Genomic analysis and characterization of new loci associated with seed protein and oil content in soybeans.

Breeding for increased protein without a reduction in oil content in soybeans [Glycine max (L.) Merr.] is a challenge for soybean breeders but an expected goal. Many efforts have been made to develop new soybean varieties with high yield in combination with desirable protein and/or oil traits. An elite line, R05-1415, was reported to be high yielding, high protein, and low oil. Several significant quantitative trait loci (QTL) for protein and oil were reported in this line, but many of them were unstable across environments or genetic backgrounds. Thus, a new study under multiple field environments using the Infinium BARCSoySNP6K BeadChips was conducted to detect and confirm stable genomic loci for these traits. Genetic analyses consistently detected a single major genomic locus conveying these two traits with remarkably high phenotypic variation explained (R2 ), varying between 24.2% and 43.5%. This new genomic locus is located between 25.0 and 26.7 Mb, distant from the previously reported QTL and did not overlap with other commonly reported QTL and the recently cloned gene Glyma.20G085100. Homolog analysis indicated that this QTL did not result from the paracentric chromosome inversion with an adjacent genomic fragment that harbors the reported QTL. The pleiotropic effect of this QTL could be a challenge for improving protein and oil simultaneously; however, a further study of four candidate genes with significant expressions in the seed developmental stages coupled with haplotype analysis may be able to pinpoint causative genes. The functionality and roles of these genes can be determined and characterized, which lay a solid foundation for the improvement of protein and oil content in soybeans.

Classification Methods and Identification of Reniform Nematode Resistance in Known Soybean Cyst Nematode-Resistant Soybean Genotypes.

Plant parasitic nematodes are a major yield-limiting factor of soybean in the United States and Canada. It has been indicated that soybean cyst nematode (SCN; Heterodera glycines Ichinohe) and reniform nematode (RN; Rotylenchulus reniformis Linford and Oliveira) resistance could be genetically related. For many years, fragmentary data have shown this relationship. This report evaluates RN reproduction on 418 plant introductions (PIs) selected from the U.S. Department of Agriculture Soybean Germplasm Collection with reported SCN resistance. The germplasm was divided into two tests of 214 PIs reported as resistant and 204 PIs reported as moderately resistant to SCN. The defining and reporting of RN resistance changed several times in the last 30 years, causing inconsistencies in RN resistance classification among multiple experiments. Comparison of four RN resistance classification methods was performed: (i) ≤10% as compared with the susceptible check, (ii) using normalized reproduction index (RI) values, and using (iii) transformed data log10(x), and (iv) transformed data log10(x + 1) in an optimal univariate k-means clustering analysis. The method of transformed data log10(x) was selected as the most accurate for classification of RN resistance. Among 418 PIs with reported SCN resistance, the log10(x) method grouped 59 PIs (15%) as resistant and 130 PIs (31%) as moderately resistant to RN. Genotyping of a subset of the most resistant PIs to both nematode species revealed their strong correlation with rhg1-a allele. This research identified genotypes with resistance to two nematode species and potential new sources of RN resistance that could be valuable to breeders in developing resistant cultivars.

Dissecting nematode resistance regions in soybean revealed pleiotropic effect of soybean cyst and reniform nematode resistance genes.

Reniform nematode (RN, Rotylenchulus reniformis Linford & Oliveira) has emerged as one of the most important plant parasitic nematodes of soybean [Glycine max (L.) Merr.]. Planting resistant varieties is the most effective strategy for nematode management. The objective of this study was to identify quantitative trait loci (QTL) for RN resistance in an exotic soybean line, PI 438489B, using two linkage maps constructed from the Universal Soybean Linkage Panel (USLP 1.0) and next-generation whole-genome resequencing (WGRS) technology. Two QTL controlling RN resistance were identified-the soybean cyst nematode (SCN, Heterodera glycines) resistance gene GmSNAP18 at the rhg1 locus and its paralog GmSNAP11. Strong association between resistant phenotype and haplotypes of the GmSNAP11 and GmSNAP18 was observed. The results indicated that GmSNAP11 possibly could have epistatic effect on GmSNAP18, or vice versa, with the presence of a significant correlation in RN resistance of rhg1-a GmSNAP18 vs. rhg1-b GmSNAP18. Most importantly, our preliminary data suggested that GmSNAP18 and GmSNAP11 proteins physically interact in planta, suggesting that they belong to the same pathway for resistance. Unlike GmSNAP18, no indication of GmSNAP11 copy number variation was found. Moreover, gene-based single nucleotide polymorphism (SNP) markers were developed for rapid detection of RN or SCN resistance at these loci. Our analysis substantiates synergic interaction between GmSNAP11 and GmSNAP18 genes and confirms their roles in RN as well as SCN resistance. These results could contribute to a better understanding of evolution and subfunctionalization of genes conferring resistance to multiple nematode species and provide a framework for further investigations.

Genetic characterization of qSCN10 from an exotic soybean accession PI 567516C reveals a novel source conferring broad-spectrum resistance to soybean cyst nematode.

KEY MESSAGE: The qSCN10 locus with broad-spectrum SCN resistance was fine-mapped to a 379-kb region on chromosome 10 in soybean accession PI 567516C. Candidate genes and potential application benefits of this locus were discussed. Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most devastating pests of soybean, causing significant yield losses worldwide every year. Genetic resistance has been the major strategy to control this pest. However, the overuse of the same genetic resistance derived primarily from PI 88788 has led to the genetic shifts in nematode populations and resulted in the reduced effectiveness in soybean resistance to SCN. Therefore, novel genetic resistance resources, especially those with broad-spectrum resistance, are needed to develop new resistant cultivars to cope with the genetic shifts in nematode populations. In this study, a quantitative trait locus (QTL) qSCN10 previously identified from a soybean landrace PI 567516C was confirmed to confer resistance to multiple SCN HG Types. This QTL was further fine-mapped to a 379-kb region. There are 51 genes in this region. Four of them are defense-related and were regulated by SCN infection, suggesting their potential role in mediating resistance to SCN. The phylogenetic and haplotype analyses of qSCN10 as well as other information indicate that this locus is different from other reported resistance QTL or genes. There was no yield drag or other unfavorable traits associated with this QTL when near-isogenic lines with and without qSCN10 were tested in a SCN-free field. Therefore, our study not only provides further insight into the genetic basis of soybean resistance to SCN, but also identifies a novel genetic resistance resource for breeding soybean for durable, broad-spectrum resistance to this pest.

Fine-mapping and characterization of qSCN18, a novel QTL controlling soybean cyst nematode resistance in PI 567516C.

KEY MESSAGE: The qSCN18 QTL from PI 56756C was confirmed and fine-mapped to improve soybean resistance to the SCN population HG Type 2.5.7 using near-isogenic lines carrying recombination crossovers within the QTL region. The QTL underlying resistance was fine-mapped to a 166-Kbp region on chromosome 18, and the candidate genes were selected based on genomic analyses. Soybean cyst nematode (SCN, Heterodera glycines, Ichinohe) is the most devastating pathogen of soybean. Understanding the genetic basis of SCN resistance is crucial for managing this parasite in the field. Two major loci, rhg1 and Rhg4, were previously characterized as valuable resources for SCN resistance. However, their continuous use has caused shifts in the virulence of SCN populations, which can overcome the resistance conferred by these two major loci. Reduced effectiveness became a major concern in the soybean industry due to continuous use of rhg1 for decades. Thus, it is imperative to identify sources of SCN resistance for durable SCN management. A novel QTL qSCN18 was identified in PI567516C. To fine-map qSCN18 and identify resistance genes, a large backcross population was developed. Nineteen near-isogenic lines (NILs) carrying recombination crossovers within the QTL region were identified. The first phase of fine-mapping narrowed the QTL region to 549-Kbp, whereas the second phase confined the region to 166-Kbp containing 23 genes. Two flanking markers, MK-1 and MK-6, were developed and validated to detect the presence of the qSCN18 resistance allele. Haplotype analysis clustered the fine-mapped qSCN18 region from PI 567516C with the cqSCN-007 locus previously mapped in the wild soybean accession PI 468916. The NILs were developed to further characterize the causal gene(s) harbored in this QTL. This study also confirmed the previously identified qSCN18. The results will facilitate marker-assisted selection (MAS) introducing the qSCN18 locus from PI 567516C into high-yielding soybean cultivars with durable resistance to SCN.

Whole-genome re-sequencing reveals the impact of the interaction of copy number variants of the rhg1 and Rhg4 genes on broad-based resistance to soybean cyst nematode.

Soybean cyst nematode (SCN) is the most devastating plant-parasitic nematode. Most commercial soybean varieties with SCN resistance are derived from PI88788. Resistance derived from PI88788 is breaking down due to narrow genetic background and SCN population shift. PI88788 requires mainly the rhg1-b locus, while 'Peking' requires rhg1-a and Rhg4 for SCN resistance. In the present study, whole genome re-sequencing of 106 soybean lines was used to define the Rhg haplotypes and investigate their responses to the SCN HG-Types. The analysis showed a comprehensive profile of SNPs and copy number variations (CNV) at these loci. CNV of rhg1 (GmSNAP18) only contributed towards resistance in lines derived from PI88788 and 'Cloud'. At least 5.6 copies of the PI88788-type rhg1 were required to confer SCN resistance, regardless of the Rhg4 (GmSHMT08) haplotype. However, when the GmSNAP18 copies dropped below 5.6, a 'Peking'-type GmSHMT08 haplotype was required to ensure SCN resistance. This points to a novel mechanism of epistasis between GmSNAP18 and GmSHMT08 involving minimum requirements for copy number. The presence of more Rhg4 copies confers resistance to multiple SCN races. Moreover, transcript abundance of the GmSHMT08 in root tissue correlates with more copies of the Rhg4 locus, reinforcing SCN resistance. Finally, haplotype analysis of the GmSHMT08 and GmSNAP18 promoters inferred additional levels of the resistance mechanism. This is the first report revealing the genetic basis of broad-based resistance to SCN and providing new insight into epistasis, haplotype-compatibility, CNV, promoter variation and its impact on broad-based disease resistance in plants.

Dissecting genomic hotspots underlying seed protein, oil, and sucrose content in an interspecific mapping population of soybean using high-density linkage mapping.

The cultivated [Glycine max (L) Merr.] and wild [Glycine soja Siebold & Zucc.] soybean species comprise wide variation in seed composition traits. Compared to wild soybean, cultivated soybean contains low protein, high oil, and high sucrose. In this study, an interspecific population was derived from a cross between G. max (Williams 82) and G. soja (PI 483460B). This recombinant inbred line (RIL) population of 188 lines was sequenced at 0.3× depth. Based on 91 342 single nucleotide polymorphisms (SNPs), recombination events in RILs were defined, and a high-resolution bin map was developed (4070 bins). In addition to bin mapping, quantitative trait loci (QTL) analysis for protein, oil, and sucrose was performed using 3343 polymorphic SNPs (3K-SNP), derived from Illumina Infinium BeadChip sequencing platform. The QTL regions from both platforms were compared, and a significant concordance was observed between bin and 3K-SNP markers. Importantly, the bin map derived from next-generation sequencing technology enhanced mapping resolution (from 1325 to 50 Kb). A total of five, nine, and four QTLs were identified for protein, oil, and sucrose content, respectively, and some of the QTLs coincided with soybean domestication-related genomic loci. The major QTL for protein and oil were mapped on Chr. 20 (qPro_20) and suggested negative correlation between oil and protein. In terms of sucrose content, a novel and major QTL were identified on Chr. 8 (qSuc_08) and harbours putative genes involved in sugar transport. In addition, genome-wide association using 91 342 SNPs confirmed the genomic loci derived from QTL mapping. A QTL-based haplotype using whole-genome resequencing of 106 diverse soybean lines identified unique allelic variation in wild soybean that could be utilized to widen the genetic base in cultivated soybean.

Advancements in breeding, genetics, and genomics for resistance to three nematode species in soybean.

KEY MESSAGE: Integration of genetic analysis, molecular biology, and genomic approaches drastically enhanced our understanding of genetic control of nematode resistance and provided effective breeding strategies in soybeans. Three nematode species, including soybean cyst (SCN, Heterodera glycine), root-knot (RKN, Meloidogyne incognita), and reniform (RN, Rotylenchulus reniformis), are the most destructive pests and have spread to soybean growing areas worldwide. Host plant resistance has played an important role in their control. This review focuses on genetic, genomic studies, and breeding efforts over the past two decades to identify and improve host resistance to these three nematode species. Advancements in genetics, genomics, and bioinformatics have improved our understanding of the molecular and genetic mechanisms of nematode resistance and enabled researchers to generate large-scale genomic resources and marker-trait associations. Whole-genome resequencing, genotyping-by-sequencing, genome-wide association studies, and haplotype analyses have been employed to map and dissect genomic locations for nematode resistance. Recently, two major SCN-resistant loci, Rhg1 and Rhg4, were cloned and other novel resistance quantitative trait loci (QTL) have been discovered. Based on these discoveries, gene-specific DNA markers have been developed for both Rhg1 and Rhg4 loci, which were useful for marker-assisted selection. With RKN resistance QTL being mapped, candidate genes responsible for RKN resistance were identified, leading to the development of functional single nucleotide polymorphism markers. So far, three resistances QTL have been genetically mapped for RN resistance. With nematode species overcoming the host plant resistance, continuous efforts in the identification and deployment of new resistance genes are required to support the development of soybean cultivars with multiple and durable resistance to these pests.

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.

Identification of quantitative trait loci underlying resistance to southern root-knot and reniform nematodes in soybean accession PI 567516C.

Soybean cyst nematode (SCN, Heterodera glycine Ichinohe), southern root-knot nematode [SRKN, Meloidogyne incognita (Kofoid and White) Chitwood] and reniform nematode (RN, Rotylenchulus reniformis Linford and Oliveira) are three important plant-parasitic pests in soybean. Previous study showed that plant introduction (PI) 567516C harbored novel quantitative trait loci (QTL) conferring SCN resistance to soybean. However, QTL underlying resistance to SRKN and RN in PI 567516C remain unknown. The objectives of this study were to identify QTL for resistance to SRKN and RN in PI 567516C. Two hundred and forty-seven F6:9 recombinant inbred lines, derived from a cross between cultivar Magellan and PI 567516C, were evaluated for resistance to SRKN and RN. Two hundred and thirty-eight simple sequence repeats and 687 single nucleotide polymorphism markers were used to construct a genetic linkage map. Three significant QTL associated with resistance to SRKN were mapped on chromosomes (Chrs.) 10, 13 and 17. Two significant QTL associated with resistance to RN were detected on Chrs. 11 and 18. Whole-genome resequencing revealed that there might be Peking-type Rhg1 in PI 567516C. Our study provides useful information to employ PI 567516C in soybean breeding in order to develop new cultivars with resistance to multiple nematodes.

Identification and evaluation of quantitative trait loci underlying resistance to multiple HG types of soybean cyst nematode in soybean PI 437655.

KEY MESSAGE: We performed QTL analysis for SCN resistance in PI 437655 in two mapping populations, characterized CNV of Rhg1 through whole-genome resequencing and evaluated the effects of QTL pyramiding to enhance resistance. Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most serious pests of soybean worldwide. PI 437655 has broader resistance to SCN HG types than PI 88788. The objectives of this study were to identify quantitative trait loci (QTL) underlying SCN resistance in PI 437655, and to evaluate the QTL for their contribution to SCN resistance. Two F6:7 recombinant inbred line populations, derived from cv. Williams 82 × PI 437655 and cv. Hutcheson × PI 437655 crosses, were evaluated for resistance to SCN HG types 1.2.5.7 (PA2), 0 (PA3), 1.3.5.6.7 (PA14), and 1.2.3.4.5.6.7 (LY2). The 1,536 SNP array was used to genotype the mapping populations and construct genetic linkage maps. Two significant QTL were consistently mapped on chromosomes (Chr.) 18 and 20 in these two populations. One QTL on Chr. 18, which corresponds to the known Rhg1 locus, contributed resistance to SCN HG types 1.2.5.7, 0, 1.3.5.6.7, and 1.2.3.4.5.6.7 (PA2, PA3, PA14, and LY2, respectively). Copy number variation (CNV) analysis by whole-genome resequencing showed that PI 437655 and PI 88788 had similar CNV at the Rhg1 locus. The QTL on Chr. 20 contributed resistance to SCN HG types 1.3.5.6.7 (PA14) and 1.2.3.4.5.6.7 (LY2). Evaluation of both QTL showed that pyramiding of Rhg1 and the QTL on Chr. 20 significantly improved the resistance to SCN HG types 1.3.5.6.7 (PA14) and 1.2.3.4.5.6.7 (LY2) in both populations. Our studies provided useful information for deploying PI 437655 as a donor for SCN resistance in soybean breeding through marker-assisted selection.

Novel quantitative trait loci for broad-based resistance to soybean cyst nematode (Heterodera glycines Ichinohe) in soybean PI 567516C.

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most destructive pest of soybean worldwide. Host plant resistance is an effective approach to control this pest. Plant introduction PI 567516C has been reported to be highly resistant to multiple-HG types of SCN. The objectives of this study were to identify and map novel quantitative trait loci (QTL) for SCN resistance to six HG types (also known as races 1, 2, 3, 5, 14, and LY1). Mapping was conducted using 250 F(2:3) progeny derived from a Magellan (susceptible) × PI 567516C (resistant) cross. F(6:7) recombinant inbred lines (RILs) developed from the F(2:3) progeny were employed to confirm the putative QTL identified. A total of 927 polymorphic simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) markers were genotyped. Following the genetic linkage analysis, permutation tests and composite interval mapping were performed to identify and map QTL. Four QTL were associated with resistance to either multiple- or single-SCN HG types. Two QTL for resistance to multiple-SCN HG types were mapped to Chromosomes 10 and 18 and have not been reported in other SCN resistance sources. New QTL were confirmed by analysis of 250 F(6:7) RILs from the same population. SSR and SNP markers closely associated with these QTL can be useful for the development of near-isogenic lines for fine-mapping and positional cloning of candidate genes for SCN resistance.