Molecular marker related to soybean cyst nematode disease resistance, primer group, kit and application

Abstract

The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a molecular marker related to soybean cyst nematode resistance, a primer group, a kit and application. The invention provides two QTLs (Quantitative Trap Like): qSCN3-15 and qSCN3-18 related to the resistance of the soybean cyst nematode disease for the first time, develops a molecular marker SCN15.2 closely linked with the qSCN3-15, and two molecular markers SCN18.1 and SCN18.2 closely linked with the qSCN3-18, the molecular markers have high correlation with the resistance of the soybean cyst nematode disease, and when the molecular markers are used for identifying the soybean cyst nematode-resistant soybean variety, the molecular markers can be used for identifying the soybean cyst nematode-resistant soybean variety. The identification efficiency of the SCN15.2 reaches 66.7%, the identification efficiency of the SCN18.1 reaches 80%, and the identification efficiency of the SCN18.2 reaches 69.2%.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and genetic breeding, and specifically relates to molecular markers, primer sets, kits and applications related to soybean cyst nematode disease resistance. Background Art

[0002] Soybean cyst nematode disease is one of the main diseases of soybean. When the disease is severe, it causes a yield reduction of more than 80%. In addition, soybeans cannot be planted in severely diseased soil within 4 to 5 years, which is extremely harmful to soybean production.

[0003] The selection of new soybean varieties with resistance to soybean cyst nematode disease can fundamentally solve this problem. Conventional phenotypic identification of soybean cyst nematode resistance is time-consuming and expensive, easily affected by the environment, and destructive to the roots of the materials. Transplanted plants are difficult to survive, resulting in slow progress in the identification of resistance sources to cyst nematodes and the cultivation of new disease-resistant germplasm. Compared with precise phenotypic identification, molecular marker-assisted selection can not only avoid the damage caused by disease resistance identification to the root system of the variety, but also has the advantages of high efficiency and low cost. Compared with the entire soybean genome, the currently developed functional markers are relatively few and the identification efficiency is low. Satt309, which is only 0.4 cM away from the main effect site rhg1, is the most widely used molecular marker in the early molecular marker-assisted selection of soybean cyst nematode resistance. Its disadvantage is that it cannot be detected in high throughput and can only be selected for disease-resistant Peking-type materials, and cannot distinguish between disease-resistant materials PI88788 and susceptible materials Lee. Whether it is SSR, InDel or CAPS markers, they all have the problems of low throughput and high cost, and cannot meet the requirements of high-throughput detection, and have limitations in the application of molecular marker-assisted selection.

[0004] KASP (kompetitive allele specific PCR) is a new genotyping technology with low cost and high throughput. It can accurately perform double allele typing on SNP and InDel sites through specific matching of primer terminal bases. It is widely used in molecular marker-assisted selection of crops such as rice, wheat, and soybean. However, the KASP markers currently developed for soybean cyst nematode resistance are mostly concentrated in the two sites of rhg1 and Rhg4, and there are still many molecular marker sites related to soybean cyst nematode resistance that have not been explored, which limits the application of molecular marker-assisted breeding technology in breeding soybeans with resistance to soybean cyst nematode disease. Summary of the invention

[0005] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide molecular markers, primer sets, kits and applications related to soybean cyst nematode resistance. The molecular markers SCN15.2, SCN18.1 and SCN18.2, primer sets and kits for amplifying molecular markers provided by the present invention can be used for the identification, auxiliary screening and mutant breeding of cyst nematode-resistant soybean varieties.

[0006] The first aspect of the present invention provides a molecular marker related to resistance to soybean cyst nematode disease, wherein the molecular marker is at least one of SCN15.2, SCN18.1 and SCN18.2, the nucleotide sequence of the molecular marker SCN15.2 is shown in SEQ IDNO.1, and has an A / G polymorphism at 49bp of this sequence; the nucleotide sequence of the molecular marker SCN18.1 is shown in SEQ IDNO.2, and has a C / T polymorphism at 50bp of this sequence; the nucleotide sequence of the molecular marker SCN18.2 is shown in SEQ IDNO.3, and has an A / T polymorphism at 63bp of this sequence.

[0007] Furthermore, the SCN15.2 is closely associated with the QTL locus qSCN3-15, and the SCN18.1 and SCN18.2 are closely associated with the QTL locus qSCN3-18.

[0008] Furthermore, the qSCN3-15 is located in the interval of 117.54 cM to 118.278 cM of the 15th linkage group of soybean, and the qSCN3-18 is located in the interval of 0 cM to 0.495 cM of the 18th linkage group of soybean.

[0009] The second aspect of the present invention provides a primer set for amplifying the molecular marker related to resistance to soybean cyst nematode disease, the primer set consisting of two specific primers and a reverse universal primer; the nucleotide sequences of the two specific primers for amplifying SCN15.2 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.6; the nucleotide sequences of the two specific primers for amplifying SCN18.1 are shown in SEQ ID NO.7 and SEQID NO.8, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.9; the nucleotide sequences of the two specific primers for amplifying SCN18.2 are shown in SEQ ID NO.10 and SEQ ID NO.11, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.12.

[0010] The third aspect of the present invention provides a kit, which comprises any one or more of the primer set for amplifying SCN15.2, the primer set for amplifying SCN18.1, and the primer set for amplifying SCN18.2.

[0011] A fourth aspect of the present invention provides a use of the molecular marker associated with soybean cyst nematode resistance or the primer set in identifying soybean varieties resistant to cyst nematodes.

[0012] A fifth aspect of the present invention provides an application of the molecular marker associated with soybean cyst nematode resistance or the primer set in assisting the screening of soybean varieties resistant to cyst nematodes.

[0013] Furthermore, the molecular marker associated with soybean cyst nematode resistance or the primer set is used for mutant breeding of cyst nematode-resistant soybean varieties.

[0014] A sixth aspect of the present invention provides a method for identifying soybean varieties resistant to cyst nematodes, comprising the following steps: Extracting DNA from soybeans to be tested; Performing PCR amplification on the extracted soybean DNA to be tested using any one of the primer set for amplifying SCN15.2, the primer set for amplifying SCN18.1, or the primer set for amplifying SCN18.2 to obtain a PCR product; The genotyping of the PCR product at the polymorphic site is detected, and whether the soybean to be tested is a soybean variety resistant to cyst nematodes is determined based on the genotyping result.

[0015] Further, when the genotype at the polymorphic site of SCN15.2 is GG, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of SCN18.1 is CC, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of SCN18.2 is AA, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes.

[0016] Furthermore, each 1 μL PCR amplification reaction system includes: 4 ng DNA template, 0.5 μL 2×KASP Master mix, 0.014 μL 72×Assay mix, and deionized water. The 72×KASP Assay Mix is ​​composed of two specific primers and one reverse universal primer, and the concentrations of the two specific primers are both 0.168 μM, and the concentration of the reverse universal primer is 0.42 μM.

[0017] Furthermore, the reaction procedure of the PCR amplification is as follows: (1) thermal activation; (2) denaturation at 94°C for 20 s; annealing: 61°C~55°C for 60 s; decreasing by 0.6°C per cycle, for a total of 10 cycles; (3) denaturation: 94°C for 20 s; annealing: 55°C for 60 s; for a total of 26 cycles.

[0018] In summary, compared with the prior art, the present invention has the following advantages and effects: (1) The present invention develops three molecular markers SCN15.2, SCN18.1 and SCN18.2 related to the soybean cyst nematode resistance trait, and provides primer sets and kits for amplifying molecular markers. The molecular markers, primer sets and kits provided by the present invention can be used to assist in screening or identifying soybean varieties resistant to cyst nematodes, wherein the identification efficiency of the molecular marker SCN15.2 is 66.7%, the identification efficiency of the molecular marker SCN18.1 is 80%, and the identification efficiency of the molecular marker SCN18.2 is 69.2%. The combined identification efficiency of the two molecular markers SCN15.2 and SCN18.1 is 95.2%. When the three molecular markers SCN15.2, SCN18.1 and SCN18.2 are combined for identification, the identification efficiency is as high as 100%.

[0019] (2) The LOD range of the two QTL loci related to soybean cyst nematode resistance provided by the present invention is greater than 5.730, with a maximum LOD value of 12.033. The contribution rates of qSCN3-15 and qSCN3-18 to the phenotype are 20.731% and 24.476%, respectively, which can assist in mining related functional genes. The present invention also provides a method for identifying soybean varieties with resistance to cyst nematodes, which can quickly, accurately and effectively identify soybean materials with resistance to cyst nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the linkage map. The horizontal axis is the linkage group number, the vertical axis is the position of the marker on the chromosome, and the black line represents the marker in the figure.

[0021] Figure 2 This is the QTL distribution map related to soybean cyst nematode resistance, the horizontal axis is the linkage group number, the vertical axis is the LOD value, and the gray and white interval background is different linkage groups; Figure 2 In Figure A, the black line is the LOD value distribution of molecular markers and traits, and the light gray line is the LOD threshold; Figure 2 The lines in panel B represent the additive effects at the corresponding positions in panel A. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0023] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0024] The biological materials involved in the embodiments are: Bainong 10, Jiyu 86 and the identification host of soybean cyst nematode resistance were obtained from the Soybean Research Institute of Jilin Academy of Agricultural Sciences. Bainong 10 was marked as BN10 and Jiyu 86 was marked as JY86. The identification hosts of soybean cyst nematode resistance were Peking, PI209332, PI437654, PI548316, PI88788, PI89772 and PI90763.

[0025] Soybean cyst nematode disease is extremely harmful to soybean production. Breeding new soybean varieties that are resistant to soybean cyst nematode disease can fundamentally solve this problem. Conventional identification of resistance phenotypes to soybean cyst nematode disease is time-consuming and expensive, easily affected by the environment, and destructive to the roots of the material, making it difficult for transplanted plants to survive. Among the currently reported molecular marker-assisted breeding technologies, whether it is SSR, InDel or CAPS markers, there are problems of low throughput and high cost, and they cannot meet the requirements of high-throughput detection.

[0026] The invention provides molecular markers, primer sets, kits and applications related to soybean cyst nematode disease resistance. The present invention uses a disease-resistant soybean variety Bainong 10, a disease-susceptible soybean variety Jiyu 86, and F6 and F7 recombinant inbred line populations containing 150 families constructed with Bainong 10 and Jiyu 86 as parents as materials to construct a linkage map, obtains two QTL loci qSCN3-15 and qSCN3-18 related to the soybean cyst nematode resistance trait by performing trait association analysis on phenotypic values ​​of soybean resistance to cyst nematodes and SNP markers, then develops three molecular markers SCN15.2, SCN18.1 and SCN18.2 according to the qSCN3-15 and qSCN3-18 loci, and provides primer sets for amplifying SCN15.2, SCN18.1 and SCN18.2, respectively, and a kit comprising amplifying SCN15.2, SCN18.1 and / or SCN18.2.

[0027] Example 1: Location of QTL and determination of molecular markers 1. Group building With the disease-resistant variety BN10 as the female parent and the susceptible variety JY86 as the male parent, a hybrid combination was configured in 2014. Table 1 shows the process of obtaining the family.

[0028] Table 1 Family acquisition process

[0029] 2. Construction of linkage map Using BN10, JY86 and F6 and F7 recombinant inbred line populations containing 150 families as materials, a high-density genetic map and typing data of soybean was obtained. Taking each chromosome of soybean as a unit, HighMap software was used for analysis to obtain the arrangement order of markers on each chromosome and estimate the genetic distance between markers, and finally the following was obtained: Figure 1 The high-density genetic map shown has a total map distance of 2755.88 cM, an average map distance of 0.3 cM, and contains 9314 SNP markers distributed in 20 linkage groups.

[0030] 3. QTL positioning results The composite interval mapping method provided by R / qtl was used to conduct trait association analysis and QTL positioning between the phenotypic values ​​and SNP markers of soybean resistance to cyst nematodes. The PT test was repeated 1000 times to set the threshold. The results are as follows Figure 2 As shown in Figure 2, when the LOD value is greater than 5.730, it indicates that the association between soybean cyst nematode resistance and SNP markers is extremely significant. Two QTL loci qSCN3-15 and qSCN3-18 related to soybean cyst nematode resistance were detected on soybean chromosomes 15 and 18, and the basic information of the associated regions is shown in Table 2.

[0031] Table 2 Basic information of associated areas

[0032] qSCN3-15 was located in the range of 117.54 cM to 118.278 cM on chromosome 15, with a maximum LOD value of 11.905, a marker additive effect of -14.720, and a phenotypic contribution rate of 20.731%. It was the major QTL locus for resistance to soybean cyst nematode disease.

[0033] qSCN3-18 was located in the interval of 0 cM~0.495 cM on chromosome 18, with a maximum LOD value of 12.033 and an additive effect of -15.995 for all markers, indicating that its disease resistance allele came from BN10, with a phenotypic contribution rate of 24.476%, and was a major QTL locus for resistance to soybean cyst nematode disease.

[0034] 4. Tag development According to the qSCN3-15 and qSCN3-18 loci, 54 F 6：9Family plants, BN10, JY86, Peking, PI209332, PI437654, PI548316, PI88788, PI89772 and PI90763 were used as test materials, and KASP technology was used for SNP typing, and three molecular markers SCN15.2, SCN18.1 and SCN18.2 related to the soybean cyst nematode resistance trait were developed.

[0035] The nucleotide sequence of the molecular marker SCN15.2 is shown in SEQ ID NO.1, and the base at 49 bp of this sequence has an A / G polymorphism; The nucleotide sequence of the molecular marker SCN18.1 is shown in SEQ ID NO.2, and the base at 50 bp of this sequence has a C / T polymorphism; The nucleotide sequence of the molecular marker SCN18.2 is shown in SEQ ID NO.3, and the base at 63 bp of this sequence has an A / T polymorphism.

[0036] SEQ ID NO.1: GAAGTTCACAAACATAGGTGGAAGCTCTCTGTAGCTTTGGGTTGAATTrTTGGTTGAAAAGAGCAATTGGACGATTTTGTTGCATG, r is A / G.

[0037] SEQ ID NO.2: AATTCTAATAGCCAGAGTAAGAAACCAAATTACAGAAAAGACCCGAACAyTAAAGAAACTTTTTAGAATATAATTAAGCTACACTTGTACTCA, y is C / T.

[0038] SEQ ID NO.3: TGTACTATACAGATTTCATTTGAACCCTGAATAGCTTCTGCAGTTACTTTAAGTCTTCATTCwTCAACCATGTGTTAGTTCTATCATTTTAAGCGTGCA, w is A / T.

[0039] Example 2: Design of Molecular Marker Primer Set Specific primer combinations were designed for the nucleotide sequences of SCN15.2, SCN18.1 and SCN18.2, respectively.

[0040] The primer set for amplifying SCN15.2 consists of two specific primers and a reverse universal primer. The nucleotide sequences of the two specific primers are shown in SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.6.

[0041] SEQ ID NO. 4: 5'-ATCGTCCAATTGCTCTTTTCAACCAAT-3'.

[0042] SEQ ID NO. 5: 5'-CGTCCAATTGCTCTTTTCAACCAAC-3'.

[0043] SEQ ID NO. 6: 5'-GAAGCTCTCTGTAGCTTTGGGTTGAA-3'.

[0044] The primer set for amplifying SCN18.1 consists of two specific primers and a reverse universal primer. The nucleotide sequences of the two specific primers are shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.9.

[0045] SEQ ID NO. 7: 5'-CCAAATTACAGAAAAGACCCGAACAC-3'.

[0046] SEQ ID NO. 8: 5'-ACCAAATTACAGAAAAGACCCGAACAT-3'.

[0047] SEQ ID NO. 9: 5'-CATAATGCTTTGAGTACAAGTGTAGCTTAA-3'.

[0048] The primer set for amplifying SCN18.2 consists of two specific primers and a reverse universal primer. The nucleotide sequences of the two specific primers are shown in SEQ ID NO.10 and SEQ ID NO.11, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.12.

[0049] SEQ ID NO. 10: 5'-TAAAATGATAGAACTAACACATGGTTGAT-3'.

[0050] SEQ ID NO. 11: 5'-CTTAAAATGATAGAACTAACACATGGTTGAA-3'.

[0051] SEQ ID NO. 12: 5'-ACCCTGAATAGCTTCTGCAGTTACTTTAA-3'.

[0052] Example 3: Validation and application of molecular markers Select 54 portions of F in Example 1 6：9 Family plants, BN10, JY86, Peking, PI209332, PI437654, PI548316, PI88788, PI89772 and PI90763 are soybean samples to be tested. DNA of the soybean samples to be tested is extracted, and the extracted DNA is PCR amplified using the primer set for amplifying SCN15.2, the primer set for amplifying SCN18.1 and the primer set for amplifying SCN18.2 in Example 2, respectively, to obtain PCR amplification products.

[0053] Each 1 μL PCR amplification reaction system includes: 4 ng DNA template, 0.5 μL 2×KASP Master mix, 0.014 μL 72×Assay mix, and deionized water. The 72×KASP Assay Mix includes two specific primers and one reverse universal primer. The concentrations of the two specific primers are both 0.168 μM, and the concentration of the reverse universal primer is 0.42 μM.

[0054] The PCR amplification reaction procedure was as follows: (1) thermal activation: 94°C, 15 min; (2) denaturation: 94°C, 20 s; annealing: 61°C~55°C, 60 s; the temperature was reduced by 0.6°C each cycle, for a total of 10 cycles; (3) denaturation: 94°C, 20 s; annealing: 55°C, 60 s; for a total of 26 cycles.

[0055] The PCR product is detected in a fluorescent quantitative PCR instrument, the fluorescence intensity signal value is read, and cluster analysis is performed to obtain the genotyping at the polymorphic site. According to the genotyping result, it is determined whether the soybean to be tested is a soybean variety resistant to cyst nematodes.

[0056] The judgment criteria are: when the genotype at the polymorphic site of the molecular marker SCN15.2 is GG, the soybean sample to be tested is judged to be a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of the molecular marker SCN18.1 is CC, the soybean sample to be tested is judged to be a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of the molecular marker SCN18.2 is AA, the soybean sample to be tested is judged to be a soybean variety resistant to cyst nematodes.

[0057] The molecular marker disease resistance identification efficiency was calculated according to the following formula.

[0058] ; According to calculations, the identification efficiency of the molecular marker SCN15.2 reached 66.7%, the identification efficiency of the molecular marker SCN18.1 reached 80%, the identification efficiency of the molecular marker SCN18.2 reached 69.2%, and the combined identification efficiency of the two molecular markers SCN15.2 and SCN18.1 was 95.2%. When the three molecular markers SCN15.2, SCN18.1 and SCN18.2 were combined for identification, the identification efficiency was as high as 100%.

Claims

1. A molecular marker associated with resistance to soybean cyst nematode disease, characterized in that: The molecular marker is at least one of SCN15.2, SCN18.1 and SCN18.2, The nucleotide sequence of the molecular marker SCN15.2 is shown in SEQ ID NO. 1, and there is an A / G polymorphism at 49 bp of this sequence; The nucleotide sequence of the molecular marker SCN18.1 is shown in SEQ ID NO. 2, and there is a C / T polymorphism at 50 bp of this sequence; The nucleotide sequence of the molecular marker SCN18.2 is shown in SEQ ID NO.3, and there is an A / T polymorphism at 63 bp of this sequence.

2. A primer set for amplifying the molecular marker associated with soybean cyst nematode resistance according to claim 1, characterized in that: The primer set is composed of two specific primers and one reverse universal primer; The nucleotide sequences of the two specific primers for amplifying SCN15.2 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.6; The nucleotide sequences of the two specific primers for amplifying SCN18.1 are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.9; The nucleotide sequences of the two specific primers for amplifying SCN18.2 are shown in SEQ ID NO.10 and SEQ ID NO.11 respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.

12.

3. A kit, characterized in that: The kit comprises any one or more of the primer set for amplifying SCN15.2, the primer set for amplifying SCN18.1, and the primer set for amplifying SCN18.2 described in claim 2.

4. Use of the molecular marker associated with soybean cyst nematode resistance according to claim 1 or the primer set according to claim 2 in identifying soybean varieties resistant to cyst nematodes.

5. Use of the molecular marker associated with soybean cyst nematode resistance according to claim 1 or the primer set according to claim 2 in assisting the screening of soybean varieties resistant to cyst nematodes.

6. The use according to claim 5, characterized in that: The molecular marker or the primer set is used for mutant breeding of cyst nematode-resistant soybean varieties.

7. A method for identifying soybean varieties resistant to cyst nematodes, characterized in that: The following steps are involved: Extracting DNA from soybeans to be tested; Using any one of the primer set for amplifying SCN15.2, the primer set for amplifying SCN18.1, or the primer set for amplifying SCN18.2 in claim 2 to perform PCR amplification on the extracted soybean DNA to obtain a PCR product; The genotyping of the PCR product at the polymorphic site is detected, and whether the soybean to be tested is a soybean variety resistant to cyst nematodes is determined based on the genotyping result.

8. The method for identifying cyst nematode-resistant soybean varieties according to claim 7, characterized in that: When the genotype at the polymorphic site of SCN15.2 is GG, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of SCN18.1 is CC, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of SCN18.2 is AA, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes.

9. The method for identifying cyst nematode-resistant soybean varieties according to claim 7, characterized in that: Each 1 μL PCR amplification reaction system includes: 4 ng DNA template, 0.5 μL 2×KASP Master mix, 0.014 μL 72×Assay mix, and deionized water. The 72×KASP Assay Mix is ​​composed of two specific primers and one reverse universal primer. The concentrations of the two specific primers are both 0.168 μM, and the concentration of the reverse universal primer is 0.42 μM.

10. The method for identifying cyst nematode-resistant soybean varieties according to claim 7, characterized in that: The reaction procedure of the PCR amplification is as follows: (1) heat activation; (2) denaturation at 94°C for 20 s; annealing at 61°C to 55°C for 60 s; decreasing by 0.6°C in each cycle, for a total of 10 cycles; (3) denaturation at 94°C for 20 s; annealing at 55°C for 60 s; for a total of 26 cycles.

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