Genetic Mapping of a Resistance Locus to Phytophthora sojae in the Korean Soybean Cultivar Daewon.

Phytophthora root and stem rot reduce soybean yields worldwide. The use of R-gene type resistance is currently crucial for protecting soybean production. The present study aimed to identify the genomic location of a gene conferring resistance to Phytophthora sojae isolate 2457 in the recombinant inbred line population developed by a cross of Daepung × Daewon. Single-marker analysis identified 20 single nucleotide polymorphisms associated with resistance to the P. sojae isolate 2457, which explained ~67% of phenotypic variance. Daewon contributed a resistance allele for the locus. This region is a well-known location for Rps1 and Rps7. The present study is the first, however, to identify an Rps gene locus from a major soybean variety cultivated in South Korea. Linkage analysis also identified a 573 kb region on chromosome 3 with high significance (logarithm of odds = 13.7). This genomic region was not further narrowed down due to lack of recombinants within the interval. Based on the latest soybean genome, ten leucine-rich repeat coding genes and four serine/threonine protein kinase-coding genes are annotated in this region, which all are well-known types of genes for conferring disease resistance in crops. These genes would be candidates for molecular characterization of the resistance in further studies. The identified R-gene locus would be useful in developing P. sojae resistant varieties in the future. The results of the present study provide foundational knowledge for researchers who are interested in soybean-P. sojae interaction.

Korean soybean core collection: Genotypic and phenotypic diversity population structure and genome-wide association study.

A core collection is a subset that represents genetic diversity of the total collection. Soybean (Glycine max (L.) Merr.) is one of major food and feed crops. It is the world's most cultivated annual herbaceous legume. Constructing a core collection for soybean could play a pivotal role in conserving and utilizing its genetic variability for research and breeding programs. To construct and evaluate a Korean soybean core collection, genotypic and phenotypic data as well as population structure, were analyzed. The Korean soybean core collection consisted of 430 accessions selected from 2,872 collections based on Affymetrix Axiom® 180k SoyaSNP array data. The core collection represented 99% of genotypic diversity of the total collection. Analysis of population structure clustered the core collection into five subpopulations. Accessions from South Korea and North Korea were distributed across five subpopulations. Analysis of molecular variance indicated that only 2.01% of genetic variation could be explained by geographic origins while 16.18% of genetic variation was accounted for by subpopulations. Genome-wide association study (GWAS) for days to flowering, flower color, pubescent color, and growth habit confirmed that the core collection had the same genetic diversity for tested traits as the total collection. The Korean soybean core collection was constructed based on genotypic information of the 180k SNP data. Size and phenotypic diversity of the core collection accounted for approximately 14.9% and 18.1% of the total collection, respectively. GWAS of core and total collections successfully confirmed loci associated with tested traits. Consequently, the present study showed that the Korean soybean core collection could provide fundamental and practical material and information for both soybean genetic research and breeding programs.

GmBRC1 is a Candidate Gene for Branching in Soybean (Glycine max (L.) Merrill).

Branch number is one of the main factors affecting the yield of soybean (Glycine max (L.)). In this study, we conducted a genome-wide association study combined with linkage analysis for the identification of a candidate gene controlling soybean branching. Five quantitative trait nucleotides (QTNs) were associated with branch numbers in a soybean core collection. Among these QTNs, a linkage disequilibrium (LD) block qtnBR6-1 spanning 20 genes was found to overlap a previously identified major quantitative trait locus qBR6-1. To validate and narrow down qtnBR6-1, we developed a set of near-isogenic lines (NILs) harboring high-branching (HB) and low-branching (LB) alleles of qBR6-1, with 99.96% isogenicity and different branch numbers. A cluster of single nucleotide polymorphisms (SNPs) segregating between NIL-HB and NIL-LB was located within the qtnBR6-1 LD block. Among the five genes showing differential expression between NIL-HB and NIL-LB, BRANCHED1 (BRC1; Glyma.06G210600) was down-regulated in the shoot apex of NIL-HB, and one missense mutation and two SNPs upstream of BRC1 were associated with branch numbers in 59 additional soybean accessions. BRC1 encodes TEOSINTE-BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS 1 and 2 transcription factor and functions as a regulatory repressor of branching. On the basis of these results, we propose BRC1 as a candidate gene for branching in soybean.

Genetic diversity patterns and domestication origin of soybean.

KEY MESSAGE: Genotyping data of a comprehensive Korean soybean collection obtained using a large SNP array were used to clarify global distribution patterns of soybean and address the evolutionary history of soybean. Understanding diversity and evolution of a crop is an essential step to implement a strategy to expand its germplasm base for crop improvement research. Accessions intensively collected from Korea, which is a small but central region in the distribution geography of soybean, were genotyped to provide sufficient data to underpin population genetic questions. After removing natural hybrids and duplicated or redundant accessions, we obtained a non-redundant set comprising 1957 domesticated and 1079 wild accessions to perform population structure analyses. Our analysis demonstrates that while wild soybean germplasm will require additional sampling from diverse indigenous areas to expand the germplasm base, the current domesticated soybean germplasm is saturated in terms of genetic diversity. We then showed that our genome-wide polymorphism map enabled us to detect genetic loci underlying flower color, seed-coat color, and domestication syndrome. A representative soybean set consisting of 194 accessions was divided into one domesticated subpopulation and four wild subpopulations that could be traced back to their geographic collection areas. Population genomics analyses suggested that the monophyletic group of domesticated soybeans was likely originated at a Japanese region. The results were further substantiated by a phylogenetic tree constructed from domestication-associated single nucleotide polymorphisms identified in this study.

GenoCore: A simple and fast algorithm for core subset selection from large genotype datasets.

Selecting core subsets from plant genotype datasets is important for enhancing cost-effectiveness and to shorten the time required for analyses of genome-wide association studies (GWAS), and genomics-assisted breeding of crop species, etc. Recently, a large number of genetic markers (>100,000 single nucleotide polymorphisms) have been identified from high-density single nucleotide polymorphism (SNP) arrays and next-generation sequencing (NGS) data. However, there is no software available for picking out the efficient and consistent core subset from such a huge dataset. It is necessary to develop software that can extract genetically important samples in a population with coherence. We here present a new program, GenoCore, which can find quickly and efficiently the core subset representing the entire population. We introduce simple measures of coverage and diversity scores, which reflect genotype errors and genetic variations, and can help to select a sample rapidly and accurately for crop genotype dataset. Comparison of our method to other core collection software using example datasets are performed to validate the performance according to genetic distance, diversity, coverage, required system resources, and the number of selected samples. GenoCore selects the smallest, most consistent, and most representative core collection from all samples, using less memory with more efficient scores, and shows greater genetic coverage compared to the other software tested. GenoCore was written in R language, and can be accessed online with an example dataset and test results at https://github.com/lovemun/Genocore.

Identification of haplotypes at the Rsv4 genomic region in soybean associated with durable resistance to soybean mosaic virus.

KEY MESSAGE: Discovery of new germplasm sources and identification of haplotypes for the durable Soybean mosaic virus resistance gene, Rsv 4, provide novel resources for map-based cloning and genetic improvement efforts in soybean. The Soybean mosaic virus (SMV) resistance locus Rsv4 is of interest because it provides a durable type of resistance in soybean [Glycine max (L.) Merr.]. To better understand its molecular basis, we used a population of 309 BC3F2 individuals to fine-map Rsv4 to a ~120 kb interval and leveraged this genetic information in a second study to identify accessions 'Haman' and 'Ilpumgeomjeong' as new sources of Rsv4. These two accessions along with three other Rsv4 and 14 rsv4 accessions were used to examine the patterns of nucleotide diversity at the Rsv4 region based on high-depth resequencing data. Through a targeted association analysis of these 19 accessions within the ~120 kb interval, a cluster of four intergenic single-nucleotide polymorphisms (SNPs) was found to perfectly associate with SMV resistance. Interestingly, this ~120 kb interval did not contain any genes similar to previously characterized dominant disease resistance genes. Therefore, a haplotype analysis was used to further resolve the association signal to a ~94 kb region, which also resulted in the identification of at least two Rsv4 haplotypes. A haplotype phylogenetic analysis of this region suggests that the Rsv4 locus in G. max is recently introgressed from G. soja. This integrated study provides a strong foundation for efforts focused on the cloning of this durable virus resistance gene and marker-assisted selection of Rsv4-mediated SMV resistance in soybean breeding programs.

Development, validation and genetic analysis of a large soybean SNP genotyping array.

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under-use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high-density genetic mapping and genome-wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome-wide association analyses. More than four million high-quality SNPs identified from high-depth genome re-sequencing of 16 soybean accessions and low-depth genome re-sequencing of 31 soybean accessions were used to select 180,961 SNPs for creation of the Axiom(®) SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170,223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene-rich chromosomal regions suggest that this array may be suitable for genome-wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.

Population genetic structure and genetic diversity of soybean aphid collections from the USA, South Korea, and Japan.

Following its recent invasion of North America, the soybean aphid (Aphis glycines Matsumura) has become the number one insect pest of soybean (Glycine max L. Merr.) in the north central states of the USA. A few studies have been conducted on the population genetic structure and genetic diversity of the soybean aphid and the source of its invasion in North America. Molecular markers, such as simple sequence repeats (SSRs) are very useful in the evaluation of population structure and genetic diversity. We used 18 SSR markers to assess the genetic diversity of soybean aphid collections from the USA, South Korea, and Japan. The aphids were collected from two sites in the USA (Indiana and South Dakota), two sites in South Korea (Yeonggwang district and Cheonan city), and one site in Japan (Utsunomiya). The SSR markers were highly effective in differentiating among aphid collections from different countries. The level of differentiation within each population and among populations from the same country was limited, even in the case of the USA where the two collection sites were more than 1200 km apart.

Genetic mapping of the powdery mildew resistance gene in soybean PI 567301B.

Powdery mildew (PMD) of soybean [Glycine max (L.) Merr.] is caused by the fungus Microsphaera diffusa. Severe infection of PMD on susceptible varieties often causes premature defoliation and chlorosis of the leaves, which can result in considerable yield losses under favorable environmental conditions for disease development in the field. A total of 334 F(7)-derived recombinant inbred lines (RILs) from a cross of a PMD susceptible soybean cultivar Wyandot and PMD-resistant PI 567301B were used for genetic mapping of PMD resistance in PI 567301B and for development of molecular markers tightly linked to the gene. The result of the PMD screening for each line in the field was in agreement with that in the greenhouse test. The genetic map containing the PMD resistance gene was constructed in a 3.3 cM interval flanked by two simple sequence repeat (SSR) markers on chromosome 16. The PMD resistance gene was mapped at the same location with SSR marker BARCSOYSSR_16_1291, indicating that there was no recombination between the 334 RILs and this marker. In addition, a single nucleotide polymorphism (SNP) marker developed by high-resolution melting curve analysis and a cleaved amplified polymorphic sequence (CAPS) marker with Rsa1 recognition site were used for the genetic mapping. These two markers were also mapped to the same genomic location with the PMD resistance gene. We validated three tightly linked markers to the PMD resistance gene using 38 BC(6)F(2) lines and corresponding BC(6)F(2:3) families. The three marker genotypes of the backcross lines predicted the observed PMD phenotypes of the lines with complete accuracy. We have mapped a putatively novel single dominant PMD resistance gene in PI 567301B and developed three new molecular markers closely linked to the gene. Molecular markers developed from this study may be used for high-throughput marker-assisted breeding for PMD resistance with the gene from PI 567301B.

Genetic map of lps3: a new short petiole gene in soybeans.

The short petiole trait is valuable for the development of plant ideotypes with high yield by improving the plant canopy. The soybean breeding line SS98206SP has shown extremely short petioles in the greenhouse and fields. A new single recessive gene designated as lps3 confers the short petiole trait in SS98206SP. The objectives of this study were to map the short petiole gene in SS98206SP with PCR-based markers. In total, 187 F(2) plants and their F(2:3) families from a cross between the short petiole line SS98206SP and the long petiole cultivar 'Taekwang' along with the two parental lines were evaluated for their petiole lengths in a greenhouse. An SSR marker from each 10-cM section of a consensus soybean map was selected for bulked segregant analysis (BSA) to identify the tentative genomic location of the gene. The BSA technique was useful to localize the gene to a genomic region in the soybean linkage group F (chromosome 13). A linkage map with six SSR and two SNP markers flanking the gene was constructed. We positioned the gene between two SSR markers, Sat_234 and Sct_033, at distances of 8.5 and 3.5 cM from the marker, respectively. The makers flanking the gene (lps3) were located within 3-4 cM of the gene. These markers will be useful for maker-assisted selection in the development of new ideotype soybean plants.

Genetic map of the powdery mildew resistance gene in soybean PI 243540.

Powdery mildew (caused by Microsphaera diffusa Cooke & Peck) is a common disease of soybean in many soybean-growing regions of the world and under greenhouse conditions. The previously reported Rmd locus of soybean for resistance to powdery mildew was mapped on soybean molecular linkage group J (chromosome 16). We have discovered a single dominant gene in PI 243540 that provides season-long resistance to powdery mildew. The objective of this study was to map the powdery mildew resistance gene in PI 243540 with PCR-based molecular markers. One hundred eighty-four F2 plants and their F(2:3) families from a cross between the powdery mildew susceptible cultivar 'Wyandot' and PI 243540 were screened with M. diffusa in greenhouses. Bulked segregant analysis (BSA) with SSR markers was used to identify the tentative genomic location of the gene. The BSA localized the gene to a genomic region in soybean chromosome 16. A linkage map with seven SSR and six SNP markers flanking the gene was constructed. We positioned the gene between SSR marker Sat_224 and SNP marker BARC-021875-04228 at distances of 9.6 and 1.3 cM from the markers, respectively. The map position of the gene was slightly different from previously reported map positions of the only known Rmd locus. We have mapped a single dominant gene, tentatively called Rmd_PI243540, near the previously known Rmd locus on chromosome 16. The molecular markers flanking the gene will be useful for marker-assisted selection of this gene.

Population genetic structure of Aphis glycines.

The soybean aphid (Aphis glycines Matsumura) is an invasive pest of cultivated soybean (Glycine max L.) in North America. After the initial invasion in 2000, the aphid has quickly spread across most of the United States and Canada, suggesting large-scale dispersal and rapid adaptation to new environments. Using microsatellite markers from closely related species, we compared the genetic diversity and the amount of genetic differentiation within and among 2 South Korean and 10 North American populations. Overall allelic polymorphism was low, never exceeding four alleles per locus. However, differences in genetic diversity were seen among South Korean and North American populations in terms of heterozygote excesses and genotypic richness. Within North America, two populations (Michigan and Ontario), had lower genetic diversities and exhibited high genetic differentiation compared with the remaining eight populations. The earlier collection time of Michigan and Ontario samples explained the genetic differences better than geographic subdivisions. These data indicate a pattern of small colonizing populations on soybeans, followed by rapid clonal amplification and subsequent large-scale dispersal across North America.

Cross-species amplification and polymorphism of microsatellite loci in the soybean aphid, Aphis glycines.

We tested the utility of 18 previously characterized Aphis spp. microsatellite loci for polymorphism and differentiation among populations of the soybean aphid, Aphis glycines. Loci were chosen from a closely related species (Aphis gossypii) and a more distantly related species (Aphis fabae). We found nine loci to be polymorphic among Korean and North American populations. Overall expected heterozygosity was moderate (average: 0.47; range: 0-1), although populations substantially differed in deviations from Hardy-Weinberg equilibrium. These loci will be valuable in characterizing population differentiation, migration and adaptation in an economically important pest of soybeans.

Genetic linkage mapping of the soybean aphid resistance gene in PI 243540.

The soybean aphid (Aphis glycines Matsumura) is a pest of soybean [Glycine max (L.) Merr.] in many soybean growing countries of the world, mainly in Asia and North America. A single dominant gene in PI 243540 confers resistance to the soybean aphid. The objectives of this study were to identify simple sequence repeat (SSR) markers closely linked to the gene in PI 243540 and to position the gene on the consensus soybean genetic map. One hundred eighty-four F2 plants and their F2:3 families from a cross between the susceptible cultivar Wyandot and PI 243540, and the two parental lines were screened with the Ohio biotype of soybean aphid using greenhouse choice tests. A SSR marker from each 10-cM section of the consensus soybean map was selected for bulked segregant analysis (BSA) to identify the tentative genomic location of the gene. The BSA technique was useful to localize the gene to a genomic region in soybean linkage group (LG) F. The entire F2 population was then screened with polymorphic SSR markers from this genomic region and a linkage map with nine SSR markers flanking the gene was constructed. The aphid resistance gene was positioned in the interval between SSR markers Satt334 and Sct_033 on LG F. These SSR markers will be useful for marker assisted selection of this gene. The aphid resistance gene from PI 243540 mapped to a different linkage group than the only named soybean aphid resistance gene, Rag1, from 'Dowling'. Also, the responses of the two known biotypes of the soybean aphid to the gene from PI 243540 and Rag1 were different. Thus, the aphid resistance gene from PI 243540 was determined to be a new and independent gene that has been named Rag2.

Mapping of putative quantitative trait loci controlling the total oligosaccharide and sucrose content of Glycine max seeds.

Although oligosaccharides and sucrose are very important nutritional components of soybean seeds, little information is available about inheritance of oligosaccharide and sucrose content. The objective of this study was to identify quantitative trait loci (QTLs) that determine the oligosaccharide and sucrose content of soybean. The 117 F(2:10) recombinant inbred lines developed from a cross of "Keunolkong" and "Shinpaldalkong" were used. Narrow-sense heritability estimates, on a plot mean basis, of oligosaccharide and sucrose content were 79.07 and 74.84%, respectively. Four QTLs for oligosaccharide content were located on linkage groups (LG) C2, H, J, and L. Sucrose content was related with two QTLs located on LG H and J. Total oligosaccharide and sucrose content have two common QTLs on LG H and J.