide valuable resources for high-density molecular genetic mapping in chilli pepper and Quantitative trait loci analysis related to fruit qualities. These markers for pepper will be highly valuable for marker-assisted breeding and other genetic studies.

Newly developed SNP markers related to genes for leaf morphological traits and disease resistance in Chinese cabbage ( Brassica rapa L. ssp. pekinensis )

Based on the sequences of Brassica rapa ssp. pekinensis, we carried out a single-nucleotide polymorphism (SNP) study for genes controlling disease resistance (clubroot, turnip mosaic virus and soft rot) and leaf traits in B. rapa leafy genotypes ‘VC40’ and ‘SR5’. In total, 7645 SNP markers were obtained based on the annotation of a B. rapa database for disease resistance. Among these 7645 SNP markers, 141 were related to 125 genes linked to leaf traits. From these 141 SNP markers, 63 were screened and High Resolution Melt (HRM) primers were designed for genomic studies. A total of 20 polymorphic SNP primers were finally obtained. Eventually, these markers will be further used for the detection of quantitative trait loci and mapping studies.