SNP molecular marker linked with peanut southern blight resistance and application of SNP molecular marker

Abstract

The invention relates to a molecular marker linked with peanut southern blight resistance, which is an SNP (Single Nucleotide Polymorphism) site positioned at the 9189944th site of the fifth chromosome of peanut, the polymorphism of the molecular marker is G / C, and a reference peanut genome is the genome of cultivated peanut Tifrunner and comes from a database Peanut Base. The molecular marker linked with southern blight resistance developed by the invention can be used for detecting the genotype of peanut so as to judge whether a material has southern blight resistance or not, through high-throughput genotyping detection, manpower and material resources are greatly saved, the detection is not influenced by environmental factors, the result is accurate and reliable, and the molecular marker can be used for early screening of peanut southern blight resistant varieties and can be applied to early screening of peanut southern blight resistant varieties. The breeding period can be shortened and the breeding efficiency is improved. Based on the developed KASP molecular marker primer combination, the southern blight resistance of cultivated peanut varieties can be identified, genotyping can be accurately carried out on the southern blight resistance of peanuts, layout of field peanut varieties and prevention and control of southern blight are guided, and the KASP molecular marker primer combination has wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of peanut molecular breeding, and more particularly to SNP molecular markers linked to peanut white mold disease resistance and their applications. Background Technology

[0002] peanut( Arachis hypogaea As a globally important source of plant oils and proteins, peanut white mold rot occupies a vital position in the world's agricultural economy. In the past decade or so, peanut white mold has risen from a minor disease to a major one, and its occurrence has become increasingly severe.

[0003] Peanut white mold is a soil-borne fungal disease. The pathogen mainly reproduces by producing a large number of sclerotia, which can survive in the soil for many years. White mold mainly affects the base of the plant stem. Conventional chemical control by foliar spraying is not very effective and poses risks of environmental pollution and pesticide residues. Currently, there are no effective biocontrol products for peanut white mold that can be applied in the field. In comparison, breeding and planting disease-resistant varieties is the most economical and effective means of controlling this disease.

[0004] Because *Sclerotium affine* is a necrotrophic pathogen, peanut resistance to this disease is low, and resistance is a quantitative trait controlled by multiple genes. Currently, research on QTLs for peanut resistance to *Sclerotium affine* has been conducted both domestically and internationally. QTLs discovered using populations constructed from resistant materials with different genetic backgrounds are located on different chromosomes. Bera et al. (2016) located it on chromosome A01, Dodia et al. (2019) located it on chromosomes B03, B04, B06, B08, and B10, Cui et al. (2020) located it on chromosomes A1 and A5, and Agnom (2022) located it on chromosomes A03, A07, B03, and B05. Yan et al. (2023) located it on 10 chromosomes, including A01 and A04, using natural populations, but so far, no stable major-effect QTLs have been reported. These studies also indicate that resistance to *Sclerotium affine* is complex, and different resistance sources may have different resistance loci. This study used a RIL population constructed by crossing the disease-resistant germplasm "Zhonghua 212" with the susceptible variety "Zhonghua 21" as the research object. The resistance locus for white mold was located on chromosome A05, and this QTL is a stable major-effect locus. Given the current lack of molecular markers for white mold-resistant assisted selection breeding, the development of KASP molecular markers related to white mold resistance is an urgent need in white mold resistance breeding. KASP markers for white mold resistance can provide strong technical support for assisting in white mold resistance breeding of peanuts and identifying white mold-resistant germplasm. Summary of the Invention

[0005] This invention provides a molecular marker linked to resistance to peanut white mold disease, which is an SNP locus located at position 9189944 on chromosome 5 of peanut, with a polymorphism of G / C. The reference peanut genome is the genome of the cultivated peanut Tifrunner, obtained from the PeanutBase database.

[0006] The present invention also provides a primer set for identifying resistance to white mold disease in peanut germplasm materials, wherein the primer set is a primer set capable of identifying the polymorphism of the molecular marker.

[0007] In one specific implementation, the primer set includes at least two upstream primers and at least one downstream primer.

[0008] In one specific implementation, the primer set includes at least two upstream primers with sequences as shown in SEQ ID NO:1 and NO:2.

[0009] In one specific implementation, the primer set includes at least a downstream primer with a sequence as shown in SEQ ID NO:3.

[0010] This invention also provides the application of the above-mentioned molecular markers or primer sets in identifying the resistance of peanut germplasm materials to white mold disease.

[0011] This invention also provides a method for identifying resistance to white mold disease in peanut germplasm materials, comprising the following steps: S1: Extract the genome from the plant sample of the peanut germplasm material to be tested; S2: Detect polymorphisms at the aforementioned molecular markers in the genome; S3: Peanut germplasm materials with homozygous C polymorphism at the molecular markers in the genome are resistant to white mold; peanut germplasm materials with homozygous G polymorphism at the molecular markers in the genome are susceptible to white mold.

[0012] In one specific implementation, the above-described primer set is used to detect polymorphisms at the molecular markers described in the genome via PCR.

[0013] In one specific implementation, the PCR reaction program is as follows: 94℃, 15 min; 94℃, 20 s, 65-57℃ gradient PCR, 1 min, decreasing by 0.8℃ per cycle, for 10 cycles; 94℃, 20 s, 57℃, 1 min, for 28 cycles.

[0014] The molecular markers linked to white mold resistance developed in this invention can be used to detect peanut genotypes, thereby determining whether materials possess white mold resistance. High-throughput genotyping detection significantly saves manpower and resources, is unaffected by environmental factors, and provides accurate and reliable results. It can be used for early screening of white mold-resistant peanut varieties, shortening breeding cycles and improving breeding efficiency. Based on the developed KASP molecular marker primer combination, it can identify white mold resistance traits in cultivated peanut varieties, accurately genotype white mold resistance, and guide the field layout of peanut varieties and the control of white mold in peanuts, showing broad application prospects. Attached Figure Description

[0015] Figure 1 Main effect QTL qSRA05 Positioning.

[0016] Figure 2 The typing results of the F9 generation of the RIL population constructed based on "Zhonghua 21" and "Zhonghua 212".

[0017] Figure 3 The disease severity index of white sclerotium in the above RIL population carrying the "CC" and "GG" genotypes under four environmental conditions. A, Positive identification in 2022 (2022YL), B, Positive identification in 2023 (2023YL), C, Positive identification in 2023 (2023WC), and D, Positive identification in 2024 (2024YL).

[0018] Figure 4 The disease index of white sclerotium var. sarcodactylis was determined for 44 accessions carrying the “CC” genotype and 151 accessions carrying the “GG” genotype from 203 natural populations under two different environmental conditions. A. Identification in Yangluo, 2021 (2021YL), B. Identification in Wuchang, 2021 (2021WC). Detailed Implementation

[0019] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0020] 1. Identification of major QTL sites for resistance to white sclerotium wilt Using the white mold-resistant peanut germplasm "Zhonghua 212" as the male parent and the susceptible variety "Zhonghua 21" as the female parent, a RIL population was constructed through hybridization. The population was propagated to the F9 generation using the single-seed method. Genome resequencing was performed on samples from the F9 RIL families. By comparing and analyzing the genome with that of the cultivated peanut variety "Tifrunner" (https: / / www.peanutbase.org / tools / browsers / gbrowse.html?iframe_pathname_suffix=arahy.Tifrunner.gnm1), a total of 275,821 variant sites were obtained, of which 246,047 were SNP markers and 29,774 were InDel markers. A high-density genetic linkage map containing 4,448 bins was constructed.

[0021] Meanwhile, F7-F9 generation plants obtained RIL population phenotypes resistant to white rot under four environmental conditions after artificial inoculation with white rot fungus in the field.

[0022] Genetic linkage analysis was performed using the high-density genetic linkage map and phenotypic data described above, and a stable major-effect QTL was obtained on chromosome 5. qSRA05 ( Figure 1 The QTL is located in the range of 6.33 Mb to 10.31 Mb of A05, and its contribution rate is 5.40% to 12.50%.

[0023] according to qSRA05 Twenty SNP markers (PA01–PA20) were developed for the reliability validation of QTL intervals, targeting candidate genes within the interval. By marker genotyping of the RIL population and combining this with analysis of four environmental resistance phenotypes to white sclerotium, the region controlling white sclerotium resistance was further localized to the area between SNP markers PA04 and PA20. This interval explained 7.27%–13.94% of the phenotypic variation, with the physical distance reduced to 3.26 Mb (7.00 Mb–10.26 Mb).

[0024] 2. Development of molecular markers linked to resistance to peanut white mold disease Based on the whole-genome resequencing results of “Zhonghua 21” and “Zhonghua 212”, in qSRA05 Within the specified interval, candidate genes were screened for SNP sites with polymorphism between parents. KASP markers closely linked to major QTLs were developed. Based on the genotyping results of the KASP markers, a KASP marker A05_9189944 with good genotyping performance was obtained, and its polymorphism was G / C.

[0025] The primer sequence for detecting this molecular marker was used in the following experiments: primer1: 5'-GAAGGTGACCAAGTTCATGCTCCTTGTTGACGTTCTATTGAGAGTC-3' (SEQ IDNO: 1); primer2: 5'-GAAGGTCGGAGTCAACGGATTCCTTGTTGACGTTCTATTGAGAGTG-3' (SEQ IDNO: 2); Reverse primer: 5'-CCAATGACCGCTTTGACGTG-3' (SEQ ID NO:3).

[0026] The amplified fragment size is 84 bp.

[0027] Genotyping analysis was performed on RIL populations constructed using the molecular marker A05_9189944 in the 'Zhonghua 21' and 'Zhonghua 212' strains. The method is as follows: Genomic DNA was collected from peanut leaves or cotyledons, and PCR amplification was performed on the extracted genomic DNA using the primer set described above. The total reaction volume for PCR amplification was 5 μl, which included 1.5 μl of genomic DNA, 2.5 μl of 2×KASP MasterMix, and 0.35 μl of primer mixing solution. The primer mixing solution included: FAM fluorescently bound specific sequence forward primer primer1, HEX fluorescently bound specific sequence forward primer primer1, and a reverse primer.

[0028] The PCR reaction program was as follows: Step 1, 94℃, 15 min; Step 2, 94℃, 20 s, 65-57℃ gradient PCR, 1 min, decreasing the temperature by 0.8℃ per cycle, for 10 cycles; Step 3, 94℃, 20 s, 57℃, 1 min, for 28 cycles; Step 4, 25℃, 1 min. After the reaction, the samples were removed and stored at 4℃.

[0029] The results are as follows Figure 2 As shown, among the F9 generation plants of 242 RIL population families, 97 families were identified carrying the "CC" genotype at the A05_9189944 locus, and 125 families carried the "GG" genotype. Figure 3As shown, the disease index of white sclerotium in families with the "CC" genotype was 44.72, 54.89, 63.93, and 52.01 in four environments: Yangluo (2022YL), Yangluo (2023YL), Wuchang (2023WC), and Yangluo (2024YL) in 2022, 2023, and 2024, respectively. Meanwhile, the disease index of the 125 families carrying the "GG" genotype was 49.66, 60.97, 68.84, and 58.47 in the corresponding four environments. The disease indices of families carrying the "CC" and "GG" genotypes were measured using a T-test. p The values ​​are 4.98E-05, 3.39D-06, 1.05E-4 and 8.43E-05, respectively.

[0030] 3. Genotyping analysis of molecular marker A05_9189944 in 203 cultivated peanut germplasm materials. Genotyping analysis was performed on 203 cultivated peanut germplasm materials using the molecular marker A05_9189944 and its primers.

[0031] The results are as follows Figure 4 As shown, 44 accessions carrying the "CC" genotype were identified from the 203 natural population germplasm materials. In two environments, the average disease index in Yangluo (2021YL) and Wuchang (2021WC) in 2021 was 46.82 and 55.59, respectively. Meanwhile, the 151 accessions carrying the "GG" genotype had average disease indices of 53.66 and 60.81 in the corresponding two environments, respectively. T-tests confirmed these indicators. p The values ​​were 0.0018 and 0.015, respectively, indicating that the phenotypes corresponding to the two genotypes differed significantly in both environments.

[0032] The above experiments demonstrate that the successful development of white mold resistance markers provides important theoretical basis and practical technical support for the targeted breeding and molecular marker-assisted breeding of white mold resistant peanut varieties, which is of great significance for promoting the high-quality development of the peanut industry.

[0033] Although specific primer sets for detecting SNPs at target locations were used above for illustrative purposes, these primer set sequences are merely illustrative and should not be used to limit the invention. Those skilled in the art, knowing the polymorphism at the site and the upstream and downstream sequences, can design primer sets as needed to perform PCR detection for identification. All such primer sets should be included within the scope of protection of this invention.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A molecular marker linked to resistance to peanut white mold disease, characterized in that, The SNP locus is located at position 9189944 on chromosome 5 of peanut, with a polymorphism of G / C. The reference peanut genome is the genome of the cultivated peanut species Tifrunner, obtained from the PeanutBase database.

2. A primer set for peanut germplasm materials capable of identifying resistance to white mold disease, characterized in that, The primer set is a primer set capable of identifying the polymorphism of the molecular marker described in claim 1.

3. The primer set according to claim 2, characterized in that, The primer set includes at least two upstream primers and at least one downstream primer.

4. The primer set according to claim 3, characterized in that, It includes at least two upstream primers with sequences as shown in SEQ ID NO:1 and 2.

5. The primer set according to claim 3, characterized in that, It includes at least a downstream primer with a sequence as shown in SEQ ID NO:

3.

6. The application of the molecular marker of claim 1 or the primer set of any one of claims 2-5 in identifying the resistance of peanut germplasm materials to white mold disease.

7. A method for identifying resistance to white mold disease in peanut germplasm materials, characterized in that, Includes the following steps: S1: Extract the genome from the plant sample of the peanut germplasm material to be tested; S2: Detect polymorphisms at the molecular markers described in claim 1 in the genome; S3: Peanut germplasm with a homozygous C polymorphism at the molecular marker in the genome is resistant to white mold disease, while peanut germplasm with a homozygous G polymorphism at the molecular marker in the genome is susceptible to white mold disease.

8. The method according to claim 7, characterized in that, Using the primer set described in any one of claims 2-5, polymorphisms at the molecular markers described herein are detected by PCR.

9. The method according to claim 8, characterized in that, The PCR reaction program was as follows: 94℃, 15 min; 94℃, 20 s, 65-57℃ gradient PCR, 1 min, decreasing by 0.8℃ for each cycle, for 10 cycles; 94℃, 20 s, 57℃, 1 min, for 28 cycles.

Images