Soybean cyst nematode (SCN) is a major yield-limiting factor of soybean. This disease has spread to at least 24 soybean-producing counties in North Dakota (ND). Host resistance is the primary management practice. SCN is known to be genetically diverse populations and can develop new virulent forms over time due to continuous use of the same resistance. Hence, it is imperative to screen soybean varieties and breeding lines to identify resistant soybeans against SCN. We screened soybean varieties, germplasm, and breeding lines for SCN and found most of them were not resistant. New seed samples from growers and new breeding lines from the NDSU soybean breeding program need to be tested to help the growers select resistant soybean varieties before planting in infested fields and to assist the breeder in developing varieties with SCN resistance. Molecular markers to detect rhg1 resistance allele and identify PI 88788-type resistance have been developed for high-throughput marker-assisted selection. The markers need to be validated for accurately detecting the resistance gene and copy number in the soybean germplasm and breeding lines to develop new soybean varieties with improved genetic resistance to SCN.

• Evaluate 40 commercial soybean varieties for their resistance responses to two common SCN populations detected in North Dakota.
• Evaluate 100 NDSU breeding lines for their resistance levels to two common SCN populations detected in North Dakota.
• Assess their copy number variation for rapidly selecting the lines with the rhg1 resistance gene against SCN.

• Resistance reactions of 140 varieties and breeding lines to SCN will be disclosed.
• Copy number variation at the rhg1 locus will be assessed to facilitate marker-assisted selection.
• The results will be summarized and made available to soybean farmers and breeder.

Update:
2024 Mid-Year report

Evaluation of soybean varieties and breeding lines for resistance to soybean cyst nematode and their copy number variation at Rhg1 locus

Principle Investigator: Dr. Guiping Yan
Co-investigators: Dr. Carrie Miranda and Dr. Sam Markell


Research Overview and Objectives

Soybean cyst nematode (SCN) is a major yield-limiting factor of soybean. Host resistance is the primary management practice. Hence, it is imperative to screen soybean varieties and breeding lines to identify resistant soybeans against SCN. We screened soybean varieties, germplasm, and breeding lines for SCN and found most of them were not resistant. New seed samples from growers and new breeding lines from the NDSU soybean breeding program need to be tested to help the growers select resistant soybean varieties before planting in infested fields and to assist the breeder in developing varieties with SCN resistance. Molecular markers to detect rhg1 resistance gene and identify PI 88788-type resistance have been reported for high-throughput marker-assisted selection. The markers need to be validated before application for accurately detecting the resistance gene and copy number in the soybean germplasm and breeding lines. The specific objectives of this research are as follows.

1. Evaluate 40 commercial soybean varieties for their resistance responses to two common SCN populations detected in North Dakota.
2. Evaluate 100 NDSU breeding lines for their resistance levels to two common SCN populations detected in North Dakota.
3. Assess their copy number variation for rapidly selecting the lines with the rhg1 resistance gene against SCN.

Completed Work

A total of 117 soybean breeding lines were acquired from the NDSU soybean breeding program and they are currently undergoing testing against a SCN population, HG type 7, collected from a soybean field in North Dakota (ND). These 117 breeding lines were divided into three lots and experiments were set up for all three lots. Barnes, a soybean cultivar from ND, was included as a susceptible check in all screening experiments. Plant introduction lines, PI 548408, PI 88788, PI 209332, and PI 548316 were used as controls in all the experiments. To increase the population of HG type 7 obtained from Traill County ND, the susceptible cultivar, Barnes, was used for inoculation under controlled greenhouse conditions. Pregerminated seeds of each soybean breeding line, the susceptible check, and controls were individually planted in cone-tainers filled with 100 cm3 of pasteurized river-sand soil, and this setup was replicated four times. Each plant was inoculated with 2,000 SCN eggs and juveniles at the time of planting and was subsequently placed in a growth chamber maintained at 27 °C with a 16-hour daylight cycle (Figure 1). The second population, HG type 2.5.7 collected from Richland County, ND has been maintained and increased under greenhouse conditions.

For copy number assessment, a real-time quantitative PCR (qPCR) assay from previously published paper Lee et al. (2015) was adopted and optimized. Genomic DNA was isolated from leaf tissues of soybean plants using a FastDNA® Spin Kit (MP Biomedicals), and qPCR was performed. A heat-shock protein gene (hsp) was used as an internal control. Relative quantification using the 2 -??CT technique was calculated to determine the copy number based on the reference check, Williams 82 with a single copy. To validate the SYBR green-based qPCR assay for detection of the rhg1 gene, 11 soybean accessions with known copy numbers were acquired from the USDA-ARS Soybean Germplasm Collection in Illinois. Correlation analysis, melting curve and amplification curve analysis were done to validate and check the specificity of the qPCR assay. Standard curves were generated to determine the qPCR assay efficiency. The optimized and validated qPCR assay will be used to detect the copy number of the breeding lines obtained from the NDSU soybean breeding program.

Progress of Work and Results to Date

The reference copy numbers, as identified through whole-genome sequencing (WGS) in Lee et al. (2015), fell within the range defined by the minimum and maximum bounds of relative copy numbers determined by our optimized qPCR assay (Figure 2). A high degree of correlation (r = 0.994) was observed between the copy numbers detected by the optimized qPCR assay and copy numbers determined by the whole genome sequencing in Lee et al. (2015) (Figure 3). These results indicated that our qPCR assay was validated. Furthermore, the standard curve generated from the data obtained with serial 2-fold dilutions of genomic DNA of Williams 82 revealed a high degree of correlation between the Cq values and log10 values of serial dilutions for both the target gene (R2 = 0.998, slope = -3.465, E = 94.37%) and reference gene (R2 = 0.999, slope = -3.289, E = 101.39%), indicating the primers were applicable to the qPCR assay (Figure 4A and 4B). Amplification reactions of the DNA samples for the target gene Glyma18g02590 and endogenous control gene Hsp were displayed by amplification curves, where no amplification was observed in the control reactions as indicated by straight lines below the threshold level. A single melting peak at 82°C and 76.5 °C for the target gene and control gene, respectively, was observed from this qPCR assay, indicating that a single specific amplicon was amplified.

Work to be Completed

The experiment of 117 breeding lines screened for HG type 7 will be harvested after 30 days. SCN white females (cysts) from plant roots and soil in each cone-tainer will be extracted and then counted under a microscope. The mean number of white females produced on the roots of each line will be used for calculating the female index (FI) by comparing to the susceptible check Barnes. Based on the female index, the resistance response of each line will be classified as resistant (FI: < 10%), moderately resistant (FI: 10-30%), moderately susceptible (FI: 30-60%), or susceptible (FI: > 60%) as described by Schmitt and Shannon (1992). Similarly, the 117 breeding lines will be screened for another SCN population, HG type 2.5.7. Similar protocols will be followed for HG type 2.5.7. Fifty of the breeding lines will be chosen for copy number evaluation using the validated qPCR assay. The genotypic data from the qPCR will be compared with the phenotypic data from our SCN resistance evaluation to further validate the accuracy of the qPCR assay.

We have received 32 commercial soybean varieties from the growers and companies and these seed samples will tested using two SCN populations and the similar protocols.

Other relevant information

For a large scale of germplasm screening, space and inoculum are a problem. We have reserved and used two growth chambers for SCN resistance evaluation. A number of susceptible soybean plants were used for producing SCN inoculum. The project is going well as proposed.

Summary

In summary, this report highlights the significant progress made in understanding soybean resistance to SCN. We used molecular techniques for precise copy number assessment, establishing correlations between genetic findings and resistance levels in breeding lines. Currently, we are actively conducting experiments, having set up 117 new breeding lines for HG type 7 and planning to assess their resistance response to HG type 2.5.7. The optimization and validation of the real-time quantitative PCR (qPCR) assay will enable us to determine copy numbers at the Rhg1 locus among the breeding lines. Our ongoing efforts involve expanding this analysis to include more breeding lines and cultivars to enhance our understanding of soybean resistance and its mechanisms. The copy number assessment will facilitate screening of soybean germplasm and breeding lines to efficiently select or develop soybean varieties with resistance to SCN.

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Updated June 29, 2024:
Evaluation of Soybean Varieties and Breeding Lines for Resistance to Soybean Cyst Nematode and Their Copy Number Variation at Rhg1 Locus

TECHNICAL REPORT

NORTH DAKOTA SOYBEAN COUNCIL
JUNE 2024

Dr. Guiping Yan, Principal Investigator, Dept. Plant Pathology, NDSU
Co-investigators: Dr. Carrie Miranda, Dept. Plant Sciences, NDSU and Dr. Sam Markell, Dept. Plant Pathology, NDSU

a. Background Information
Soybean cyst nematode (SCN; Heterodera glycines), a major pest affecting soybean, significantly impacts yield in infested fields. Effective management of SCN relies heavily on the development and deployment of resistant soybean varieties, which is considered as a sustainable and environmentally friendly strategy. In North Dakota, SCN populations HG type 0, HG type 7 and HG type 2.5.7 are prevalent, posing a challenge to soybean production. Major sources of SCN resistance that are widely used to develop resistant soybean varieties include Peking and PI 88788. However, the overuse of these limited resistance sources has led to the emergence of more virulent SCN populations capable of overcoming this resistance. So, it is crucial to explore new resistance sources and thoroughly evaluate soybean breeding lines and commercial varieties for resistance to SCN. The Rhg1 locus, derived from PI 88788, is known to confer strong resistance to SCN. More than 90% of soybean varieties in the United States utilize PI 88788 as their resistance source against SCN. Previous research has shown that the copy number at the Rhg1 locus determines the level of resistance to SCN. Therefore, evaluation of the resistance responses of soybean breeding lines and commercial varieties to SCN populations help to select SCN resistant soybeans, and assessing copy number at Rhg1 locus facilitate rapid selection of soybean lines with increased SCN resistance.

b. Research Objectives

1. Evaluate 40 commercial soybean varieties for their resistance responses to two common SCN populations detected in North Dakota.
2. Evaluate 100 NDSU breeding lines for their resistance levels to two common SCN populations detected in North Dakota.
3. Assess their copy number variation for rapidly selecting the lines with the rhg1 resistance gene against SCN.

c. Materials and Methods

A total of 152 soybean breeding lines from the NDSU breeding program (117) and commercial soybean varieties (35) from various companies and growers were acquired and used. Out of these, 12 varieties and all breeding lines were screened for resistance reactions to SCN population HG type 7 and 23 varieties were screened for HG type 0. All of these soybean breeding lines and varieties were screened for resistance reactions to SCN population HG type 2.5.7. The HG type 2.5.7 population, which has a higher ability to reproduce on the major SCN resistance source PI 88788 and its derivative lines, was collected from Richland County, ND, and HG type 0 and 7 was collected from Traill County, ND.
Five days old pre-germinated seedlings from each of the soybean breeding lines and varieties were planted in 100 cc of pasteurized river sand in cone-tainers, which were arranged in a completely randomized design with four replicates. Each plant was inoculated with 2,000 SCN eggs at the time of planting and grown in a controlled growth chamber maintained at 27°C with a 16-hour daylight period for 32 days. Then white females formed on each plant were extracted and counted, and the numbers of white females in the four replicates were averaged to determine the mean number of white females. This mean was then used to calculate the Female Index (FI) according to the formula: FI = (mean number of white females produced on a tested soybean line / mean number of white females on the susceptible check, Barnes) × 100%. Based on the FI values, soybean varieties and lines were categorized for their resistance responses into four groups, as described by Schmitt and Shannon (1992): resistant (R) (FI < 10%), moderately resistant (MR) (FI = 11% to < 30%), moderately susceptible (MS) (FI = 30% to < 60%), or susceptible (S) (FI = 60%).
To determine copy number variations at the Rhg1 locus, a real-time quantitative PCR (qPCR) assay was used. This assay was optimized for annealing temperature, primer concentration, and DNA template concentration, and validated using 12 soybean accessions with known copy numbers at the Rhg1 locus. Genomic DNA was extracted from the leaf tissues of 10-day-old soybean plants. A total of 50 DNA samples were prepared from the soybean breeding lines from the NDSU breeding program.
The qPCR was conducted using an internal control gene, a heat-shock protein gene (hsp). The qPCR reaction was performed in a 10 µl volume (containing 5 µl of 2× Sso Advanced SYBR Mastermix, 0.2 µl each of forward and reverse primers (10 mM), 3.1 µl of nuclease-free H2O, and 1.5 µl of template DNA). The amplification program included an initial denaturation step at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 30 seconds, and annealing at 60°C for 1 minute. Relative quantification was determined using the 2 -??CT method to calculate the copy number based on the reference check, Williams 82 with a single copy of Rhg1 repeat. The copy numbers of the 50 breeding lines obtained by qPCR assay were then compared with the resistance responses to two SCN populations, HG type 7 and HG type 2.5.7, to assess their degree of association. Correlation analysis was conducted between the female indexes of the respective HG types and the copy numbers from qPCR assay to evaluate if the copy number could serve as an indicator to determine the resistance level to SCN.

d. Research Results and Discussion
Among the 35 commercial soybean varieties tested for HG type 0 or 7, nine varieties were resistant (FI: 0.0 to 9.6%), eight varieties were moderately resistant (FI: 14.5 to 27.1%), 13 varieties were moderately susceptible (FI: 33.5 to 58.5%), and the remaining five varieties were susceptible (FI: 60.2 to 89.6%) (Figure 1). For another SCN population HG type 2.5.7, six varieties were resistant (FI: 2.4 to 8.7%), seven varieties were moderately resistant (FI: 15.2 to 29.9%), 10 varieties were moderately susceptible (FI: 32.3 to 59.8%), and the remaining 12 varieties were susceptible (FI: 60.5 to 120.2%) (Figure 1). Interestingly, 12 of the varieties were resistant or moderately resistant to both the SCN populations (HG type 2.5.7 and HG type 0/7).
Likewise, among the 117 NDSU breeding lines screened for HG type 7, six lines were resistant (FI: 7.8 to 9.9%), 34 lines were moderately resistant (FI: 12.9 to 29.9%), 22 lines were moderately susceptible (FI: 34.8 to 59.6%), and the remaining 55 lines were susceptible (FI: 60.0 to 91.3%) (Figure 2). For HG type 2.5.7, none of the lines were resistant, 25 lines were moderately resistant (FI: 22.0 to 28.7%), 18 lines were moderately susceptible (FI: 30.5 to 59.8%), and the remaining 74 lines were susceptible (FI: 60.1 to 90.1%) (Figure 2). ND21-11516(GT) had the lowest female index value for both HG type 7 (7.8%) and HG type 2.5.7 (22.0%). A total of 25 breeding lines among the 117 breeding lines showed resistant or moderately resistant reaction to both the HG types (Figure 3).
The majority of the 152 soybean lines and varieties tested for the two SCN populations were susceptible or moderately susceptible. Comparatively, more resistant and moderately resistant soybeans with lower female index values were identified when screened for HG type 0/7 compared to HG type 2.5.7. This disparity is likely due to the different genetic diversities of the SCN populations, each exhibiting unique capabilities in parasitizing soybean lines. The higher susceptibility observed in lines screened for HG type 2.5.7 indicates a direct association between pathogen virulence and the SCN population's ability to overcome resistance, leading to an increased presence of SCN white females on the root system.
The Rhg1 locus copy numbers among the 50 breeding lines ranged from 1 to 11. Six lines had 11 copies, seven had 10 copies, and the remaining 37 had a single copy (Figure 4). Lines with high copy numbers (10 or 11) were either resistant or moderately resistant to HG type 7 and moderately resistant or moderately susceptible to HG type 2.5.7 (Figure 5). Majority of the lines with a single copy exhibited moderately susceptible or susceptible response to HG type 7 and exhibited susceptible response to HG type 2.5.7. A strong negative correlation was observed between the female indexes and copy numbers for both HG type 7 (r = -0.780) and HG type 2.5.7 (r = -0.922), suggesting that an increased copy number at Rhg1 is indicative of greater resistance to SCN. One line with a single copy at Rhg1 locus was moderately resistant to HG type 2.5.7 and two lines with a single copy at Rhg1 locus were moderately resistant to HG type 7. This suggests further experiments on SCN resistance phenotyping and investigation into the potential co-expression or genetic interaction of different SCN resistance genes in these lines.

e. Benefits to ND Soybean Farmers and Industry
This study identified commercial varieties and breeding lines with resistance to SCN. Twelve commercial varieties among the 35 commercial varieties showed resistant or moderately resistant response to both HG types, offering valuable options for farmers to select resistant varieties for infested fields, reducing yield losses and ensuring stable production. Additionally, 25 breeding lines among the 117 breeding lines were resistant or moderately resistant to both HG types, providing breeders with a pool of candidates for developing new SCN-resistant varieties. The determination of Rhg1 copy number further aids in the rapid selection of breeding lines with increased resistance to SCN. The integration of both phenotypic and molecular methodologies in this research has facilitated the exploration of potential correlations between the resistance response and copy number variation at the Rhg1 locus. The findings of these two approaches appear to be mutually corroborative. A manuscript on this topic has been prepared for submission to a journal for publication for unrestricted use to disseminate the research findings.





Figure 1. Resistance responses of 35 commercial soybean varieties from companies and growers to HG type 0/HG type 7 and HG type 2.5.7 isolated from soybean fields in ND.




Figure 2. Distribution of 117 soybean breeding lines across four resistance categories: resistant (R) (FI < 10%), moderately resistant (MR) (FI = 10 to <30%), moderately susceptible (MS) (FI = 30 to <60%), and susceptible (S) (FI = 60%), based on the criteria established by Schmitt and Shannon (1992), for HG types 2.5.7 and 7 isolated from soybean fields in ND.


Figure 3. Resistance responses of 25 breeding lines that showed resistant or moderately resistant response to both HG type 7 and 2.5.7 collected from soybean fields in ND.




Figure 4. Copy number variation at the Rhg1 locus among 50 randomly selected breeding lines from the NDSU soybean breeding program.




Figure 5. Relationship between female indexes (%) and copy numbers at Rhg1 locus of 10 randomly selected breeding lines. The primary y-axis represents the female index, while the secondary y-axis represents the copy number at the Rhg1 locus.

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Evaluation of Soybean Varieties and Breeding Lines for Resistance to Soybean Cyst Nematode and Their Copy Number Variation at Rhg1 Locus

EXECUTIVE SUMMARY

NORTH DAKOTA SOYBEAN COUNCIL
JUNE 2024

Dr. Guiping Yan, Principal Investigator, Dept. Plant Pathology, NDSU
Co-investigators: Dr. Carrie Miranda, Dept. Plant Sciences, NDSU and Dr. Sam Markell, Dept. Plant Pathology, NDSU

a. Importance of the Research
Soybean cyst nematode (SCN) causes a significant yield loss in soybean. Effective SCN management relies on resistant soybean varieties. However, overuse of limited resistance sources has led to the emergence of more virulent SCN populations. Evaluating breeding lines and commercial varieties for SCN resistance helps in selecting resistant soybeans. Most SCN-resistant varieties have PI 88788-type resistance, particularly involving genes at the Rhg1 locus. Copy number variations (CNVs) at Rhg1 determine the level of resistance to SCN. Therefore, screening of soybean lines and varieties and analyzing CNVs are important.

b. Research Conducted
This research aimed to evaluate soybean varieties and breeding lines for resistance responses to two common SCN populations in ND and to assess copy number variations (CNVs) at the Rhg1 locus. A total of 152 soybean breeding lines and commercial varieties have been tested against HG type 2.5.7 (higher ability to reproduce on PI 88788), and HG type 0/7 (less to no ability to reproduce on PI 88788). Each line was inoculated with 2,000 SCN eggs, and grown under controlled growth chamber conditions (Fig. 1). After 32 days, white females were extracted and counted. Then female index was calculated, and resistance response was classified. CNVs at the Rhg1 locus were detected using an optimized and validated qPCR assay.

c. Research Findings
Among 35 commercial soybean varieties, nine were found resistant to HG type 0/7, while six varieties were resistant to HG type 2.5.7. Five varieties showed resistance to both HG types. Among 117 breeding lines tested, six were resistant to HG type 7, while none of the lines was resistant to HG type 2.5.7. Twenty-five breeding lines were moderately resistant or resistant to both HG types. Rhg1 copy numbers ranged from 1 to 11, with higher copies generally linked to greater resistance (Fig. 2). There was a strong negative correlation between the female indexes and copy numbers for both the populations.

d. Benefits
We identified soybean varieties and breeding lines with resistance to SCN from this research. Five of the commercial varieties tested were resistant to both HG types, offering valuable options for farmers to select resistant varieties to reduce yield losses. Additionally, 25 of the breeding lines were resistant or moderately resistant to both HG types, providing a pool for developing new SCN-resistant varieties. Determining Rhg1 copy number helps in the rapid selection of soybean lines with increased SCN resistance.




Fig 1. Soybean plants were tested for SCN resistance in a controlled growth chamber maintained at 27°C, ensuring optimal testing conditions.



Fig. 2. Relationship between female indexes (%) and copy numbers at Rhg1 locus of 20 selected breeding lines including 11 resistant or moderately resistant lines (bold) to both SCN populations and 9 susceptible or moderately susceptible lines to both populations. The primary y-axis represents the female index, while the secondary y-axis represents the copy number at the Rhg1 locus.

SCN is an important disease in soybean. The resistant soybean varieties identified in this proposed research will be recommended to farmers for suppressing SCN in infested fields. The soybean varieties with resistance to SCN are desirable for managing the disease. The resistant breeding lines identified will be provided to the NDSU soybean breeding program for transferring the resistance to locally adapted susceptible varieties to develop and release new varieties with improved genetic resistance. Molecular markers to identify the rhg1 resistance allele will be validated to predict rhg1 copy number in soybean lines. This will help improve resistance selection and breeding accuracy and efficiency. Thus, this research is important to navigate the resistance sources that should be used in the soybean breeding program for developing new resistant varieties as well as help growers select the resistant varieties for controlling the nematode disease to increase soybean yield.