Genotyping-by-sequencing (GBS) is a cost-efficient method which can be useful for SNP marker discovery in a population of interest. GBS is genome reduction sequencing method using restriction enzyme. The quality of DNA is a key factor which could have an influence in downstream analysis. However, there have not been many studies which investigated the impact of DNA degradation and the quality of the data on marker discovery. In this study, GBS data of 6 Hanwoo samples (H1~6) showing differing level of DNA degradation were compared. Re-sequencing pipeline was followed to investigate the impact of DNA degradation on marker discovery. As a result, we found that the quantity and quality of SNPs were not affected in the sample H5 and H6 with moderately degraded DNA. On the other hand, marker discovery was greatly affected in samples with severe DNA degradation (H3 and H4). The findings in this study support that GBS is a robust genotyping method towards moderate DNA degradation.

Random amplified polymorphic DNA (RAPD) and fluorescent amplified fragment length polymorphism (FAFLP) analyses were executed on a total of 28 Salmonella spp., including 6 ATCC reference strains, 2 isolates from outbreaks of food poisoning in Gwangju, and 20 isolates from carcasses. For RAPD analysis, four primers, named P1254, 23L, OPA-4, OPB-17 were used producing amplification fragments ranged from 0.18kb to 2.6kb. As a result, 5 types using P1254, 5 types using 23L, 3 types using OPA-4, and 6 types using OPB-17 and a total of 18 RAPD types were achieved. For FAFLP analysis, bacterial genomic DNA was digested with endonucleases EcoRⅠ and MseⅠ, site-specific adaptors were ligated, and PCR amplification was carried out with an EcoR1 adaptor-specific primer labelled with fluorescent dye. Amplified fragments, which were separated on a polyacrylamide sequencing gel ranged from 35bp to 300bp were analysed. Results were displayed as a dendrogram with genetic distance. Twenty two Salmonella isolates and 6 reference strains were divided into 14 groups in a level of 0.136 genetic distance. In conclusion, Salmonella isolates of chicken carcasses have different genetic properties when compared to reference strains and isolates from outbreak of food poisoning.

Pepper (Capsicum spp.) germplasm shows diverse phenotypic variations including fruit size, color, pungency, and many other horticultural traits. Traditional markers including SSR, AFLP, and RFLP have been used to construct genetic maps using biparental populations. However to assess the genetic diversity of large number of germplasm, a robust and rapid marker development and genotyping approach is needed. We used six pepper accessions including C. annuum, C. chinense, C. baccatum and C. frutescens and performed genotyping-by-sequencing (GBS). To select the most appropriate condition, eight different 2 bp selective nucleotides were used to make GBS libraries. Selective nucleotide ‘OO’ showed the largest number of reads in all samples, and 11,026 to 47,957 high-quality SNPs were called in six accessions. When C. annuum ‘CM334’ genome sequence was used as a reference, C. annuum showed the smallest number of SNPs, while C. baccatum which was known to be a different Capsicum clade showed the largest number of SNPs. Pepper core collection chosen to represent the genetic diversity of whole germplasm will be genotyped by high-density SNPs developed from GBS. We will perform genome-wide association study (GWAS) using genetic and phenotypic variation to identify the functional genetic loci controlling horticultural traits.

Foxglove aphid, Aulacorthum solani (Kaltenbach), is a Hemipteran insect that infected a wide variety of plants worldwide and caused serious yield losses in crops. The foxglove aphid resistance gene, Raso2 was previously mapped from PI 366121 (Glycine soja Sieb. and Zucc.) to a 26cM marker interval on soybean chromosome 7. The development of additional genetic markers, which are mapped closer to Raso2 were required to accurately position the gene to improve the effectiveness of marker assisted selection. The objective of this study was to narrow down the putative QTL region, which is responsible to foxglove aphid resistance in PI366121 using recently developed high-density 180K Axiom SoyaSNP genotyping array. One hundred and forty one F8-derived F12 recombinant inbred lines developed from a cross of susceptible Williams 82 and resistant PI 366121, were used to generate a fine map of Raso2 interval. The phenotyping of antibiosis and antixenosis was done through choice and no-choice assays with total plant damage (TPD) and primary infestation leaf damage (PLD). The composite interval mapping analysis showed that the physical interval between two flanking makers, which was corresponding to Raso2, was narrowed down to 500kb on the Williams 82 genome assembly (Glyma2.0), instead of 4Mb in the previous report using Goldengate assay. In the Raso2 interval, there are about 60 candidate genes, including 4 of NBS-containing putative R genes. This result could be useful in breeding for new foxglove aphid resistant soybean cultivars.

Clubroot is a devastating disease caused by Plasmodiophora brassicae and results in severe losses of yield and quality in Brassica crops including Brassica oleracea. Therefore, it is important to identify resistance gene for CR disease and apply it to breeding of Brassica crops. In this study, we applied genotyping-by-sequencing (GBS) technique to construct high resolution genetic map and mapping of clubroot resistance (CR) genes. A total of 18,187 GBS markers were identified between two parent lines resistant and susceptible to the disease, of which 4,103 markers were genotyped in all 78 F2 plants generated from crossing of both parent lines. The markers were clustered into nine linkage groups spanning 879.9 cM, generating high resolution genetic map enough to refine reported reference genome of cabbage. In addition, through QTL analysis using 78 F2:3 progenies and mapping based on the genetic map, two and single major QTLs were identified for resistance of race 2 and race 9 of P. brassicae, respectively. These QTLs did not show collinearity with CR loci found in Chinese cabbage (Brassica rapa) but roughly overlapped with CR loci identified in cabbage for resistance to race 4. Taken together, genetic map and QTLs obtained in this study will provide valuable information to improve reference genome and clubroot resistance in cabbage.

Soybean is an important worldwide crop of dietary protein and oil resources for human foods and animal feeds. However, soybean breeding and improvement has been experienced challenges by a narrow germplasms. SNP genotyping array is regarded as a promising tool for dissecting wild and cultivated germplasms to find important genes by high-density genetic mapping and genome-wide association studies (GWAS). Here, we present the establishment of a large soyaSNP array and its use for diversity analysis and high density linkage mapping. More than 4 million high-quality SNPs identified from 16 high-depth and 31 low-depth soybean genome resequencing data were used to select 180,961 SNPs for the Axiom® SoyaSNP array. Our validation analysis for a set of 222 diverse soybean lines showed that a total of 171,161 markers were good quality for genotyping. Of the converted SNPs, 82.6% SNPs had a marker spacing of less than 9 kb and 17.4% SNPs greater than 9 kb, thereby suggesting that our array is likely suitable for GWAS of soybean germplasms. In the GWAS for seed protein content in the wild soybean germplasms with the size of 1,135 accessions, 22 loci on 12 chromosomes showed significant association (-logP>4). The highest associated peaks were shown at the 28 Mbp region on Gm05 (-logP=5.89), at 45 Mbp on Gm03 (-logP=5.32), and at 2.8 Mbp on Gm17 (-logP=5.00). Of the 22 associations, 8 corresponded with the location of previously reported seed protein QTLs and 14 regions is thought to be new QTLs for seed protein content in wild soybean. This array is being used to construct high-density genetic maps in two recombinant inbred lines and nested-association mapping populations with 30 combinations used Daepung cultivar as hub-parents, with an objective to confirm large structural variations of chromosomes using the ultra-high-density maps.

An important worldwide plant source of dietary protein and oil, modern breeding and improvement of soybean is suffered from a narrow cultivated germplasm relative to other crop species likely because of underuse of wild soybeans as breeding resources. SNP genotyping array is regarded as a promising tool for dissecting wild and cultivated germplasms to find important adaptive genes by high-density genetic mapping and genome-wide association studies (GWAS). Here, we present the establishment of a large soybean SNP array and its use for diversity analysis and high density linkage mapping. More than 4 million high-quality SNPs identified from 16 high-depth and 31 low-depth soybean genome resequencing data were used to select 180,961 SNPs for the AxiomÒ SoyaSNP array. Our validation analysis for a set of 222 diverse soybean lines showed that a total of 171,161markers were of good quality for genotyping. Of the converted SNPs, 82.6% 82.6% SNPs had a marker spacing of less than 9 kb and 17.4% SNPs greater than 9 kb with the 297 inter-SNP spacings of >100 kb and with 812 kb of the largest spacing, thereby suggesting that our array is likely suitable for GWAS of soybean germplasm. This array is being used to construct high-density genetic map in populations generated from intermatings of two cultivated and two wild soybeans, with an objective to confirm large structural variations of chromosomes using the ultra-high-density maps

Apple is a typical climacteric fruit, whose loss of firmness during storage is associated with internal levels of ethylene. MdACS1 (1-aminocyclopropane-1-carboxylate synthase) and MdACO1 (1-aminocyclopropane-1-carboxylate oxidase) genes are characterized well as functional markers for the ethylene production of ripening apple fruit. Presence of two alleles for each gene are commonly reported in cultivated apples. MdACS1-1/1, 1-1/2 and 1-2/2 generally induce high, medium, and low ethylene production, respectively. Homozygosity of MdACO1-1 resulted in low levels of ethylene production than MdACO1-1/2 and 1-2/2. It was reported that cultivars homozygous for MdACS1-2 and MdACO1-1 had superior shelf-life with lowest ethylene production. In this study, genotypes of 42 apple cultivars including Korean-developed ones at MdACS1 and MdACO1 loci were determined. Polymorphisms were detected by PCR and were separated by electrophoresis in a 2.0% agarose gel. Of PCR products of MdACS1, the fragrament of 490 bp was corresponded to the MdACS1-1 allele and 640 bp corresponded to the MdACS1-2 allele. ACO1-1 and ACO1-2 alleles had the fragment size of 525 and 587 bp, respectively. The 42 cultivars could be grouped into three classes to each gene, MdACS1-1/1, MdACS1-1/2 and MdACS1-2/2; MdACO1-1/1, MdACO1-1/2 and MdACO1-2/2.