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

콩에서 유전자지도 작성은 마커간의 상대적인 위치를 설정하여 금후 genome 상에 목표 유전자 위치를 확보하고 이를 표지화하는데 매우 요긴한 수단으로 활용된다. 최근 많은 연구자들이 콩에서 고밀도의 유전자지도 작성을 위해 노력하였고, 현재 soybean consensus map이 soybase(www.soybase.org) 에 공유되고 있다. 본 연구에서는 고밀 도의 콩 유전자 지도 작성을 위해 genotyping은 한국과 중국의 콩 genome resequencing결과 얻어진 약 4백만 개의 SNP중에서 선정된 약 180만개의 SNP로 만들어진 180k Axiom SoyaSNP array를 이용하였고, 실험집단은 큰올콩/신 팔달콩 및 큰올콩/익산10호의 교배후대로 작성된 F12 RIL집단을 활용하였다. 유전자지도 작성결과 큰올콩/신팔달 콩 집단에서 사용된 166,279개의 SNP 중 27,308개의 SNP가 다형성을 보였고 이중 6,535개의 SNP가 유전자지도상 에 표기되어 지도의 총 거리는(total coverage)는 약 3,313cM을 나타내었다. 한편 큰올콩/익산10호 집단에서는 사용 된 166,279개의 SNP 중 23,581개의 SNP가 다형성을 보였고 이중 6,597개의 SNP가 유전자지도상에 표기되어 지도 의 총 거리는(total coverage)는 약 5,017cM을 나타내었다. 본 연구를 통해 작성된 유전자지도에서 마커간 평균거리 는 큰올콩/신팔달콩 0.51cM, 큰올콩/익산10호 0.76cM으로 나타나 매우 고밀도의 유전자 지도가 작성되었음을 나 타내었다.

Single nucleotide polymorphisms (SNPs) are the most abundant variation in plant genomes. As DNA markers, SNPs are rapidly replacing simple sequence repeats (SSRs) and sequence tagged sites (STSs) markers, because SNPs are more abundant, stable, easy to automation, efficient, and increasingly cost-effective. We developed a 96-plex indica/japonica SNP genotyping set for genetic analysis and molecular breeding in rice using Fluidigm platform. Informative SNPs for indica/japonica populations were selected from 1536 Illumina SNPs and 44K Affymetrix SNP chip data of Rice Diversity and our resequencing data sets. Selected SNPs were evenly distributed across 12 chromosomes and average physical distance between adjacent SNP markers was 4.38Mb. We conducted genetic diversity analysis of 49 Bangladesh germplasm and check varieties to test a 96-plex indica/japonica SNP genotyping set we developed. High-throughput Fluidigm SNP genotyping system will serve a more efficient and valuable tool for genetic diversity analysis, DNA fingerprinting, quantitative trait locus (QTL) mapping and background selection for crosses between indica and japonica in rice. This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ008125), Rural Development Administration, Republic of Korea.

We are currently developing a high-throughput single nucleotide polymorphism (SNP) genotyping service at IRRI to accelerate progress in rice breeding by providing rapid and cost-effective marker services. SNP marker development and validation is being performed based on cloned genes and QTLs, GWAS hits, and whole genome sequence data to identify predictive SNP markers at important genes for key traits for the breeding programs. Trait-based and targeted SNPs are being deployed in sets of 24 and 96 SNPs on a Fluidigm EP1 system. At the same time, 384 SNP sets and a 6K SNP chip developed by Susan McCouch at Cornell University are being used for higher density genome scans on an Illumina system. Genotyping by sequencing (GBS) approaches with 96 and 384 barcoded samples per sequence lane are also being evaluated in comparison to SNP array technology based on the number of loci, call rates, turnaround times, and cost per sample. An efficient sample processing workflow with an integrated LIMS is also being optimized to enable high throughput genotyping with sample tracking to minimize errors. Moreover, web-based SNP data analysis tools have been deployed through the IRRI Galaxy workbench to speed up SNP data analysis. Future efforts will focus on large-scale deployment of GBS across breeding materials to enable QC genotyping, tracking of donor introgressions, and integration of genome-wide prediction into the variety development pipelines. The large-scale application of high-density markers will help transform IRRI’s rice breeding programs and increase the rate of genetic gain towards developing high-yielding, stress-tolerant varieties for target environments and market segments

Recently whole genome SNP genotyping has been used to do association analysis and to map a gene of interest. Here we report application of bulked segregant analysis(BSA) using Infinium HD assay with ‘BARC Bean6K_3’, a SNP genotyping beadchip containing 5,399 SNPs for common bean to locate a target gene. We used BSA using Infinium HD assay was performed to find the candidate region of a single dominant rust resistant gene in PI310762, a common bean cultivar. And SSR markers were identified and mapped on the candidate region using F2 population derived from the cross of susceptible Pinto114 x resistant PI310762. BSA revealed the candidate region of the resistant gene is on chromosome 4 where we developed nine SSR markers. Three SSR markers (beanssr1170, beanssr1168, and beanssr1167) of them appeared closely linked to the resistant gene which is located between beanssr1167 at 0.1cM and beanssr1170 at 0.5cM on chromosome 4. This study showed BSA using high-throughput whole genome SNP genotyping is a very fast and efficient method to locate a gene of interest on chromosome.

Single nucleotide polymorphisms (SNPs) are valuable DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed. In this report, one of effective and high throughput SNP genotyping assays, which was named the template-directed dye-terminator incorporation with fluorescence polarization detection (FP-TDI) was described. Although the most of this assay succeed, the objective of this work was to deter­mine the reasons for the failures, find ways to improve the assay and reduce the running cost. Ninety F2 -derived soybean, Glycine max (L.) Merr., RILs from a cross between 'Pureunkong' and 'Jinpumkong 2' were genotyped at four SNPs. FP measurement was done on Victot3 microplate reader (perkinelmer Inc., Boston, MA, USA). Increasing the number of thermal cycles in the single-base extension step increased the separation of the FP values between the products corresponding to different genotypes. But in some assays, excess of heterozygous genotypes was observed with increase of PCR cycles. We discovered that the excess heterozygous was due to misincorporation of one of the dye­terminators during the primer extension reaction. After pyrophosphatase incubation and thermal cycle control, misincoporation can be effectively prevented. Using long amplicons instead of short amplicons for SNP genotyping and decreasing the amount of dye terminator and Acyclopol Taq polymerase to 1/2 or 1/3 decreased the cost of the assay. With these minor adjustments, the FP-TDI assay can be used more accurately and cost-effectively.