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.
View uploaded report 