Update:
a. Background information
Soybean cyst nematode (SCN; Heterodera glycines) is the most significant yield-reducing factor in the United States. Host resistance is considered the most efficient and eco-friendly practice for managing SCN, and the primary sources of resistance to SCN are PI 88788 and PI 548402 (Peking). However, the continued use of these limited sources of resistance has resulted in the emergence of virulent SCN populations capable of breaking the resistance. Consequently, there is a need to explore new sources of resistance and broaden the genetic basis for resistance. Over 90% of SCN-resistant cultivars in the United States rely on a PI-88788-type resistance, with the Rhg1 resistance locus derived from PI 88788 conferring the strong and effective resistance. Research has demonstrated that the copy number of the 31.2 kb tandem repeat at the Rhg1 locus plays a dominant role in determining the level of resistance to SCN. Therefore, screening of soybean breeding lines and analyzing their diversity at the Rhg1 locus could help determine the extent of SCN resistance.
b. Research objectives
1. Evaluate 40 commercial soybean cultivars for their resistance reactions to two prevalent SCN populations detected in North Dakota.
2. Evaluate 100 NDSU breeding lines for their resistance reactions to two prevalent SCN populations detected in North Dakota.
3. Validate molecular markers for rapidly detecting the SCN resistance gene rhg1.
To achieve the objectives, 100 soybean breeding lines from the NDSU soybean breeding program and 58 commercial soybean cultivars from companies and growers were screened for their resistance reactions to two prevalent SCN populations; HG type 2.5.7, which can reproduce on the PI 88788 line, and HG type 0 or 7, which cannot reproduce on the PI 88788 line. HG type 2.5.7 was collected from Richland County, ND and HG type 0/7 was collected from Traill County, ND.
Pregerminated seeds from each of the soybean cultivars and lines were planted in 100 cc of pasteurized river bank sand in cone-tainers arranged in completely randomized design with four replicates. Each plant was inoculated with 2,000-2,500 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 30-32 days. SCN white females were then extracted from both the roots and soil of individual plants. The numbers of white females in the four replicates were averaged to determine the mean number of white females, which was used to calculate the Female Index (FI) according to the formula FI = (mean no. of white females produced on a tested soybean line/mean no. of white females on the susceptible check Barnes) x 100%. Based on the FI values, soybean cultivars and lines were classified for their resistance responses, as described by Schmitt and Shannon (1992), into four categories: resistant (R) (FI < 10), moderately resistant (MR) (10% < FI < 30%), moderately susceptible (MS) (30% < FI < 60%), or susceptible (S) (FI > 60%).
To distinguish between the "Williams 82" type rhg1-c, "Peking" type rhg1-a, or "PI 88788" type rhg1-b locus (Figure 1) for 24 NDSU breeding lines, the genomic region containing the two single nucleotide polymorphisms (SNPs) at 10,978 and 10,995 positions was amplified using a forward primer (5'-CTAGTTAGAGCATGAACTGC) and a reverse primer (5'-GTAGTAACAGGGCTATCAC) and then the purified PCR products were sequenced (McLab, San Francisco, CA).
For the assessment of copy number variation, 12 soybean accessions with known copy numbers of the Rhg1 repeat were selected to validate molecular markers for detecting copy number variation at the Rhg1 locus. A SYBR Green-based quantitative real-time PCR (qPCR) assay was adopted using the primers originally designed in the paper published by Lee et al. (2015). Genomic DNA was extracted from leaf tissues of 10-day old soybean plants, and qPCR was performed using an internal control gene, a heat-shock protein gene (hsp). A qPCR reaction was performed in a 10 µl volume using 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 reaction was carried out using an amplification program consisting of 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. Fluorescence data were collected after each annealing step.
To visualize specific amplicons, melting curve profiles were generated by increasing the temperature from 60 to 95°C in increments of 0.1°C per 0.4 to 0.5 fluorescence units. Relative quantification using the 2 -??CT technique was used to determine the copy number based on the reference check, Williams 82. The assay efficiency was calculated by using the formula, E = 10(1/–m) – 1; where m is the slope of the standard curve generated by plotting the Cq values against log of the DNA concentrations of Williams 82 by sequential two-fold dilutions. The copy numbers obtained from qPCR were compared to the standard values obtained by whole genome sequencing (Lee et al., 2015) to validate the molecular markers. Once validated, the same qPCR assay was used to investigate copy number variation among the 100 NDSU breeding lines at the Rhg1 locus and its association with resistance responses.
c. Research findings
Among the 100 breeding lines tested for HG type 2.5.7, 32 lines were moderately resistant (FI: 16.1 to 29.9%), 12 lines were moderately susceptible (FI: 31.4 to 58.7%), and the remaining 56 lines were susceptible (FI: 62.2 to 117.9%). For another SCN population HG type 7, one line was found to be resistant with FI 7.0%, 41 lines were moderately resistant (FI: 11.7 to 24.4%), 23 lines were moderately susceptible (FI: 41.6 to 59.7%), and the remaining 35 lines were susceptible (FI: 60.1 to 143.8%). Among the 58 commercial soybean cultivars screened for HG type 2.5.7, 3% were resistant (FI: 2.7 to 9.7%), 10% were moderately resistant (FI: 11.7 to 29.9%), 35% were moderately susceptible (FI: 35.2 to 59.2%), and 52% were susceptible (FI: 60.3 to 167.9%) (Figure 2). For HG type 7 or 0, 7% were resistant (FI: 5.3 to 9.7%), 12% were moderately resistant (FI: 10.7 to 28.2%), 47% were moderately susceptible (FI: 32.6 to 58.9%), and 34% were susceptible (FI: 60.8 to 110.9%) (Figure 2). Among the 58 commercial cultivars, JF30-93N and P001 were resistant to both HG type 2.5.7 and HG type 7.
Among 24 breeding lines sequenced to identify the type of Rhg1 locus, three had rhg1-b locus, 21 had rhg1-c locus, and none had rhg1-a locus as (Figure 1). Regarding the copy number variation among the 100 NDSU breeding lines, 20% had 11 copies, 21% lines had 10 copies, 1% had 6 copies, 1% had 3 copies, and the remaining 57% lines had only one copy of the Rhg1 repeat as shown (Figure 3). The lines that had higher copy number (= 6) had female index less than 40% for both HG types (Figure 4). For HG type 7, 42 lines that had copy number = 6 were either resistant or moderately resistant while the remaining lines with copy number = 3 were either moderately susceptible or susceptible. There was a high negative correlation (r = -0.86) between the female index (HG type 7) and copy number values. Therefore, a higher copy number at the Rhg1 locus was associated with greater resistance to the SCN population, which cannot attack the PI 88788 line.
d. Benefits to ND soybean farmers and Industry
SCN is a devastating disease in soybean. Among 100 breeding lines tested, 32 exhibited resistant or moderately resistant to both HG types, from which the breeder can choose to develop new SCN-resistant cultivars. Additionally, two commercial cultivars demonstrated resistance to both SCN populations, offering farmers in ND valuable choices to select resistant cultivars for infested fields. Furthermore, optimization of the copy number assessment assay will facilitate rapid detection of the resistance gene rhg1 and confirm the level of resistance to SCN. Both resistance screening and copy number assessment experiments will be repeated to validate the resistance responses and the association with copy numbers to identity or develop SCN-resistant cultivars more efficiently to manage this nematode disease.
Figure 1. Single nucleotide polymorphisms (SNPs) identified at Glyma18g02590 at 10,978 and 10,995 positions that help to distinguish the types of Rhg1 locus as rhg1-a, rhg1-b or rhg1-c.
Figure 2. Resistance responses of 58 commercial soybean cultivars from companies and growers to two SCN populations, HG type 2.5.7 and HG type 7/0 isolated from soybean fields in ND.
Figure 3. Copy number variation at the Rhg1 locus among the 100 breeding lines from the NDSU soybean breeding program.
Figure 4. Resistance responses of breeding lines from the NDSU soybean breeding program with female index < 40% to both HG type 2.5.7 and HG type 7 isolated from soybean fields in ND.
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a. Research Project Description
Soybean cyst nematode (SCN) is a major yield-limiting factor of soybean. Using host resistance is one of the best methods for managing SCN, but continuous use of the same resistance sources resulted in emergence of more virulent SCN populations capable of breaking resistance. New resistance sources need to be explored. Most of the SCN-resistant cultivars rely on PI 88788-type resistance, specifically the resistance genes at Rhg1 locus. Copy number of the genes determines the level of resistance. Therefore, screening of soybeans and analyzing variation at Rhg1 are important for identifying resistance.
b. Research Conducted
This research aimed to evaluate 140 soybean cultivars and breeding lines for resistance responses to two common SCN populations and validate molecular markers for detecting the resistance genes at Rhg1. In total, 158 cultivars and breeding lines were screened for two SCN populations, HG type 2.5.7 (more virulent) and HG type 7/0 (less virulent). Each plant was inoculated with approximately 2,000 eggs and grown in a growth chamber (Fig. 1). After harvest, white females (cysts) were extracted, female index (FI) was calculated, and resistance reactions were categorized. Copy numbers at Rhg1 among 100 breeding lines were determined using a SYBR Green-based qPCR assay.
c. Research Results
Two commercial cultivars were found to be resistant to both HG types. Among 100 breeding lines, 32 were moderately resistant to HG type 2.5.7 (FI < 30%), while one line was resistant (FI < 10%) and 41 were moderately resistant to HG type 7 (Fig. 2). 41 lines had 10-11 copies, 1 had 6 copies, and the remaining had 1-3 copies of the Rhg1 repeat. Lines with copy number = 6 were either resistant or moderately resistant to HG type 7, while the lines with copy number = 3 were moderately susceptible or susceptible. There was a strong negative correlation (r = -0.86) between female index values (HG type 7) and copy numbers.
d. Benefits
SCN is a devastating disease in soybean. Among 100 breeding lines tested for SCN, 32 exhibited resistance or moderate resistance to both HG types, from which the breeder can choose to develop new SCN-resistant cultivars. Additionally, two commercial cultivars demonstrated resistance to both SCN populations, offering farmers in ND valuable choices to select resistant cultivars for infested fields. Furthermore, the copy number assessment assay will facilitate rapid detection of the resistance gene rhg1 and confirm the level of resistance to SCN.
Fig 1. Dr. Guiping Yan checking soybean plants in a controlled growth chamber maintained at 27°C to ensure SCN resistance testing was performed under the optimal conditions.
Fig. 2. Resistance responses of breeding lines from the NDSU soybean breeding program with female index < 40% to both HG types 2.5.7 and 7 isolated from soybean fields in ND.