Molecular marker for identifying soybean rhizoctonia webbl disease and application thereof

Abstract

The present application relates to the technical field of molecular marker assisted breeding, and particularly discloses a molecular marker for assisting in identifying the property of soybean Rhizoctonia web-blight and application. The present application develops a molecular marker S13_32595026-KASP related to the resistance of soybean to Rhizoctonia web-blight, and provides a primer set and a kit for amplifying the molecular marker. The molecular marker, the primer set and the kit provided by the present application can be used for assisting in screening or identifying soybean varieties resistant to Rhizoctonia web-blight, and the KASP marker is developed, and the selection efficiency of the KASP marker on the resistant material in the typing material is 65.00%.

Description

Technical Field

[0001] This invention relates to the field of molecular marker-assisted breeding technology, specifically to a molecular marker for assisting in the identification of soybean root rot and its application. Background Technology

[0002] Soybeans possess significant nutritional and economic value, making them a widely cultivated dual-purpose crop for both food and oilseed production globally. In recent years, soybean root rot has become one of the major diseases affecting yield and quality, especially when caused by *Rhizoctonia solani* (…). Rhizoctonia solani Root rot caused by Rhizoctonia solani is showing a trend of increasing severity and scope of occurrence year by year. This pathogen is highly adaptable to various environments, able to survive under a variety of adverse conditions and repeatedly infect hosts, posing a continuous threat to stable soybean production. Therefore, breeding soybean varieties resistant to Rhizoctonia solani root rot has become a key direction in current soybean breeding and disease control research.

[0003] The persistent occurrence of root rot has become a significant factor limiting stable and high soybean yields. The complexity and diverse population structure of its pathogens further complicate disease control. In agricultural production, the control of soil-borne diseases often relies on chemical fertilizers and pesticides, but long-term application can lead to environmental risks and pesticide resistance. Rhizoctonia solani possesses a wide host range, strong survival ability, and diverse infection modes, allowing it to survive in the soil for extended periods and repeatedly infect plants, making conventional control measures ineffective. Therefore, to combat Rhizoctonia solani root rot, it is necessary to explore more efficient and sustainable control methods from the perspectives of genetic improvement and green control to mitigate its harm to soybean production.

[0004] Currently, prevention is still the primary method for controlling Rhizoctonia solani root rot in agricultural production. However, the pathogen's strong adaptability and long survival period make conventional control measures difficult to achieve stable and lasting effects. In contrast, variety improvement using genetic resistance is considered the most economical and sustainable approach to controlling this disease. Therefore, systematically exploring new resistant germplasm resources and developing molecular markers closely linked to them to provide technical support for the precise selection of resistance traits is of great significance for improving the level of molecular-assisted breeding for Rhizoctonia solani resistance in soybeans in my country. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a molecular marker for assisting in the identification of root rot and stem rot traits in soybeans and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a molecular marker for assisting in the identification of root rot and stem rot traits in soybean. The molecular marker is located at 32595026 bp on chromosome 13 of the soybean genome, and the SNP site has C / T polymorphism; or the nucleotide sequence shown in SEQ ID NO.1 has C / T polymorphism at position 169.

[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 root rot and stem rot, the kit comprising the above-mentioned primer set or primer pair, Flu-Arms2×PCR Mix, and DNA template buffer.

[0009] This invention provides the application of the above-mentioned molecular markers in assisting the screening of soybean varieties resistant to damping-off root rot, identifying soybean varieties resistant to damping-off root rot, or in the breeding of soybean varieties resistant to damping-off root rot.

[0010] This invention provides the application of the above primer set in assisting the screening of soybean varieties resistant to damping-off root rot, identifying soybean varieties resistant to damping-off root rot, or in the breeding of soybean varieties resistant to damping-off root rot.

[0011] This invention provides the application of the above-mentioned kit in assisting the screening of soybean varieties resistant to damping-off root rot, identifying soybean varieties resistant to damping-off root rot, or in the breeding of soybean varieties resistant to damping-off root rot.

[0012] This invention also provides a method for identifying soybean varieties resistant to damping-off root rot, 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 S13_32595026 polymorphic site was detected, and the genotype results were used to determine whether the soybean to be tested was a soybean variety resistant to root rot and stem rot. When the genotype at the molecular marker polymorphism site is TT, the soybean sample to be tested is determined to be a soybean variety resistant to root rot.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention develops a molecular marker, S13_32595026-KASP, associated with soybean resistance to damping-off and root rot, and provides a primer set and kit for amplifying this marker. The molecular marker, primer set, and kit provided by this invention can be used to assist in screening or identifying soybean varieties resistant to damping-off and root rot. Simultaneously, the KASP marker was developed, and its selection efficiency for disease-resistant materials in genotyping materials was 65.00%. Attached Figure Description

[0014] Figure 1 PCR amplification diagram of the variant site; M: DL 2000 (+) DNA molecular weight standard; 1: L-334, 2: Hujiao 04-528, 3: Dengke No. 5, 4: Dongnong 50, 5: Liao 98072, 6: Hefeng 52, 7: Qingan Black Bean, 8: DY2004-5, 9: Jiyu 204, 10: Suinong 25, 11: Suinong 1, 12: Hujiao 11-322, 13: Mufeng No. 1, 14: Dunajika, 15: Zhonghuang No. 10, 16: Zhongpin 03-5355, 17: Ji 06B7, 18: Datun Small Black Bean, 19: Haguang 1657, 20: Suinong 35; Figure 2 This is a sequence alignment diagram of variant sites in resistant and resistant materials; Figure 3 This is the molecular marker typing diagram for S13_32595026-KASP. Note: Red markers represent T / T type, and blue markers represent C / C type. Figure 4 The chart shows the significance of the KASP typing results for S13_32595026. Note: This indicates that it is significant at the 0.01 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: 96 domestic and foreign soybean germplasm resources are shown in Table 1.

[0018] Table 1 Test strain: The Rhizoctonia solani strain used in this study was numbered RS23 (mycelial fusion group AG-4), which was isolated from the root system of soybean plants exhibiting root rot symptoms collected in Harbin, Heilongjiang Province, China.

[0019] Example 1: Preparation of genomic DNA from soybean germplasm resources and amplification and sequencing of target sequences Genomic DNA was extracted from 96 soybean accessions grown in a greenhouse using the CTAB method. The genomic sequence of the S13_32595026 locus was downloaded from the Phytozome database. Specific primers for the superior allelic variants were designed using Premier 5.0 software, targeting these superior allelic variants. The nucleotide sequences of these specific primers are as follows: F-GTTCTGGACGTAAATAACAACATTGC, as shown in SEQ ID NO.5; R-CTTGCTAGAGAAACGATCTAACAAACTG, as shown in SEQ ID NO.6.

[0020] 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 2, and the reaction procedure is shown in Table 3. The amplification product of the S13_32595026 site was 570 bp in length, and the results are as follows: Figure 1 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.

[0021] Table 2 PCR amplification system Table 3 PCR amplification program The successfully amplified products were purified and sent to Beijing Ruiboxingke Sequencing Co., Ltd. for first-generation sequencing. The DNA sequence was then assembled and aligned using DNAMANV6.0 software. The results are as follows: The S13_32595026 locus exhibits C / T genotyping between resistant and susceptible materials. The variant carries a T allelic variant in resistant materials with a disease index less than 20, such as Suinong 1, Hujiao 11-322, Mufeng 1, Dunajika, and Zhonghuang 10; while it carries a C allelic variant in susceptible materials with a disease index greater than 30, such as Jindou 33, Hujiao 04-528, Dengke 5, Dongnong 50, Liao 98072, and Suinong 25.

[0022] Example 2: KASP molecular markers and their application S13_32595026, C / T classification SEQ ID NO.1 r is C or T.

[0023] KASP marker primers were designed based on the upstream and downstream sequences of the S13_32595026 site and synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd. The primer sequences are as follows: KASP-tagged primers: F1-GAAGGTGACCAAGTTCATGCTACAATGTCTGCAAATCACGGGATGC (SEQ ID NO. 2); F2-GAAGGTCGGAGTCAACGGATTACAATGTCTGCAAATCACGGGATGT (SEQ ID NO.3) R-TCAGACAATGTCCAGTGGAGTGGAAGCCT (SEQ ID NO.4) The KASP reaction system is shown in Table 4, and the reaction procedure is shown in Table 5. The genotyping data were acquired and analyzed using ABI7500 Software v2.3 quantitative fluorescence software.

[0024] Table 4 KASP Labeling Reaction System Table 5 KASP Labelling Reaction Procedure Example 3: Validation and Application of Molecular Markers DNA was extracted from 96 soybean samples listed in Table 1. The extracted soybean DNA was amplified by PCR using the primer set described below and the reaction conditions described in Example 2 to obtain the PCR product. F1-GAAGGTGACCAAGTTCATGCTACAATGTCTGCAAATCACGGGATGC (SEQ ID NO. 2); F2-GAAGGTCGGAGTCAACGGATTACAATGTCTGCAAATCACGGGATGT (SEQ ID NO.3) R-TCAGACAATGTCCAGTGGAGTGGAAGCCT (SEQ ID NO.4) The genotype of the PCR product at the S13_32595026 site was detected, and the genotyping results were used to determine whether the soybean to be tested was a soybean variety resistant to root rot.

[0025] When the genotype at the molecular marker polymorphism site is TT, the soybean sample to be tested is determined to be a soybean variety resistant to root rot.

[0026] The KASP marker results for site S13_32595026 are as follows: Figure 3 The blue markers near the Y-axis represent soybean materials carrying the CC homozygous genotype, while the red markers near the X-axis represent soybean materials carrying the TT homozygous genotype. Analysis of all experimental materials revealed that 79.17% of soybean materials carried the CC homozygous genotype, and 20.83% carried the TT homozygous genotype. T-test analysis showed that germplasm resources with the superior TT genotype exhibited significantly higher disease resistance than those with the CC genotype. These results indicate that the KASP molecular marker for superior allelic variation sites has been successfully developed, providing a valuable tool for molecularly assisted breeding against damping-off root rot.

[0027] The efficiency of molecular marker disease resistance identification is calculated using the following formula.

[0028] 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 allele TT carried by the S13_32595026-KASP molecular marker at the S13_32595026 site had a selection efficiency of 65.00% for anti-disease materials, indicating that the superior allele of the molecular marker is effective in assisting the selection of anti-disease materials.

[0029] 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 assisting in the identification of soybean root rot and stem rot traits, characterized in that, The molecular marker is located at 32595026 bp on chromosome 13 of the soybean genome, and the SNP site exhibits C / T polymorphism; or the nucleotide sequence shown in SEQ ID NO.1 has C / T polymorphism at position 169.

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 characteristics of soybean root rot and stem rot, characterized in that, The kit includes the primer set as described in claim 2, Flu-Arms2×PCR Mix, and DNA template buffer.

4. The application of the molecular markers as described in claim 1 in assisting the screening of soybean varieties resistant to damping-off root rot, identifying soybean varieties resistant to damping-off root rot, or in the breeding of soybean varieties resistant to damping-off root rot.

5. The application of the primer set as described in claim 2 in assisting in the screening of soybean varieties resistant to damping-off root rot, the identification of soybean varieties resistant to damping-off root rot, or in the breeding of soybean varieties resistant to damping-off root rot.

6. The application of the kit as described in claim 3 in assisting the screening of soybean varieties resistant to damping-off root rot, identifying soybean varieties resistant to damping-off root rot, or in the breeding of soybean varieties resistant to damping-off root rot.

7. A method for identifying soybean varieties resistant to damping-off and root rot, 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; Genotyping of PCR products at the S13_32595026 site was detected, and the genotyping results were used to determine whether the soybean to be tested was a soybean variety resistant to root rot and stem rot. When the genotype at the molecular marker polymorphism site is TT, the soybean sample to be tested is determined to be a soybean variety resistant to root rot.

Images