A soybean cyst nematode resistance GmSBPC molecular marker and application thereof

Abstract

The application relates to the technical field of molecular marker assisted breeding, and particularly discloses a soybean cyst nematode disease resistance GmSBPC molecular marker and application, a molecular marker Glyma.07-rs36760079 related to the soybean cyst nematode disease resistance is developed, and a primer group and a kit for amplifying the molecular marker are provided, the molecular marker, the primer group and the kit provided by the application can be used for assisting in screening or identifying soybean varieties resistant to cyst nematodes, and a KASP marker is developed, and the selection efficiency of the KASP marker on the disease-resistant materials in the typing materials is 77.8%.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, specifically to a molecular marker for identifying soybean resistance to cyst nematode disease, GmSBPC, and its application. Background Technology

[0002] Soybeans are a core dual-purpose crop for both grain and oil in my country and globally. They are a core source of plant protein and high-quality edible oil in the human diet, and an important supplier of high-quality protein feed for the livestock industry. Their production and supply are directly related to national food security, the stability of the agricultural product supply chain, and the high-quality development of the agricultural industry. With the upgrading of residents' dietary structure and the rapid development of animal husbandry, the market demand for soybeans is showing a rigid growth trend. Developing new soybean varieties that combine high yield, high quality, and multiple resistances has become a core task and urgent need in the field of soybean breeding.

[0003] Soybean cyst nematode (SCN) is a soil-borne disease that restricts high and stable soybean yields. It is widely distributed in major soybean-producing areas of Northeast my country and the Huang-Huai-Hai Plain. Infected fields typically experience yield reductions of 30-50%, with severely affected fields suffering reductions exceeding 60% or even total crop failure. The soybean cyst nematode is characterized by a wide host range, rapid physiological race differentiation, and frequent pathogenic variations, making its control extremely difficult. Traditional control methods mainly include crop rotation, application of chemical nematicides, and breeding of resistant varieties. While crop rotation can reduce nematode populations in the short term, it is insufficient to address the rapid differentiation of physiological races and cannot fundamentally curb the spread of the disease. Chemical agents, while effective quickly, can easily cause soil microecological imbalances, pesticide residue exceeding standards, and other environmental problems, and are inconsistent with the concept of green agriculture. Therefore, breeding and promoting SCN-resistant soybean varieties is the optimal approach that balances economic, ecological, and control effectiveness.

[0004] Although domestic and international researchers have conducted extensive research on the SCN resistance mechanism of soybeans, and the promotion of resistant varieties has achieved certain results, the long-term application of a single resistance gene can easily induce the directed evolution of nematode physiological races, leading to a rapid loss of varietal resistance and severely restricting the sustainability of disease-resistant breeding. Against this backdrop, exploring new SCN-resistant germplasm resources and developing molecular markers closely linked to resistance genes can not only achieve rapid and accurate screening of resistant germplasm and significantly shorten the identification cycle of resistant materials, but also promote the large-scale application of molecular marker-assisted breeding technology in the breeding of new SCN-resistant soybean varieties, breaking through the bottleneck of "low phenotypic selection efficiency and long cycle" in traditional breeding. This is of vital strategic significance for improving the efficiency of SCN-resistant soybean breeding in my country, enriching disease-resistant breeding gene resources, and ensuring soybean production security. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a molecular marker for identifying soybean resistance to cyst nematode disease and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a molecular marker for identifying soybean resistance to cyst nematode disease GmSBPC, wherein the molecular marker has an A / C polymorphism at position 36760079 of soybean Glyma.07; or has an A / C polymorphism at position 736 of the nucleotide sequence as shown in SEQ ID NO.1.

[0007] The present invention provides a primer set for amplifying the above-mentioned molecular markers, the primer set consisting of two specific primers and one reverse universal primer, the nucleotide sequences of the two specific primers are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.4.

[0008] This invention provides a kit for identifying soybean resistance to cyst nematode disease, the kit comprising the above-mentioned primer set or primer pair, 10×PCR Buffer, dNTPs and Taq enzyme.

[0009] This invention provides the application of the above-mentioned molecular markers in assisting the screening of soybean varieties resistant to cyst nematodes, identifying soybean varieties resistant to cyst nematode disease, or in the breeding of soybean varieties resistant to cyst nematodes.

[0010] This invention provides the application of the above primer set in assisting the screening of soybean varieties resistant to cyst nematodes, identifying soybean varieties resistant to cyst nematode disease, or in the breeding of soybean varieties resistant to cyst nematodes.

[0011] This invention provides the application of the above-mentioned kit in assisting the screening of soybean varieties resistant to cyst nematodes, identifying soybean varieties resistant to cyst nematode disease, or in the breeding of soybean varieties resistant to cyst nematodes.

[0012] This invention also provides a method for identifying soybean varieties resistant to cyst nematodes, comprising the following steps: DNA was extracted from the soybeans to be tested; The extracted soybean DNA was amplified by PCR using the primer set described above to obtain the PCR product. The genotype of the PCR product at the polymorphic site was detected, and the genotype results were used to determine whether the soybean to be tested was a soybean variety resistant to cyst nematodes. When the genotype at the molecular marker polymorphism site is CC, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention developed a molecular marker Glyma.07-rs36760079 associated with soybean resistance to cyst nematodes, and provided primer sets and kits for amplifying the molecular marker. The molecular marker, primer set and kits provided by this invention can be used to assist in screening or identifying soybean varieties resistant to cyst nematodes. At the same time, KASP markers were developed, and the selection efficiency of KASP markers for disease-resistant materials in genotyping materials was 77.8%. Attached Figure Description

[0014] Figure 1 After nematode stress GmSBPC Gene expression pattern diagrams: a: DN-L10-T: Dongnong-L10 treatment; DN-L10-CK: Dongnong-L10 control; b: Dongnong50-T: Dongnong50 treatment; Dongnong50-CK: Dongnong50 control; Figure 2 This is a map of soybean genomic DNA. Note: M: DL 2000 (+) DNA molecular weight standard; 1: Liaodou 3, 2: Tiejiazi, 3: Dongnong 44, 4: Suinong 1, 5: Fengshou 10, 6: Hefeng 29, 7: Dongnong 55, 8: Heilongjiang 41, 9: Fengshou 6, 10: Keshan Silijia, 11: Fengshou 10, 12: Haguang 1657, 13: Shisheng Changye, 14: Hefeng 2320, 15: Suinong 8, 16: Kenjiandou, 17: Chamoshidou, 18: Beijiang 9, 19: Chundou 1, 20: Henong 95, 21: Heihe 54, 22: Heinong 58, 23: Huajiang 1, 24: Hujiao 423, 25: Negative control - water; Figure 3 This is a PCR amplification diagram of the variant site. M: DL 2000 (+) DNA molecular weight standard; 1: Zhongpin 95-5388, 2: Tiejiazi, 3: Jiunong 21, 4: Suinong 1, 5: Fengshou 10, 6: Beifeng No. 4, 7: Baicheng Moshi Bean, 8: Mengdou 21, 9: Zhongpin 03-5334, 10: Suinong 11, 11: Neidou 4, 12: Suinong 22, 13: Dongnong L-13, 14: Longpin 03-311, 15: Jingshanpu, 16: Kenjiandou 15, 17: Chamoshidou, 18: Hujiao 423, 19: Dongnong 66, 20: Suinong 8; Figure 4 This is a sequence alignment diagram of variant sites in resistant and resistant materials; Figure 5 KASP tagging diagram for Glyma.07-rs36760079. Note: Blue tags represent type A / A, yellow tags represent type C / C, and green tags represent type C / A. Figure 6The chart shows the significance of the KASP typing results for Glyma.07-rs36760079. Note: This indicates that it is significant at the 0.03 level. Detailed Implementation

[0015] The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0016] It should be noted that, unless otherwise specified, all chemical reagents involved in this invention were purchased through commercial channels.

[0017] Plant varieties: 300 domestic and foreign soybean germplasm resources are shown in Table 1.

[0018] Table 1 Diseased soil species: Soybean cyst nematode HG type 1.2.3.5.7 (physiological race 3) was recorded in the article by Wu Depeng, Zhao Yue, Sheng Bihan, et al. Genetic model analysis of soybean cyst nematode race 3 by Dongnong L-10 [J]. Soybean Science, 2016, 35(3):6.DOI:10.11861 / j.issn.1000-9841.2016.03.0367, collected from the experimental field of Soybean Research Institute of Northeast Agricultural University.

[0019] Example 1: Obtaining the soybean cyst nematode resistance-related gene GmSBPC Based on RNA-seq sequencing data of the resistant variety Dongnong-L10 and the susceptible variety Suinong 14 in response to SCN physiological race 3 stress, one differentially expressed gene after SCN stress was identified and named [gene name missing]. GmSBPC RT-qPCR technology analysis GmSBPC Expression dynamics of this gene during infection with SCN race 3. Results showed that in the root system of the resistant material Dongnong-L10, the expression level of this gene reached its peak 12 h after infection, and was significantly higher than that of the susceptible material Dongnong 50 (e.g., ...). Figure 1 (As shown).

[0020] Table 2 GmSBPC Gene sequence variation analysis Genotypic data from 300 soybean germplasm resources were resequencing and analyzed using TASSEL software. GmSBPCSequence variation of the gene. The results showed that there were four allelic variation sites in the gene region: Chr.7:36760079 and Chr.7:36761897 were located in the 5'UTR region, Chr.7:36761089 was located in the 3'UTR region, and Chr.7:36761064 was located within an intron region (Table 2). Using the GLM model in TASSEL software, the association between these variation sites and the resistance phenotype of SCN 3 physiological race was analyzed based on genotypic and phenotypic data from 300 soybean germplasm resources. The results showed that the Chr.7:36760079 site located in the 5'UTR region was most significantly associated with resistance to SCN 3 physiological race.

[0021] Example 2: Preparation of genomic DNA from soybean germplasm resources and amplification and sequencing of target sequences Genomic DNA was extracted from 300 soybean accessions grown in a greenhouse using the CTAB method, and DNA bands were then detected by agarose gel electrophoresis. The results are as follows: Figure 2 As shown, the results indicate that the extracted genomic DNA bands are clear, correctly positioned, and highly pure, indicating successful extraction of genomic DNA from the test material. This DNA can be used for further sequencing verification of superior allelic variant sites and development of molecular markers.

[0022] The genomic sequences of key candidate genes were downloaded from the Phytozome database. Using superior allelic variant sites as target sites, specific primers for these sites were designed using Premier 5.0 software. The nucleotide sequences of these specific primers are as follows: F-AGAGCCGCTGAGAAACAGCG, as shown in SEQ ID NO.5; R-GTCTGCTGAATAATCGGAAG, as shown in SEQ ID NO.6.

[0023] Specific primer sequences were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd. DNA from 10 highly resistant and 10 highly susceptible samples were selected for PCR amplification. The PCR amplification system is shown in Table 3, and the reaction procedure is shown in Table 4. The amplified product from the rs36760079 site of Chr.07 was 1286 bp in length, as shown in the results. Figure 3 As shown, the amplification results indicate that the PCR amplification bands are single and accurately positioned, with no other non-specific amplification bands. The amplification products can be used for the next sequencing operation.

[0024] Table 3 PCR amplification system Table 4 PCR Amplification Program The successfully amplified variant sites were amplified and purified in large quantities. The purified products were sent to Beijing Ruiboxingke Sequencing Co., Ltd. for first-generation sequencing. The DNA sequence sequencing results were assembled and compared using DNAMAN V6.0 software. The results showed that the excellent variant site of this candidate gene has genotyping. The detailed comparison results are as follows: Locus 36760079 of Chr.07 showed an A / C genotype between resistant and susceptible materials. Detailed comparison results are as follows: Figure 4 As shown, the disease-resistant materials, including *Iron Bean*, *Suinong 1*, *Beifeng 4*, *Suinong 11*, and *Fengshou 10*, carry the C allelic variant; while the disease-susceptible materials, including *Hujiao 423*, *Dongnong 66*, *Suinong 22*, *Chamoshi Dou*, and *Longpin 03-311*, carry the A allelic variant. Sequencing analysis results indicate that the variant sites exhibit genotyping between the disease-resistant and disease-susceptible materials, paving the way for the development of molecular markers for superior allelic variant sites.

[0025] Example 3: KASP molecular markers and their application Glyma.07-rs36760079 A / C classification SEQ ID NO.1 r is either A or C.

[0026] KASP marker primers were designed based on the upstream and downstream sequences of superior allelic variant sites and synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd. The primer sequences are as follows: KASP-tagged primers: F1-GAAGGTGACCAAGTTCATGCTAGGTGAGTGAGGTGGGTTTTGGAGC (SEQ ID NO. 2); F2-GAAGGTCGGAGTCAACGGATTAGGTGAGTGAGGTGGGTTTTGGAGA (SEQ ID NO. 3); R-GATCTCTCATTACTCTCTTATTCCTCTT (SEQ ID NO. 4).

[0027] The KASP reaction system is shown in Table 5, and the reaction procedure is shown in Table 6. The genotyping data were acquired and analyzed using ABI7500 Software v2.3 quantitative fluorescence software.

[0028] Table 5 KASP Labeling Reaction System Table 6 KASP Labelling Reaction Procedure Example 4: Validation and Application of Molecular Markers DNA was extracted from 300 soybean samples listed in Table 1. The extracted soybean DNA was amplified by PCR using the following primer set and the reaction conditions described in Example 2 to obtain the PCR product; F1-GAAGGTGACCAAGTTCATGCTAGGTGAGTGAGGTGGGTTTTGGAGC; F2-GAAGGTCGGAGTCAACGGATTAGGTGAGTGAGGTGGGTTTTGGAGA; R-GATCTCTCATTACTCTCTTATTCCTTCTT; The genotype of the PCR product at the polymorphic site was detected, and the genotype results were used to determine whether the soybean to be tested was a soybean variety resistant to cyst nematodes. When the genotype at the molecular marker polymorphism site is CC, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes.

[0029] The KASP marker results for the rs36760079 locus of Glyma.07 are as follows: Figure 5 As shown, the blue markers near the Y-axis represent soybean materials carrying the homozygous AA genotype, the yellow markers near the X-axis represent soybean materials carrying the homozygous CC genotype, and the green markers in the middle represent the heterozygous genotypes A / C. Analysis of all experimental materials revealed that 18% of the soybean materials carried the homozygous CC genotype, and 75.3% carried the homozygous AA genotype. A t-test analysis of the two groups showed the following results: Figure 6As shown, the female insect index of soybean materials carrying the CC homozygous genotype was significantly lower than that of materials carrying the AA homozygous genotype. This marker can accurately distinguish soybean materials carrying A / C allelic variations, indicating successful KASP marker genotyping. The results demonstrate the successful development of the KASP molecular marker for superior allelic variation sites, and molecular markers can provide a valuable tool for molecularly assisted breeding of soybeans cyst nematode resistance.

[0030] The efficiency of molecular marker disease resistance identification is calculated using the following formula.

[0031] Disease resistance identification efficiency = (Number of disease-resistant samples among those carrying the disease-resistant genotype) / (Number of samples carrying the disease-resistant genotype) × 100%; Calculations showed that the superior allelic CC carried by the Glyma.07-rs36760079 marker had a selection efficiency of 77.8% for anti-disease materials, while the selection efficiency of AA for susceptible materials was 25.3%. These results indicate that the superior allelic molecular marker is effective in assisting the selection of anti-disease materials.

[0032] Finally, it should be noted that the above embodiments do not limit the present invention in any way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A molecular marker for identifying soybean resistance to cyst nematode disease GmSBPC, characterized in that, The molecular marker exhibits an A / C polymorphism at position 36760079 of soybean Glyma.07; or an A / C polymorphism at position 736 of the nucleotide sequence shown in SEQ ID NO.

1.

2. A primer set for amplifying the molecular marker as described in claim 1, characterized in that, The primer set consists of two specific primers and one reverse universal primer. The nucleotide sequences of the two specific primers are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.

4.

3. A kit for identifying soybean resistance to cyst nematode disease, characterized in that, The kit includes the primer set as described in claim 2, 10×PCR Buffer, dNTPs, and Taq enzyme.

4. The application of the molecular markers as described in claim 1 in assisting the screening of soybean varieties resistant to cyst nematodes, identifying soybean varieties resistant to cyst nematode disease, or in the breeding of soybean varieties resistant to cyst nematodes.

5. The application of the primer set as described in claim 2 in assisting the screening of soybean varieties resistant to cyst nematodes, identifying soybean varieties resistant to cyst nematode disease, or in the breeding of soybean varieties resistant to cyst nematodes.

6. The application of the kit as described in claim 3 in assisting the screening of soybean varieties resistant to cyst nematodes, identifying soybean varieties resistant to cyst nematode disease, or in the breeding of soybean varieties resistant to cyst nematodes.

7. A method for identifying soybean varieties resistant to cyst nematodes, characterized in that, Includes the following steps: DNA was extracted from the soybeans to be tested; The extracted soybean DNA to be tested was amplified by PCR using the primer set described in claim 2 to obtain PCR products; The genotype of the PCR product at the polymorphic site was detected, and the genotype results were used to determine whether the soybean to be tested was a soybean variety resistant to cyst nematodes. When the genotype at the molecular marker polymorphism site is CC, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes.

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