Molecular marker closely linked with peanut bacterial wilt resistance QTL and application thereof

Abstract

The invention belongs to the technical field of peanut disease resistance breeding and molecular biology, and particularly relates to a molecular marker closely linked with peanut bacterial wilt resistance QTL and application of the molecular marker. The invention discloses three groups of peanut plant anti-bacterial wilt QTL (quantitative trait loci) associated molecular marker systems, which comprise an SSR (simple sequence repeat) marker, an Indel marker, an SNP (single nucleotide polymorphism) marker and a matched specific nucleic acid sequence for detection. Peanut genome DNA (deoxyribonucleic acid) is used as a template, and 293bp (ZHSSR2), 169bp (ZHIndel9) and 1275bp (ZHSNP1) characteristic products can be respectively obtained through PCR (polymerase chain reaction) amplification. Wherein amplification products of the ZHSSR2 and the ZHIndel9 are detected through polypropylene gel electrophoresis, and the ZHSNP1 needs to be subjected to sequencing and base variation analysis. The system can carry out molecular marker-assisted selection in a peanut seedling stage, realizes rapid identification and screening of bacterial wilt resistance, and effectively shortens a new peanut variety breeding period.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of peanut disease resistance breeding and molecular biology, and particularly relates to a molecular marker closely linked to a peanut bacterial wilt resistance QTL and application thereof. BACKGROUND

[0002] Peanut (Arachis hypogaea L.) is an important oil and economic crop in the world, and plays an important role in agricultural production. However, peanut cultivation is often affected by various biological and non-biological stresses, among which bacterial wilt (BW) caused by Ralstonia solanacearum is a major bacterial disease. Ralstonia solanacearum is a gram-negative bacterium with a broad host range, which can infect more than 400 plant species, including key crops such as peanut, tomato, tobacco, potato, pepper and soybean. The occurrence of bacterial wilt not only reduces peanut yield, but also affects peanut quality and cultivation benefits. Therefore, in-depth study of the mechanism of peanut resistance to bacterial wilt is of great significance to enhance resistance and promote the sustainable development of peanut industry.

[0003] The resistance of plants to Ralstonia solanacearum is mainly manifested as inhibition of pathogen reproduction and spread, delay of plant wilting and reduction of plant mortality. This resistance is usually determined by quantitative traits. Therefore, locating quantitative trait loci (QTLs) of peanut resistance to bacterial wilt is crucial for molecular marker-assisted selection (MAS) breeding of disease-resistant varieties. So far, only a few major QTLs of bacterial wilt resistance have been identified from different cultivated varieties, such as Yueyou 92 (qBW-1 and qBW-2), Yuanza 9102 (qBWB02.1 and qBWA12), and Zhonghua 6 (qBWRB02-1). However, with the co-evolution of Ralstonia solanacearum and peanut, the pathogenicity of the pathogen may vary, which may gradually weaken the defense effect of the original resistance genes in peanut. Therefore, it is necessary to continuously explore new disease resistance loci (QTLs / genes), analyze their resistance mechanisms, and develop corresponding molecular markers to promote the innovation and application of molecular breeding technology for bacterial wilt resistance, so as to ensure the sustainable development of peanut industry. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the application provides a set of molecular markers for identifying peanut resistance to bacterial wilt. The application of the molecular markers to breeding work can solve the problem of lack of peanut bacterial wilt resistance-related markers, and has important significance for selective breeding of peanuts.

[0005] In order to achieve the above object, the technical scheme adopted by the present application is:

[0006] The application provides the application of a peanut anti-rhizome rot QTL associated molecular marker in identifying a peanut rhizome rot resistance phenotype or peanut rhizome rot resistance breeding, and the peanut anti-rhizome rot QTL associated molecular marker comprises ZH_SSR2, ZH_Indel9 or ZH_SNP1, is located at the positions of 133,899,227 bp, 134,544,638 bp and 133,937,916 bp of a peanut B05 chromosome respectively, the nucleotide sequence of the ZH_SSR2 is shown as SEQ ID NO. 1, the nucleotide sequence of the ZH_Indel9 is shown as SEQ ID NO. 2, and the ZH_SNP1 is a Guanine (Guanine, G) / Adenine (Adenine, A) variation, wherein the disease-resistant material is A at the site, and the disease-susceptible material is G.

[0007] The application provides the application of a peanut anti-rhizome rot QTL associated molecular marker in identifying a peanut rhizome rot resistance phenotype or peanut rhizome rot resistance breeding, and the peanut anti-rhizome rot QTL associated molecular marker comprises ZH_SSR2, ZH_Indel9 or ZH_SNP1, is located at the positions of 133,899,227 bp, 134,544,638 bp and 133,937,916 bp of a peanut B05 chromosome respectively, the nucleotide sequence of the ZH_SSR2 is shown as SEQ ID NO. 1, the nucleotide sequence of the ZH_Indel9 is shown as SEQ ID NO. 2, and the ZH_SNP1 is a Guanine (Guanine, G) / Adenine (Adenine, A) variation, wherein the disease-resistant material is A at the site, and the disease-susceptible material is G.

[0008] Preferably, the detection primer of the ZH_SSR2 comprises ZH_SSR2-F shown in SEQ ID NO: 4 and ZH_SSR2-R shown in SEQ ID NO: 5, the detection primer of the ZH_InDel9 comprises ZH_InDel9-F shown in SEQ ID NO: 6 and ZH_InDel9-R shown in SEQ ID NO: 7, and the detection primer of the ZH_SNP1 comprises ZH_SNP1-F shown in SEQ ID NO: 8 and ZH_SNP1-R shown in SEQ ID NO: 9.

[0009] The third aspect of the present application provides a kit for identifying the resistance phenotype of peanut bacterial wilt, which comprises an upstream primer ZH_SSR2-F shown in SEQ ID NO: 4 and a downstream primer ZH_SSR2-R shown in SEQ ID NO: 5, or an upstream primer ZH_InDel9-F shown in SEQ ID NO: 6 and a downstream primer ZH_InDel9-R shown in SEQ ID NO: 7, or an upstream primer ZH_SNP1-F shown in SEQ ID NO: 8 and a downstream primer ZH_SNP1-R shown in SEQ ID NO: 9.

[0010] The fourth aspect of the present application provides a method for identifying the resistance phenotype of peanut bacterial wilt, which comprises the following steps:

[0011] S1, extracting the DNA of the peanut to be identified, and using the DNA as a template to amplify the ZH_SSR2, ZH_Indel9 or ZH_SNP1 molecular marker by the kit of claim 4;

[0012] S2, judging the resistance phenotype of the peanut to be identified according to the genotype in the PCR amplification product, wherein: the ZH_SSR2 and ZH_InDel9 markers can detect the fragment length polymorphism of 293bp / 277bp and 169bp / 166bp by 7% polyacrylamide gel electrophoresis, the high-molecular-weight amplification fragment is associated with the resistant phenotype, and the low-molecular-weight fragment is associated with the susceptible phenotype; the ZH_SNP1 marker presents G / A single nucleotide polymorphism, the resistant material is A at the site, and the susceptible material is G.

[0013] Preferably, the DNA concentration is diluted to 40-70 ng / μL.

[0014] Preferably, when amplifying the molecular markers ZH_SSR2 and ZH_InDel9 by PCR, a 10 μL reaction system is used, including 1.0 μL of template DNA, 5.0 μL of Tap enzyme, 3.0 μL of ddH2O, and 0.5 μL of each of the upstream and downstream primers.

[0015] Preferably, the amplification reaction conditions when amplifying the molecular markers ZH_SSR2 and ZH_InDel9 by PCR are as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; and 72℃ extension for 10 min.

[0016] Preferably, when amplifying the molecular marker ZH_SNP1 by PCR, a 20 μL reaction system is used, including 1.0 μL of template DNA, 10.0 μL of Tap enzyme, 8.0 μL of ddH2O, and 0.5 μL of each of the upstream and downstream primers.

[0017] Preferably, the amplification reaction conditions for PCR amplification of the molecular marker ZH_SNP1 are as follows: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 60 s, 30 cycles; and extension at 72℃ for 10 min.

[0018] Preferably, the specific operation for analyzing the genotype of the ZH_SNP1 molecular marker is as follows: the amplification product is first subjected to 1% agarose gel electrophoresis, and then recovered and sequenced.

[0019] Compared with the prior art, the present application has the following advantages:

[0020] The present application discloses three groups of molecular marker systems closely related to Quantitative Trait Locus (QTL) of peanut bacterial wilt resistance, and specific nucleic acid sequences for detecting peanut plant QTL-associated markers for bacterial wilt resistance. The molecular marker system comprises one Simple Sequence Repeats (SSR), one Insertion-Deletion (Indel) and one Single Nucleotide Polymorphism (SNP) marker. With peanut genomic nucleotides as templates, the designed specific nucleic acid sequences are used for PCR amplification, and characteristic amplification products of 293 bp / 277 bp, 169 bp / 166 bp and 1275 bp can be obtained, respectively, and the corresponding markers are named as ZH_SSR2, ZH_Indel9 and ZH_SNP1, respectively. Among them, the amplification products of ZH_SSR2 and ZH_Indel9 are detected by polyacrylamide gel electrophoresis, the amplification product of ZH_SNP1 is sequenced and analyzed for base variation, and the resistance and susceptibility screening efficiencies of the three are 77.5%, 70.5% and 73.6%, respectively. By using the molecular marker system established in the present application, rapid identification and screening of peanut bacterial wilt resistance can be realized by implementing molecular marker-assisted selection in the seedling stage of peanut, and the breeding cost is significantly reduced. Moreover, the molecular marker system provided by the present application can provide an efficient and accurate screening tool for breeding peanut varieties resistant to bacterial wilt, and can effectively accelerate the breeding cycle of high-yield, high-quality and disease-resistant peanut new varieties.

[0021] Specifically, the present application has the following advantages:

[0022] (1) The application first identifies and verifies three novel peanut bacterial wilt resistance related molecular markers (ZH_SSR2, ZH_InDel9, ZH_SNP1), which are located on peanut B05 chromosome. Among them: ZH_SSR2 and ZH_InDel9 markers can detect 293bp / 277bp and 169bp / 166bp fragment length polymorphism by 7% polyacrylamide gel electrophoresis, respectively, and the high molecular weight amplified fragment is associated with the disease resistance phenotype, and the low molecular weight fragment is associated with the disease susceptible phenotype; ZH_SNP1 marker presents G / A single nucleotide polymorphism, and the disease resistant material is A at this site, and the disease susceptible material is G.

[0023] (2) The molecular marker system established by the application provides an efficient technical means for peanut bacterial wilt resistance identification, which has the outstanding advantages of significantly reducing breeding cost, greatly improving selection accuracy, providing reliable molecular detection tools for breeding new varieties of disease-resistant peanuts, and effectively shortening the breeding cycle of peanuts. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Flow chart for locating peanut bacterial wilt resistance major QTL.

[0025] Figure 2 Fine mapping of peanut bacterial wilt resistance major QTL; (A) Fine mapping of peanut variety H108 high bacterial wilt resistance major QTL-qBWR15, (B) Genotype of molecular markers ZH_SSR2 and ZH_InDel9 in 16 high susceptible RIL lines and 16 high resistant RIL lines.

[0026] Figure 3 Phenotypic trait analysis of H107 and H108 and RILs population; (A) Genotype of molecular marker ZH_SNP1 in H107, H108 and part of RILs population, (B) Haplotype analysis of ZH_SNP1 in RILs population. DETAILED DESCRIPTION

[0027] The specific embodiments of the application will be further described below. It should be noted that the description of these embodiments is used to help understand the application, but does not constitute a limitation on the application. In addition, the technical features involved in each embodiment of the application described below can be combined with each other as long as they do not conflict with each other.

[0028] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0029] Unless otherwise specified, all instruments and reagents used in the following examples are conventional equipment in the field, and all reagents are commercially available analytical grade products. Unless otherwise specified, all detection methods used are standard methods in the field. Primer synthesis and sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd. (hereinafter referred to as "Sangon").

[0030] Example 1: Localization of QTL-qBWR15, which is the main active ingredient for high resistance to bacterial wilt in peanut variety H108.

[0031] (1) Construction of genetic segregation population

[0032] The highly resistant peanut variety “Nongda Hua 108” (H108) and the highly susceptible variety “Nongda Hua 107” (H107), both bred by the peanut breeding team of the College of Agriculture of Henan Agricultural University, were selected as parents. Figure 1 Using H108 as the female parent, H107 was crossed to obtain 24 true hybrids. After further propagation at the Hainan base, 432 F2 generation seeds were obtained.

[0033] (2) Identification of bacterial wilt resistance in genetically segregating populations

[0034] Peanut seedlings from populations H107, H108, and F2 were planted in a greenhouse under conditions of 16 hours of light (28°C) and 8 hours of darkness (26°C). The planting containers were 10cm x 10cm plastic pots. After obtaining three-week-old peanut seedlings, they were inoculated with *Ralstonia solanacearum* isolated and preserved in our laboratory (specific isolation methods are referenced in "Dai Xiaoqiu et al., 2022. Isolation and genetic diversity identification of the pathogen of peanut bacterial wilt in Guangdong Province," *Journal of Zhongkai University of Agriculture and Engineering*, 2022, 35(03): 26-33). The specific method is as follows: The *Ralstonia solanacearum* strain was transferred to 2,3,5-triphenyltetrazolium chloride (TTC) agar medium (5.0 g / L peptone, 2.0 g / L glucose, 0.5 g / L 2,3,5-triphenyltetrazolium chloride, 0.1 g / L casein hydrolysate, and 15.0 g / L agar) and incubated at 28°C for 48 hours. Afterwards, viable colonies were picked with sterile toothpicks and incubated in TTC liquid medium at 28°C and 200 rpm for 48 hours to obtain a concentration of approximately 10... 8 cfu / mL (OD) 600= 0.5) of the bacterial solution. Then the wounded root method was used to inoculate the peanut seedlings with P. solanacearum(Zhao K, Ren R, Ma X, et al. Genome-wide investigation of defensin genes in peanut (Arachis hypogaea L.) reveals AhDef2.2 conferring resistance to bacterial wilt [J]. The Crop Journal, 2022, 10(3): 809-819.). The control plants were inoculated with distilled water.

[0035] The resistance level of infected peanut plants was scored 7 days after inoculation: high resistance (HR, no wilting or only partial wilting of infected leaf blades); medium resistance (MR, wilting of uncut leaf blades and stems of inoculated branches); medium susceptibility (MS, wilting of uncut leaf blades and stems of non-inoculated branches); high susceptibility (S, wilting of the whole plant or death of the whole plant).

[0036] (3) Mapping of the major QTL-qBWR15 for peanut resistance to bacterial wilt

[0037] Thirty individuals with extreme phenotypes of resistance and susceptibility were selected from the F2 population to construct the resistant bulk (RB) and susceptible bulk (SB), respectively. Then whole-genome resequencing and bulked segregant analysis-sequencing (BSA-seq) were performed on the two bulk pools and the parents. The specific method was as follows: 30 ER individuals (peanut resistant to bacterial wilt) and 30 ES individuals (peanut susceptible to bacterial wilt) were selected as the basic samples of RB and SB, respectively. The genomic DNA was extracted from the leaf tissues of H107, H108, RB and SB plants using RapidPure Plant DNA Isolation Mini Kit (DC104, Nanjing Weishaim Biotechnology Co., Ltd.). The DNA of RB and SB plants was mixed and broken, and then BSA-seq sequencing was performed on the Illumina HiSeq platform (Beijing Boyma Biotechnology Co., Ltd.). The SNP index and Δ(SNP index) profiles between SB and RB were analyzed by sliding window analysis, and the genomic regions above the threshold value (p < 0.01) were considered as the candidate regions of the major QTL-qBWR15 for peanut variety H108 high resistance to bacterial wilt.

[0038] H107, H108, SB and RB generated 54.34 Gb, 50.14 Gb, 77.62 Gb and 76.28 Gb sequencing data, respectively. By comparative genomic analysis of the four samples, a total of 1,502,829 SNPs (single nucleotide polymorphisms) and 103,996 high-quality InDels (insertion / deletion) mutations were detected. Based on the peanut Shitouqi.gnm1.L4VP genome as the reference sequence, association analysis was performed based on SNP-index, ΔSNP-index and Euclidean distance algorithm, and finally the qBWR15 candidate interval was located in a new genomic region, which was different from the previously reported body weight resistance QTL, specifically in the interval of about 5.9 Mb on B05 chromosome, corresponding to the genomic coordinates of Chr.B05: 131975428-137765046.

[0039] Example 2: Development of molecular markers and fine mapping of qBWR15

[0040] (1) Development of molecular markers

[0041] A total of 1214 SNPs and 832 InDels were detected on the peanut B05 chromosome. From the distribution of mutations, most of the mutations (1300, accounting for 63.5%) occurred in the intergenic region, and the remaining mutations were scattered in different functional regions, specifically: 145 in the upstream region (accounting for 7.1%), 95 in the downstream region (accounting for 4.6%), 71 in the intron region (accounting for 3.5%), and 70 in the exon region (accounting for 3.5%). To precisely locate the 5.9 Mb genomic region where qBWR15 is located, 20 InDels, 1 SNP markers were developed based on the mutations in the region adjacent to qBWR15. At the same time, the reference genome sequence of the region adjacent to qBWR15 was obtained from the Peanutbase database (https: / / dev.peanutbase.org / genomics / #hypogaea), and according to the target region DNA sequence (http: / / www.phytozome.net / soybean), SSRHunter 1.3 software (Li and Wan, 2005, SSRHunter: development of a local searching software for SSR sites. Hereditas 27:808-810, https: / / europepmc.org / article / med / 16257914.) was used to find SSR characteristic sequences. Primer Premier 5.0 software (Premier, Palo Alto, CA) was used to design primer sequences, and 3 SSR molecular markers were obtained.

[0042] (2) Extraction of genomic DNA from peanut

[0043] Genomic DNA of peanut H107 and H108 was extracted by CTAB method. The reagents and formulations involved are as follows:

[0044] Tris-HCl solution (1M, pH 8.0): 121.1 g of Tris base was dissolved in 800 mL of ddH2O, and the pH value was adjusted to 8.0 with concentrated hydrochloric acid. EDTA solution (0.5M, pH 8.0): 186.1 g of disodium ethylenediaminetetraacetate was added to 800 mL of ddH2O, and the pH value was adjusted to 8.0 with NaOH (about 20 g of NaOH particles) after stirring. The volume was made up to 1 L, and sterilized. CTAB solution: 2.0 g of CTAB, 8.2 g of sodium chloride, and 0.5 g of polyvinylpyrrolidone were dissolved in 70 mL of ddH2O, and then 10 mL of Tris-HCl solution (1M, pH 8.0) and 10 mL of EDTA solution (0.5M, pH 8.0) were added. Finally, the volume was made up to 100 mL with ddH2O.

[0045] The specific operation steps are as follows:

[0046] 1) Take about 2 g of fresh peanut leaves and put them in a mortar. Add liquid nitrogen and grind quickly until the leaves are powdered. Put the two pre-cooled centrifuge tubes in a -20°C refrigerator for standby.

[0047] 2) Add 650 μL of CTAB solution preheated at 65°C for about 30 min and 20 μL of β-mercaptoethanol to the centrifuge tube containing the leaf powder, mix well, and place it in a 65°C water bath for about 60 min. Shake gently every 10 min or so during the period.

[0048] 3) After the water bath, add 650 μL of chloroform-isoamyl alcohol (24:1 / v:v) and mix gently for 5 min. Let it stand for 15 min to react fully. After balancing, centrifuge at 4°C and 12000 rpm for 15 min.

[0049] 4) Gently take out and slowly suck up 400 μL of supernatant with a cut 200 μL syringe tip and transfer it to another centrifuge tube. Add 650 μL of chloroform-isoamyl alcohol (24:1 / v:v), mix gently, and let it stand for 10 min. Centrifuge at 4°C and 8000 rpm for 10 min.

[0050] 5) Take out, same as step 4) above, aspirate the supernatant into another centrifuge tube. Add 150 μL of ice-cold isopropanol and freeze overnight.

[0051] 6) Centrifuge at 12000 rpm for 10 min at 4°C.

[0052] 7) Discard the supernatant, add 800 μL of 70% ethanol, centrifuge at 12000 rpm for 5 min at 4°C.

[0053] 8) Discard the supernatant, place in a fume hood to dry, then add 200 μL of ddH2O.

[0054] 9) After dissolving in a 4°C refrigerator for 1 day, store in a -20°C refrigerator for later use.

[0055] Dilute the extracted DNA solution to 5 times, 10 times, 15 times, 20 times and 30 times, respectively, to 5 concentration gradients, and perform electrophoresis on 1% agarose gel. Use a gel imaging system to detect the bands, estimate the quality of the DNA and obtain the required DNA concentration.

[0056] (3) Polymorphic molecular marker screening

[0057] Use the specific primers of 20 InDel and 3 SSR markers to perform PCR amplification on the genomic DNA of H107 and H108. The reaction system (10 μL) contains: 5 μL Taq DNA polymerase, 3 μL double distilled water, 0.5 μL each forward and reverse primer and 1.0 μL template DNA. The amplification program is: 94°C pre-denaturation for 2 min; 30 cycles of 94°C denaturation for 30 s / 55°C annealing for 30 s / 72°C extension for 30 s; 72°C final extension for 10 min. Then use 7% polyacrylamide gel for electrophoretic separation, and the specific steps are as follows:

[0058] 1) Soak the glass plate in water and add detergent. After about 30 min, repeatedly scrub the glass plate to remove foreign matter and dirt on the surface of the glass plate, and then rinse it with pure water. After the glass plate is dried, pair it and fix it to the electrophoresis tank, and seal it with 1% agarose;

[0059] 2) Add 10% ammonium persulfate and tetramethyl ethylenediamine to the triangular flask containing 8% polyacrylamide solution, mix well, and stand for about 2 min. Gently pour the glue along the glue pouring port, and if there are bubbles in the glue, remove them in time with a fine needle. Insert the comb and wait for the glue to coagulate;

[0060] 3) Add 1 x TBE electrophoresis buffer to the electrophoresis tank and pull out the comb. At the same time, add 2 μL of bromophenol blue buffer to the PCR amplification product and spot 3 μL. When spotting, the first hole at both ends of the gel is not spotted, 48 holes are spotted per gel plate, and the molecular weight marker is spotted in the middle, 0.8 μL. The amount of marker should not be too much, otherwise the bands of large molecular weight cannot be separated. The electrophoresis uses constant power, 10 W / gel plate. It can be electrophoresed for about 50-70 min. The specific situation depends on the size of the molecular weight of the amplification product.

[0061] 4) After electrophoresis, pour out the electrophoresis buffer and remove the gel. Place the gel plate in the fixing solution, shake for 30 min on the shaker; pour out the fixing solution, add the permeating solution, permeate for 15 min; then rinse twice with pure water (the first time for 50 s; the second time for 40 s), add the color developing solution for color development until the bands can be clearly seen, stop color development, rinse twice with pure water, and record the polymorphic bands through the gel imaging system.

[0062] Finally, among the 20 InDels and 3 SSR markers, 6 InDels and 2 SSR markers including ZH_SSR2 and ZH_Indel9 were screened out, and there were polymorphic difference bands between the parent lines H107 and H108.

[0063] (4) Fine mapping of qBWR15

[0064] H108 as the female parent, 151 recombinant inbred lines (RIL, F5) were derived by single-seed descent method. H107 and H108, and the RIL population were planted in the peanut bacterial wilt disease nursery of Henan Agricultural University, and inoculated with bacterial wilt bacteria (strain isolation and extraction according to the literature “Zhao K, Ren R, Ma X, et al. Genome-wide investigation of defensin genes in peanut (Arachis hypogaea L.) reveals AhDef2.2 conferring resistance to bacterial wilt [J]. The Crop Journal, 2022, 10(3): 809-819.”) to investigate the resistance level. The resistance level of RIL lines was evaluated according to the survival rate (SR) at the harvest stage: high resistance (SR>90%), resistance (80%<SR<90%), medium resistance (65%<SR<80%), medium susceptibility (35%<SR<65%) and high susceptibility (SR<35%). Finally, these phenotypes can be used as morphological markers for genetic linkage analysis, where RIL with SR(%) value greater than 65% is identified as R line, and RIL with SR(%) value less than 65% is identified as S line.

[0065] SR(%) = number of surviving plants / total number of plants in the line x 100%.

[0066] The CTAB method was used to extract the leaf genomic DNA of the RIL population. The specific primers of the screened 6 InDel and 2 SSR markers were used to amplify the genomic DNA of H107 and H108 and the RIL population as templates. The reaction system (10 μL) contained 5 μL Taq DNA polymerase, 3 μL double distilled water, 0.5 μL each forward and reverse primer, and 1.0 μL template DNA. The amplification program was as follows: 94°C pre-denaturation for 2 min; 30 cycles of 94°C denaturation for 30 s / 55°C annealing for 30 s / 72°C extension for 30 s; 72°C final extension for 10 min. Then 7% polyacrylamide gel electrophoresis and band analysis were performed.

[0067] The phenotype and genotype data of RIL were analyzed by genetic linkage analysis using JoinMap 4.0 software. The minimum log-likelihood ratio 3.0 was used for linkage analysis test. Finally, qBWR15 was accurately located in a 645 kb region between markers ZH_SSR2 and ZH_InDel9 Figure 2 A).

[0068] Example 3: Confirmation and sequence analysis of molecular markers

[0069] According to the fine mapping results of qBWR15, two molecular markers closely associated with the resistance gene qBWR15 were identified, named ZH_SSR2 and ZH_InDel9. In addition, a key SNP site was found in the above marker interval, named ZH_SNP1. Among them, ZH_SSR2, ZH_Indel9 and ZH_SNP1 are located at the positions of 133,899,227bp, 134,544,638bp and 133,937,916bp of peanut B05 chromosome, respectively.

[0070] Among them, the marker sequence of ZH_SSR2 (SEQ ID NO: 1) is as follows:

[0071] ACTCTCCATTCTCCATCTATCTGATAGTTCTAACTAATTATTAATTTTCATATGAATAGAACTGCAAGTATCCATCCTATACAAAAACAGTAAGATTTTATACAAAAAGTCTATTAGGAATTTTAGGTTTATATATATATATATATATATATATATATATATATATATATGCGTGAAAGGACTAAAAAACATATATGAGAAATAAATTATTAAGGACAATTCAGTAAATTTATGAATTTTGAGGATTAGCGGGGATGAGACGGAGATCCCTGTTC.

[0072] The marker sequence of ZH_InDel9 (SEQ ID NO: 2) is as follows:

[0073] CAACAAGTGAAAAGAGCGAAAAAGGGTAATTACCACTAAACTATTTGCTCATTTTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTAGTTAAGGTAATTTCCTCTATTAGTAACATATCCTCTCTGTGTTG.

[0074] The marker sequence containing ZH_SNP1 (SEQ ID NO: 3) is as follows:

[0075]

[0076] To clarify the specific sequence differences of the above molecular markers in the parent materials H107 and H108, the sequences corresponding to the three molecular markers were cloned in H107 and H108, respectively. The specific steps are as follows:

[0077] (1) Extraction of peanut genomic DNA

[0078] The CTAB method was used to extract peanut leaf genomic DNA, and it was diluted to 50 ng / μL.

[0079] (2) Cloning and sequence analysis of ZH_SSR2, ZH_InDel9 and ZH_SNP1 markers

[0080] PCR primers were designed, and ZH_SSR2-F and ZH_SSR2-R were as follows:

[0081] ZH_SSR2-F: 5’-ACTCTCCATTCTCCATCTATCTGA-3’ (SEQ ID NO: 4);

[0082] ZH_SSR2-R: 5’-GAACAGGGATCTCCGTCTCAT-3’ (SEQ ID NO: 5).

[0083] PCR primers were designed, and ZH_InDel9-F and ZH_InDel9-R were as follows:

[0084] ZH_InDel9-F: 5’-CAACAAGTGAAAAGAG-3’ (SEQ ID NO: 6);

[0085] ZH_InDel9-R: 5’-CAACACAGAGAGGATA-3’ (SEQ ID NO: 7).

[0086] PCR primers were designed, and ZH_SNP1-F and ZH_SNP1-R were as follows:

[0087] ZH_SNP1-F: 5’-GTGGCTGCGGGTATTGC-3’ (SEQ ID NO: 8);

[0088] ZH_SNP1-R: 5’-GGTACTCAACGGCGCGAGCAGCT-3’ (SEQ ID NO: 9).

[0089] The genomic DNA of H107 and H108 was amplified by PCR using specific primers ZH_SSR2-F / R and ZH_InDel9-F / R, and the reaction system (10 μL) contained 5 μL Taq DNA polymerase, 3 μL double distilled water, 0.5 μL each forward and reverse primer, and 1.0 μL template DNA. The amplification procedure was as follows: 94°C pre-denaturation for 2 min; 30 cycles of 94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 30 s; and 72°C final extension for 10 min. Subsequently, 7% polyacrylamide gel electrophoresis was used for separation.

[0090] The genomic DNA of H107 and H108 was amplified by PCR using specific primers ZH_SNP1-F / ZH_SNP1-R, and the reaction system (20 μL) contained 1.0 μL template DNA, 10.0 μL Tap enzyme, 8.0 μL ddH2O, 0.5 μL ZH_SNP1-F, and 0.5 μL ZH_SNP1-R. The PCR amplification reaction conditions were as follows: 94°C pre-denaturation for 2 min; 30 cycles of 94°C denaturation for 30 s, 56°C annealing for 60 s, and 72°C extension for 60 s; and 72°C extension for 10 min. After the amplification product was separated by 1% agarose gel electrophoresis for 20 min, the target band was cut under ultraviolet light, purified using a Hailing (Shanghai) agarose gel recovery kit, and finally sent to Shengong for sequence determination.

[0091] Polyacrylamide gel electrophoresis analysis showed that when the genomic DNA of H107 and H108 was amplified using ZH_SSR2 and ZH_InDel9 markers, obvious band size polymorphism was produced Figure 2 B), in which the resistant variety H108 showed high molecular weight bands (293 bp and 169 bp, respectively), and the susceptible variety H107 showed low molecular weight bands (277 bp and 166 bp, respectively). The sequencing analysis of the ZH_SNP1 marker further confirmed Figure 3 that the SNP site was A allele in H108 and G allele in H107, which was completely consistent with the phenotype of the resistance to Ralstonia solanacearum.

[0092] Example 4: Verification of resistance screening of molecular markers in the progeny population

[0093] To verify whether the resistance of peanut to Ralstonia solanacearum is associated with the ZH_SSR2, ZH_InDel9, and ZH_SNP1 markers, the markers were verified in the RIL population. The specific steps were as follows:

[0094] (1) Extraction of DNA

[0095] DNA of RIL population was extracted by CTAB method and the concentration of DNA was diluted to 50 ng / μL.

[0096] (2) Detection of molecular marker genotypes

[0097] PCR amplification was performed with the primer pairs of the three markers using the DNA of RIL population as template, and the PCR reaction system and amplification procedure were as described in Example 3.

[0098] Genetic analysis showed that among the 16 high-susceptible lines and 16 high-resistant lines of RIL population, ZH_SSR2 and ZH_InDel9 markers could effectively distinguish the extreme phenotype materials, and the resistant lines all showed high molecular weight bands, while the susceptible lines all showed low molecular weight bands Figure 2 B) Combined with the phenotype investigation results of population materials, the screening efficiency of ZH_SSR2 and ZH_InDel9 in peanut hybrid progeny population was 77.5% and 70.5%, respectively. Further verification of 151 known resistant lines in the population by ZH_SNP1 marker showed that 76% of the susceptible lines were G base at this site, while 64% of the resistant lines were A base, and the screening efficiency of resistance and susceptibility was 73.6% Figure 3 , Table 1).

[0099] Table 1 Molecular marker genotypes and resistance-susceptibility phenotypes of peanut resistance-susceptibility parents and RIL population of progeny

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] In summary, the peanut fusarium wilt resistance molecular marker system provided by the application can realize efficient screening of the fusarium wilt resistance phenotype in the early stage of breeding. Meanwhile, the molecular marker system can significantly reduce the breeding cost and improve the screening accuracy by means of the molecular marker assisted selection technology, and provides important technical support for peanut breeding practice. In addition, the peanut fusarium wilt resistance related molecular marker provided by the application can not only be used for screening the peanut fusarium wilt resistance phenotype in the early stage of peanut breeding, but also be used for identifying the peanut fusarium wilt resistance phenotype, so as to greatly save the production cost and improve the selection efficiency, and has important application value in peanut breeding.

[0108] The embodiments of the application are described in detail above, but the application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the application, and still fall within the protection scope of the application.

Claims

1. Use of a peanut anti-Ralstonia solanacearum QTL association molecular marker in identifying a peanut Ralstonia solanacearum resistance phenotype or in breeding for peanut Ralstonia solanacearum resistance, characterized in that, The peanut anti-Ralstonia solanacearum QTL associated molecular marker comprises ZH_SSR2, ZH_Indel9 or ZH_SNP1, the nucleotide sequence of ZH_SSR2 is shown as SEQ ID NO. 1, the nucleotide sequence of ZH_Indel9 is shown as SEQ ID NO. 2, and ZH_SNP1 is G / A variation, wherein the disease-resistant material is A at the site, and the disease-susceptible material is G.

2. The use of the detection primer of the peanut anti-Ralstonia solanacearum QTL associated molecular marker in identifying peanut Ralstonia solanacearum resistance phenotype or peanut Ralstonia solanacearum resistance breeding, characterized in that, The peanut anti-Ralstonia solanacearum QTL associated molecular marker comprises ZH_SSR2, ZH_Indel9 or ZH_SNP1, the nucleotide sequence of ZH_SSR2 is shown as SEQ ID NO. 1, the nucleotide sequence of ZH_Indel9 is shown as SEQ ID NO. 2, and ZH_SNP1 is G / A variation, wherein the disease-resistant material is A at the site, and the disease-susceptible material is G.

3. Use according to claim 2, characterized in that, The detection primer of ZH_SSR2 comprises ZH_SSR2-F shown in SEQ ID NO: 4 and ZH_SSR2-R shown in SEQ ID NO: 5, the detection primer of ZH_InDel9 comprises ZH_InDel9-F shown in SEQ ID NO: 6 and ZH_InDel9-R shown in SEQ ID NO: 7, and the detection primer of ZH_SNP1 comprises ZH_SNP1-F shown in SEQ ID NO: 8 and ZH_SNP1-R shown in SEQ ID NO:

9.

4. A kit for identifying a resistance phenotype to peanut bacterial wilt, characterized in that, The kit comprises the upstream primer ZH_SSR2-F shown in SEQ ID NO: 4 and the downstream primer ZH_SSR2-R shown in SEQ ID NO: 5, or the upstream primer ZH_InDel9-F shown in SEQ ID NO: 6 and the downstream primer ZH_InDel9-R shown in SEQ ID NO: 7, or the upstream primer ZH_SNP1-F shown in SEQ ID NO: 8 and the downstream primer ZH_SNP1-R shown in SEQ ID NO:

9.

5. A method of identifying a resistance phenotype to peanut bacterial wilt, characterized in that, The method comprises the following steps: S1, extracting the DNA of the peanut to be identified, and using the DNA as a template to amplify the ZH_SSR2, ZH_Indel9 or ZH_SNP1 molecular marker by PCR with the kit of claim 4; S2, judging the Ralstonia solanacearum resistance phenotype of the peanut to be identified according to the genotype in the PCR amplification product, wherein: the ZH_SSR2 and ZH_InDel9 markers can detect 293bp / 277bp and 169bp / 166bp fragment length polymorphisms by 7% polyacrylamide gel electrophoresis, the high-molecular-weight amplification fragment is associated with the disease-resistant phenotype, and the low-molecular-weight fragment is associated with the disease-susceptible phenotype; the ZH_SNP1 marker presents G / A single nucleotide polymorphism, the disease-resistant material is A at the site, and the disease-susceptible material is G.

6. The method of identifying resistance phenotype to Ralstonia solanacearum in peanut as claimed in claim 5, wherein, The DNA concentration is diluted to 40-70 ng / μL.

7. The method of identifying resistance phenotype to Ralstonia solanacearum in peanut as claimed in claim 5, wherein, The PCR amplification of the molecular markers ZH_SSR2 and ZH_InDel9 was performed in a 10 μL reaction system, including 1.0 μL template DNA, 5.0 μL Tap enzyme, 3.0 μL ddH2O, 0.5 μL upper primer and 0.5 μL lower primer.

8. The method of identifying resistance phenotype to Ralstonia solanacearum in peanut as claimed in claim 5, wherein, The amplification reaction conditions for the PCR amplification of the molecular markers ZH_SSR2 and ZH_InDel9 were as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 10 min.

9. The method of identifying resistance phenotype to Ralstonia solanacearum in peanut as claimed in claim 5, wherein, The PCR amplification of the molecular marker ZH_SNP1 was performed in a 20 μL reaction system, including 1.0 μL template DNA, 10.0 μL Tap enzyme, 8.0 μL ddH2O, 0.5 μL upper primer and 0.5 μL lower primer.

10. The method of identifying resistance phenotype to Ralstonia solanacearum in peanut as claimed in claim 5 wherein, The amplification reaction conditions for the PCR amplification of the molecular marker ZH_SNP1 were as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 60 s, 30 cycles; 72℃ extension for 10 min.

Images