Development of an SNP marker set for marker-assisted backcrossing using genotyping-by-sequencing in tetraploid perilla.

High-quality molecular markers are essential for marker-assisted selection to accelerate breeding progress. Compared with diploid species, recently diverged polyploid crop species tend to have highly similar homeologous subgenomes, which is expected to limit the development of broadly applicable locus-specific single-nucleotide polymorphism (SNP) assays. Furthermore, it is particularly challenging to make genome-wide marker sets for species that lack a reference genome. Here, we report the development of a genome-wide set of kompetitive allele specific PCR (KASP) markers for marker-assisted recurrent selection (MARS) in the tetraploid minor crop perilla. To find locus-specific SNP markers across the perilla genome, we used genotyping-by-sequencing (GBS) to construct linkage maps of two F2 populations. The two resulting high-resolution linkage maps comprised 2326 and 2454 SNP markers that spanned a total genetic distance of 2133 cM across 16 linkage groups and 2169 cM across 21 linkage groups, respectively. We then obtained a final genetic map consisting of 22 linkage groups with 1123 common markers from the two genetic maps. We selected 96 genome-wide markers for MARS and confirmed the accuracy of markers in the two F2 populations using a high-throughput Fluidigm system. We confirmed that 91.8% of the SNP genotyping results from the Fluidigm assay were the same as the results obtained through GBS. These results provide a foundation for marker-assisted backcrossing and the development of new varieties of perilla.

Transcriptome Analysis of Differentially Expressed Genes Associated with Salt Stress in Cowpea (Vigna unguiculata L.) during the Early Vegetative Stage.

Cowpea (Vigna unguiculata (L.), 2n = 22) is a tropical crop grown in arid and semiarid regions that is tolerant to abiotic stresses such as heat and drought. However, in these regions, salt in the soil is generally not eluted by rainwater, leading to salt stress for a variety of plant species. This study was conducted to identify genes related to salt stress using the comparative transcriptome analysis of cowpea germplasms with contrasting salt tolerance. Using the Illumina Novaseq 6000 platform, 1.1 billion high-quality short reads, with a total length of over 98.6 billion bp, were obtained from four cowpea germplasms. Of the differentially expressed genes identified for each salt tolerance type following RNA sequencing, 27 were shown to exhibit significant expression levels. These candidate genes were subsequently narrowed down using reference-sequencing analysis, and two salt stress-related genes (Vigun_02G076100 and Vigun_08G125100) with single-nucleotide polymorphism (SNP) variation were selected. Of the five SNPs identified in Vigun_02G076100, one that caused significant amino acid variation was identified, while all nucleotide variations in Vigun_08G125100 was classified as missing in the salt-resistant germplasms. The candidate genes and their variation, identified in this study provide, useful information for the development of molecular markers for cowpea breeding programs.

Identification of quantitative trait loci associated with flowering time in perilla using genotyping-by-sequencing.

Understanding the transition to the reproductive period is important for crop breeding. This information can facilitate the production of novel varieties that are better adapted to local environments or changing climatic conditions. Here, we report the development of a high-density linkage map based on genotyping-by-sequencing (GBS) for the genus perilla. Through GBS library construction and Illumina sequencing of an F2 population, a total of 9607 single-nucleotide polymorphism (SNP) markers were developed. The ten-group linkage map of 1309.39 cM contained 2518 markers, with an average marker density of 0.56 cM per linkage group (LG). Using this map, a total of six QTLs were identified. These quantitative trait loci (QTLs) are associated with three traits related to flowering time: days to visible flower bud, days to flowering, and days to maturity. Ortholog analysis conducted with known genes involved in the regulation of flowering time among different crop species identified GI, CO and ELF4 as putative perilla orthologs that are closely linked to the QTL regions associated with flowering time. These results provide a foundation that will be useful for future studies of flowering time in perilla using fine mapping, and marker-assisted selection for the development of new varieties of perilla.

A comparative synteny analysis tool for target-gene SNP marker discovery: connecting genomics data to breeding in Solanaceae.

It is necessary for molecular breeders to overcome the difficulties in applying abundant genomic information to crop breeding. Candidate orthologs would be discovered more efficiently in less-studied crops if the information gained from studies of related crops were used. We developed a comparative analysis tool and web-based genome viewer to identify orthologous genes based synteny as well as sequence similarity between tomato, pepper and potato. The tool has a step-by-step interface with multiple viewing levels to support the easy and accurate exploration of functional orthologs. Furthermore, it provides access to single nucleotide-polymorphism markers from the massive genetic resource pool in order to accelerate the development of molecular markers for candidate orthologs in the Solanaceae. This tool provides a bridge between genome data and breeding by supporting effective marker development, data utilization and communication.Database URL: http://tgsol.seeders.co.kr/scomp/.

Molecular characterization of proton beam-induced mutations in soybean using genotyping-by-sequencing.

Proton beam irradiation is a next-generation technique to develop mutant crop varieties. The mutagenic effects and molecular mechanisms of radiation are important multi-disciplinary research subjects. This study was conducted to investigate the types of mutations induced in the soybean genome by proton beam irradiation. In total, 22 plants, including 10 M2 plants treated with proton beam irradiation at 118 and 239 Gy, each, and two wild-type plants (Daepung) were sequenced by genotyping-by-sequencing (GBS). In total, 7453 single nucleotide polymorphisms (SNPs) were detected in the 20 M2 plants, compared with the two wild-type controls. The SNP frequency was 1/36,976 bp with proton beam irradiation at 118 Gy, and 1/32,945 bp at 239 Gy. Of these, 3569 SNPs were detected in genic regions. We observed that proton beam irradiation induced more substitutions than small insertion-deletions (INDELs). Based on the mutagenic effect of proton beam irradiation, the frequency of transition mutations was shown to be higher than that of transversions. The proton beam-induced SNPs were distributed uniformly in most of the chromosomes. Gene ontology (GO) analysis showed that there were many genes involved in protein metabolic process under biological process, intracellular membrane-bounded organelle under cellular component, and nucleic acid binding under molecular function. This study could provide valuable information for investigating the potential mechanisms of mutation, and guidance for developing soybeans cultivars using mutation breeding.

Whole-transcriptome analyses of the Sapsaree, a Korean natural monument, before and after exercise-induced stress.

BACKGROUND: The Sapsaree (Canis familiaris) is a Korean native dog that is very friendly, protective, and loyal to its owner, and is registered as a natural monument in Korea (number: 368). To investigate large-scale gene expression profiles and identify the genes related to exercise-induced stress in the Sapsaree, we performed whole-transcriptome RNA sequencing and analyzed gene expression patterns before and after exercise performance. RESULTS: We identified 525 differentially expressed genes in ten dogs before and after exercise. Gene Ontology classification and KEGG pathway analysis revealed that the genes were mainly involved in metabolic processes, such as programmed cell death, protein metabolic process, phosphatidylinositol signaling system, and cation binding in cytoplasm. The ten Sapsarees could be divided into two groups based on the gene expression patterns before and after exercise. The two groups were significantly different in terms of their basic body type (p ≤ 0.05). Seven representative genes with significantly different expression patterns before and after exercise between the two groups were chosen and characterized. CONCLUSIONS: Body type had a significant effect on the patterns of differential gene expression induced by exercise. Whole-transcriptome sequencing is a useful method for investigating the biological characteristics of the Sapsaree and the large-scale genomic differences of canines in general.

Identification of candidate genes for an early-maturing soybean mutant by genome resequencing analysis.

Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In this study, we performed whole genome resequencing by Illumina HiSeq to identify changes in flowering genes using an early-flowering phenotype of soybean mutant line Josaengserori (JS) derived from Korean landrace, Seoritae (SR), and we obtained mapped reads of 131,769,690 and 167,669,640 bp in JS and SR, respectively. From the whole genome sequencing results between JS and SR, we identified 332,821 polymorphic SNPs and 65,178 indels, respectively. Among these, 30 flowering genes were in SNPs and 25 were in indels. Among 30 flowering genes detected in SNPs, Glyma02g33040, Glyma06g22650, Glyma10g36600, Glyma13g01290, Glyma14g10530, Glyma16g01980, Glyma17g11040, Glyma18g53690, and Glyma20g29300 were non-synonymous substitutions between JS and SR. Changes in Glyma10g36600 (GI), Glya02g33040 (AGL18), Glyma17g11040 (TOC1), and Glyma14g10530 (ELF3) in JS affected the expression of GmFT2a and resulted in early flowering. These results provide insight into the regulatory pathways of flowering in soybean mutants and help to improve our knowledge of soybean mutation breeding.

De Novo Transcriptome Analysis of Cucumis melo L. var. makuwa.

Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family.

High-throughput sequencing and de novo assembly of Brassica oleracea var. Capitata L. for transcriptome analysis.

BACKGROUND: The cabbage, Brassica oleracea var. capitata L., has a distinguishable phenotype within the genus Brassica. Despite the economic and genetic importance of cabbage, there is little genomic data for cabbage, and most studies of Brassica are focused on other species or other B. oleracea subspecies. The lack of genomic data for cabbage, a non-model organism, hinders research on its molecular biology. Hence, the construction of reliable transcriptomic data based on high-throughput sequencing technologies is needed to enhance our understanding of cabbage and provide genomic information for future work. METHODOLOGY/PRINCIPAL FINDINGS: We constructed cDNAs from total RNA isolated from the roots, leaves, flowers, seedlings, and calcium-limited seedling tissues of two cabbage genotypes: 102043 and 107140. We sequenced a total of six different samples using the Illumina HiSeq platform, producing 40.5 Gbp of sequence data comprising 401,454,986 short reads. We assembled 205,046 transcripts (≥ 200 bp) using the Velvet and Oases assembler and predicted 53,562 loci from the transcripts. We annotated 35,274 of the loci with 55,916 plant peptides in the Phytozome database. The average length of the annotated loci was 1,419 bp. We confirmed the reliability of the sequencing assembly using reverse-transcriptase PCR to identify tissue-specific gene candidates among the annotated loci. CONCLUSION: Our study provides valuable transcriptome sequence data for B. oleracea var. capitata L., offering a new resource for studying B. oleracea and closely related species. Our transcriptomic sequences will enhance the quality of gene annotation and functional analysis of the cabbage genome and serve as a material basis for future genomic research on cabbage. The sequencing data from this study can be used to develop molecular markers and to identify the extreme differences among the phenotypes of different species in the genus Brassica.

Genome-wide SNP calling using next generation sequencing data in tomato.

The tomato (Solanum lycopersicum L.) is a model plant for genome research in Solanaceae, as well as for studying crop breeding. Genome-wide single nucleotide polymorphisms (SNPs) are a valuable resource in genetic research and breeding. However, to do discovery of genome-wide SNPs, most methods require expensive high-depth sequencing. Here, we describe a method for SNP calling using a modified version of SAMtools that improved its sensitivity. We analyzed 90 Gb of raw sequence data from next-generation sequencing of two resequencing and seven transcriptome data sets from several tomato accessions. Our study identified 4,812,432 non-redundant SNPs. Moreover, the workflow of SNP calling was improved by aligning the reference genome with its own raw data. Using this approach, 131,785 SNPs were discovered from transcriptome data of seven accessions. In addition, 4,680,647 SNPs were identified from the genome of S. pimpinellifolium, which are 60 times more than 71,637 of the PI212816 transcriptome. SNP distribution was compared between the whole genome and transcriptome of S. pimpinellifolium. Moreover, we surveyed the location of SNPs within genic and intergenic regions. Our results indicated that the sufficient genome-wide SNP markers and very sensitive SNP calling method allow for application of marker assisted breeding and genome-wide association studies.

Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species.

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.

Molecular mapping and characterization of a single dominant gene controlling CMV resistance in peppers (Capsicum annuum L.).

Cucumber mosaic virus (CMV) is one of the most destructive viruses in the Solanaceae family. Simple inheritance of CMV resistance in peppers has not previously been documented; all previous studies have reported that resistance to this virus is mediated by several partially dominant and recessive genes. In this study, we showed that the Capsicum annuum cultivar 'Bukang' contains a single dominant resistance gene against CMV(Korean) and CMV(FNY) strains. We named this resistance gene Cmr1 (Cucumber mosaic resistance 1). Analysis of the cellular localization of CMV using a CMV green fluorescent protein construct showed that in 'Bukang,' systemic movement of the virus from the epidermal cell layer to mesophyll cells is inhibited. Genetic mapping and FISH analysis revealed that the Cmr1 gene is located at the centromeric region of LG2, a position syntenic to the ToMV resistance locus (Tm-1) in tomatoes. Three SNP markers were developed by comparative genetic mapping: one intron-based marker using a pepper homolog of Tm-1, and two SNP markers using tomato and pepper BAC sequences mapped near Cmr1. We expect that the SNP markers developed in this study will be useful for developing CMV-resistant cultivars and for fine mapping the Cmr1 gene.

Evolution of ribosomal DNA-derived satellite repeat in tomato genome.

BACKGROUND: Tandemly repeated DNA, also called as satellite DNA, is a common feature of eukaryotic genomes. Satellite repeats can expand and contract dramatically, which may cause genome size variation among genetically-related species. However, the origin and expansion mechanism are not clear yet and needed to be elucidated. RESULTS: FISH analysis revealed that the satellite repeat showing homology with intergenic spacer (IGS) of rDNA present in the tomato genome. By comparing the sequences representing distinct stages in the divergence of rDNA repeat with those of canonical rDNA arrays, the molecular mechanism of the evolution of satellite repeat is described. Comprehensive sequence analysis and phylogenetic analysis demonstrated that a long terminal repeat retrotransposon was interrupted into each copy of the 18S rDNA and polymerized by recombination rather than transposition via an RNA intermediate. The repeat was expanded through doubling the number of IGS into the 25S rRNA gene, and also greatly increasing the copy number of type I subrepeat in the IGS of 25-18S rDNA by segmental duplication. Homogenization to a single type of subrepeat in the satellite repeat was achieved as the result of amplifying copy number of the type I subrepeat but eliminating neighboring sequences including the type II subrepeat and rRNA coding sequence from the array. FISH analysis revealed that the satellite repeats are commonly present in closely-related Solanum species, but vary in their distribution and abundance among species. CONCLUSION: These results represent that the dynamic satellite repeats were originated from intergenic spacer of rDNA unit in the tomato genome. This result could serve as an example towards understanding the initiation and the expansion of the satellite repeats in complex eukaryotic genome.

Integration of cytogenetic and genetic linkage maps unveils the physical architecture of tomato chromosome 2.

We report the integration of the linkage map of tomato chromosome 2 with a high-density bacterial artificial chromosome fluorescence in situ hybridization (BAC-FISH)-based cytogenetic map. The euchromatic block of chromosome 2 resides between 13 and 142 cM and has a physical length of 48.12 microm, with 1 microm equivalent to 540 kb. BAC-FISH resolved a pair of loci that were 3.7-3.9 Mb apart and were not resolved on the linkage map. Most of the regions had crossover densities close to the mean of approximately 200 kb/cM. Relatively hot and cold spots of recombination were unevenly distributed along the chromosome. The distribution of centimorgan/micrometer values was similar to the previously reported recombination nodule distribution along the pachytene chromosome. FISH-based physical maps will play an important role in advanced genomics research for tomato, including map-based cloning of agronomically important traits and whole-genome sequencing.

Transluminal removal of a fractured and embolized indwelling central venous catheter in the pulmonary artery.

Vascular catheters are associated with complications such as infection, thrombosis and stenosis. The embolization of a venous catheter fragment is a rare complication. A 39-year-old woman underwent placement of a totally implantable venous access device for chemotherapy to treat a recurrent liposarcoma of the left thigh. The "pinch-off sign" was noted on a routine chest X-ray but that was ignored. Three-months after implantation of the intravenous access device, the indwelling central catheter was fractured and embolized to the pulmonary trunk. The catheter in the pulmonary trunk was successfully removed through a percutaneous femoral vein approach using a pigtail catheter and goose neck snare.