Crop DNA extraction with lab-made magnetic nanoparticles.

Molecular breeding methods, such as marker-assisted selection and genomic selection, require high-throughput and cost-effective methods for isolating genomic DNA from plants, specifically from crop tissue or seed with high polysaccharides, lipids, and proteins. A quick and inexpensive high-throughput method for isolating genomic DNA from seed and leaf tissue from multiple crops was tested with a DNA isolation method that combines CTAB extraction buffer and lab-made SA-coated magnetic nanoparticles. This method is capable of isolating quality genomic DNA from leaf tissue and seeds in less than 2 hours with fewer steps than a standard CTAB extraction method. The yield of the genomic DNA was 582-729 ng per 5 leaf discs or 216-1869 ng per seed in soybean, 2.92-62.6 ng per 5 leaf discs or 78.9-219 ng per seed in wheat, and 30.9-35.4 ng per 5 leaf discs in maize. The isolated DNA was tested with multiple molecular breeding methods and was found to be of sufficient quality and quantity for PCR and targeted genotyping by sequencing methods such as molecular inversion probes (MIPs). The combination of SA-coated magnetic nanoparticles and CTAB extraction buffer is a fast, simple, and environmentally friendly, high-throughput method for both leaf tissues and seed(s) DNA preparation at low cost per sample. The DNA obtained from this method can be deployed in applied breeding programs for marker-assisted selection or genomic selection.

Recombination hotspots in soybean [Glycine max (L.) Merr.].

Recombination allows for the exchange of genetic material between two parents, which plant breeders exploit to make improved cultivars. This recombination is not distributed evenly across the chromosome. Recombination mostly occurs in euchromatic regions of the genome and even then, recombination is focused into clusters of crossovers termed recombination hotspots. Understanding the distribution of these hotspots along with the sequence motifs associated with them may lead to methods that enable breeders to better exploit recombination in breeding. To map recombination hotspots and identify sequence motifs associated with hotspots in soybean [Glycine max (L.) Merr.], two biparental recombinant inbred lines populations were genotyped with the SoySNP50k Illumina Infinium assay. A total of 451 recombination hotspots were identified in the two populations. Despite being half-sib populations, only 18 hotspots were in common between the two populations. While pericentromeric regions did exhibit extreme suppression of recombination, 27% of the detected hotspots were located in the pericentromeric regions of the chromosomes. Two genomic motifs associated with hotspots are similar to human, dog, rice, wheat, drosophila, and arabidopsis. These motifs were a CCN repeat motif and a poly-A motif. Genomic regions spanning other hotspots were significantly enriched with the tourist family of mini-inverted-repeat transposable elements that resides in <0.34% of the soybean genome. The characterization of recombination hotspots in these two large soybean biparental populations demonstrates that hotspots do occur throughout the soybean genome and are enriched for specific motifs, but their locations may not be conserved between different populations.

Development of molecular inversion probes for soybean progeny genomic selection genotyping.

Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single-nucleotide polymorphisms (SNPs). The main limitation of current methods is the cost is too high to screen breeding populations. Molecular inversion probes (MIPs) are a targeted genotyping-by-sequencing (GBS) method that could be used for soybean [Glycine max (L.) Merr.] that is both cost-effective, high-throughput, and provides high data quality to screen breeder's germplasm for genomic selection. A 1K MIP SNP set was developed for soybean with uniformly distributed markers across the genome. The SNPs were selected to maximize the number of informative markers in germplasm being tested in soybean breeding programs located in the northern-central and middle-southern regions of the United States. The 1K SNP MIP set was tested on diverse germplasm and a recombinant inbred line (RIL) population. Targeted sequencing with MIPs obtained an 85% enrichment for the targeted SNPs. The MIP genotyping accuracy was 93% overall, whereas homozygous call accuracy was 98% with <10% missing data. The accuracy of MIPs combined with its low per-sample cost makes it a powerful tool to enable genomic selection within soybean breeding programs.

Genotyping Platforms for Genome-Wide Association Studies: Options and Practical Considerations.

Genome-wide association studies (GWAS) in crops requires genotyping platforms that are capable of producing accurate high density genotyping data on hundreds of plants in a cost-effective manner. Currently there are multiple commercial platforms available that are being effectively used across crops. These platforms include genotyping arrays such as the Illumina Infinium arrays and the Applied Biosystems Axiom Arrays along with a variety of resequencing methods. These methods are being used to genotype tens of thousands of markers up to millions of markers on GWAS panels. They are being used on crops with simple genomes to crops with very complex, large, polyploid genomes. Depending on the crop and the goal of the GWAS, there are several options and practical considerations to take into account when selecting a genotyping technology to ensure that the right coverage, accuracy, and cost for the study is achieved.

Candidate Genes Modulating Reproductive Timing in Elite US Soybean Lines Identified in Soybean Alleles of Arabidopsis Flowering Orthologs With Divergent Latitude Distribution.

Adaptation of soybean cultivars to the photoperiod in which they are grown is critical for optimizing plant yield. However, despite its importance, only the major loci conferring variation in flowering time and maturity of US soybean have been isolated. By contrast, over 200 genes contributing to floral induction in the model organism Arabidopsis thaliana have been described. In this work, putative alleles of a library of soybean orthologs of these Arabidopsis flowering genes were tested for their latitudinal distribution among elite US soybean lines developed in the United States. Furthermore, variants comprising the alleles of genes with significant differences in latitudinal distribution were assessed for amino acid conservation across disparate genera to infer their impact on gene function. From these efforts, several candidate genes from various biological pathways were identified that are likely being exploited toward adaptation of US soybean to various maturity groups.

Comparing a Mixed Model Approach to Traditional Stability Estimators for Mapping Genotype by Environment Interactions and Yield Stability in Soybean [Glycine max (L.) Merr.].

Identifying genetic loci associated with yield stability has helped plant breeders and geneticists begin to understand the role and influence of genotype by environment (GxE) interactions in soybean [Glycine max (L.) Merr.] productivity, as well as other crops. Quantifying a genotype's range of performance across testing locations has been developed over decades with dozens of methodologies available. This includes directly modeling GxE interactions as part of an overall model for yield, as well as methods which generate overall yield "stability" values from multi-environment trial data. Correspondence between these methods as it pertains to the outcomes of genome wide association studies (GWAS) has not been well defined. In this study, the GWAS results for yield and yield stability were compared in 213 soybean lines across 11 environments to determine their utility and potential intersection. Both univariate and multivariate conventional stability estimates were considered alongside a mixed model for yield that fit marker by environment interactions as a random effect. One-hundred and six total QTL were discovered across all mapping results, however, genetic loci that were significant in the mixed model for grain yield that fit marker by environment interactions were completely distinct from those that were significant when mapping using traditional stability measures as a phenotype. Furthermore, 73.21% of QTL discovered in the mixed model were determined to cause a crossover interaction effect which cause genotype rank changes between environments. Overall, the QTL discovered via explicitly mapping GxE interactions also explained more yield variance that those QTL associated with differences in traditional stability estimates making their theoretical impact on selection greater. A lack of intersecting results between mapping approaches highlights the importance of examining stability in multiple contexts when attempting to manipulate GxE interactions in soybean.

Soybean BARCSoySNP6K: An assay for soybean genetics and breeding research.

The limited number of recombinant events in recombinant inbred lines suggests that for a biparental population with a limited number of recombinant inbred lines, it is unnecessary to genotype the lines with many markers. For genomic prediction and selection, previous studies have demonstrated that only 1000-2000 genome-wide common markers across all lines/accessions are needed to reach maximum efficiency of genomic prediction in populations. Evaluation of too many markers will not only increase the cost but also generate redundant information. We developed a soybean (Glycine max) assay, BARCSoySNP6K, containing 6000 markers, which were carefully chosen from the SoySNP50K assay based on their position in the soybean genome and haplotype block, polymorphism among accessions and genotyping quality. The assay includes 5000 single nucleotide polymorphisms (SNPs) from euchromatic and 1000 from heterochromatic regions. The percentage of SNPs with minor allele frequency >0.10 was 95% and 91% in the euchromatic and heterochromatic regions, respectively. Analysis of progeny from two large families genotyped with SoySNP50K versus BARCSoySNP6K showed that the position of the common markers and number of unique bins along linkage maps were consistent based on the SNPs genotyped with the two assays; however, the rate of redundant markers was dramatically reduced with the BARCSoySNP6K. The BARCSoySNP6K assay is proven as an excellent tool for detecting quantitative trait loci, genomic selection and assessing genetic relationships. The assay is commercialized by Illumina Inc. and being used by soybean breeders and geneticists and the list of SNPs in the assay is an ideal resource for SNP genotyping by targeted amplicon sequencing.

Generating High Density, Low Cost Genotype Data in Soybean [Glycine max (L.) Merr.].

Obtaining genome-wide genotype information for millions of SNPs in soybean [Glycine max (L.) Merr.] often involves completely resequencing a line at 5X or greater coverage. Currently, hundreds of soybean lines have been resequenced at high depth levels with their data deposited in the NCBI Short Read Archive. This publicly available dataset may be leveraged as an imputation reference panel in combination with skim (low coverage) sequencing of new soybean genotypes to economically obtain high-density SNP information. Ninety-nine soybean lines resequenced at an average of 17.1X were used to generate a reference panel, with over 10 million SNPs called using GATK's Haplotype Caller tool. Whole genome resequencing at approximately 1X depth was performed on 114 previously ungenotyped experimental soybean lines. Coverages down to 0.1X were analyzed by randomly subsetting raw reads from the original 1X sequence data. SNPs discovered in the reference panel were genotyped in the experimental lines after aligning to the soybean reference genome, and missing markers imputed using Beagle 4.1. Sequencing depth of the experimental lines could be reduced to 0.3X while still retaining an accuracy of 97.8%. Accuracy was inversely related to minor allele frequency, and highly correlated with marker linkage disequilibrium. The high accuracy of skim sequencing combined with imputation provides a low cost method for obtaining dense genotypic information that can be used for various genomics applications in soybean.

Genetic Architecture of Soybean Yield and Agronomic Traits.

Soybean is the world's leading source of vegetable protein and demand for its seed continues to grow. Breeders have successfully increased soybean yield, but the genetic architecture of yield and key agronomic traits is poorly understood. We developed a 40-mating soybean nested association mapping (NAM) population of 5,600 inbred lines that were characterized by single nucleotide polymorphism (SNP) markers and six agronomic traits in field trials in 22 environments. Analysis of the yield, agronomic, and SNP data revealed 23 significant marker-trait associations for yield, 19 for maturity, 15 for plant height, 17 for plant lodging, and 29 for seed mass. A higher frequency of estimated positive yield alleles was evident from elite founder parents than from exotic founders, although unique desirable alleles from the exotic group were identified, demonstrating the value of expanding the genetic base of US soybean breeding.

Genetic Characterization of the Soybean Nested Association Mapping Population.

A set of nested association mapping (NAM) families was developed by crossing 40 diverse soybean [ (L.) Merr.] genotypes to the common cultivar. The 41 parents were deeply sequenced for SNP discovery. Based on the polymorphism of the single-nucleotide polymorphisms (SNPs) and other selection criteria, a set of SNPs was selected to be included in the SoyNAM6K BeadChip for genotyping the parents and 5600 RILs from the 40 families. Analysis of the SNP profiles of the RILs showed a low average recombination rate. We constructed genetic linkage maps for each family and a composite linkage map based on recombinant inbred lines (RILs) across the families and identified and annotated 525,772 high confidence SNPs that were used to impute the SNP alleles in the RILs. The segregation distortion in most families significantly favored the alleles from the female parent, and there was no significant difference of residual heterozygosity in the euchromatic vs. heterochromatic regions. The genotypic datasets for the RILs and parents are publicly available and are anticipated to be useful to map quantitative trait loci (QTL) controlling important traits in soybean.

Genome-wide Association Mapping of Qualitatively Inherited Traits in a Germplasm Collection.

Genome-wide association (GWA) has been used as a tool for dissecting the genetic architecture of quantitatively inherited traits. We demonstrate here that GWA can also be highly useful for detecting many major genes governing categorically defined phenotype variants that exist for qualitatively inherited traits in a germplasm collection. Genome-wide association mapping was applied to categorical phenotypic data available for 10 descriptive traits in a collection of ∼13,000 soybean [ (L.) Merr.] accessions that had been genotyped with a 50,000 single nucleotide polymorphism (SNP) chip. A GWA on a panel of accessions of this magnitude can offer substantial statistical power and mapping resolution, and we found that GWA mapping resulted in the identification of strong SNP signals for 24 classical genes as well as several heretofore unknown genes controlling the phenotypic variants in those traits. Because some of these genes had been cloned, we were able to show that the narrow GWA mapping SNP signal regions that we detected for the phenotypic variants had chromosomal bp spans that, with just one exception, overlapped the bp region of the cloned genes, despite local variation in SNP number and nonuniform SNP distribution in the chip set.

Molecular Characterization of Resistance to Soybean Rust (Phakopsora pachyrhizi Syd. & Syd.) in Soybean Cultivar DT 2000 (PI 635999).

Resistance to soybean rust (SBR), caused by Phakopsora pachyrhizi Syd. & Syd., has been identified in many soybean germplasm accessions and is conferred by either dominant or recessive genes that have been mapped to six independent loci (Rpp1 -Rpp6), but No U.S. cultivars are resistant to SBR. The cultivar DT 2000 (PI 635999) has resistance to P. pachyrhizi isolates and field populations from the United States as well as Vietnam. A F6:7 recombinant inbred line (RIL) population derived from Williams 82 × DT 2000 was used to identify genomic regions associated with resistance to SBR in the field in Ha Noi, Vietnam, and in Quincy, Florida, in 2008. Bulked segregant analysis (BSA) was conducted using the soybean single nucleotide polymorphism (SNP) USLP 1.0 panel along with simple sequence repeat (SSR) markers to detect regions of the genome associated with resistance. BSA identified four BARC_SNP markers near the Rpp3 locus on chromosome (Chr.) 6. Genetic analysis identified an additional genomic region around the Rpp4 locus on Chr. 18 that was significantly associated with variation in the area under disease progress curve (AUDPC) values and sporulation in Vietnam. Molecular markers tightly linked to the DT 2000 resistance alleles on Chrs. 6 and 18 will be useful for marker-assisted selection and backcrossing in order to pyramid these genes with other available SBR resistance genes to develop new varieties with enhanced and durable resistance to SBR.

Multi-Population Selective Genotyping to Identify Soybean [Glycine max (L.) Merr.] Seed Protein and Oil QTLs.

Plant breeders continually generate ever-higher yielding cultivars, but also want to improve seed constituent value, which is mainly protein and oil, in soybean [Glycine max (L.) Merr.]. Identification of genetic loci governing those two traits would facilitate that effort. Though genome-wide association offers one such approach, selective genotyping of multiple biparental populations offers a complementary alternative, and was evaluated here, using 48 F2:3 populations (n = âˆ¼224 plants) created by mating 48 high protein germplasm accessions to cultivars of similar maturity, but with normal seed protein content. All F2:3 progeny were phenotyped for seed protein and oil, but only 22 high and 22 low extreme progeny in each F2:3 phenotypic distribution were genotyped with a 1536-SNP chip (ca 450 bimorphic SNPs detected per mating). A significant quantitative trait locus (QTL) on one or more chromosomes was detected for protein in 35 (73%), and for oil in 25 (52%), of the 48 matings, and these QTL exhibited additive effects of ≥ 4 g kg(-1) and R(2) values of 0.07 or more. These results demonstrated that a multiple-population selective genotyping strategy, when focused on matings between parental phenotype extremes, can be used successfully to identify germplasm accessions possessing large-effect QTL alleles. Such accessions would be of interest to breeders to serve as parental donors of those alleles in cultivar development programs, though 17 of the 48 accessions were not unique in terms of SNP genotype, indicating that diversity among high protein accessions in the germplasm collection is less than what might ordinarily be assumed.

Construction of high resolution genetic linkage maps to improve the soybean genome sequence assembly Glyma1.01.

BACKGROUND: A landmark in soybean research, Glyma1.01, the first whole genome sequence of variety Williams 82 (Glycine max L. Merr.) was completed in 2010 and is widely used. However, because the assembly was primarily built based on the linkage maps constructed with a limited number of markers and recombinant inbred lines (RILs), the assembled sequence, especially in some genomic regions with sparse numbers of anchoring markers, needs to be improved. Molecular markers are being used by researchers in the soybean community, however, with the updating of the Glyma1.01 build based on the high-resolution linkage maps resulting from this research, the genome positions of these markers need to be mapped. RESULTS: Two high density genetic linkage maps were constructed based on 21,478 single nucleotide polymorphism loci mapped in the Williams 82 x G. soja (Sieb. & Zucc.) PI479752 population with 1083 RILs and 11,922 loci mapped in the Essex x Williams 82 population with 922 RILs. There were 37 regions or single markers where marker order in the two populations was in agreement but was not consistent with the physical position in the Glyma1.01 build. In addition, 28 previously unanchored scaffolds were positioned. Map data were used to identify false joins in the Glyma1.01 assembly and the corresponding scaffolds were broken and reassembled to the new assembly, Wm82.a2.v1. Based upon the plots of the genetic on physical distance of the loci, the euchromatic and heterochromatic regions along each chromosome in the new assembly were delimited. Genomic positions of the commonly used markers contained in BARCSOYSSR_1.0 database and the SoySNP50K BeadChip were updated based upon the Wm82.a2.v1 assembly. CONCLUSIONS: The information will facilitate the study of recombination hot spots in the soybean genome, identification of genes or quantitative trait loci controlling yield, seed quality and resistance to biotic or abiotic stresses as well as other genetic or genomic research.

SNP Assay Development for Linkage Map Construction, Anchoring Whole-Genome Sequence, and Other Genetic and Genomic Applications in Common Bean.

A total of 992,682 single-nucleotide polymorphisms (SNPs) was identified as ideal for Illumina Infinium II BeadChip design after sequencing a diverse set of 17 common bean (Phaseolus vulgaris L) varieties with the aid of next-generation sequencing technology. From these, two BeadChips each with >5000 SNPs were designed. The BARCBean6K_1 BeadChip was selected for the purpose of optimizing polymorphism among market classes and, when possible, SNPs were targeted to sequence scaffolds in the Phaseolus vulgaris 14× genome assembly with sequence lengths >10 kb. The BARCBean6K_2 BeadChip was designed with the objective of anchoring additional scaffolds and to facilitate orientation of large scaffolds. Analysis of 267 F2 plants from a cross of varieties Stampede × Red Hawk with the two BeadChips resulted in linkage maps with a total of 7040 markers including 7015 SNPs. With the linkage map, a total of 432.3 Mb of sequence from 2766 scaffolds was anchored to create the Phaseolus vulgaris v1.0 assembly, which accounted for approximately 89% of the 487 Mb of available sequence scaffolds of the Phaseolus vulgaris v0.9 assembly. A core set of 6000 SNPs (BARCBean6K_3 BeadChip) with high genotyping quality and polymorphism was selected based on the genotyping of 365 dry bean and 134 snap bean accessions with the BARCBean6K_1 and BARCBean6K_2 BeadChips. The BARCBean6K_3 BeadChip is a useful tool for genetics and genomics research and it is widely used by breeders and geneticists in the United States and abroad.

Fingerprinting Soybean Germplasm and Its Utility in Genomic Research.

The United States Department of Agriculture, Soybean Germplasm Collection includes 18,480 domesticated soybean and 1168 wild soybean accessions introduced from 84 countries or developed in the United States. This collection was genotyped with the SoySNP50K BeadChip containing greater than 50K single-nucleotide polymorphisms. Redundant accessions were identified in the collection, and distinct genetic backgrounds of soybean from different geographic origins were observed that could be a unique resource for soybean genetic improvement. We detected a dramatic reduction of genetic diversity based on linkage disequilibrium and haplotype structure analyses of the wild, landrace, and North American cultivar populations and identified candidate regions associated with domestication and selection imposed by North American breeding. We constructed the first soybean haplotype block maps in the wild, landrace, and North American cultivar populations and observed that most recombination events occurred in the regions between haplotype blocks. These haplotype maps are crucial for association mapping aimed at the identification of genes controlling traits of economic importance. A case-control association test delimited potential genomic regions along seven chromosomes that most likely contain genes controlling seed weight in domesticated soybean. The resulting dataset will facilitate germplasm utilization, identification of genes controlling important traits, and will accelerate the creation of soybean varieties with improved seed yield and quality.

A reference genome for common bean and genome-wide analysis of dual domestications.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for human consumption and has a role in sustainable agriculture owing to its ability to fix atmospheric nitrogen. We assembled 473 Mb of the 587-Mb genome and genetically anchored 98% of this sequence in 11 chromosome-scale pseudomolecules. We compared the genome for the common bean against the soybean genome to find changes in soybean resulting from polyploidy. Using resequencing of 60 wild individuals and 100 landraces from the genetically differentiated Mesoamerican and Andean gene pools, we confirmed 2 independent domestications from genetic pools that diverged before human colonization. Less than 10% of the 74 Mb of sequence putatively involved in domestication was shared by the two domestication events. We identified a set of genes linked with increased leaf and seed size and combined these results with quantitative trait locus data from Mesoamerican cultivars. Genes affected by domestication may be useful for genomics-enabled crop improvement.

Identification and validation of quantitative trait loci for seed yield, oil and protein contents in two recombinant inbred line populations of soybean.

Soybean seeds contain high levels of oil and protein, and are the important sources of vegetable oil and plant protein for human consumption and livestock feed. Increased seed yield, oil and protein contents are the main objectives of soybean breeding. The objectives of this study were to identify and validate quantitative trait loci (QTLs) associated with seed yield, oil and protein contents in two recombinant inbred line populations, and to evaluate the consistency of QTLs across different environments, studies and genetic backgrounds. Both the mapping population (SD02-4-59 × A02-381100) and validation population (SD02-911 × SD00-1501) were phenotyped for the three traits in multiple environments. Genetic analysis indicated that oil and protein contents showed high heritabilities while yield exhibited a lower heritability in both populations. Based on a linkage map constructed previously with the mapping population and using composite interval mapping and/or interval mapping analysis, 12 QTLs for seed yield, 16 QTLs for oil content and 11 QTLs for protein content were consistently detected in multiple environments and/or the average data over all environments. Of the QTLs detected in the mapping population, five QTLs for seed yield, eight QTLs for oil content and five QTLs for protein content were confirmed in the validation population by single marker analysis in at least one environment and the average data and by ANOVA over all environments. Eight of these validated QTLs were newly identified. Compared with the other studies, seven QTLs for seed yield, eight QTLs for oil content and nine QTLs for protein content further verified the previously reported QTLs. These QTLs will be useful for breeding higher yield and better quality cultivars, and help effectively and efficiently improve yield potential and nutritional quality in soybean.

A genome-wide association study of seed protein and oil content in soybean.

BACKGROUND: Association analysis is an alternative to conventional family-based methods to detect the location of gene(s) or quantitative trait loci (QTL) and provides relatively high resolution in terms of defining the genome position of a gene or QTL. Seed protein and oil concentration are quantitative traits which are determined by the interaction among many genes with small to moderate genetic effects and their interaction with the environment. In this study, a genome-wide association study (GWAS) was performed to identify quantitative trait loci (QTL) controlling seed protein and oil concentration in 298 soybean germplasm accessions exhibiting a wide range of seed protein and oil content. RESULTS: A total of 55,159 single nucleotide polymorphisms (SNPs) were genotyped using various methods including Illumina Infinium and GoldenGate assays and 31,954 markers with minor allele frequency >0.10 were used to estimate linkage disequilibrium (LD) in heterochromatic and euchromatic regions. In euchromatic regions, the mean LD (r2) rapidly declined to 0.2 within 360 Kbp, whereas the mean LD declined to 0.2 at 9,600 Kbp in heterochromatic regions. The GWAS results identified 40 SNPs in 17 different genomic regions significantly associated with seed protein. Of these, the five SNPs with the highest associations and seven adjacent SNPs were located in the 27.6-30.0 Mbp region of Gm20. A major seed protein QTL has been previously mapped to the same location and potential candidate genes have recently been identified in this region. The GWAS results also detected 25 SNPs in 13 different genomic regions associated with seed oil. Of these markers, seven SNPs had a significant association with both protein and oil. CONCLUSIONS: This research indicated that GWAS not only identified most of the previously reported QTL controlling seed protein and oil, but also resulted in narrower genomic regions than the regions reported as containing these QTL. The narrower GWAS-defined genome regions will allow more precise marker-assisted allele selection and will expedite positional cloning of the causal gene(s).