Method for identifying soybean cyst nematode resistant germplasm by using transcription detection technology

Abstract

The invention discloses a method for identifying soybean cyst nematode resistant germplasm by using a transcription detection technology. The invention belongs to the technical field of biology, and particularly relates to a method for identifying soybean cyst nematode resistant germplasm by using a transcription detection technology. The method for detecting the resistance of soybeans to cyst nematode comprises the following steps: 1) extracting total RNA (Ribonucleic Acid) of a sample to be detected, and reversely transcribing the RNA into cDNA (Complementary Deoxyribose Nucleic Acid); (2) analyzing the comprehensive relative transcriptional level sigma T of the target gene GmRhg1 of the soybean to be detected by utilizing RT-PCR (Reverse Transcription-Polymerase Chain Reaction); the comprehensive relative transcriptional level sigma T of the target gene is equal to A * 0.25 + B * 0.50 + C * 0.25, A is the ratio of GmRhg1-1 / GmActin, B is the ratio of GmRhg1-2 / GmActin, and C is the ratio of GmRhg1-3 / GmActin; and (3) judging the resistance capability of the soybean cyst nematode according to the comprehensive relative transcriptional level of the obtained target gene.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for identifying soybean cyst nematode resistant germplasm by using transcriptional detection technology. Background Art

[0002] Soybean cyst nematode (SCN) is a worldwide soybean disease that occurs in all soybean production areas around the world. The yield reduction caused by soybean cyst nematode is more serious than that of other soybean diseases. Generally, it reduces soybean yield by 10% - 30%, and in severe plots, it can reach 70% - 90%, or even cause a complete crop failure. In the United States, the annual yield loss due to SCN reaches 1.5 billion US dollars per year, accounting for about 36% of the total soybean disease losses. Soybean cyst nematodes belong to obligate, sedentary, endoparasites, mainly damaging the roots of soybeans. The damaged plants are significantly dwarfed, the leaves turn yellow and fall early, the flowering period is delayed, the flower organs are clustered, the flowers and young pods shrink, the pods are few and small, and even no pods are formed; the root systems of diseased plants are underdeveloped, the lateral roots are reduced, the fine roots are increased, the root nodules are scarce, and in the initial stage of the disease, white or yellowish-brown granules the size of millet grains are attached to the roots of the diseased plants.

[0003] In the past few decades, only two resistance sources, namely PI88788 and Peking (PI548402), have been widely used in soybean breeding. The single resistance source (more than 90% of SCN-resistant varieties come from PI88788) and the planting of single varieties have led to the reduction of the effectiveness of resistance or the loss of resistance. Coupled with the continuous differentiation of SCN races and the emergence of more virulent races, 76% of the soybean fields in northern United States have been infected, indicating an urgent need to explore new resistance sources and apply them to soybean breeding. By using GWAS to analyze the SCN resistance of 120 Chinese soybean cultivars, 70% of them were moderately susceptible or completely susceptible to race 1, only 6 cultivars were highly resistant (IP < 10), 30 were moderately resistant (10 < IP < 30), and 4 of the 6 highly resistant cultivars had Peking bloodline in their pedigrees, indicating that the SCN resistance genes in soybean cultivars are also single, and there is also an urgent need to find new resistance sources and cultivate new SCN-resistant varieties.

[0004] Traditional methods for identifying soybean cyst nematode resistance include field disease nursery identification, potting identification with diseased soil, and greenhouse inoculation identification. However, whether it is artificial inoculation or disease nursery induction, due to climate, environmental differences, inconsistent inoculation egg concentrations among the same variety, and large temperature differences, the differences in resistance identification among different replicates of the identification materials are often large, and it is often necessary to repeat multiple times to determine the resistance. Therefore, the identification of soybean cyst nematode resistance emphasizes multi-year and multi-point identification, which results in not only a long identification cycle but also a serious waste of land and labor for traditional identification methods.

[0005] Through the continuous deepening of research on the signal transduction pathway of soybean cyst nematode resistance, transcriptional detection technology is conducive to selecting key target sites and key target genes that can represent soybean resistance to SCN. By detecting the transcriptional expression level of target genes, the disease resistance level of different soybean germplasms to soybean cyst nematode is comprehensively evaluated, thereby achieving rapid and accurate identification of soybean germplasm resources for SCN resistance. Summary of the invention

[0006] The main problem to be solved by the present invention is how to efficiently identify the resistance of soybean to cyst nematodes.

[0007] In order to solve the above problems, the present invention provides a method for detecting resistance of soybean to cyst nematodes.

[0008] The method for detecting the resistance of soybean to cyst nematodes of the present invention comprises the following steps:

[0009] 1) extracting total RNA from the soybean sample to be tested, and reverse transcribing the RNA into cDNA;

[0010] 2) RT-PCR was used to analyze the comprehensive relative transcription level ΣT of the target gene GmRhg1 in the tested soybean;

[0011] The comprehensive relative transcription level of the target gene GmRhg1 ΣT=A×0.25+B×0.50+C×0.25, wherein A is the transcription level ratio of GmRhg1-1 / GmActin gene, B is the transcription level ratio of GmRhg1-2 / GmActin gene, and C is the transcription level ratio of GmRhg1-3 / GmActin gene;

[0012] 3) According to the comprehensive relative transcription level of the target gene obtained in step 2), the resistance ability of soybean cyst nematode is determined.

[0013] The tested soybeans with a high comprehensive relative transcription level of the target gene have higher resistance to soybean cyst nematodes than the tested soybeans with a low comprehensive relative transcription level of the target gene.

[0014] Furthermore, the specific criteria are as follows:

[0015] a. When the comprehensive relative transcription level of the target gene GmRhg1 ΣT≥1.00, the soybean material is highly resistant to soybean cyst nematode;

[0016] b. When the comprehensive relative transcription level of the target gene GmRhg1 is 0.50≤ΣT<1.00, the soybean material is moderately resistant to soybean cyst nematode;

[0017] c. When the comprehensive relative transcription level of the target gene GmRhg1 is 0.30≤ΣT<0.50, the soybean material is moderately susceptible to soybean cyst nematode;

[0018] d. When the comprehensive relative transcription level ΣT of the target gene GmRhg1 is less than 0.30, the soybean material is susceptible to soybean cyst nematode.

[0019] Wherein, the nucleotide sequence of the GmRhg1-1 gene is:

[0020]

[0021] The nucleotide sequence of the GmRhg1-2 gene is:

[0022]

[0023] The nucleotide sequence of the GmRhg1-3 gene is:

[0024]

[0025]

[0026] The nucleotide sequence of the GmActin gene is:

[0027]

[0028] In the above method, the composition for analyzing the comprehensive relative transcription level ΣT of the GmRhg1 gene of the soybean to be tested in step 2) consists of composition 1, composition 2, composition 3 and composition 4, wherein composition 1 is a detection primer set for detecting the GmRhg1-1, and composition 1 consists of GmRhg1-1 / F and GmRhg1-1 / R; the nucleotide sequence of the GmRhg1-1 / F is sequence 1; the nucleotide sequence of the GmRhg1-1 / R is sequence 2;

[0029] The composition 2 is a detection primer set for detecting the GmRhg1-2, and the composition 2 is composed of GmRhg1-2 / F and GmRhg1-2 / R; the nucleotide sequence of the GmRhg1-2 / F is sequence 3; the nucleotide sequence of the GmRhg1-2 / R is sequence 4;

[0030] The composition 3 is a detection primer set for detecting the GmRhg1-3, and the composition 3 is composed of GmRhg1-3 / F and GmRhg1-3 / R; the nucleotide sequence of the GmRhg1-3 / F is sequence 5; the nucleotide sequence of the GmRhg1-3 / R is sequence 6;

[0031] The composition 4 is a detection primer set for detecting the GmActin, and the composition 4 consists of GmActin / F and GmActin / R; the nucleotide sequence of the GmActin / F is sequence 27; and the nucleotide sequence of the GmActin / R is sequence 28.

[0032] The present invention also provides the use of the above-mentioned method in identifying or assisting in identifying the resistance of soybean to cyst nematodes.

[0033] The present invention also provides the use of the above-mentioned method in preparing a product for identifying or assisting in identifying the resistance of soybean to cyst nematodes.

[0034] The present invention also provides the application of the above-mentioned method in soybean breeding, wherein the purpose of the soybean breeding is to breed soybean varieties with strong resistance to cyst nematodes.

[0035] The present invention also provides the composition described above. The composition consists of composition 1, composition 2, composition 3 and composition 4, wherein composition 1 is a detection primer set for detecting the GmRhg1-1, and the composition 1 consists of GmRhg1-1 / F and GmRhg1-1 / R; the nucleotide sequence of the GmRhg1-1 / F is sequence 1; the nucleotide sequence of the GmRhg1-1 / R is sequence 2;

[0036] The composition 2 is a detection primer set for detecting the GmRhg1-2, and the composition 2 is composed of GmRhg1-2 / F and GmRhg1-2 / R; the nucleotide sequence of the GmRhg1-2 / F is sequence 3; the nucleotide sequence of the GmRhg1-2 / R is sequence 4;

[0037] The composition 3 is a detection primer set for detecting the GmRhg1-3, and the composition 3 is composed of GmRhg1-3 / F and GmRhg1-3 / R; the nucleotide sequence of the GmRhg1-3 / F is sequence 5; the nucleotide sequence of the GmRhg1-3 / R is sequence 6;

[0038] The composition 4 is a detection primer set for detecting the GmActin, and the composition 4 consists of GmActin / F and GmActin / R; the nucleotide sequence of the GmActin / F is sequence 27; and the nucleotide sequence of the GmActin / R is sequence 28.

[0039] The present invention also provides the use of the above-mentioned composition in preparing a product for identifying or assisting in identifying the resistance of soybean to cyst nematodes.

[0040] The present invention also provides the use of the above-mentioned composition in soybean breeding, wherein the purpose of the soybean breeding is to breed soybean varieties with strong resistance to cyst nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of cyst inoculation of soybean cyst nematode-resistant materials. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0043] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0044] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0045] The plant total RNA extraction kit in the following embodiments: Coolaber, product number: RE600; reverse transcription kit HiScript III 1st Strand cDNA Synthesis kit, Nanjing Vazyme, product number R312-02; real-time fluorescence kit Taq Pro Universal SYBR qPCR Master Mix, Nanjing Vazyme, product number: Q712-02; 2×Rapid Tap Master Mix (Vazyme), product number R222-03.

[0046] The disease-resistant materials PI88788, Fayett, Sijiaoqi black bean, moderately resistant material Jin 1265, moderately susceptible material Hefeng 35 and susceptible varieties Lee and Williams 82 in the following examples have been recorded in: Patil, Whole-genome re-sequencing reveals the impact of the interaction of copy number variants of the rhg1 and Rhg4 genes on broad-based resistance to soybean cyst nematode, Plant Biotechnology Journal 17: 1595-1611; Wei He, Evaluation and Utilization of Soybean Germplasm with Resistance to Cyst Nematode No. 2, Journal of Plant Genetic Resources, 2022, 23(2): 450-459; Liu Zhangxiong, Germplasm Innovation of Soybean Resistance to Cyst Nematode No. 4, Soybean Science, 2008, 27(6); Guo Tai, Variety Characteristics and Yield Benefit of New Soybean Variety "Hefeng 35", Soybean Bulletin, 2001, 5. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0047] The soybean cyst nematode race 3 in the following examples has been recorded in: Li Haiyan, Effects of soybean resistance resources on the growth and development dynamics of soybean cyst nematode race 3, Crop Journal, 2017(1): 144-149. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0048] The 30 materials in Example 2 below are from the National Germplasm Resource Bank, the website is: https: / / www.cgris.net. The public can obtain the biological materials from the National Germplasm Resource Bank. The biological materials are only used to repeat the experiments of the present invention and cannot be used for other purposes. The specific information of the 30 materials is as follows:

[0049] 1. Bainong 9: Characteristics and cultivation techniques of the new soybean variety Bainong 9, Soybean Bulletin, 2003: 4.

[0050] 2. Weidou 7:

[0051] https: / / baike.sogou.com / v75713070.htm?fromTitle=%E6%BD%8D%E8%B1%867%E5%8F%B7.

[0052] 3. Week 9521:

[0053] https: / / baike.sogou.com / v167345768.htm?fromTitle=%E7%9A%96%E8%B1%8621116.

[0054] 4. Huaxia No. 16: https: / / baike.baidu.com / item / %E5%8D%8E%E5%A4%8F16%E5%8F%B7.

[0055] 5. Dyer: Deciphering the genetic basis of resistance to soybean cystnematode combining IBD and association mapping, Theoretical and AppliedGenetics, (2023) 136:50.

[0056] 6. Jin 1265: Germplasm innovation of soybean cyst nematode resistance race 4, Soybean Science, 2008, 27(6): 911-914.

[0057] 7. Sijiaoqi black bean: Evaluation and utilization of soybean germplasm with resistance to cyst nematode No. 2 physiological subspecies, Journal of Plant Genetic Resources, 2022, 23(2): 450-459.

[0058] 8. Chibuliuheidou: Research progress on identification and evaluation of soybean disease resistance resources, 2008, 28(4): 30-32.

[0059] 9. Round black bean: Evaluation and utilization of soybean germplasm with resistance to cyst nematode No. 2 physiological race, Journal of Plant Genetic Resources, 2022, 23(2): 450-459.

[0060] 10. Original black bean: Evaluation and utilization of soybean germplasm with resistance to cyst nematode No. 2 physiological race, Journal of Plant Genetic Resources, 2022, 23(2): 450-459.

[0061] 11. Zheng 12195: Evaluation and utilization of soybean germplasm with resistance to cyst nematode physiological race 2. Journal of Plant Genetic Resources, 2022, 23(2): 450-459.

[0062] 12. Huacaidou4: https: / / www.zhunongtong.com / ask / 482060.html.

[0063] 13. Qinong 12: Breeding and supporting cultivation technology of high gelling and line-resistant soybean variety Qinong 12, Heilongjiang Agricultural Science, 2021(7): 141-143.

[0064] 14. M85-610: ALPHA SOYBEANS.

[0065] 15. Shore: China Crop Germplasm Resources Query Network, https: / / www.cgris.net.

[0066] 16. TGX814-26D: China Crop Germplasm Resources Query Network, https: / / www.cgris.net.

[0067] 17. Life: China Crop Germplasm Resources Query Network, https: / / www.cgris.net.

[0068] 18. Dongnong 10-L: Analysis of genetic model of Dongnong L-10+ against physiological race 3+ of soybean cyst nematode, Soybean Science, 201635(3): 367-372.

[0069] 19. Brief report on the breeding of a new soybean variety Jiunong 20, 1993, 2(3): 53.

[0070] 20. Harvest No. 10: http: / / soy.cropdb.cn / variety / varis / 600225.htm.

[0071] 21. Hefeng 35: Characteristics and yield benefits of the new soybean variety “Hefeng 35”, Soybean Bulletin, 2001, 5.

[0072] 22. Black Farmer 37

[0073] https: / / baike.sogou.com / v66657478.htm?ch=frombaikevr&fromTitle=%E9%BB%91%E5%86%9C37%E5%8F%B7.

[0074] 23. Thorne: China Crop Germplasm Resources Query Network, https: / / www.cgris.net.

[0075] 24. Nanyang tall green: http: / / soy.cropdb.cn / variety / varis / 602824.htm

[0076] 25. Yukimidori, China Crop Germplasm Resources Query Network, https: / / www.cgris.net.

[0077] 26. SinoKubanskaya 4958: China crop germplasm resources enquiry website.

[0078] 27. Hill: China Crop Germplasm Resources Query Network, https: / / www.cgris.net.

[0079] 28. Black farmers 531 http: / / soy.cropdb.cn / variety / varis / 601245.htm.

[0080] 29. Environmental stability analysis of quality traits of the new soybean variety Heinong 84, Soybean Science, 2024, 43(01).

[0081] 30. Black Farmer 81

[0082] https: / / baike.baidu.com / item / %E9%BB%91%E5%86%9C81 / 56121937#::text=%E9%BB%91%E5%86%9C81%E6%98%AF%E5%A4%A7%E8%B1%86%E5%93%81%E7%A7%8D.

[0083] Example 1. Transcriptome sequencing of different soybean cyst nematode-resistant varieties and screening of target genes

[0084] Reproduction of soybean cyst nematode: Dispense the diseased soil infected with soybean cyst nematode No. 3 physiological subspecies into nutrient pots, sow the SCN susceptible variety Williams82, and cultivate in a greenhouse at 25℃, 16h / 8h light / dark. After the soybean plants have grown in the diseased soil for about 25 days, pour them out of the diseased soil, use a high-pressure water gun to wash the soybean root cysts, collect the cysts with 25 mesh, 60 mesh, and 100 mesh sieves in turn, and then gently grind the cysts to release the eggs, collect the eggs with a 500 mesh sieve and count them under a microscope, prepare the inoculation solution, and set aside.

[0085] The soybean cyst nematode-resistant varieties Sijiaoqi black beans, original black beans and susceptible variety Williams82 were sown in sterilized soil and cultured in a greenhouse at 27°C and 16h / 8h light / dark. After the soybeans grew into seedlings, one healthy seedling was left in each pot for inoculation of cysts. The number of cyst eggs inoculated per plant was 2700. The inoculation group was SCN race 3 as the experimental group and the control group was not inoculated. Each group had 3 biological replicates ( Figure 1 ).

[0086] Nine days after inoculation, the soybean plants were removed from the nutrient pots, and the root tissues of soybeans were taken and treated with liquid nitrogen for subsequent experiments. The soybean root samples (Sijiaoqi black beans, original black beans, and Williams82 inoculated and uninoculated materials, 3 biological replicates, a total of 18 samples) were provided to Beijing Aoweisen Gene Technology Co., Ltd. for transcriptome sequencing.

[0087] Based on the analysis of the transcriptome counts of six groups of inoculated and uninoculated Sijiaoqi black beans, original black beans and Williams82, 13 genes differentially expressed in soybean cyst nematode resistant and susceptible materials were preliminarily screened out, including GmRhg1-1 (Glyma18g02580), GmRhg1-2 (Glyma18g02590), GmRhg1-3 (Glyma18g02610), GmSHMT (Glyma08g109000), GmPal2.1 (Glyma18g0582 00), GmPR1a (Glyma13g251600), GmBAG6 (Glyma07g061500), GmCML (Glyma17g128900), GmIT (Glyma18g032300 ), GmPOD (Glyma09g022300), GmMYB48 (Glyma03G143600), GmPTHR (Glyma10g188200) GmMTE (Glyma10g189400).

[0088] In order to facilitate rapid and large-scale screening and identification of soybean germplasm resources, the target genes should be selected with a bias towards genes with large basal level differences and high transcript numbers. To this end, we first used PI88788, Fayett, Sijiaoqi Black Bean, Jin 1265, Hefeng 35 and Lee, Williams82 to screen the above candidate target genes.

[0089] First, the above 7 soybean materials were inoculated and verified in a greenhouse. The seeds were sown in sterilized soil and cultured in a greenhouse at 27°C and 16h / 8h light / dark. After the soybeans grew into seedlings, one healthy seedling was left in each pot for inoculation of cysts. The number of cyst eggs inoculated per plant was 2700. Ten plants were inoculated for each material, with three biological replicates. After the plants were cultured for 25 days, the materials were removed from the nutrient pots, the cyst quantity was recorded, and the cyst index was calculated.

[0090] Through inoculation identification, the varieties of soybean cyst nematode highly resistant (HR) are: PI88788, Fayett, Sijiaoqi black bean; the varieties of soybean cyst nematode moderately resistant (MR) are: Jin 1265; the varieties of soybean cyst nematode moderately susceptible (MS) are: Hefeng 35; the varieties of soybean cyst nematode highly susceptible (HS) are: Lee, Williams82.

[0091] The above 7 materials were sown in a greenhouse, and the total RNA of the leaves was extracted using a plant total RNA extraction kit. The quality of RNA was detected by 1.5% agarose gel electrophoresis, and the RNA electrophoresis bands were clear and the RNA was not significantly degraded. The RNA was reversed into cDNA using the HiScript IIQ RT SuperMix for qPCR (Vazyme, catalog number R222-01) kit.

[0092] According to the gene sequences published by NCBI, GmActin (Glyma18g290800) was used as the internal reference gene, PCR primers were designed for 13 genes (see Table 1), and RT-PCR was used to preliminarily analyze the expression of differentially expressed genes in five soybean varieties.

[0093] The RT-PCR reaction system was as follows: 2× Rapid Tap Master Mix 25 μl; 1 μl cDNA template; 1 μl each of primers AC-F / AC-R (10 μM) and differential gene F / R (10 μM); and sterile ddH2O to make up to 50 μl.

[0094] The RT-PCR reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, annealing temperature of 53°C, annealing for 15 s, 72°C for 30 min, 24 cycles; 72°C for 5 min.

[0095] Table 1. Primers and their sequences used in semi-quantitative RT-PCR analysis

[0096]

[0097] The RT-PCR products were subjected to 2.0% gel electrophoresis, and the expression level of soybean GmActin was used as the internal standard. Image J was used to analyze the relative expression levels of each differentially expressed gene. The results are shown in Table 2.

[0098] Table 2. Transcriptional expression analysis of target genes in known resistant varieties

[0099]

[0100] The results showed that five genes, GmPR1a (Glyma13g251600), GmBAG6 (Glyma07g061500), GmCML (Glyma17g128900), GmIT (Glyma18g032300), and GmPOD (Glyma09g022300), were not detected due to low transcript counts. Although five genes, GmSHMT, GmPal2.1 (Glyma10g058200), GmMYB48 (Glyma03G143600), GmPTHR (Glyma10g188200), and GmMTE (Glyma10g189400), were detected to be expressed, no significant differences were shown in the soybean cyst nematode resistant and susceptible materials. The transcription levels of three genes, GmRhg1-1 (Glyma18g02580), GmRhg1-2 (Glyma18g02590), and GmRhg1-3 (Glyma18g02610), are correlated with soybean cyst nematode resistance.

[0101] Therefore, in large-scale rapid screening, GmRhg1-1 (Glyma18g02580I), GmRhg1-2 (Glyma18g02590), and GmRhg1-3 (Glyma18g02610) can be selected as target genes for rapid screening of soybean cyst nematode-resistant materials.

[0102] Example 2: Study on the correlation between the expression levels of three target genes and resistance to soybean cyst nematodes

[0103] To further study the correlation between the transcriptional expression of the three target genes GmRhg1-1 (Glyma18g02580I), GmRhg1-2 (Glyma18g02590), and GmRhg1-3 (Glyma18g02610) and their resistance to soybean cyst nematodes, 30 soybean germplasm resources (Table 3) were selected as materials for field inoculation verification of soybean cyst nematodes and detection of their three target genes.

[0104] 1. Study on resistance phenotype of soybean cyst nematode

[0105] The above 30 soybean materials were sown in the SCN disease nursery of the Changlinghu Experimental Base of the Soybean Institute of Heilongjiang Academy of Agricultural Sciences for field disease nursery identification, with pI88788 and Peking as disease-resistant controls, and Lee and William82 as susceptible controls. The field inoculation identification method was implemented in accordance with the "Heilongjiang Provincial Local Standard "Technical Regulations for Identification of Field Disease Nursery of Soybean Resistance to Cyst Nematodes, DB 23 / T3110-2022". The disease nursery was identified as physiological race 3, with a cyst content of 80 to 149 per 100 grams of air-dried soil, which met the SCN identification requirements. Each material was set up with 3 biological replicates and sown in 3 periods, with a difference of 10 days between each sowing period.

[0106] About 45 days after soybean sowing, when the third three-leaf is complete, the first generation of cyst nematodes is in the peak period of cyst development and can be investigated. Gently pull the roots out of the diseased bed, shake off the soil on the roots, observe the white cysts attached to the roots of each plant, record the cyst quantity, and calculate the cyst index. Cyst index (FI) = (average cyst quantity of the test variety / average cyst quantity of the susceptible control variety) × 100.

[0107] The classification is based on FI, with specific standards as follows: HR: 0-10%; MR: 10.1-30.0%, MS: 30.1%-60.0%, HS: above 60.1%.

[0108] The results of the resistance identification of 30 soybean materials to soybean cyst nematodes are shown in Table 3. The results showed that among the 30 materials, there were 5 materials with high resistance (HR), 5 materials with medium resistance (MR), 2 materials with medium sensitivity (MS), and 12 materials with high sensitivity (HS).

[0109] Table 3. Field inoculation of 30 soybean materials and their resistance classification

[0110]

[0111]

[0112] 2. Detection of relative expression levels of three target genes in soybean germplasm resources

[0113] The above 30 materials and the disease-resistant control varieties PI88788 and Fayette and the susceptible varieties Lee and wiliiams were sown in sterilized soil and cultured in a greenhouse at 27°C and 16h / 8h light / darkness. The total RNA of the leaves was extracted using a plant total RNA extraction kit. The quality of RNA was detected by 1.5% agarose gel electrophoresis. The RNA electrophoresis bands were clear and there was no obvious degradation of RNA. The RNA was reversed into cDNA using the HiScript IIQ RT SuperMix for qPCR (Vazyme, catalog number R222-01) kit.

[0114] Using GmActin (Glyma18g290800) as the internal reference gene, the relative transcription levels of the three target genes GmRhg1-1 (Glyma18g02580), GmRhg1-2 (Glyma18g02590), and GmRhg1-3 (Glyma18g02610) in the above 30 soybean germplasm resources were detected.

[0115] The RT-PCR reaction system was: 2×Rapid Tap Master Mix 25μl; 1μl cDNA template; 1μl each of primers AC-F / AC-R (10μM) and differential gene F / R (10μM); sterile ddH2O to make up to 50μl. The RT-PCR reaction conditions were: 95℃3min; 95℃15s, annealing temperature 53℃, annealing 15s, 72℃30min, 24 cycles; 72℃5min.

[0116] The RT-PCR products were subjected to 2.0% gel electrophoresis, and the expression level of soybean GmActin was used as the internal standard. Image J was used to analyze the relative expression levels of each differentially expressed gene. The results are shown in Table 4.

[0117] Table 4. Target gene identification and resistance classification of 30 soybean materials

[0118]

[0119]

[0120] Based on the relative expression of the preliminary test and control varieties, the materials were evaluated by the relative transcription level of individual genes, i.e.

[0121] 1) The relative transcription level of GmRhg1-1 gene was used as the standard for identification: when the transcription level of GmRhg1-1 gene pair was ≥1.00, the soybean material was highly resistant to soybean cyst nematode (HR); when the relative transcription level of GmRhg1-1 gene was ≥0.50 and <1.00, the soybean material was moderately resistant to soybean cyst nematode (MR); when the relative transcription level of GmRhg1-1 gene was ≥0.30 and <0.50, the soybean material was moderately susceptible to soybean cyst nematode (MS); when the comprehensive relative transcription level of GmRhg1-1 gene was <0.30, the soybean material was susceptible to soybean cyst nematode (HS). Among the 30 materials, there were 4 highly resistant (HR) materials, 6 moderately resistant (MR) materials, 5 moderately susceptible materials, and 15 highly susceptible materials. The data were compared with the field identification results, and 18 identification results were consistent, with a consistency of 60.00% (Tables 3 and 4).

[0122] 2) The relative transcription level of GmRhg1-2 gene was used as the standard for identification: when the transcription level of GmRhg1-2 gene pair was ≥1.00, the soybean material was highly resistant to soybean cyst nematode (HR); when the relative transcription level of GmRhg1-2 gene was ≥0.50 and <1.00, the soybean material was moderately resistant to soybean cyst nematode (MR); when the relative transcription level of GmRhg1-2 gene was ≥0.30 and <0.50, the soybean material was moderately susceptible to soybean cyst nematode (MS); when the comprehensive relative transcription level of GmRhg1-2 gene was <0.30, the soybean material was susceptible to soybean cyst nematode (HS). Among the 30 materials, there were 5 highly resistant (HR) materials, 7 moderately resistant (MR) materials, 9 moderately susceptible materials, and 9 highly susceptible materials. The data were compared with the field identification results, and 22 identification results were consistent, with a consistency of 73.33% (Table 3 and Table 4).

[0123] 3) The relative transcription level of GmRhg1-3 genes was used as the standard for identification: when the transcription level of GmRhg1-3 genes was ≥1.00, the soybean material was highly resistant to soybean cyst nematodes (HR); when the relative transcription level of GmRhg1-3 genes was ≥0.50 and <1.00, the soybean material was moderately resistant to soybean cyst nematodes (MR); when the relative transcription level of GmRhg1-3 genes was ≥0.30 and <0.50, the soybean material was moderately susceptible to soybean cyst nematodes (MS); when the comprehensive relative transcription level of GmRhg1-3 genes was <0.30, the soybean material was susceptible to soybean cyst nematodes (HS). Among the 30 materials, there were 3 highly resistant (HR) materials, 11 moderately resistant (MR) materials, 7 moderately susceptible materials, and 9 highly susceptible materials. The data were compared with the field identification results, and 16 identification results were consistent, with a consistency of 53.33% (Tables 3 and 4).

[0124] According to the above results, when the resistance of soybean to cyst nematodes was comprehensively evaluated according to the relative transcription levels of the three target genes, the evaluation results using the comprehensive transcription levels of the three genes were more in line with expectations. The calculation formula was: GmRhg1 comprehensive transcription level ΣT = A × 0.25 + B × 0.50 + C × 0.25, where A, B and C represent the relative expression levels of GmRhg1-1 / GmActin, GmRhg1-2 / GmActin, and GmRhg1-3 / GmActin genes, respectively.

[0125] According to the comprehensive relative transcription level (ΣT) of Rhg1 gene of known materials, it is divided into three intervals, and the resistance of soybean cyst nematode can be determined according to the three intervals:

[0126] When the comprehensive relative transcription level of GmRhg1 gene ΣT≥1.00, the soybean material was highly resistant to soybean cyst nematode (HR);

[0127] When the comprehensive relative transcription level of GmRhg1 gene was 0.50≤ΣT<1.00, the soybean material was moderately resistant to soybean cyst nematode (MR);

[0128] When the comprehensive relative transcription level of GmRhg1 gene was 0.30≤ΣT<0.50, the soybean material was moderately susceptible to soybean cyst nematode (MS);

[0129] When the comprehensive relative transcription level of GmRhg1 gene ΣT<0.30, the soybean material was susceptible to soybean cyst nematode (HS).

[0130] Using this identification standard, among the 30 materials, there were 5 high resistance (HR) materials, 7 medium resistance (MR) materials, 8 medium susceptible materials, and 10 high susceptible materials. Comparing this data with the field identification results, 25 identification results were consistent, with a consistency of 83.33% (Table 3 and Table 4). The experimental results show that the accuracy of using the GmRhg1 gene comprehensive relative transcription level to identify SCN resistance in soybean germplasm is good, and this method can be used for large-scale rapid screening of soybean cyst nematode resistance.

[0131] Example 3: Application of the comprehensive relative transcription level of GmRhg1 gene in identifying resistance to soybean cyst nematode

[0132] 141 materials from the National Germplasm Resource Bank were used as test samples to test the resistance to soybean cyst nematode.

[0133] According to the method in Example 2 above, the transcriptional expression levels of GmRhg1-1, GmRhg1-2 and GmRhg1-3 genes in leaves were detected by RT-PCR method, and three biological replicates were performed for each material.

[0134] The RT-PCR reaction system was: 2×PCR mix 25ul; 1ul cDNA template, 1ul, primers AC-F / AC-R (10uM) and differential gene F / R (10uM) 1ul each; sterile ddH2O made up to 50ul.

[0135] The RT-PCR reaction conditions were as follows: 95°C, 3 min; 95°C, 15 s; 58°C, 15 s, 72°C, 30 min (cycle numbers see Table 2); 72°C, 5 min.

[0136] The obtained RT-PCR products were subjected to electrophoresis, and the expression level of soybean Actin was used as the internal standard. The relative expression levels of the differentially expressed genes were analyzed using Image J. The specific results are shown in Table 5.

[0137] The results showed that 8 highly resistant materials, 20 moderately resistant (MR) materials, 64 moderately sensitive materials and 49 highly sensitive materials were screened out from 141 materials using this method. The highly resistant materials were Nanyang Gaoganqing, Shore, TGX814-26D, Qinong No. 12, M85-610, Yuanheidou, Donnong L-10 and Jinpin No. 8.

[0138] The resistance of 141 soybean materials was identified by field inoculation identification. The soybean materials were sown in the SCN pest nursery of the Changlinghu Experimental Base of the Soybean Institute of Heilongjiang Academy of Agricultural Sciences, and three biological replicates were set for each material. About 45 days after soybean sowing, when the third three-leaf was complete, the first generation of cyst nematodes was in the peak period of cyst development. The soybean materials were dug out to investigate the number of cysts and calculate the cyst index (IF). The specific results are shown in Table 5. The results showed that 8 highly resistant materials, 27 moderately resistant (MR) materials, 50 moderately susceptible materials, and 56 highly susceptible materials were screened out from 141 materials using this method.

[0139] Comparing the identification results of the two methods, there were 118 soybean materials with completely consistent identification, and the consistency of the two methods was 83.69%. The experimental results showed that the accuracy of using the GmRhg1 gene comprehensive relative transcription level ΣT to identify SCN resistance in soybean germplasm was good, and this method could be used for large-scale rapid screening of soybean cyst nematode resistance.

[0140] Table 5. Transcriptional expression of target genes in 141 soybean germplasm resources and their SCN resistance

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for detecting resistance of soybean to cyst nematodes, comprising the following steps: 1) extracting total RNA from the soybean sample to be tested, and reverse transcribing the RNA into cDNA; 2) RT-PCR was used to analyze the comprehensive relative transcription level ΣT of the target gene in the tested soybean; The comprehensive relative transcription level of the target gene ΣT=A×0.25+B×0.50+C×0.25, wherein A is the transcription level ratio of GmRhg1-1 / GmActin gene, B is the transcription level ratio of GmRhg1-2 / GmActin gene, and C is the transcription level ratio of GmRhg1-3 / GmActin gene; 3) determining the resistance to soybean cyst nematode according to the comprehensive relative transcription level of the target gene obtained in step 2); The tested soybeans with a high comprehensive relative transcription level of the target gene have higher resistance to soybean cyst nematodes than the tested soybeans with a low comprehensive relative transcription level of the target gene.

2. The method according to claim 1, characterized in that: The composition for analyzing the comprehensive relative transcription level ΣT of the Rhg1 gene of the soybean to be tested in step 2) consists of composition 1, composition 2, composition 3 and composition 4, The composition 1 is a detection primer set for detecting the GmRhg1-1, and the composition 1 is composed of GmRhg1-1 / F and GmRhg1-1 / R; the nucleotide sequence of the GmRhg1-1 / F is sequence 1; the nucleotide sequence of the GmRhg1-1 / R is sequence 2; The composition 2 is a detection primer set for detecting the GmRhg1-2, and the composition 2 is composed of GmRhg1-2 / F and GmRhg1-2 / R; the nucleotide sequence of the GmRhg1-2 / F is sequence 3; the nucleotide sequence of the GmRhg1-2 / R is sequence 4; The composition 3 is a detection primer set for detecting the GmRhg1-3, and the composition 3 is composed of GmRhg1-3 / F and GmRhg1-3 / R; the nucleotide sequence of the GmRhg1-3 / F is sequence 5; the nucleotide sequence of the GmRhg1-3 / R is sequence 6; The composition 4 is a detection primer set for detecting the GmActin, and the composition 4 consists of GmActin / F and Actin / R; the nucleotide sequence of Actin / F is sequence 27; and the nucleotide sequence of GmActin / R is sequence 28.

3. Use of the method according to claim 1 or 2 in identifying or assisting in identifying the resistance of soybean to cyst nematodes.

4. Use of the method according to any one of claims 1 to 3 in preparing a product for identifying or assisting in identifying the resistance of soybean to cyst nematodes.

5. Use of the method according to any one of claims 1 to 3 in soybean breeding, wherein the purpose of soybean breeding is to breed soybean varieties with strong resistance to cyst nematodes.

6. The composition of claim 2.

7. Use of the composition according to claim 6 in preparing a product for identifying or assisting in identifying the resistance of soybean to cyst nematodes.

8. Use of the composition according to claim 6 in soybean breeding, wherein the purpose of soybean breeding is to breed soybean varieties with strong resistance to cyst nematodes.

Images