Jeong-Ho Kim

Transcriptome analysis of Capsicum annuum varieties Mandarin and Blackcluster: assembly, annotation and molecular marker discovery.

Next generation sequencing technologies have proven to be a rapid and cost-effective means to assemble and characterize gene content and identify molecular markers in various organisms. Pepper (Capsicum annuum L., Solanaceae) is a major staple vegetable crop, which is economically important and has worldwide distribution. High-throughput transcriptome profiling of two pepper cultivars, Mandarin and Blackcluster, using 454 GS-FLX pyrosequencing yielded 279,221 and 316,357 sequenced reads with a total 120.44 and 142.54Mb of sequence data (average read length of 431 and 450 nucleotides). These reads resulted from 17,525 and 16,341 isogroups and were assembled into 19,388 and 18,057 isotigs, and 22,217 and 13,153 singletons for both the cultivars, respectively. Assembled sequences were annotated functionally based on homology to genes in multiple public databases. Detailed sequence variant analysis identified a total of 9701 and 12,741 potential SNPs which eventually resulted in 1025 and 1059 genotype specific SNPs, for both the varieties, respectively, after examining SNP frequency distribution for each mapped unigenes. These markers for pepper will be highly valuable for marker-assisted breeding and other genetic studies.

De novo transcriptome assembly and novel microsatellite marker information in Capsicum annuum varieties Saengryeg 211 and Saengryeg 213

BackgroundPepper, Capsicum annuum L., Solanaceae, is a major staple economically important vegetable crop worldwide. Limited functional genomics resources and whole genome association studies could be substantially improved through the application of molecular approach for the characterization of gene content and identification of molecular markers. The massive parallel pyrosequencing of two pepper varieties, the highly pungent, Saengryeg 211, and the non-pungent, Saengryeg 213, including de novo transcriptome assembly, functional annotation, and in silico discovery of potential molecular markers is described. We performed 454 GS-FLX Titanium sequencing of polyA-selected and normalized cDNA libraries generated from a single pool of transcripts obtained from mature fruits of two pepper varieties.ResultsA single 454 pyrosequencing run generated 361,671 and 274,269 reads totaling 164.49 and 124.60xa0Mb of sequence data (average read length of 454 nucleotides), which assembled into 23,821 and 17,813 isotigs and 18,147 and 15,129 singletons for both varieties, respectively. These reads were organized into 20,352 and 15,781 isogroups’ for both varieties. Assembled sequences were functionally annotated based on homology to genes in multiple public databases and assigned with Gene Ontology (GO) terms. Sequence variants analyses identified a total of 3,766 and 2,431 potential (Simple Sequence Repeat) SSR motifs for microsatellite analysis for both varieties, where trinucleotide was the most common repeat unit (84%), followed by di (9.9%), hexa (4.1%) and pentanucleotide repeats (2.1%). GAA repeat (8.6%) was the most frequent repeat motif, followed by TGG (7.2%), TTC (6.5%), and CAG (6.2%).ConclusionsHigh-throughput transcriptome assembly, annotation and large scale of SSR marker discovery has been achieved using next generation sequencing (NGS) of two pepper varieties. These valuable informations for functional genomics resource shall help to further improve the pepper breeding efforts with respect to genetic linkage maps, QTL mapping and marker-assisted trait selection.

Identification and development of molecular markers linked to Phytophthora root rot resistance in pepper (Capsicum annuum L.)

Phytopthora root rot in pepper (C. annuum) is caused by Phytophthora capsici L., which exhibits a high level of pathogenic diversity. Resistance to this disease is conditioned by a number of quantitative trait loci. Pyramiding resistance alleles is desirable and could be simplified by the use of molecular markers tightly linked to the resistance genes. The purpose of this study was development of molecular markers linked to Phytophthora root rot resistance. An F8 recombinant inbred line (RIL) population derived from a cross between YCM334 and a susceptible cultivar ‘Tean’ was used in combination with bulk segregant analysis utilizing RAPD and conversion of AFLP markers linked to Phytophtora root rot resistance into sequence-characterized amplified region (SCAR) markers. In conversion: one marker was successfully converted into a co-dominant SCAR marker SA133_4 linked to the trait. In bulked segregant analysis (BSA): three RAPD primers (UBC484, 504, and 553) produced polymorphisms between DNA pools among 400 primers screened. Genetic linkage analysis showed that the SCAR and RAPD markers were located on chromosome 5 of pepper. Quantitative trait locus (QTL) analysis showed that the SA133_4 and UBC553 were linked to Phytophtora root rot resistance. These markers were correctly identified as resistant or susceptible in nine promising commercial pepper varieties. These markers will be beneficial for marker-assisted selection in pepper breeding.

Ectopic expression of Arabidopsis H+-pyrophosphatase AVP1 enhances drought resistance in bottle gourd (Lagenaria siceraria Standl.)

Bottle gourd (Lagenaria siceraria Standl.) has been used as a source of rootstock for grafting watermelon to improve its fruit quality. We report here the development of a bottle gourd with resistance to drought by ectopic expression of the ArabidopsisAVP1 gene that encodes a vacuolar H+-pyrophosphatase. The drought resistance of AVP1-expressing and wild-type plants was assessed by growing plants under drought conditions. After 12xa0days of water deprivation, both AVP1-expressing and wild-type plants demonstrated reduced growth. After 10xa0days of re-watering, wild-type plants showed minimal growth while the AVP1-expressing plants resumed rapid growth. Further, AVP1-expressing plants displayed longer primary roots and more robust root systems than wild-type plants.

Complete genome sequencing and analysis of Capsicum annuum varieties

Peppers (Capsicum spp.), economically important crops of the nightshade family, are used as vegetables, spices, and medicines. Bacterial wilt (BW), one of the most devastating bacterial plant diseases, causes heavy losses in many crops, including peppers. The aim of this study was to develop DNA-based molecular markers that could be used in future marker-assisted breeding for BW resistance. Genome-wide discovery of single-nucleotide polymorphisms (SNPs was achieved by comparing whole-genome resequencing data from two pepper varieties, Saengryeg 211 (susceptible to BW) and 82PR66 (resistant to BW) with the reference genome of Capsicum annuum cv. CM334. A total of 118,588,231 and 148,774,861 paired raw reads with an average length of 151xa0bp were recorded for Saengryeg 211 and 82PR66, respectively. Data mining revealed 6,804,889 and 4,293,534 SNPs for Saengryeg 211 and 82PR66, respectively. The SNPs, were assorted into 12 chromosomes. A total of 5,514,563 polymorphic SNPs were discovered between the two varieties, and 42,236 high-resolution melting marker primer sets were selected for use in future experiments. Our results reveal numerous DNA-based molecular markers that might be useful in molecular breeding and QTL mapping in pepper.

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.

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.