Comparison of Rps loci toward isolates, singly and combined inocula, of Phytophthora sojae in soybean PI 407985, PI 408029, PI 408097, and PI424477.

For soybean, novel single dominant Resistance to Phytophthora sojae (Rps) genes are sought to manage Phytophthora root and stem rot. In this study, resistance to P. sojae was mapped individually in four recombinant inbred line (RIL) populations derived from crosses of the susceptible cultivar Williams with PI 407985, PI 408029, PI 408097, and PI424477 previously identified as putative novel sources of disease resistance. Each population was screened for resistance with five to seven isolates of P. sojae separately over multiple F7-F10 generations. Additionally, three of the populations were screened with inoculum from the combination of three P. sojae isolates (PPR), which comprised virulence to 14 Rps genes. Over 2,300 single-nucleotide polymorphism markers were used to construct genetic maps in each population to identify chromosomal regions associated with resistance to P. sojae. Resistance segregated as one or two genes to the individual isolates and one gene toward PPR in each population and mapped to chromosomes 3, 13, or 18 in one or more of the four RIL populations. Resistance to five isolates mapped to the same chromosome 3 region are as follows: OH7 (PI 424477 and PI408029), OH12168, OH7/8, PPR (PI 407985), and 1.S.1.1 (PI408029). The resistance regions on chromosome 13 also overlapped for OH1, OH25, OH-MIA (PI424477), PPR (PI 424477, PI 407985, and PI 408097), PPR and OH0217 (PI 408097), and OH4 (PI 408029), but were distinct for each population suggesting multiple genes confer resistance. Two regions were identified on chromosome 18 but all appear to map to known loci; notably, resistance to the combined inoculum (PPR) did not map at this locus. However, there are putative new alleles in three of four populations, three on chromosome 3 and two on chromosome 13 based on mapping location but also known virulence in the isolate used. This characterization of all the Rps genes segregating in these populations to these isolates will be informative for breeding, but the combined inoculum was able to map a novel loci. Furthermore, within each of these P. sojae isolates, there was virulence to more than the described Rps genes, and the effectiveness of the novel genes requires testing in larger populations.

Identification of quantitative trait loci controlling soybean seed protein and oil content.

Soybean is a major source of seed protein and oil globally with an average composition of 40% protein and 20% oil in the seed. The goal of this study was to identify quantitative trait loci (QTL) conferring seed protein and oil content utilizing a population constructed by crossing an above average protein content line, PI 399084 to another line that had a low protein content value, PI 507429, both from the USDA soybean germplasm collection. The recombinant inbred line (RIL) population, PI 507429 x PI 399084, was evaluated in two replications over four years (2018-2021); the seeds were analyzed for seed protein and oil content using near-infrared reflectance spectroscopy. The recombinant inbred lines and the two parents were re-sequenced using genotyping by sequencing. A total of 12,761 molecular markers, which came from genotyping by sequencing, the SoySNP6k BeadChip and selected simple sequence repeat (SSR) markers from known protein QTL chromosomal regions were used for mapping. One QTL was identified on chromosome 2 explaining up to 56.8% of the variation for seed protein content and up to 43% for seed oil content. Another QTL identified on chromosome 15 explained up to 27.2% of the variation for seed protein and up to 41% of the variation for seed oil content. The protein and oil QTLs of this study and their associated molecular markers will be useful in breeding to improve nutritional quality in soybean.

Identification of Quantitative Disease Resistance Loci Toward Four Pythium Species in Soybean.

In this study, four recombinant inbred line (RIL) soybean populations were screened for their response to infection by Pythium sylvaticum, Pythium irregulare, Pythium oopapillum, and Pythium torulosum. The parents, PI 424237A, PI 424237B, PI 408097, and PI 408029, had higher levels of resistance to these species in a preliminary screening and were crossed with "Williams," a susceptible cultivar. A modified seed rot assay was used to evaluate RIL populations for their response to specific Pythium species selected for a particular population based on preliminary screenings. Over 2500 single-nucleotide polymorphism (SNP) markers were used to construct chromosomal maps to identify regions associated with resistance to Pythium species. Several minor and large effect quantitative disease resistance loci (QDRL) were identified including one large effect QDRL on chromosome 8 in the population of PI 408097 × Williams. It was identified by two different disease reaction traits in P. sylvaticum, P. irregulare, and P. torulosum. Another large effect QDRL was identified on chromosome 6 in the population of PI 408029 × Williams, and conferred resistance to P. sylvaticum and P. irregulare. These large effect QDRL will contribute toward the development of improved soybean cultivars with higher levels of resistance to these common soil-borne pathogens.

Mining germplasm panels and phenotypic datasets to identify loci for resistance to Phytophthora sojae in soybean.

Phytophthora sojae causes Phytophthora root and stem rot of soybean and has been primarily managed through deployment of qualitative Resistance to P. sojae genes (Rps genes). The effectiveness of each individual or combination of Rps gene(s) depends on the diversity and pathotypes of the P. sojae populations present. Due to the complex nature of P. sojae populations, identification of more novel Rps genes is needed. In this study, phenotypic data from previous studies of 16 panels of plant introductions (PIs) were analyzed. Panels 1 and 2 consisted of 448 Glycine max and 520 G. soja, which had been evaluated for Rps gene response with a combination of P. sojae isolates. Panels 3 and 4 consisted of 429 and 460 G. max PIs, respectively, which had been evaluated using individual P. sojae isolates with complex virulence pathotypes. Finally, Panels 5-16 (376 G. max PIs) consisted of data deposited in the USDA Soybean Germplasm Collection from evaluations with 12 races of P. sojae. Using these panels, genome-wide association (GWA) analyses were carried out by combining phenotypic and SoySNP50K genotypic data. GWA models identified two, two, six, and seven novel Rps loci with Panels 1, 2, 3, and 4, respectively. A total of 58 novel Rps loci were identified using Panels 5-16. Genetic and phenotypic dissection of these loci may lead to the characterization of novel Rps genes that can be effectively deployed in new soybean cultivars against diverse P. sojae populations.

Genetic interactions regulating seed phytate and oligosaccharides in soybean (Glycine max L.).

Two low-phytate soybean (Glycine max (L.) Merr.) mutant lines- V99-5089 (mips mutation on chromosome 11) and CX-1834 (mrp-l and mrp-n mutations on chromosomes 19 and 3, respectively) have proven to be valuable resources for breeding of low-phytate, high-sucrose, and low-raffinosaccharide soybeans, traits that are highly desirable from a nutritional and environmental standpoint. A recombinant inbred population derived from the cross CX1834 x V99-5089 provides an opportunity to study the effect of different combinations of these three mutations on soybean phytate and oligosaccharides levels. Of the 173 recombinant inbred lines tested, 163 lines were homozygous for various combinations of MIPS and two MRP loci alleles. These individuals were grouped into eight genotypic classes based on the combination of SNP alleles at the three mutant loci. The two genotypic classes that were homozygous mrp-l/mrp-n and either homozygous wild-type or mutant at the mips locus (MIPS/mrp-l/mrp-n or mips/mrp-l/mrp-n) displayed relatively similar ~55% reductions in seed phytate, 6.94 mg g -1 and 6.70 mg g-1 respectively, as compared with 15.2 mg g-1 in the wild-type MIPS/MRP-L/MRP-N seed. Therefore, in the presence of the double mutant mrp-l/mrp-n, the mips mutation did not cause a substantially greater decrease in seed phytate level. However, the nutritionally-desirable high-sucrose/low-stachyose/low-raffinose seed phenotype originally observed in soybeans homozygous for the mips allele was reversed in the presence of mrp-l/mrp-n mutations: homozygous mips/mrp-l/mrp-n seed displayed low-sucrose (7.70%), high-stachyose (4.18%), and the highest observed raffinose (0.94%) contents per gram of dry seed. Perhaps the block in phytic acid transport from its cytoplasmic synthesis site to its storage site, conditioned by mrp-l/mrp-n, alters myo-inositol flux in mips seeds in a way that restores to wild-type levels the mips conditioned reductions in raffinosaccharides. Overall this study determined the combinatorial effects of three low phytic acid causing mutations on regulation of seed phytate and oligosaccharides in soybean.

Genome-wide transcriptome analyses of developing seeds from low and normal phytic acid soybean lines.

BACKGROUND: Low phytic acid (lpa) crops are potentially eco-friendly alternative to conventional normal phytic acid (PA) crops, improving mineral bioavailability in monogastric animals as well as decreasing phosphate pollution. The lpa crops developed to date carry mutations that are directly or indirectly associated with PA biosynthesis and accumulation during seed development. These lpa crops typically exhibit altered carbohydrate profiles, increased free phosphate, and lower seedling emergence, the latter of which reduces overall crop yield, hence limiting their large-scale cultivation. Improving lpa crop yield requires an understanding of the downstream effects of the lpa genotype on seed development. Towards that end, we present a comprehensive comparison of gene-expression profiles between lpa and normal PA soybean lines (Glycine max) at five stages of seed development using RNA-Seq approaches. The lpa line used in this study carries single point mutations in a myo-inositol phosphate synthase gene along with two multidrug-resistance protein ABC transporter genes. RESULTS: RNA sequencing data of lpa and normal PA soybean lines from five seed-developmental stages (total of 30 libraries) were used for differential expression and functional enrichment analyses. A total of 4235 differentially expressed genes, including 512-transcription factor genes were identified. Eighteen biological processes such as apoptosis, glucan metabolism, cellular transport, photosynthesis and 9 transcription factor families including WRKY, CAMTA3 and SNF2 were enriched during seed development. Genes associated with apoptosis, glucan metabolism, and cellular transport showed enhanced expression in early stages of lpa seed development, while those associated with photosynthesis showed decreased expression in late developmental stages. The results suggest that lpa-causing mutations play a role in inducing and suppressing plant defense responses during early and late stages of seed development, respectively. CONCLUSIONS: This study provides a global perspective of transcriptomal changes during soybean seed development in an lpa mutant. The mutants are characterized by earlier expression of genes associated with cell wall biosynthesis and a decrease in photosynthetic genes in late stages. The biological processes and transcription factors identified in this study are signatures of lpa-causing mutations.

Metabolite Profiling of Soybean Seed Extracts from Near-Isogenic Low and Normal Phytate Lines Using Orthogonal Separation Strategies.

Untargeted metabolomic profiling using liquid chromatography-mass spectrometry (LC-MS) was applied to lipid-depleted methanolic extracts of soybean seeds utilizing orthogonal chromatographic separations (reversed-phase and hydrophilic interaction) in both positive and negative ionization modes. Four near-isogenic lines (NILs) differing in mutations for two genes encoding highly homologous multidrug resistant proteins (MRPs) were evaluated. The double mutant exhibited a low phytate phenotype, whereas the other three NILs, the two single mutants and the wild type, did not. Principal component analysis (PCA) of the four LC-MS data sets fully separated the low phytate line from the other three. While the levels of neutral oligosaccharides were the same for all lines, there were significant metabolite differences residing in the levels of malonyl isoflavones, soyasaponins, and arginine. Two methanol-soluble polypeptides were also found as differing in abundance levels, one of which was identified as the allergen Gly m 1.

Genetic variants in root architecture-related genes in a Glycine soja accession, a potential resource to improve cultivated soybean.

BACKGROUND: Root system architecture is important for water acquisition and nutrient acquisition for all crops. In soybean breeding programs, wild soybean alleles have been used successfully to enhance yield and seed composition traits, but have never been investigated to improve root system architecture. Therefore, in this study, high-density single-feature polymorphic markers and simple sequence repeats were used to map quantitative trait loci (QTLs) governing root system architecture in an inter-specific soybean mapping population developed from a cross between Glycine max and Glycine soja. RESULTS: Wild and cultivated soybean both contributed alleles towards significant additive large effect QTLs on chromosome 6 and 7 for a longer total root length and root distribution, respectively. Epistatic effect QTLs were also identified for taproot length, average diameter, and root distribution. These root traits will influence the water and nutrient uptake in soybean. Two cell division-related genes (D type cyclin and auxin efflux carrier protein) with insertion/deletion variations might contribute to the shorter root phenotypes observed in G. soja compared with cultivated soybean. Based on the location of the QTLs and sequence information from a second G. soja accession, three genes (slow anion channel associated 1 like, Auxin responsive NEDD8-activating complex and peroxidase), each with a non-synonymous single nucleotide polymorphism mutation were identified, which may also contribute to changes in root architecture in the cultivated soybean. In addition, Apoptosis inhibitor 5-like on chromosome 7 and slow anion channel associated 1-like on chromosome 15 had epistatic interactions for taproot length QTLs in soybean. CONCLUSION: Rare alleles from a G. soja accession are expected to enhance our understanding of the genetic components involved in root architecture traits, and could be combined to improve root system and drought adaptation in soybean.