J. Grover Shannon

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait

BackgroundThe alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B.ResultsThree polymorphisms found in the FAD2-1B alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant FAD2-1B alleles were associated with an increase in oleic acid levels, although the FAD2-1B mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing FAD2-1A mutations were combined with the novel mutant FAD2-1B alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles.ConclusionsWe were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the FAD2-1B alleles were developed into highly efficient molecular markers for tracking the mutant alleles. The resources described here for the creation of high oleic acid soybeans provide a framework to efficiently develop soybean varieties to meet changing market demands.

Genetic control of soybean seed isoflavone content: importance of statistical model and epistasis in complex traits

A major objective for geneticists is to decipher genetic architecture of traits associated with agronomic importance. However, a majority of such traits are complex, and their genetic dissection has been traditionally hampered not only by the number of minor-effect quantitative trait loci (QTL) but also by genome-wide interacting loci with little or no individual effect. Soybean (Glycine max [L.] Merr.) seed isoflavonoids display a broad range of variation, even in genetically stabilized lines that grow in a fixed environment, because their synthesis and accumulation are affected by many biotic and abiotic factors. Due to this complexity, isoflavone QTL mapping has often produced conflicting results especially with variable growing conditions. Herein, we comparatively mapped soybean seed isoflavones genistein, daidzein, and glycitein by using several of the most commonly used mapping approaches: interval mapping, composite interval mapping, multiple interval mapping and a mixed-model based composite interval mapping. In total, 26 QTLs, including many novel regions, were found bearing additive main effects in a population of RILs derived from the cross between Essex and PI 437654. Our comparative approach demonstrates that statistical mapping methodologies are crucial for QTL discovery in complex traits. Despite a previous understanding of the influence of additive QTL on isoflavone production, the role of epistasis is not well established. Results indicate that epistasis, although largely dependent on the environment, is a very important genetic component underlying seed isoflavone content, and suggest epistasis as a key factor causing the observed phenotypic variability of these traits in diverse environments.

A novel FAD2-1 A allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content.

The alteration of fatty acid profiles in soybean to improve soybean oil quality has been a long-time goal of soybean researchers. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of soybean oil compared to other oils. In the lipid biosynthetic pathway, the enzyme fatty acid desaturase 2 (FAD2) is responsible for the conversion of oleic acid precursors to linoleic acid precursors in developing soybean seeds. Two genes encoding FAD2-1A and FAD2-1B were identified to be expressed specifically in seeds during embryogenesis and have been considered to hold an important role in controlling the seed oleic acid content. A total of 22 soybean plant introduction (PI) lines identified to have an elevated oleic acid content were characterized for sequence mutations in the FAD 2-1A and FAD2-1B genes. PI 603452 was found to contain a deletion of a nucleotide in the second exon of FAD2-1A. These important SNPs were used in developing molecular marker genotyping assays. The assays appear to be a reliable and accurate tool to identify the FAD 2-1A and FAD2-1B genotype of wild-type and mutant plants. PI 603452 was subsequently crossed with PI 283327, a soybean line that has a mutation in FAD2-1B. Interestingly, soybean lines carrying both homozygous insertion/deletion mutation (indel) FAD2-1A alleles and mutant FAD2-1B alleles have an average of 82–86% oleic acid content, compared to 20% in conventional soybean, and low levels of linoleic and linolenic acids. The newly identified indel mutation in the FAD2-1A gene offers a simple method for the development of high oleic acid commercial soybean varieties.

Genomic-assisted haplotype analysis and the development of high-throughput SNP markers for salinity tolerance in soybean.

Soil salinity is a limiting factor of crop yield. The soybean is sensitive to soil salinity, and a dominant gene, Glyma03g32900 is primarily responsible for salt-tolerance. The identification of high throughput and robust markers as well as the deployment of salt-tolerant cultivars are effective approaches to minimize yield loss under saline conditions. We utilized high quality (15x) whole-genome resequencing (WGRS) on 106 diverse soybean lines and identified three major structural variants and allelic variation in the promoter and genic regions of the GmCHX1 gene. The discovery of single nucleotide polymorphisms (SNPs) associated with structural variants facilitated the design of six KASPar assays. Additionally, haplotype analysis and pedigree tracking of 93 U.S. ancestral lines were performed using publically available WGRS datasets. Identified SNP markers were validated, and a strong correlation was observed between the genotype and salt treatment phenotype (leaf scorch, chlorophyll content and Na+ accumulation) using a panel of 104 soybean lines and, an interspecific bi-parental population (F8) from PI483463 x Hutcheson. These markers precisely identified salt-tolerant/sensitive genotypes (>91%), and different structural-variants (>98%). These SNP assays, supported by accurate phenotyping, haplotype analyses and pedigree tracking information, will accelerate marker-assisted selection programs to enhance the development of salt-tolerant soybean cultivars.

Recent Advances in Breeding For Drought and Salt Stress Tolerance in Soybean

Drought and salinity are two important abiotic factors limiting soybean production worldwide and drought alone accounts for about 40% crop loss. Irrigation and soil reclamation are not economically viable options for soybean production under drought and salinity. Hence, genetic improvement for drought and salt tolerance are cost effective. Conventional breeding has made a significant contribution to soybean improvement in the last 50 years. Through conventional breeding, it is easy to manipulate simply inherited qualitative traits which are less sensitive to environmental variation, but quantitative traits like yield or tolerance to abiotic stress are significantly influenced by environment. Most agronomically important traits are quantitatively inherited and are difficult to improve through conventional breeding. Molecular marker technologies can dissect quantitative traits into individual components, known as quantitative trait loci enabling marker assisted selection of desired traits in much shorter time avoiding labor intensive, conventional, phenotypic selection. A molecular breeding approach can supplement the conventional breeding system. Well developed molecular genetic maps, functional genomic resources, and other molecular tools are available for soybean. Effective use of these resources will allow a greater understanding of basic mechanisms of tolerance to abiotic stress. Integration of these genomic tools coupled with well-designed breeding strategies will help to develop soybean varieties with higher tolerance to drought and salt

Variability in distribution and virulence phenotypes of Heterodera glycines in missouri during 2005

The soybean cyst nematode, Heterodera glycines, is a serious economic threat to soybean producers in Missouri. Periodic monitoring for the presence, population densities, and virulence phenotypes of H. glycines is essential for determining crop losses and devising management strategies implementing the use of resistant cultivars. A survey using area-frame sampling was conducted to determine the distribution and virulence phenotypes of H. glycines in Missouri during 2005. Two samples from each of 125 fields representing eight geographical regions of Missouri were collected; 243 samples were processed for extraction of cysts and eggs. In all, 49% of samples had detectable cyst nematode populations, which ranged from 138 to 85,250 eggs per 250 cm3 of soil. Race and H. glycines type tests were conducted on populations from 45 samples. Nearly 80% of the populations that were tested, irrespective of the region, were virulent on the indicator line plant introduction (PI) 88788, which is the source of resistance for most H. glycines-resistant cultivars. More than 70% of populations could reproduce on the indicator lines PI 88788, PI 209332, and PI 548316 (Cloud), indicating that soybean cultivars with resistance derived from these sources need to be carefully monitored and used only in rotation with nonhost crops and soybean cultivars with resistance from other sources. Approximately one-third of the populations, primarily in the southern regions of Missouri, could reproduce on PI 548402 (Peking), another common source of resistance. Fewer than 10% of the populations could reproduce on PI 90763, PI 437654, PI 89772, or PI 438489B, suggesting that these sources of resistance should be used in soybean breeding programs to develop H. glycines-resistant soybean cultivars.

Inheritance of Salt Tolerance in Wild Soybean (Glycine soja Sieb. and Zucc.) Accession PI483463

Tolerant soybean (Glycine max [L.] Merr.) cultivars aid in reducing salt damage in problem fields. New genes are important to reduce losses from salt injury. Objectives of this study were to determine inheritance of salt tolerance in wild soybean (Glycine soja Sieb. and Zucc.) PI483463 and to test allelism of tolerance genes from genotypes PI483463 and S-100, a common ancestor of southern in US cultivars. Tolerant (T) PI483463 was crossed to sensitive (S) cultivar Hutcheson to study inheritance. PI483463 (T) was crossed with S-100 (T) to test for allelism. Parents, F(1) plants, F(2) populations, and F(2:3) lines were assayed in a 100 mM salt solution to determine tolerance. F(2) from T x S cross segregated 3(T):1 (S) and the F(2:3) lines responded 1 (T): 2 (segregating):1 (S). F(2) plants from PI483463 (T) x S-100 (T) segregated 15 (T):1 (S) indicating different genes from the 2 sources. Results showed that G. soja line PI483463 had a single dominant gene for salt tolerance, which was different than the gene in G. max line S-100. The symbol, Ncl2, was designated for this new salt tolerance allele.

Landscape of genomic diversity and trait discovery in soybean

Cultivated soybean [Glycine max (L.) Merr.] is a primary source of vegetable oil and protein. We report a landscape analysis of genome-wide genetic variation and an association study of major domestication and agronomic traits in soybean. A total of 106 soybean genomes representing wild, landraces, and elite lines were re-sequenced at an average of 17x depth with a 97.5% coverage. Over 10 million high-quality SNPs were discovered, and 35.34% of these have not been previously reported. Additionally, 159 putative domestication sweeps were identified, which includes 54.34 Mbp (4.9%) and 4,414 genes; 146 regions were involved in artificial selection during domestication. A genome-wide association study of major traits including oil and protein content, salinity, and domestication traits resulted in the discovery of novel alleles. Genomic information from this study provides a valuable resource for understanding soybean genome structure and evolution, and can also facilitate trait dissection leading to sequencing-based molecular breeding.

Loss-of-function mutations affecting a specific Glycine max R2R3 MYB transcription factor result in brown hilum and brown seed coats

BackgroundAlthough modern soybean cultivars feature yellow seed coats, with the only color variation found at the hila, the ancestral condition is black seed coats. Both seed coat and hila coloration are due to the presence of phenylpropanoid pathway derivatives, principally anthocyanins. The genetics of soybean seed coat and hilum coloration were first investigated during the resurgence of genetics during the 1920s, following the rediscovery of Mendels work. Despite the inclusion of this phenotypic marker into the extensive genetic maps developed for soybean over the last twenty years, the genetic basis behind the phenomenon of brown seed coats (the R locus) has remained undetermined until now.ResultsIn order to identify the gene responsible for the r gene effect (brown hilum or seed coat color), we utilized bulk segregant analysis and identified recombinant lines derived from a population segregating for two phenotypically distinct alleles of the R locus. Fine mapping was accelerated through use of a novel, bioinformatically determined set of Simple Sequence Repeat (SSR) markers which allowed us to delimit the genomic region containing the r gene to less than 200 kbp, despite the use of a mapping population of only 100 F6 lines. Candidate gene analysis identified a loss of function mutation affecting a seed coat-specific expressed R2R3 MYB transcription factor gene (Glyma09g36990) as a strong candidate for the brown hilum phenotype. We observed a near perfect correlation between the mRNA expression levels of the functional R gene candidate and an UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) gene, which is responsible for the final step in anthocyanin biosynthesis. In contrast, when a null allele of Glyma09g36990 is expressed no upregulation of the UF3GT gene was found.ConclusionsWe discovered an allelic series of four loss of function mutations affecting our R locus gene candidate. The presence of any one of these mutations was perfectly correlated with the brown seed coat/hilum phenotype in a broadly distributed survey of soybean cultivars, barring the presence of the epistatic dominant I allele or gray pubescence, both of which can mask the effect of the r allele, resulting in yellow or buff hila. These findings strongly suggest that loss of function for one particular seed coat-expressed R2R3 MYB gene is responsible for the brown seed coat/hilum phenotype in soybean.

Genomic-assisted phylogenetic analysis and marker development for next generation soybean cyst nematode resistance breeding.

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is a serious soybean pest. The use of resistant cultivars is an effective approach for preventing yield loss. In this study, 19,652 publicly available soybean accessions that were previously genotyped with the SoySNP50K iSelect BeadChip were used to evaluate the phylogenetic diversity of SCN resistance genes Rhg1 and Rhg4 in an attempt to identify novel sources of resistance. The sequence information of soybean lines was utilized to develop KASPar (KBioscience Competitive Allele-Specific PCR) assays from single nucleotide polymorphisms (SNPs) of Rhg1, Rhg4, and other novel quantitative trait loci (QTL). These markers were used to genotype a diverse set of 95 soybean germplasm lines and three recombinant inbred line (RIL) populations. SNP markers from the Rhg1 gene were able to differentiate copy number variation (CNV), such as resistant-high copy (PI 88788-type), low copy (Peking-type), and susceptible-single copy (Williams 82) numbers. Similarly, markers for the Rhg4 gene were able to detect Peking-type (resistance) genotypes. The phylogenetic information of SCN resistance loci from a large set of soybean accessions and the gene/QTL specific markers that were developed in this study will accelerate SCN resistance breeding programs.