Response of soybean genotypes from Northeast China to Heterodera glycines races 4 and 5, and characterisation of rhg1 and Rhg4 genes for soybean resistance

Summary Soybean cyst nematode (SCN, Heterodera glycines) is a devastating plant-parasitic nematode worldwide. Two SCN races, race 4 (HG Type 1.2.3.5.6.7) and race 5 (HG Type 2.5.7), with increased virulence were previously identified in Northeast China. To obtain new resistance sources to these SCN populations, the response of 62 genotypes, including 51 local varieties and breeding lines, and 11 indicator lines for SCN race and HG Type identification, were evaluated. Four new primers in the regions of two loci of GmSHMT08 (Rhg4) and GmSNAP18 (rhg1) were designed for PCR amplification and subsequent sequencing to characterise haplotypes instead of genome resequencing. Results indicated three haplotypes among 51 local genotypes; there were 26 lines in Haplotype I carrying both the rhg1-a and Rhg4-a resistant loci as in ‘Peking’, 13 lines in Haplotype II containing only the resistant rhg1-a locus but Rhg4-b susceptible loci, and 12 lines in Haplotype III with rhg1-c and Rhg4-b susceptible loci. Interestingly, there was no ‘PI 88788’-type resistance identified in Northeast China, although it accounts for 90% of sources in the USA. Two local breeding lines in Haplotype I displayed resistance to both SCN races. The resistance lines carried higher copy number (>1) of the tandem duplication at the rhg1 locus compared with susceptible lines (⩽1). The combination of the two microsatellite markers, Sat_162 on Chr 8 and 590 on Chr 18, distinguished the three haplotypes and predicted the resistance/susceptibility for SCN race 5. The knowledge of the phenotypes and molecular characteristics of 51 local breeding lines in Northeast China will accelerate the utilisation of sources for broad-based SCN resistance and marker-assisted selection.

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 has shown this relationship. This report evaluates RN reproduction on 418 plant introductions (PIs) selected from the USDA Soybean Germplasm Collection with reported SCN resistance. The germplasm was divided into two tests of 214 PIs reported as resistant, and 204 PIs 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: (1) ≤10% as compared to the susceptible check, (2) using normalized reproduction index (RI) values, and transformed data (3) log10 (x) and (4) 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.

Soybean cyst nematode resistance QTL cqSCN-006 alters the expression of a ɣ-SNAP protein

Soybean cyst nematode is the most economically damaging pathogen of soybean and host resistance is a core management strategy. The SCN resistance QTL cqSCN-006, introgressed from the wild relative Glycine soja, provides intermediate resistance against nematode populations including those with increased virulence on the heavily used rhg1-b resistance locus. cqSCN-006 was previously fine-mapped to a genome interval on chromosome 15. The present study determined that Glyma.15G191200 at cqSCN-006, encoding a ɣ-SNAP (gamma-SNAP), contributes to SCN resistance. CRISPR/Cas9-mediated disruption of the cqSCN-006 allele reduced SCN resistance in transgenic roots. There are no encoded amino acid polymorphisms between resistant and susceptible alleles. However, other cqSCN-006-specific DNA polymorphisms in the Glyma.15G191200 promoter and gene body were identified, and we observed differing induction of ɣ-SNAP protein abundance at SCN infection sites between resistant and susceptible roots. We identified alternative RNA splice forms transcribed from the Glyma.15G191200 ɣ-SNAP gene and observed differential expression of the splice forms two days after SCN infection. Heterologous overexpression of ɣ-SNAPs in plant leaves caused moderate necrosis, suggesting that careful regulation of this protein is required for cellular homeostasis. Apparently, certain G. soja evolved quantitative SCN resistance through altered regulation of ɣ-SNAP. Previous work has demonstrated SCN resistance impacts of the soybean α-SNAP proteins encoded by Glyma.18G022500 (Rhg1) and Glyma.11G234500. The present study shows that a different type of SNAP protein can also impact SCN resistance. Little is known about ɣ-SNAPs in any system, but the present work suggests a role for ɣ-SNAPs during susceptible responses to cyst nematodes.

Genome-Wide Association Study and Genomic Prediction for Soybean Cyst Nematode Resistance in USDA Common Bean (Phaseolus vulgaris) Core Collection

Soybean cyst nematode (SCN, Heterodera glycines) has become the major yield-limiting biological factor in soybean production. Common bean is also a good host of SCN, and its production is challenged by this emerging pest in many regions such as the upper Midwest USA. The use of host genetic resistance has been the most effective and environmentally friendly method to manage SCN. The objectives of this study were to evaluate the SCN resistance in the USDA common bean core collection and conduct a genome-wide association study (GWAS) of single nucleotide polymorphism (SNP) markers with SCN resistance. A total of 315 accessions of the USDA common bean core collection were evaluated for resistance to SCN HG Type 0 (race 6). The common bean core set was genotyped with the BARCBean6K_3 Infinium BeadChips, consisting of 4,654 SNPs. Results showed that 15 accessions were resistant to SCN with a Female Index (FI) at 4.8 to 9.4, and 62 accessions were moderately resistant (10 < FI < 30) to HG Type 0. The association study showed that 11 SNP markers, located on chromosomes Pv04, 07, 09, and 11, were strongly associated with resistance to HG Type 0. GWAS was also conducted for resistance to HG Type 2.5.7 and HG Type 1.2.3.5.6.7 based on the public dataset (N = 276), consisting of a diverse set of common bean accessions genotyped with the BARCBean6K_3 chip. Six SNPs associated with HG Type 2.5.7 resistance on Pv 01, 02, 03, and 07, and 12 SNPs with HG Type 1.2.3.5.6.7 resistance on Pv 01, 03, 06, 07, 09, 10, and 11 were detected. The accuracy of genomic prediction (GP) was 0.36 to 0.49 for resistance to the three SCN HG types, indicating that genomic selection (GS) of SCN resistance is feasible. This study provides basic information for developing SCN-resistant common bean cultivars, using the USDA core germ plasm accessions. The SNP markers can be used in molecular breeding in common beans through marker-assisted selection (MAS) and GS.

Novel resistance strategies to soybean cyst nematode (SCN) in wild soybean

Soybean cyst nematode (SCN, Heterodera glycine Ichinohe) is the most damaging soybean pest worldwide and management of SCN remains challenging. The current SCN resistant soybean cultivars, mainly developed from the cultivated soybean gene pool, are losing resistance due to SCN race shifts. The domestication process and modern breeding practices of soybean cultivars often involve strong selection for desired agronomic traits, and thus, decreased genetic variation in modern cultivars, which consequently resulted in limited sources of SCN resistance. Wild soybean (Glycine soja) is the wild ancestor of cultivated soybean (Glycine max) and it’s gene pool is indisputably more diverse than G. max. Our aim is to identify novel resistant genetic resources from wild soybean for the development of new SCN resistant cultivars. In this study, resistance response to HG type 2.5.7 (race 5) of SCN was investigated in a newly identified SCN resistant ecotype, NRS100. To understand the resistance mechanism in this ecotype, we compared RNA seq-based transcriptomes of NRS100 with two SCN-susceptible accessions of G. soja and G. max, as well as an extensively studied SCN resistant cultivar, Peking, under both control and nematode J2-treated conditions. The proposed mechanisms of resistance in NRS100 includes the suppression of the jasmonic acid (JA) signaling pathway in order to allow for salicylic acid (SA) signaling-activated resistance response and polyamine synthesis to promote structural integrity of root cell walls. Our study identifies a set of novel candidate genes and associated pathways involved in SCN resistance and the finding provides insight into the mechanism of SCN resistance in wild soybean, advancing the understanding of resistance and the use of wild soybean-sourced resistance for soybean improvement.

Resistance Gene Pyramiding and Rotation to Combat Widespread Soybean Cyst Nematode Virulence

Soybean cyst nematode (SCN) is an important pathogen of soybean causing more than $1 billion in yield losses annually in the United States. Planting SCN resistant soybean cultivars is the primary management strategy. Resistance genes derived from the plant introductions (PI) 88788 (rhg1-b) and PI 548402 (Peking; rhg1-a and Rhg4) are the main types of resistance available in commercial cultivars. The PI 88788 rhg1-b resistance allele is found in the majority of SCN resistant cultivars in the north central US. The widespread use of PI 88788 rhg1-b has led to limited options for farmers to rotate resistance sources to manage SCN. Consequently, an over reliance on a single type of resistance has resulted in the selection of SCN populations that have adapted to reproduce on these resistant cultivars. Here we evaluated the effectiveness of rotating soybean lines with different combinations of resistance genes to determine the best strategy for combating the widespread increase in virulent SCN and limit future nematode adaptation to resistant cultivars. Eight SCN populations were developed by either continuous selection of a virulent SCN field population (HG type 1.2.5.7) on a single resistance source or in rotation with soybean pyramiding different resistance gene alleles derived from PI 88788 (rhg1-b), PI 437654 (rhg1-a and Rhg4), PI 468916 (cqSCN-006 and cqSCN-007) and PI 567516C (Chr10). SCN population densities were determined for eight generations. HG type tests were conducted after the eighth generation to evaluate population shifts. The continued use of rhg1-b or 006/007 had limited effectiveness for reducing SCN type 1.2.5.7 population density, whereas rotation to the use of rhg1-a/Rhg4 resistance significantly reduced SCN population density, but selected for broader SCN virulence (HG type 1.2.3.5.6.7). A rotation of rhg1-a/Rhg4 with a pyramid of rhg1-b/006/007/Chr10 was the most effective combination at both reducing population density and minimizing selection pressure. Our results provide guidance for implementation of a strategic SCN resistance rotation plan to manage the widespread virulence on PI 88788 and sustain the future durability of SCN resistance genes.

Identification of novel haplotype of a cyst nematode resistance gene, GmSNAP18 in soybean [Glycine max (L.) Merr.]

Soybean cyst nematode (SCN) is one of the most damaging pest of soybean. Discovery and characterization of the genes involved in SCN resistance are important in soybean breeding. Soluble NSF attachment protein (SNAP) genes are related to SCN resistance in soybean. SNAP genes include five gene families, and 2 haplotypes of exons 6 and 9 of SNAP18 are considered resistant to the SCN. In present study the haplotypes of GmSNAP18 were surveyed and chacterized in a total of 60 diverse soybean genotypes including Korean cultivars, landraces, and wild-types. The target region of exons 6 and 9 in GmSNAP18 region was amplified and sequenced to examine nucleotide variation. Characterization of 5 haplotypes identified in present study for the GmSNAP18 gene revealed two haplotypes as resistant, 1 as susceptible and two as novel. A total of twelve genotypes showed resistant haplotypes, and 45 cultivars were found susceptible. Interestingly, the two novel haplotypes were present in 3 soybean lines. The information provided here about the haplotypic variation of GmSNAP18 gene can be further explored for soybean breeding to develop resistant varieties.

The soybean Rhg1 amino acid transporter protein becomes abundant along the SCN penetration path and impacts ROS generation

ABSTRACTRhg1 mediates soybean resistance to soybean cyst nematode. Glyma.18G022400, one of three resistance-conferring genes at the complex Rhg1 locus, encodes the putative amino acid transporter AATRhg1 whose mode of action is largely unknown. We discovered that AATRhg1 protein abundance increases 7- to 15-fold throughout root cells penetrated by SCN. These root cells develop increased abundance of vesicles and larger vesicle-like bodies. AATRhg1 was often associated with these vesicles. AATRhg1 abundance remained low in syncytia (plant reprogrammed feeding cells), unlike the Rhg1 α-SNAP protein whose abundance was previously shown to increase in syncytia. In N. benthamiana, if soybean AATRhg1 was present, oxidative stress promoted formation of larger macrovesicles and they contained AATRhg1. AATRhg1 was found to interact with GmRBOHC2, a soybean ortholog of Arabidopsis RBOHD previously found to exhibit upregulated expression upon SCN infection. Reactive oxygen species (ROS) generation was more elevated when AATRhg1 and GmRBOHC2 abundance were co-expressed. These findings suggest that AATRhg1 contributes to SCN resistance along the penetration path as SCN invades the plant, and does so at least in part by interactions with GmRBOHC2 that increase ROS production. The study also shows that Rhg1 resistance functions via at least two spatially and temporally separate modes of action.

Therhg1-a(Rhg1low-copy) nematode resistance source harbors a copia-family retrotransposon within theRhg1-encoded α-SNAP gene

Soybean growers widely use theResistance toHeteroderaglycines1 (Rhg1) locus to reduce yield losses caused by soybean cyst nematode (SCN).Rhg1is a tandemly repeated four gene block. Two classes of SCN resistance-conferringRhg1haplotypes are recognized:rhg1-a(“Peking-type”, low copy number, 3 or fewerRhg1repeats) andrhg1-b(“PI 88788-type”, high copy number, 4 or moreRhg1repeats). Therhg1-aandrhg1-bhaplotypes encode α-SNAP (alpha-SolubleNSFAttachmentProtein) variants α-SNAPRhg1LC and α-SNAPRhg1HC respectively, with differing atypical C-terminal domains, that contribute to SCN-resistance. Here we report thatrhg1-asoybean accessions harbor a copia retrotransposon within theirRhg1 Glyma.18G022500(α-SNAP-encoding) gene. We termed this retrotransposon “RAC”, forRhg1alpha-SNAPcopia. Soybean carries multipleRAC-like retrotransposon sequences. TheRhg1 RACinsertion is in theGlyma.18G022500genes of all truerhg1-ahaplotypes we tested and was not detected in any examinedrhg1-borRhg1WT(single-copy) soybeans.RACis an intact element residing within intron 1, anti-sense to therhg1-a α-SNAPopen reading frame.RAChas intrinsic promoter activities, but overt impacts ofRACon transgenic α-SNAPRhg1LC mRNA and protein abundance were not detected. From the nativerhg1-a RAC+genomic context, elevated α-SNAPRhg1LC protein abundance was observed in syncytium cells, as was previously observed for α-SNAPRhg1HC (whoserhg1-bdoes not carryRAC). Using a SoySNP50K SNP corresponding withRACpresence, just ∼42% of USDA accessions bearing previously identifiedrhg1-aSoySNP50K SNP signatures harbor theRACinsertion. Subsequent analysis of several of these putativerhg1-aaccessions lackingRACrevealed that none encodedα-SNAPRhg1LC, and thus they are notrhg1-a.rhg1-ahaplotypes are of rising interest, withRhg4, for combating SCN populations that exhibit increased virulence against the widely usedrhg1-bresistance. The present study reveals another unexpected structural feature of manyRhg1loci, and a selectable feature that is predictive ofrhg1-ahaplotypes.

Effect of Soybean Cyst Nematode Resistance Source and Seed Treatment on Population Densities of Heterodera glycines, Sudden Death Syndrome, and Yield of Soybean

A three-year study was conducted in Illinois, Indiana, Iowa, Michigan, and Ontario, Canada, from 2013 through 2015 to determine the effect of soybean (Glycine max) cultivars’ source of soybean cyst nematode (SCN; Heterodera glycines) resistance on SCN population densities, sudden death syndrome (SDS; caused by Fusarium virguliforme), and yield of soybean. Five cultivars were evaluated with and without fluopyram seed treatment at each location. Cultivars with no SCN resistance had greater SDS severity, greater postharvest SCN egg counts (Pf), and lower yield than cultivars with plant introduction (PI) 548402 (Peking) and PI 88788-type of SCN resistance (P < 0.05). Cultivars with Peking-type resistance had lower Pf than those with PI 888788-type and no SCN resistance. In two locations with HG type 1.2-, cultivars with Peking-type resistance had greater foliar disease index (FDX) than cultivars with PI 88788-type. Fluopyram seed treatment reduced SDS and improved yield compared with a base seed treatment but did not affect SCN reproduction and Pf (P > 0.05). FDX and Pf were positively correlated in all three years (P < 0.01). Our results indicate that SDS severity may be influenced by SCN population density and HG type, which are important to consider when selecting cultivars for SCN management.