A chromosome 16 deletion conferring a high sucrose phenotype in soybean.

KEY MESSAGE: Sucrose in soybean seeds is desirable for many end-uses. Increased sucrose contents were discovered to associate with a chromosome 16 deletion resulting from fast neutron irradiation. Soybean is one of the most economically important crops in the United States. A primary end-use of soybean is for livestock feed. Therefore, genetic improvement of seed composition is one of the most important goals in soybean breeding programs. Sucrose is desired in animal feed due to its role as an easily digestible energy source. An elite soybean line was irradiated with fast neutrons and the seed from plants were screened for altered seed composition with near-infrared spectroscopy (NIR). One mutant line, G15FN-54, was found to have higher sucrose content (8-9%) than the parental line (5-6%). Comparative genomic hybridization (CGH) revealed three large deletions on chromosomes (Chrs) 10, 13, and 16 in the mutant, which were confirmed through whole genome sequencing (WGS). A bi-parental population derived from the mutant G15FN-54 and the cultivar Benning was developed to conduct a bulked segregant analysis (BSA) with SoySNP50K BeadChips, revealing that the deletion on Chr 16 might be responsible for the altered phenotype. The mapping result using the bi-parental population confirmed that the deletion on Chr 16 conferred elevated sucrose content and a total of 21 genes are located within this Chr 16 deletion. NIR and high-pressure liquid chromatography (HPLC) were used to confirm the stability of the phenotype across generations in the bi-parental population. The mutation will be useful to understand the genetic control of soybean seed sucrose content.

Mining QTLs for elevated protein and other major seed composition traits from diverse soybean germplasm.

Soybean is the world's largest source of protein for animal feed and the second largest source of vegetable oil. Improving the seed protein of soybean without negatively affecting yield and oil content is an important goal for soybean breeders. A population consisting of 132 recombinant inbred lines (RILs) was developed by crossing an elite breeding line, G00-3213 with a plant introduction, PI 594458A, with elevated protein content. In 2016 and 2017, each of the RILs was grown as a single row in Watkinsville, GA, while in 2018, the population was grown at two locations. The seed composition of RILs was analyzed with near-infrared (NIR) spectroscopy. The RIL population was genotyped using the SoySNP6k BeadChip for quantitative trait locus (QTL) mapping. Significant genotype × environment interaction was observed. QTL analyses in and across four environments identified 16, 10, 10, 16, and 5 QTLs for protein, oil, sucrose, and normalized cysteine and methionine contents, respectively. QTLs for protein content identified on chromosomes (Chrs) 3, 6, 13, and 20 were detected in multiple environments. Eight genomic regions on Chrs 3, 6, 8, 10, 13, 17, and 20 were detected that influenced two to four traits, indicating that pleiotropic or linkage effects of these loci may influence multiple seed composition traits. The results of this research provide additional genomic resources for genetic improvement of seed composition and help breeders to better understand the environmental impacts on these QTLs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01242-z.

Identification of SNP markers associated with soybean fatty acids contents by genome-wide association analyses.

Composition of fatty acids (FAs) in soybean seed is important for the quality and uses of soybean oil. Using gas chromatography, we have measured soybean FAs profiles of 621 soybean accessions (maturity groups I through IV) grown in five different environments; Columbus, OH (2015), Wooster, OH (2014 and 2015), Plymouth, NC (2015), and Urbana, IL (2015). Using publicly available SoySNP50K genotypic data and the FA profiles from this study, a genome-wide association analysis was completed with a compressed mixed linear model to identify 43 genomic regions significantly associated with a fatty acid at a genome wide significance threshold of 5%. Among these regions, one and three novel genomic regions associated with palmitic acid and stearic acid, respectively, were identified across all five environments. Additionally, nine novel environment-specific FA-related genomic regions were discovered providing new insights into the genetics of soybean FAs. Previously reported FA-related loci, such as FATB1a, SACPD-C, and KASIII, were also confirmed in this study. Our results will be useful for future functional studies and marker-assisted breeding for soybean FAs. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01216-1.

Workflow for the Quantification of Soluble and Insoluble Carbohydrates in Soybean Seed.

Soybean seed composition has a profound impact on its market value and commercial use as an important commodity. Increases in oil and protein content have been historically pursued by breeders and genetic engineers; consequently, rapid methods for their quantification are well established. The interest in complete carbohydrate profiles in mature seeds, on the other hand, has recently increased due to numerous attempts to redirect carbohydrates into oil and protein or to offer specialty seed with a specific sugar profile to meet animal nutritional requirements. In this work, a sequential protocol for quantifying reserve and structural carbohydrates in soybean seed was developed and validated. Through this procedure, the concentrations of soluble sugars, sugar alcohols, starch, hemicellulose, and crystalline cellulose can be determined in successive steps from the same starting material using colorimetric assays, LC-MS/MS, and GC-MS. The entire workflow was evaluated using internal standards to estimate the recovery efficiency. Finally, it was successfully applied to eight soybean genotypes harvested from two locations, and the resulting correlations of carbohydrate and oil or protein are presented. This methodology has the potential not only to guide soybean cultivar optimization processes but also to be expanded to other crops with only slight modifications.

Identification and characterization of a fast-neutron-induced mutant with elevated seed protein content in soybean.

KEY MESSAGE: Protein content of soybean is critical for utility of soybean meal. A fast-neutron-induced deletion on chromosome 12 was found to be associated with increased protein content. Soybean seed composition affects the utility of soybean, and improving seed composition is an essential breeding goal. Fast neutron radiation introduces genomic mutations resulting in novel variation for traits of interest. Two elite soybean lines were irradiated with fast neutrons and screened for altered seed composition. Twenty-three lines with altered protein, oil, or sucrose content were selected based on near-infrared spectroscopy data from five environments and yield tested at five locations. Mutants with significantly increased protein averaged 19.1-36.8 g kg-1 more protein than the parents across 10 environments. Comparative genomic hybridization (CGH) identified putative mutations in a mutant, G15FN-12, that has 36.8 g kg-1 higher protein than the parent genotype, and whole genome sequencing (WGS) of the mutant has confirmed these mutations. An F2:3 population was developed from G15FN-12 to determine association between genomic changes and increased protein content. Bulked segregant analysis of the population using the SoySNP50K BeadChip identified a CGH- and WGS-confirmed deletion on chromosome 12 to be responsible for elevated protein content. The population was genotyped using a KASP marker designed at the mutation region, and significant association (P < 0.0001) between the deletion on chromosome 12 and elevated protein content was observed and confirmed in the F3:4 generation. The F2 segregants homozygous for the deletion averaged 27 g kg-1 higher seed protein and 8 g kg-1 lower oil than homozygous wild-type segregants. Mutants with altered seed composition are a new resource for gene function studies and provide elite materials for genetic improvement of seed composition.

Genome-wide association study of seed protein, oil and amino acid contents in soybean from maturity groups I to IV.

KEY MESSAGE: Genomic regions associated with seed protein, oil and amino acid contents were identified by genome-wide association analyses. Geographic distributions of haplotypes indicate scope of improvement of these traits. Soybean [Glycine max (L.) Merr.] protein and oil are used worldwide in feed, food and industrial materials. Increasing seed protein and oil contents is important; however, protein content is generally negatively correlated with oil content. We conducted a genome-wide association study using phenotypic data collected from five environments for 621 accessions in maturity groups I-IV and 34,014 markers to identify quantitative trait loci (QTL) for seed content of protein, oil and several essential amino acids. Three and five genomic regions were associated with seed protein and oil contents, respectively. One, three, one and four genomic regions were associated with cysteine, methionine, lysine and threonine content (g kg-1 crude protein), respectively. As previously shown, QTL on chromosomes 15 and 20 were associated with seed protein and oil contents, with both exhibiting opposite effects on the two traits, and the chromosome 20 QTL having the most significant effect. A multi-trait mixed model identified trait-specific QTL. A QTL on chromosome 5 increased oil with no effect on protein content, and a QTL on chromosome 10 increased protein content with little effect on oil content. The chromosome 10 QTL co-localized with maturity gene E2/GmGIa. Identification of trait-specific QTL indicates feasibility to reduce the negative correlation between protein and oil contents. Haplotype blocks were defined at the QTL identified on chromosomes 5, 10, 15 and 20. Frequencies of positive effect haplotypes varied across maturity groups and geographic regions, providing guidance on which alleles have potential to contribute to soybean improvement for specific regions.

Transcriptomic dynamics in soybean near-isogenic lines differing in alleles for an aphid resistance gene, following infestation by soybean aphid biotype 2.

BACKGROUND: Genetic resistance of soybean [Glycine max (L.) Merr] against Aphis glycines provides effective management of this invasive pest, though the underlying molecular mechanisms are largely unknown. This study aimed to investigate genome-wide changes in gene expressions of soybean near-isogenic lines (NILs) either with the Rag5 allele for resistance or the rag5 allele for susceptibility to the aphid following infestation with soybean aphid biotype 2. RESULTS: The resistant (R)-NIL responded more rapidly to aphid infestation than the susceptible (S)-NIL, with differential expressions of 2496 genes during first 12 h of infestation (hai), compared to the aphid-free control. Although the majority of the differentially expressed genes (DEGs) in the R-NIL also responded to aphid infestation in S-NIL, overall the response time was longer and/or the magnitude of change was smaller in the S-NIL. In addition, 915 DEGs in R-NIL continued to be regulated at all time points (0, 6, 12, and 48 hai), while only 20 DEGs did so in S-NIL. Enriched gene ontology of the 2496 DEGs involved in plant defense responses including primary metabolite catalysis, oxidative stress reduction, and phytohormone-related signaling. By comparing R- vs. S-NIL, a total of 556 DEGs were identified. Of the 13 genes annotated in a 120-kb window of the Rag5 locus, two genes (Glyma.13 g190200 and Glyma.13 g190600) were differentially expressed (upregulated in S- or R-NIL), and another gene (Glyma.13 g190500) was induced up to 4-fold in the R-NIL at 6 and 12 h following aphid infestation. CONCLUSIONS: This study strengthens our understanding of the defense dynamics in compatible and incompatible interactions of soybean and soybean aphid biotype 2. Several DEGs (e.g., Glyma.13 g190200, Glyma.13 g190500, and Glyma.13 g190600) near the Rag5 locus are strong candidate genes for further investigations.

Identification of Soybean Proteins and Genes Differentially Regulated in Near Isogenic Lines Differing in Resistance to Aphid Infestation.

Soybean aphid is an important pest causing significant yield losses. The Rag2 locus confers resistance to soybean aphid biotypes 1 and 2. Transcriptomic and proteomic analyses were done over a 48 h period after aphid infestation using near isogenic lines (NILs) differing at the Rag2 locus. Comparing the Rag2 and/or rag2 lines identified 3445 proteins, of which 396 were differentially regulated between the two lines, including proteins involved in cell wall metabolism, carbohydrate metabolism, and stress response. RNA-seq transcriptomic analysis identified 2361 genes significantly regulated between the resistant and susceptible lines. Genes upregulated in the Rag2 line were annotated as being involved in cell wall, secondary, and hormone metabolism as well as in stress, signaling, and transcriptional responses. Genes downregulated in the Rag2 line were annotated as being involved in photosynthesis and carbon metabolism. Interestingly, two genes (unknown and mitochondrial protease) located within the defined Rag2 locus were expressed significantly higher in the resistant genotype. The expression of a putative NBS-LRR resistant gene within the Rag2 locus was not different between the two soybean lines, but a second NBL-LRR gene located just at the border of the defined Rag2 locus was. Therefore, this gene may be a candidate R gene controlling aphid resistance.

Joint linkage QTL analyses for partial resistance to Phytophthora sojae in soybean using six nested inbred populations with heterogeneous conditions.

Partial resistance to Phytophthora sojae in soybean is controlled by multiple quantitative trait loci (QTL). With traditional QTL mapping approaches, power to detect such QTL, frequently of small effect, can be limited by population size. Joint linkage QTL analysis of nested recombinant inbred line (RIL) populations provides improved power to detect QTL through increased population size, recombination, and allelic diversity. However, uniform development and phenotyping of multiple RIL populations can prove difficult. In this study, the effectiveness of joint linkage QTL analysis was evaluated on combinations of two to six nested RIL populations differing in inbreeding generation, phenotypic assay method, and/or marker set used in genotyping. In comparison to linkage analysis in a single population, identification of QTL by joint linkage analysis was only minimally affected by different phenotypic methods used among populations once phenotypic data were standardized. In contrast, genotyping of populations with only partially overlapping sets of markers had a marked negative effect on QTL detection by joint linkage analysis. In total, 16 genetic regions with QTL for partial resistance against P. sojae were identified, including four novel QTL on chromosomes 4, 9, 12, and 16, as well as significant genotype-by-isolate interactions. Resistance alleles from PI 427106 or PI 427105B contributed to a major QTL on chromosome 18, explaining 10-45% of the phenotypic variance. This case study provides guidance on the application of joint linkage QTL analysis of data collected from populations with heterogeneous assay conditions and a genetic framework for partial resistance to P. sojae.

The use of refuge in host plant resistance systems for the control of virulent biotype adaptation in the soybean aphid (Hemiptera: Aphididae.

Host plant resistant (HPR) crop varieties offer control of many insect pest species. However, the evolution of virulent biotypes capable of overcoming plant resistance poses challenges for the implementation of HPR. Widespread planting of HPR crops further reduces HPR efficacy by increasing selection pressure on pests, favoring the rapid proliferation of virulence. An analogous situation occurs in managing insect resistance to transgenic Bt crops, where planting of susceptible refuges effectively delays the evolution and spread of Bt resistance. We investigated the applicability of susceptible refuges in HPR as a tactic to manage virulent biotypes, using the soybean aphid (Aphis glycines Matsumura) as a model system. The virulent biotype 3 and avirulent biotype 1 were reared in greenhouse microcosms using a variety of refuge size, HPR gene, and biotype mixture treatments, allowing us to discern how the presence of a refuge alters the relative fitness and movement of biotypes both by themselves and in competition. The virulent biotype had greater relative fitness in 10 of 12 tested microcosms, with the greatest advantage observed in refuge-free microcosms. In microcosms with a refuge, avirulent fitness increased significantly as these biotypes moved to and used refuge plants. When the two biotypes were reared in the same microcosm, biotype 3's fitness increased significantly relative to when reared in isolation, while biotype 1's fitness was slightly, but not significantly, increased. Our findings suggested that while susceptible refuges would be incapable of reversing the proliferation of virulent biotypes, they could slow the spread of virulence by maintaining avirulence.

Novel quantitative trait loci for partial resistance to Phytophthora sojae in soybean PI 398841.

Phytophthora root and stem rot caused by Phytophthora sojae Kaufmann and Gerdemann is one of the most severe soybean [Glycine max (L.) Merr] diseases in the USA. Partial resistance is as effective in managing this disease as single-gene (Rps gene)-mediated resistance and is more durable. The objective of this study was to identify quantitative trait loci (QTL) associated with partial resistance to P. sojae in PI 398841, which originated from South Korea. A population of 305 F7:8 recombinant inbred lines derived from a cross of OX20-8 × PI 398841 was used to evaluate partial resistance against P. sojae isolate C2S1 using a tray test. Composite interval mapping using a genome-wide logarithm of odd (LOD) threshold detected three QTL on chromosomes 1, 13, and 18, which individually explained 4-16 % of the phenotypic variance. Seven additional QTL, accounting for 2-3 % of phenotypic variance each, were identified using chromosome-wide LOD thresholds. Seven of the ten QTL for resistance to P. sojae were contributed by PI 398841. Seven QTL co-localized with known Rps genes and previously reported QTL for soil-borne root pathogens, isoflavone, and seed oil. Three QTL on chromosomes 3, 13, and 18 co-localized with known Rps genes, but PI 398841 did not exhibit an Rps gene-mediated resistance response following inoculation with 48 different isolates of P. sojae. PI 398841 is potentially a source of novel genes for improving soybean cultivars for partial resistance to P. sojae.

Population genetic structure and genetic diversity of soybean aphid collections from the USA, South Korea, and Japan.

Following its recent invasion of North America, the soybean aphid (Aphis glycines Matsumura) has become the number one insect pest of soybean (Glycine max L. Merr.) in the north central states of the USA. A few studies have been conducted on the population genetic structure and genetic diversity of the soybean aphid and the source of its invasion in North America. Molecular markers, such as simple sequence repeats (SSRs) are very useful in the evaluation of population structure and genetic diversity. We used 18 SSR markers to assess the genetic diversity of soybean aphid collections from the USA, South Korea, and Japan. The aphids were collected from two sites in the USA (Indiana and South Dakota), two sites in South Korea (Yeonggwang district and Cheonan city), and one site in Japan (Utsunomiya). The SSR markers were highly effective in differentiating among aphid collections from different countries. The level of differentiation within each population and among populations from the same country was limited, even in the case of the USA where the two collection sites were more than 1200 km apart.

Molecular characterization and expression analysis of soluble trehalase gene in Aphis glycines, a migratory pest of soybean.

In insects, the enzyme trehalase plays a crucial role in energy metabolism, chitin synthesis and possibly during plant-insect interactions. We have characterized a soluble trehalase gene (Tre-1) from cDNA of Aphis glycines, a serious migratory pest of soybean. The full-length cDNA of Tre-1 in A. glycines (AyTre-1) was 2550 bp long with an open reading frame of 1770 bp that encoded for a 589 amino acid residues protein. Sequence assessment and phylogenetic analysis of the putative protein suggested that the selected cDNA belongs to soluble trehalase group. Quantitative PCR (qPCR) analysis in different tissues and developmental stages revealed peak mRNA levels of AyTre-1 in the gut (compared with other tissues assayed) and highest expression in the second instar compared with the other developmental stages assayed. Interestingly, a significantly increased expression of AyTre-1 (1.9-fold, P < 0.05) was observed in the alate morphs compared with that in apterate morphs. However, there was no significant difference in AyTre-1 expression in A. glycines-nymphs fed with resistant and susceptible plants. Expression patterns identified in this study provide a platform to investigate the role of AyTre-1 in physiological activities such as flight and feeding in A. glycines. The characterization of soluble trehalase gene may help to develop novel strategies to manage A. glycines using trehalase inhibitors and using RNA interference for knock-down of AyTre-1 expression.

Soybean genetics, genomics, and breeding for improving nutritional value and reducing antinutritional traits in food and feed.

Soybean [Glycine max (L.) Merr.] is a globally important crop due to its valuable seed composition, versatile feed, food, and industrial end-uses, and consistent genetic gain. Successful genetic gain in soybean has led to widespread adaptation and increased value for producers, processors, and consumers. Specific focus on the nutritional quality of soybean seed composition for food and feed has further elucidated genetic knowledge and bolstered breeding progress. Seed components are historical and current targets for soybean breeders seeking to improve nutritional quality of soybean. This article reviews genetic and genomic foundations for improvement of nutritionally important traits, such as protein and amino acids, oil and fatty acids, carbohydrates, and specific food-grade considerations; discusses the application of advanced breeding technology such as CRISPR/Cas9 in creating seed composition variations; and provides future directions and breeding recommendations regarding soybean seed composition traits.

Genetic mapping of the powdery mildew resistance gene in soybean PI 567301B.

Powdery mildew (PMD) of soybean [Glycine max (L.) Merr.] is caused by the fungus Microsphaera diffusa. Severe infection of PMD on susceptible varieties often causes premature defoliation and chlorosis of the leaves, which can result in considerable yield losses under favorable environmental conditions for disease development in the field. A total of 334 F(7)-derived recombinant inbred lines (RILs) from a cross of a PMD susceptible soybean cultivar Wyandot and PMD-resistant PI 567301B were used for genetic mapping of PMD resistance in PI 567301B and for development of molecular markers tightly linked to the gene. The result of the PMD screening for each line in the field was in agreement with that in the greenhouse test. The genetic map containing the PMD resistance gene was constructed in a 3.3 cM interval flanked by two simple sequence repeat (SSR) markers on chromosome 16. The PMD resistance gene was mapped at the same location with SSR marker BARCSOYSSR_16_1291, indicating that there was no recombination between the 334 RILs and this marker. In addition, a single nucleotide polymorphism (SNP) marker developed by high-resolution melting curve analysis and a cleaved amplified polymorphic sequence (CAPS) marker with Rsa1 recognition site were used for the genetic mapping. These two markers were also mapped to the same genomic location with the PMD resistance gene. We validated three tightly linked markers to the PMD resistance gene using 38 BC(6)F(2) lines and corresponding BC(6)F(2:3) families. The three marker genotypes of the backcross lines predicted the observed PMD phenotypes of the lines with complete accuracy. We have mapped a putatively novel single dominant PMD resistance gene in PI 567301B and developed three new molecular markers closely linked to the gene. Molecular markers developed from this study may be used for high-throughput marker-assisted breeding for PMD resistance with the gene from PI 567301B.

Validation of reference genes for gene expression studies in Aphis glycines (Hemiptera: Aphididae).

Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs. We evaluated seven potential HKGs (SDFS, succinate dehydrogenase flavoprotein subunit; EF1a, elongation factor-la; HEL, helicase; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; RPS9, ribosomal protein S9; TBP, TATA-box binding protein; and UBQ, ubiquitin-conjugating protein) to determine the most efficient HKGs that have stable expression among tissues, developmental stages, and aphids fed on susceptible and host plant-resistant soybean. HKG stability was determined using GeNorm and NormFinder. Results from three different experimental conditions revealed high stability of TBP compared with the other HKGs profiled across the samples assayed. RPS9 showed stable expression among aphids on susceptible and resistant plants, whereas EF1a showed stable expression in tissues and developmental stages. Therefore, we recommend the TBP as a suitable HKG for efficient normalization among treatments, tissues, and developmental stages of A. glycines. In addition, RPS9 may be used for host-plant resistance experiments and EF1a could be considered for testing differential expression across tissues or developmental stages. These results will enable a more accurate and reliable normalization of qRT-PCR data in A. glycines.

Genome-Wide Association Study and Genomic Selection for Proteinogenic Methionine in Soybean Seeds.

Soybean [Glycine max (L.) Merr.] seeds have an amino acid profile that provides excellent viability as a food and feed protein source. However, low concentrations of an essential amino acid, methionine, limit the nutritional utility of soybean protein. The objectives of this study were to identify genomic associations and evaluate the potential for genomic selection (GS) for methionine content in soybean seeds. We performed a genome-wide association study (GWAS) that utilized 311 soybean accessions from maturity groups IV and V grown in three locations in 2018 and 2019. A total of 35,570 single nucleotide polymorphisms (SNPs) were used to identify genomic associations with proteinogenic methionine content that was quantified by high-performance liquid chromatography (HPLC). Across four environments, 23 novel SNPs were identified as being associated with methionine content. The strongest associations were found on chromosomes 3 (ss715586112, ss715586120, ss715586126, ss715586203, and ss715586204), 8 (ss715599541 and ss715599547) and 16 (ss715625009). Several gene models were recognized within proximity to these SNPs, such as a leucine-rich repeat protein kinase and a serine/threonine protein kinase. Identification of these linked SNPs should help soybean breeders to improve protein quality in soybean seeds. GS was evaluated using k-fold cross validation within each environment with two SNP sets, the complete 35,570 set and a subset of 248 SNPs determined to be associated with methionine through GWAS. Average prediction accuracy (r 2) was highest using the SNP subset ranging from 0.45 to 0.62, which was a significant improvement from the complete set accuracy that ranged from 0.03 to 0.27. This indicated that GS utilizing a significant subset of SNPs may be a viable tool for soybean breeders seeking to improve methionine content.

Identification of Candidate Genes for a Major Quantitative Disease Resistance Locus From Soybean PI 427105B for Resistance to Phytophthora sojae.

Phytophthora root and stem rot is a yield-limiting soybean disease caused by the soil-borne oomycete Phytophthora sojae. Although multiple quantitative disease resistance loci (QDRL) have been identified, most explain <10% of the phenotypic variation (PV). The major QDRL explaining up to 45% of the PV were previously identified on chromosome 18 and represent a valuable source of resistance for soybean breeding programs. Resistance alleles from plant introductions 427105B and 427106 significantly increase yield in disease-prone fields and result in no significant yield difference in fields with less to no disease pressure. In this study, high-resolution mapping reduced the QDRL interval to 3.1 cm, and RNA-seq analysis of near-isogenic lines (NILs) varying at QDRL-18 pinpointed a single gene of interest which was downregulated in inoculated NILs carrying the resistant allele compared to inoculated NILs with the susceptible allele. This gene of interest putatively encodes a serine-threonine kinase (STK) related to the AtCR4 family and may be acting as a susceptibility factor, based on the specific increase of jasmonic acid concentration in inoculated NILs. This work facilitates further functional analyses and marker-assisted breeding efforts by prioritizing candidate genes and narrowing the targeted region for introgression.

Development of soybean aphid genomic SSR markers using next generation sequencing.

Simple sequence repeats (SSRs) or microsatellites are very useful molecular markers, owing to their locus-specific codominant and multiallelic nature, high abundance in the genome, and high rates of transferability across species. The soybean aphid (Aphis glycines Matsumura) has become the most damaging insect pest of soybean (Glycine max (L.) Merr.) in North America, since it was first found in the Midwest of the United States in 2000. Biotypes of the soybean aphid capable of colonizing newly developed aphid-resistant soybean cultivars have been recently discovered. Genetic resources, including molecular markers, to study soybean aphids are severely lacking. Recently developed next generation sequencing platforms offer opportunities for high-throughput and inexpensive genome sequencing and rapid marker development. The objectives of this study were (i) to develop and characterize genomic SSR markers from soybean aphid genomic sequences generated by next generation sequencing technology and (ii) to evaluate the utility of the SSRs for genetic diversity or relationship analyses. In total 128 SSR primer pairs were designed from sequences generated by Illumina GAII from a reduced representation library of A. glycines. Nearly 94% (120) of the primer pairs amplified SSR alleles of expected size and 24 SSR loci were polymorphic among three aphid samples from three populations. The polymorphic SSRs were successfully used to differentiate among 24 soybean aphids from Ohio and South Dakota. Sequencing of PCR products of two SSR markers from four aphid samples revealed that the allelic polymorphism was due to variation in the SSR repeats among the aphids. These markers should be particularly useful for genetic differentiation among aphids collected from soybean fields at different localities and regions. These SSR markers provide the soybean aphid research community with the first set of PCR-based codominant markers developed from the genomic sequences of A. glycines.

Characterizing Resistance to Soybean Aphid (Hemiptera: Aphididae): Antibiosis and Antixenosis Assessment.

Host-plant resistance (HPR) remains a vital tool to manage soybean aphid (Aphis glycines Matsumura), a major pest of soybean in Midwestern United States and southern Canada. HPR can be overcome by virulent biotypes of A. glycines; thus, in order to increase the durability of resistant cultivars, HPR needs to be deployed strategically. To improve the strategic deployment, a complete understanding of HPR in existing resistant germplasm will help ensure HPR success. In this study, we characterized HPR soybean to determine antibiosis and antixenosis categories of resistance to different biotypes of A. glycines. No-choice and free-choice tests were performed on 11 previously reported plant introductions (PIs) possessing resistance to at least one A. glycines biotype (1, 2, and 3). Overall, we found that the PIs manifested differences of a particular resistance category in response to infestation by different biotypes. Our data from no-choice tests indicate that all tested PIs possess antibiosis-based resistance to three biotypes. However, the strength of antibiosis was variable as some PIs showed stronger antibiosis toward a given biotype than others. All tested PIs manifested antixenosis, in addition to antibiosis. Furthermore, detached leaf assays revealed that resistance to A. glycines was not retained in excised soybean leaves. Characterization of resistance in this study can contribute to develop strategies for future deployment of resistant cultivars developed from these PIs.