• Proceedings of the Korean Society of Crop Science Conference
• /
• 2017.06a
• /
• pp.117-117
• /
• 2017

The onion (Allium cepa L.) is the most widely cultivated species of the genus Allium, especially it has been valued because of the pungent flavor and aroma. Allium species including onion has very large genome sizes ranging from approximately 10 to 20 Gbp, which have complicated genomic studies and precluded genome sequencing until recently. A population of 186 F2 individuals derived from a cross of 'Umjinara' ${\times}$ 'Sinsunhwang' and the two parental lines were used for this study. For the development of framework map, various types of markers including SSRs, RAPD, SNPs, and CAPS makers have been used for polymorphism test. Especially, a lot of SNP and CAPS loci were developed from the onion transcriptome sequence by RNASEQ of two parental lines. The GBS libraries have been constructed based on a modified protocol from Poland Lab using a two-enzyme system. We have been developing markers showing polymorphism between two parental lines, and genotyping for all F2 individuals were finished for a number of polymorphic markers. For the construction of GBS libraries, a set of 192 barcoded adapters were generated from complementary oligonucleotides with XhoI overhang sequence and unique barcodes of length 4-8 bp and they have been tested using two parental linesto determine the optimum conditions for GBS analysis.

• Korean Journal of Agricultural Science
• /
• v.44 no.4
• /
• pp.495-503
• /
• 2017

Genotyping-by-sequencing (GBS) is an outstanding technology for genotyping and single nucleotide polymorphism (SNP) discovery compared to next generation sequencing (NGS) because it can save time when analyzing large-scale samples and carries a low cost per sample. Recently, studies using GBS have been conducted on major crops and, to a greater extent, on fruit crops. However, many researchers have some problems due to low GBS efficiency resulting from low quality GBS libraries. To overcome this limitation, we developed an efficient GBS library construction method that regulates important conditions such as restriction enzymes (RE) digestion and a PCR procedure for grapevine. For RE digestion, DNA samples are digested with ApeKI (3.6U) at $75^{\circ}C$ for 5 hours and adapters are ligated to the ends of gDNA products. To produce suitable PCR fragments for sequencing, we modified the PCR amplification conditions; temperature cycling consisted of $72^{\circ}C$ (5 min), $98^{\circ}C$ (30 s), followed by 16 cycles of $98^{\circ}C$ (30 s), $65^{\circ}C$ (30 s), $72^{\circ}C$ (20 s) with a final extension step. As a result, we had obtained optimal library construct sizes (200 to 400 bp) for GBS analysis. Furthermore, it not only increased the mapping efficiency by approximately 10.17% compared to the previous method, but also produced mapped reads which were distributed equally on the19 chromosomes in the grape genome. Therefore, we suggest that this system can be used for various fruit crops and is expected to increase the efficiency of various genomic analysis performed.

• Proceedings of the Korean Society of Crop Science Conference
• /
• 2022.10a
• /
• pp.192-192
• /
• 2022

Sweetpotato is rich in starch, which is converted to sugar during storage due to enzymatic hydrolysis. The sugar content of sweetpotato is a component related to taste and storability. In this study, the sugar content (fructose, glucose, maltose, sucrose and total sugar content) of 94 genotypes was evaluated and the GWAS (Genome-Wide Association Study) was conducted to search for candidate genes for sugar content. The fructose and glucose content were 0.2 ~ 8.8 and 0.2 ~ 9.4 g/100g, respectively. The maltose, sucrose and total sugar content were 0.2 ~ 9.1,3.2 - 30.0 and 7.9 ~ 40.2 g/100g, respectively. The fructose and glucose showed a positive correlation (0.98). The 94 genotypes were genotyped with genotyping-by-sequencing (GBS) and aligned against the reference genome sequences of sweetpotato. The GBS libraries from 94 genotypes were sequenced on an Illumina HiSeqXten system, and 1,339,892 SNPs (Single Nucleotide Polymorphism) were generated. Filtering for < 60% missing rate and > 0.05 minor allele frequency resulted in a total of 44,255 SNPs used in GWAS. The GAPIT (Genome Association and Prediction Integrated Tool) was used to conduct based on the mean of sugar content with a Bonferroni-corrected chromosome-wide significance threshold with a -logio(P) of 5.95. The significant SNPs were obtained with fructose (seven), glucose (six), maltose (four) and sucrose (nine). There were several genes related to sugar content around the significant SNPs such as sugar transport protein 8-like, probable galactose-1 -phosphate uridyltransferase-like and beta-amylase. These results will contribute to understanding of sugar content and conversion in sweetpotato.

• Proceedings of the Plant Resources Society of Korea Conference
• /
• 2018.10a
• /
• pp.73-73
• /
• 2018

Bacterial leaf blight(BLB), caused by X. oryzae pv. oryzae(Xoo), is one of the most destructive diseases of rice due to its high epidemic potential. Understanding BLB resistance at a genetic level is important to further improve the rice breeding that provides one of the best approaches to control BLB disease. In the present investigation, a collection of 192 accessions was used in the genome-wide association study (GWAS) for BLB resistance loci against four Korean races of Xoo that were represented by the prevailing BLB isolates under Xoo differential system. A total of 192 accessions of rice germplasm were selected on the basis of the bioassay using four isolated races of Xoo such as K1, K2, K3 and K3a. The selected accessions was used to prepare 384-plex genotyping by sequencing (GBS) libraries and Illumina HiSeq 2000 paired- end read was used for GBS sequencing. GWAS was conducted using T ASSEL 5.0. The T ASSEL program uses a mixed linear model (MLM). T he results of the bioassay using a selected set of 192 accessions showed that a large number of accessions (93.75%) were resistant to K1 race, while the least number of accessions (34.37%) resisted K3a race. For races K2 and K3, the resistant germplasm proportion remained between 66.67 to 70.83%. T he genotypic data produced SNP matrix for a total of 293,379 SNPs. After imputation the missing data was removed, which exhibited 34,724 SNPs for association analysis. GWAS results showed strong signals of association at a threshold of [-log10(P-value)] more than5 (K1 and K2) and more than4 (K3 and K3a) for nine of the 39 SNPs, which are plausible candidate loci of resistance genes. T hese SNP loci were positioned on rice chromosome 2, 9, and 11 for K1 and K2 races, whereas on chromosome 4, 6, 11, and 12 for K3 and K3a races. The significant loci detected have also been illustrated, NBS-LRR type disease resistance protein, SNARE domain containing protein, Histone deacetylase 19, NADP-dependent oxidoreductase, and other expressed and unknown proteins. Our results provide a better understanding of the distribution of genetic variation of BLB resistance to Korean pathogen races and breeding of resistant rice.

• Proceedings of the Plant Resources Society of Korea Conference
• /
• 2019.04a
• /
• pp.62-62
• /
• 2019

Bacterial leaf blight(BLB), caused by X. oryzae pv. oryzae(Xoo), is one of the most destructive diseases of rice due to its high epidemic potential. Understanding BLB resistance at a genetic level is important to further improve the rice breeding that provides one of the best approaches to control BLB disease. In the present investigation, a collection of 192 accessions was used in the genome-wide association study (GWAS) for BLB resistance loci against four Korean races of Xoo that were represented by the prevailing BLB isolates under Xoo differential system. A total of 192 accessions of rice germplasm were selected on the basis of the bioassay using four isolated races of Xoo such as K1 and K2. The selected accessions was used to prepare 384-plex genotyping by sequencing (GBS) libraries and Illumina HiSeq 2000 pairedend read was used for GBS sequencing. GWAS was conducted using TASSEL 5.0. The TASSEL program uses a mixed linear model (MLM). The results of the bioassay using a selected set of 192 accessions showed that a large number of accessions (93.75%) were resistant to K1 race and K2 resistant germplasm proportion remained between 66.67. The genotypic data produced SNP matrix for a total of 293,379 SNPs. After imputation the missing data was removed, which exhibited 34,724 SNPs for association analysis. GWAS results showed strong signals of association at a threshold of [-log10(P-value)] more than 5 (K1 and K2) for nine of the 39 SNPs, which are plausible candidate loci of resistance genes. These SNP loci were positioned on rice chromosome 2, 9, and 11 for K1 and K2 races. The significant loci detected have also been illustrated and make the CPAS markers for NBS-LRR type disease resistance protein, SNARE domain containing protein, Histone deacetylase 19, NADP-dependent oxidoreductase, and other expressed and unknown proteins. Our results provide a better understanding of the distribution of genetic variation of BLB resistance to Korean pathogen races and breeding of resistant rice.