Genome-wide association study and genomic selection for soybean chlorophyll content associated with soybean cyst nematode tolerance.

BACKGROUND: Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, has been one of the most devastating pathogens affecting soybean production. In the United States alone, SCN damage accounted for more than $1 billion loss annually. With a narrow genetic background of the currently available SCN-resistant commercial cultivars, high risk of resistance breakdown can occur. The objectives of this study were to conduct a genome-wide association study (GWAS) to identify QTL, SNP markers, and candidate genes associated with soybean leaf chlorophyll content tolerance to SCN infection, and to carry out a genomic selection (GS) study for the chlorophyll content tolerance. RESULTS: A total of 172 soybean genotypes were evaluated for the effect of SCN HG Type 1.2.3.5.6.7 (race 4) on soybean leaf chlorophyll. The soybean lines were genotyped using a total of 4089 filtered and high-quality SNPs. Results showed that (1) a large variation in SCN tolerance based on leaf chlorophyll content indices (CCI); (2) a total of 22, 14, and 16 SNPs associated with CCI of non-SCN-infected plants, SCN-infected plants, and reduction of CCI SCN, respectively; (3) a new locus of chlorophyll content tolerance to SCN mapped on chromosome 3; (4) candidate genes encoding for Leucine-rich repeat protein, plant hormone signaling molecules, and biomolecule transporters; and (5) an average GS accuracy ranging from 0.31 to 0.46 with all SNPs and varying from 0.55 to 0.76 when GWAS-derived SNP markers were used across five models. This study demonstrated the potential of using genome-wide selection to breed chlorophyll-content-tolerant soybean for managing SCN. CONCLUSIONS: In this study, soybean accessions with higher CCI under SCN infestation, and molecular markers associated with chlorophyll content related to SCN were identified. In addition, a total of 15 candidate genes associated with chlorophyll content tolerance to SCN in soybean were also identified. These candidate genes will lead to a better understanding of the molecular mechanisms that control chlorophyll content tolerance to SCN in soybean. Genomic selection analysis of chlorophyll content tolerance to SCN showed that using significant SNPs obtained from GWAS could provide better GS accuracy.

'MN1606SP' by 'Spencer' filial soybean population reveals novel quantitative trait loci and interactions among loci conditioning SDS resistance.

KEY MESSAGE: Four novel QTL and interactions among QTL were identified in this research, using as a parent line the most SDS-resistant genotype within soybean cultivars of the US early maturity groups. Soybean sudden death syndrome (SDS) reduces soybean yield in most of the growing areas of the world. The causal agent of SDS, soilborne fungus Fusarium virguliforme (Fv), releases phytotoxins taken up by the plant to produce chlorosis and necrosis in the leaves. Planting resistant cultivars is the most successful management practice to control the disease. The objective of this study was to identify quantitative trait loci (QTL) associated with the resistance response of MN1606SP to SDS. A mapping population of F 2:3 lines created by crossing the highly resistant cultivar 'MN1606SP' and the susceptible cultivar 'Spencer' was phenotyped in the greenhouse at three different planting times, each with three replications. Plants were artificially inoculated using SDS infested sorghum homogeneously mixed with the soil. Data were collected on three disease criteria, foliar disease incidence (DI), foliar leaf scorch disease severity (DS), and root rot severity. Disease index (DX) was calculated as DI × DS. Ten QTL were identified for the different disease assessment criteria, three for DI, four for DX, and three for root rot severity. Three QTL identified for root rot severity and one QTL for disease incidence are considered novel, since no previous reports related to these QTL are available. Among QTL, two interactions were detected between four different QTL. The interactions suggest that resistance to SDS is not only dependent on additive gene effects. The novel QTL and the interactions observed in this study will be useful to soybean breeders for improvement of SDS resistance in soybean germplasm.

Genetic basis of soybean adaptation to North American vs. Asian mega-environments in two independent populations from Canadian × Chinese crosses.

One of the goals of plant breeding is to increase yield with improved quality characters. Plant introductions (PI) are a rich source of favorable alleles that could improve different characters in modern soybean [Glycine max (L.) Merril] including yield. The objectives of this study were to identify yield QTL underlying the genetic basis for differential adaptation of soybeans to the Canadian, United States or Chinese mega-environments (ME) and to evaluate the relationship and colocalization between yield and agronomic traits QTL. Two crosses between high-yielding Canadian cultivars and elite Chinese cultivars, OAC Millennium × Heinong 38 and Pioneer 9071 × #8902, were used to develop two recombinant inbred line (RIL) populations. Both populations were evaluated at different locations in Ontario, Canada; Minnesota, United States (US), Heilongjiang and Jilin, China, in 2009 and 2010. Significant variation for yield was observed among the RILs of both populations across the three hypothetical ME. Two yield QTL (linked to the interval Satt364-Satt591 and Satt277) and one yield QTL (linked to marker Sat_341) were identified by single-factor ANOVA and interval mapping across all ME in populations 1 and 2, respectively. The most frequent top ten high-yielding lines across all ME carried most of the high-yielding alleles of the QTL that were identified in two and three ME. Both parents contributed favorable alleles, which suggests that not only the adapted parent but also the PI parents are potential sources of beneficial alleles in reciprocal environments. Other QTL were detected also at two and one ME. Most of the yield QTL were co-localized with a QTL associated with an agronomic trait in one, two, or three ME in just one or in both populations. Results suggested that most of the variation observed in seed yield can be explained by the variation of different agronomic traits such a maturity, lodging and height. Novel alleles coming from PI can favorably contribute, directly or indirectly, to seed yield and the utilization of QTL detected across one, two or three ME would facilitate the new allele introgression into breeding populations in both North America and China.

Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean.

Mutagenized populations have become indispensable resources for introducing variation and studying gene function in plant genomics research. In this study, fast neutron (FN) radiation was used to induce deletion mutations in the soybean (Glycine max) genome. Approximately 120,000 soybean seeds were exposed to FN radiation doses of up to 32 Gray units to develop over 23,000 independent M2 lines. Here, we demonstrate the utility of this population for phenotypic screening and associated genomic characterization of striking and agronomically important traits. Plant variation was cataloged for seed composition, maturity, morphology, pigmentation, and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. The application of comparative genomic hybridization (CGH) to lesion-induced mutants for deletion mapping was validated on a midoleate x-ray mutant, M23, with a known FAD2-1A (for fatty acid desaturase) gene deletion. Using CGH, a subset of mutants was characterized, revealing deletion regions and candidate genes associated with phenotypes of interest. Exome resequencing and sequencing of PCR products confirmed FN-induced deletions detected by CGH. Beyond characterization of soybean FN mutants, this study demonstrates the utility of CGH, exome sequence capture, and next-generation sequencing approaches for analyses of mutant plant genomes. We present this FN mutant soybean population as a valuable public resource for future genetic screens and functional genomics research.

Genome-wide association study and genomic selection for tolerance of soybean biomass to soybean cyst nematode infestation.

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is one of the most devastating pathogens affecting soybean production in the U.S. and worldwide. The use of SCN-resistant soybean cultivars is one of the most affordable strategies to cope with SCN infestation. Because of the limited sources of SCN resistance and changes in SCN virulence phenotypes, host resistance in current cultivars has increasingly been overcome by the pathogen. Host tolerance has been recognized as an additional tool to manage the SCN. The objectives of this study were to conduct a genome-wide association study (GWAS), to identify single nucleotide polymorphism (SNP) markers, and to perform a genomic selection (GS) study for SCN tolerance in soybean based on reduction in biomass. A total of 234 soybean genotypes (lines) were evaluated for their tolerance to SCN in greenhouse using four replicates. The tolerance index (TI = 100 × Biomass of a line in SCN infested / Biomass of the line without SCN) was used as phenotypic data of SCN tolerance. GWAS was conducted using a total of 3,782 high quality SNPs. GS was performed based upon the whole set of SNPs and the GWAS-derived SNPs, respectively. Results showed that (1) a large variation in soybean TI to SCN infection among the soybean genotypes was identified; (2) a total of 35, 21, and 6 SNPs were found to be associated with SCN tolerance using the models SMR, GLM (PCA), and MLM (PCA+K) with 6 SNPs overlapping between models; (3) GS accuracy was SNP set-, model-, and training population size-dependent; and (4) genes around Glyma.06G134900, Glyma.15G097500.1, Glyma.15G100900.3, Glyma.15G105400, Glyma.15G107200, and Glyma.19G121200.1 (Table 4). Glyma.06G134900, Glyma.15G097500.1, Glyma.15G100900.3, Glyma.15G105400, and Glyma.19G121200.1 are best candidates. To the best of our knowledge, this is the first report highlighting SNP markers associated with tolerance index based on biomass reduction under SCN infestation in soybean. This research opens a new approach to use SCN tolerance in soybean breeding and the SNP markers will provide a tool for breeders to select for SCN tolerance.

Identification and Fine-Mapping of a Soybean Quantitative Trait Locus on Chromosome 5 Conferring Tolerance to Iron Deficiency Chlorosis.

CORE IDEAS: 'Fiskeby III' harbors a combination of abiotic stress traits, including iron deficiency chlorosis (IDC) tolerance. An IDC quantitative trait locus on chromosome Gm05 was identified in genome-wide association studies and biparental populations. Fine-mapping resolved a 137-kb interval containing strong candidate genes. Iron deficiency chlorosis (IDC) is an important nutrient stress for soybean [Glycine max (L.) Merr.] grown in high-pH soils. Despite numerous agronomic attempts to alleviate IDC, genetic tolerance remains the most effective preventative measure against symptoms. In this study, two association mapping populations and a biparental mapping population were used for genetic mapping of IDC tolerance. Quantitative trait loci (QTLs) were identified on chromosomes Gm03, Gm05, and Gm06. Heterogenous inbred families were developed to fine-map the Gm05 QTL, which was uniquely supported in all three mapping populations. Fine-mapping resulted in a QTL with an interval size of 137 kb on the end of the short arm of Gm05, which produced up to a 1.5-point reduction in IDC severity on a 1 to 9 scale in near isogenic lines.

Identification of differentially expressed genes between developing seeds of different soybean cultivars.

Soybean is a major source of protein and oil and a primary feedstock for biodiesel production. Research on soybean seed composition and yield has revealed that protein, oil and yield are controlled quantitatively and quantitative trait loci (QTL) have been identified for each of these traits. However, very limited information is available regarding the genetic mechanisms controlling seed composition and yield. To help address this deficiency, we used Affymetrix Soybean GeneChips® to identify genes that are differentially expressed between developing seeds of the Minsoy and Archer soybean cultivars, which differ in seed weight, yield, protein content and oil content. A total of 700 probe sets were found to be expressed at significantly different (defined as having an adjusted p-value below or equal to 0.05 and an at least 2-fold difference) levels between the two cultivars at one or more of the three developmental stages and in at least one of the two years assayed. Comparison of data from soybeans collected in two different years revealed that 97 probe sets were expressed at significantly different levels in both years. Functional annotations were assigned to 78% of these 97 probe sets based on the SoyBase Affymetrix™ GeneChip® Soybean Genome Array Annotation. Genes involved in receptor binding/activity and protein binding are overrepresented among the group of 97 probe sets that were differentially expressed in both years assayed. Probe sets involved in growth/development, signal transduction, transcription, defense/stress response and protein and lipid metabolism were also identified among the 97 probe sets and their possible implications in the regulation of agronomic traits are discussed. As the Minsoy and Archer soybean cultivars differ with respect to seed size, yield, protein content and lipid content, some of the differentially expressed probe sets identified in this study may thus play important roles in controlling these traits. Others of these probe sets may be involved in regulation of general seed development or metabolism. All microarray data and expression values after GCRMA are available at the Gene Expression Omnibus (GEO) at NCBI (http://www.ncbi.nlm.nih.gov/geo), under accession number GSE21598.

Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants.

The soybean [Glycine max (L.) Merr.] chlorophyll-deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a nonsynonymous nucleotide substitution in the third exon of a Mg-chelatase subunit gene (ChlI1a) on chromosome 13. This gene was selected as a candidate for a different yellow foliage mutant, T219H (Y11y11), that had been previously mapped to chromosome 13. Although the phenotypes of MinnGold and T219H are clearly distinct, sequencing of ChlI1a in T219H identified a different nonsynonymous mutation in the third exon, only six base pairs from the MinnGold mutation. This information, along with previously published allelic tests, were used to identify and clone a third yellow foliage mutation, CD-5, which was previously mapped to chromosome 15. This mutation was identified in the ChlI1b gene, a paralog of ChlI1a. Sequencing of the ChlI1b allele in CD-5 identified a nonsynonymous substitution in the third exon that confers an identical amino acid change as the T219H substitution at ChlI1a. Protein sequence alignments of the two Mg-chelatase subunits indicated that the sites of amino acid modification in MinnGold, T219H, and CD-5 are highly conserved among photosynthetic species. These results suggest that amino acid alterations in this critical domain may create competitive inhibitory interactions between the mutant and wild-type ChlI1a and ChlI1b proteins.

Genome resilience and prevalence of segmental duplications following fast neutron irradiation of soybean.

Fast neutron radiation has been used as a mutagen to develop extensive mutant collections. However, the genome-wide structural consequences of fast neutron radiation are not well understood. Here, we examine the genome-wide structural variants observed among 264 soybean [Glycine max (L.) Merrill] plants sampled from a large fast neutron-mutagenized population. While deletion rates were similar to previous reports, surprisingly high rates of segmental duplication were also found throughout the genome. Duplication coverage extended across entire chromosomes and often prevailed at chromosome ends. High-throughput resequencing analysis of selected mutants resolved specific chromosomal events, including the rearrangement junctions for a large deletion, a tandem duplication, and a translocation. Genetic mapping associated a large deletion on chromosome 10 with a quantitative change in seed composition for one mutant. A tandem duplication event, located on chromosome 17 in a second mutant, was found to cosegregate with a short petiole mutant phenotype, and thus may serve as an example of a morphological change attributable to a DNA copy number gain. Overall, this study provides insight into the resilience of the soybean genome, the patterns of structural variation resulting from fast neutron mutagenesis, and the utility of fast neutron-irradiated mutants as a source of novel genetic losses and gains.

Abnormal leaf formation in soybean: genetic and environmental effects.

Soybean is trifoliolate, but 4-, 5-, or 6-foliolate leaves have been reported and expression of such multi-foliolate (MF) leaf forms has been shown to be heritable. Here we analyze the genetic complexity of the MF phenotype and the dependence of its expression on the environment. Recombinant inbred (RI) segregants of soybean were grown in different environments. The frequency of plants expressing the MF phenotype as well as the frequency of nodes exhibiting MF leaves varied with both the environment and the RI segregant genotype. Growth chamber experiments supported field observations suggesting that environment (day length, temperature, etc.) at emergence influenced expression of MF during subsequent growth. Marker facilitated analyses of three RI segregant populations identified quantitative trait loci (QTLs) in 17 regions of the soybean genome. These either directly regulated MF phenotype expression, or were involved in interactions with such loci. Loci, identified in one RI population also were identifiable in another, different, RI population. Most of the loci affected both the frequency of plants expressing MF, and the number of nodes on MF plants that expressed the phenotype. However, a few loci differentiated between these two effects. Many loci affected plants in both field experiments, however, a few differentiated between the two environments. Similar patterns were observed for interactions between loci. QTLs regulating the MF phenotype were located in genome regions that also contained QTLs regulating major agronomic traits-e.g. yield, lodging, etc. This suggests that the loci involved regulate plant growth at some over-arching level, controlling multiple phenotypes or traits.