Application of the major peanut early spot resistance gene AhELSR1 and its molecular markers
By identifying and applying the main effect gene AhELSR1 and its molecular marker for peanut premature spot disease, the problem of long breeding cycle of resistant varieties in the prior art has been solved, and efficient resistance breeding and breeding efficiency has been achieved.
Patent Information
- Application Number
- CN202310588509.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-24
- Publication Date
- 2025-04-29
- Estimated Expiration
- 2043-05-24
AI Technical Summary
The prior art is difficult to effectively identify and utilize the main effect gene for resistance to peanut premature spot disease, resulting in a long breeding cycle of resistant varieties and a slow breeding progress.
By identifying and applying the main effect gene AhELSR1 and its molecular marker for peanut premature spot disease, primer pairs were designed with specific SNP sites for PCR amplification and sequencing to assist in the selection of peanut materials that resist premature spot disease.
It has achieved efficient identification and breeding assistance for peanut premature spot resistance, shortened the breeding cycle of resistant varieties, and improved breeding efficiency.
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Figure CN116515858B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular genetics, and in particular to an application of a major gene for peanut early spot resistance and a molecular marker thereof. Background Art
[0002] peanut( Arachis hypogaea ) is an important oil and cash crop in my country. Cercospora arachidicola The peanut early spot disease caused by it is one of the important fungal diseases that endangers peanut production. The affected area in my country is more than 1 million hectares each year. The production of affected peanuts generally decreases by 10% to 20%, and in severe cases, it can reach more than 50%.
[0003] Breeding and planting resistant peanut varieties is the most economical and effective way to prevent and control early spot disease. However, when using traditional breeding methods for resistance breeding, the field identification of resistance is easily affected by multiple factors such as light, temperature, humidity, rainfall, etc., the results fluctuate greatly and need to be repeated for many years, which leads to a longer breeding cycle for resistant varieties and very slow breeding progress. The core foundation of molecular breeding technology is to identify the key gene loci of related traits and clarify the mechanism of action of genes to traits. With its advantages of being directional, precise, and efficient, it has shown broad application prospects in improving the breeding efficiency of peanuts. Identifying and cloning major resistance genes is a prerequisite for molecular breeding of peanut early spot resistance.
[0004] At present, although the positioning of peanut early spot disease resistance genes has been carried out at home and abroad, and a number of early spot disease resistance QTLs have been identified, most QTLs are minor QTLs, or cannot be detected repeatedly, or the genetic distance between the QTL marker and the target gene is far, and it is difficult to use them for the targeted improvement of early spot disease resistance. In order to further improve the efficiency of early spot disease resistance breeding, it is of great significance to identify major resistance genes and develop functional markers. Summary of the invention
[0005] The purpose of the present invention is to provide a peanut early spot disease resistance major gene AhELSR1 and application of its molecular marker.
[0006] Specifically, the present invention provides the use of the gene AhELSR1 in early spot disease resistance peanuts: the CDS sequence of the gene is shown in SEQ ID NO.1.
[0007] The second aspect of the present invention provides the use of molecular markers for detecting specific SNPs in identifying or assisting in identifying resistance to peanut early spot disease; the specific SNPs are nucleotides 4186273, 4187545, 4187547, 4187575 and 4187586 on chromosome B02 of the peanut genome; the specific SNPs are G / A, C / G, A / G, C / A, C / G polymorphisms.
[0008] In the third aspect of the present invention, a primer pair for detecting SNP markers in AhELSR1 is provided, the sequence of which is shown in SEQ ID NO.2-3, the specific SNPs are nucleotides 4186273, 4187545, 4187547, 4187575 and 4187586 on chromosome B02 of the peanut genome; the specific SNPs are G / A, C / G, A / G, C / A, C / G polymorphisms.
[0009] In a fourth aspect of the present invention, the present invention seeks protection for a reagent or a kit containing the above-mentioned primer pair.
[0010] According to the understanding of those skilled in the art, the present invention also requests protection for the use of the primer pairs for SNP markers in the above-mentioned AhELSR1, or the primer pairs as shown in SEQ ID NO.2-3, or the reagents or kits containing the primer pairs described in SEQ ID NO.2-3 in detecting the major effect gene AhELSR1 for resistance to peanut early spot disease.
[0011] In a fifth aspect of the present invention, the above-mentioned primer pair, a reagent or a kit containing the above-mentioned primer pair is provided for use in any of the following:
[0012] (1) Application in identification, improvement or molecular marker-assisted breeding of peanut germplasm resources;
[0013] (2) Application in early prediction of peanut early spot resistance;
[0014] (3) Application in screening peanuts resistant to early spot disease.
[0015] A sixth aspect of the present invention provides a method for identifying or assisting in identifying resistance to peanut early spot disease, comprising the following steps:
[0016] (1) using the primer pairs shown in SEQ ID NOs. 2-3, PCR amplification of the DNA of the peanut to be tested;
[0017] (2) sequencing the PCR product, analyzing the genotype of the SNP marker in the PCR amplification product, and determining the resistance of the peanut to be identified to early spot disease based on the genotype;
[0018] (3) If the nucleotides at positions 4186273, 4187545, 4187547, 4187575 and 4187586 on chromosome B02 of the SNP molecular marker are G, C, A, C, C, the peanut to be identified has strong resistance to early blight. If the nucleotides at positions 4186273, 4187545, 4187547, 4187575 and 4187586 on chromosome B02 of the SNP molecular marker are A, G, G, A, G, the peanut to be identified has weak resistance to early blight.
[0019] The beneficial effects of the present invention are:
[0020] (1) The present invention identified for the first time the major peanut early spot resistance gene AhELSR1.
[0021] (2) Based on the discovered major effect gene AhELSR1, the present invention screened out specific SNP sites that distinguish peanut early spot resistance in the CDS region of the gene. The nucleotides of the SNPs at positions 4186273, 4187545, 4187547, 4187575 and 4187586 on chromosome B02 are G, C, A, C, C.
[0022] (3) The use of primer pairs that detect specific SNPs can assist in the selection of peanut materials resistant to early spot disease, which is conducive to promoting the selection and breeding of peanut varieties resistant to early spot disease and improving breeding efficiency.
[0023] (4) The SNP molecular marker of the present invention is mainly used to detect whether the major effect gene AhELSR1 of early spot disease resistance is contained. Peanut materials containing AhELSR1 loci have a stronger ability to resist early spot disease. In breeding applications, materials with AhELSR1 can be used as parents for hybridization, and many individuals with or without AhELSR1 will be produced in their offspring. Individual materials containing AhELSR1 are retained through marker-assisted selection, and then high-yield and high-quality materials are selected from these individual materials, and finally a new peanut variety resistant to early spot disease can be formed. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 To locate the peanut early spot resistance gene using BSR-seq and linkage analysis.
[0025] Figure 2 Fine mapping of the early spot resistance gene AhELSR1.
[0026] Figure 3 CDS sequence alignment of AhELSR1 sequence and its mutant sequence ahelsr1. DETAILED DESCRIPTION
[0027] It should be noted that the following detailed description is illustrative and is intended to provide further description of the application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the application belongs. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.
[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0029] Example 1: Localization of the major peanut early spot resistance gene AhELSR1
[0030] Test materials: The highly susceptible variety Zhonghua 10 was used as the male parent, and the highly resistant variety ICG12625 was used as the male parent. A recombinant inbred line (RIL) population containing 140 individuals was obtained through single-seed transmission.
[0031] Phenotypic identification: In 2019, 2020 and 2021, the early spot resistance of 140 strains of Zhonghua No. 10, ICG12625 and RIL populations was continuously identified in the field at the research base of Zhanjiang Experimental Station of Chinese Academy of Tropical Agricultural Sciences. A completely randomized block experimental design was adopted with 3 replications. Each material was planted in 1 row with 15 plants per replication, with a row spacing of 30 cm and a plant spacing of 10 cm. Standard field management methods were adopted, and no fungicides were used during the entire growth period. Before peanut harvest, the disease grade of each material was identified in accordance with the Peanut Germplasm Resource Description Specifications and Data Standards. The higher the disease grade, the worse the resistance.
[0032] QTL positioning: According to the results of phenotypic identification, 15 extremely resistant and 15 extremely susceptible families to early spot disease were selected from the RIL population to construct two mixed pools (R-buLk and S-buLk). Thirty families were planted in the greenhouse. After inoculation with 105 / ml early spot disease spore suspension at the seedling stage for 1 day, young leaves were taken and mixed in equal amounts according to the mixed pool. Total RNA was extracted to construct a library and paired-end 150bp (PE150) sequencing was performed using the Illumina HiSeq platform. After quality control of the raw data off the machine, high-quality reads were obtained. The reads were aligned to the reference genome of peanut (https: / / www.peanutbase.org / data / public / Arachis_hypogaea / Tifrunner.gnm2.J5K5 / ) using Hisat software, and the alignment results were sorted using samtools software (version 1.3.1), and variant detection was performed using GATK software. The detected variants were filtered according to the criteria that the sequencing depth of the mixed pool was not less than 10, and the SNP-index value of the mixed pool could not be greater than 0.8 or less than 0.2 at the same time. The Δ(SNP-index) value of each variant site was calculated using QTLseqr (R package), and its distribution on the genome was investigated with a sliding window of 2Mb. The obtained △SNP-index was subjected to 1000 permutation tests, and the 99.9% confidence interval level was selected as the screening threshold. The area exceeding the threshold line was selected as the candidate target interval of the early spot disease resistance QTL, and the early spot disease resistance QTL was located in the interval 2438877-4994316 of chromosome B02 ( Figure 1 ).
[0033] The whole genome of the parents was resequenced, and the raw data was quality controlled to obtain high-quality clean data. The clean data was then aligned to the tetraploid cultivated peanut reference genome (https: / / www.peanutbase.org / data / public / Arachis_hypogaea / Tifrunner.gnm2.J5K5 / ) using BWA software, and finally the HaplotypeCaller module of GATK was used for SNP detection. A total of 645 SNPs and InDel sites were obtained in the QTL interval. 50 SNPs were screened from the middle distance for primer design, and the RIL population was typed by capture sequencing. A QTL interval genetic map with a total length of 10.10 cM was constructed using 22 SNP markers using JOINMAP4.0 software. Combined with the phenotypic identification of the population over the past 3 years, the target QTL was further located to the interval 3580792-4929416 of chromosome B02 using WinQTLCart software and composite interval mapping ( Figure 1 ), the phenotypic variation explanation rates were 21.57%, 27.62% and 30.48% respectively.
[0034] Fine positioning of QTL: Based on the markers at both ends of the QTL and combined with the genetic map information, 16 families with QTL interval recombination were screened from the RIL population. The offspring of the recombinant families were screened using the markers at both ends of the QTL, and heterozygous recombinants in the QTL interval were obtained. The recombinants were self-pollinated to isolate the pure strains in the QTL interval. Based on the QTL interval information, 30 pairs of SNP primers were further designed, and the near-isogenic lines were typed through capture sequencing. By integrating the recombination exchange in the QTL interval, the target QTL was further narrowed down to the 3905564-4402689 interval of chromosome B02 ( Figure 2 ).
[0035] Identification of candidate genes: Variation analysis of genes in the QTL interval revealed that 4 genes had mutations in the exon region. Comparative transcriptome analysis of the results of BSR sequencing revealed that only one of the 4 genes had significant expression differences between the resistant and susceptible mixed pools. Total RNA was extracted from the parents 1, 3, and 7 days after early spot infection, and RT-PCR was performed on these 8 genes. It was found that the differentially expressed genes detected by the mixed pool transcriptome were differentially expressed in the resistant and susceptible parents, and they were used as the target genes of AhELSR1.
[0036] Example 2: Cloning and sequencing of peanut AhELSR1 gene
[0037] (1) Total DNA extraction: Take young leaves of the parent and extract total DNA using the CTAB method.
[0038] (2) Full-length amplification of AhELSR1 gene: The primer sequences were synthesized by Shanghai Bioengineering Co., Ltd., AhELSR1-F: 5'-GTATTTTTTTCCCTTTGTGTGGTT-3'; (SEQ ID NO.4) AhELSR1-R: 5'-GGCATTTTTGTTATGAAAAATGA-3'. (SEQ ID NO.5)
[0039] PCR amplification system: 20 μL of PCR mixture, including 10 μL of 2×EasyTaq PCR SuperMix, 1 μL of each primer pair mentioned above (used at a concentration of 10 μM), 2 μL of template DNA, and ddHO. 2 O 6 μL.
[0040] The PCR reaction program was as follows: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 45 s, 55°C for 40 s, and 72°C for 1 min; and extension at 72°C for 10 min.
[0041] (3) Electrophoresis detection: Take 5 μL of PCR product and add it to the well of 1% agarose gel containing 1.2% gel-red fluorescent dye; electrophoresis is performed for 20 minutes under 0.5×TAE electrophoresis buffer and 120V voltage conditions; after electrophoresis, place it under the fluorescent imaging system for observation, and a band of about 4000 bp can be detected.
[0042] After the PCR product was purified, it was sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing primers were as follows:
[0043] AhELSR1-F: 5'-GTATTTTTTTCCCTTTGTGTGGTT-3'; (SEQ ID NO.4)
[0044] AhELSR1-R: 5'-GGCATTTTTGTTATGAAAAATGA-3'; (SEQ ID NO.5)
[0045] AhELSR1-M1: 5'-GGAAGGGGTCAGAATCTCCTTC-3'; (SEQ ID NO. 6)
[0046] AhELSR1-M2: 5'-GGCCTTGTGGGCAATGATTTTA-3'; (SEQ ID NO.7)
[0047] AhELSR1-M3: 5'-TGAAAGAGTTACCATTAGCGGT-3'; (SEQ ID NO.8)
[0048] Sequence comparison analysis was performed using DNAMAN software. The results showed that the full length of the AhELSR1 gene was 3606 bp, containing 2 exons and 1 intron, and the CDS sequence was 2421 bp (SEQ ID NO.1). The full length of the AhELSR1 gene of the parent had five base nonsense mutations at positions 4186273, 4187545, 4187547, 4187575, and 4187586 on chromosome B02 (located in the first segment of CDS), and these five SNP sites in the resistant parent were G, C, A, C, C ( Figure 3 ).
[0049] Example 3: Development and application of peanut AhELSR1 gene SNP markers
[0050] According to the AhELSR1 gene sequence information and SNP site information, the primer pair was designed as follows; 5'-TTCCCAAACCTCCAATCCCAAC -3'; (SEQ ID NO.2)
[0051] 5'-GTATGGAAGCATTTGCACGAAG-3'. (SEQ ID NO.3)
[0052] The designed AhELSR1 gene specific SNP detection primer pairs were used to detect 137 families in the RIL population. The specific steps are as follows:
[0053] Using genomic DNA as template and the sequences shown in SEQ ID NO. 2-3 as primers, the PCR amplification system was as follows: 20 μL of PCR mixture, including 10 μL of 2×EasyTaq PCR SuperMix, 1 μL of each pair of primers mentioned above (used at a concentration of 10 μM), 2 μL of template DNA, and ddH 2 O 6 μL. The PCR reaction program is: 94℃ pre-denaturation for 3 minutes; 94℃ 45 seconds, 55℃ 40 seconds, 72℃ 30 seconds, 35 cycles; 72℃ extension for 10 minutes. The amplified product was sequenced, the sequence was aligned to the reference sequence, and the base of the SNP site was detected. If the SNP site base detected by the amplified fragment of RIL and AhELSR1 is consistent, it means that the family contains the early spot disease resistance gene AhELSR1. At the same time, the actual identification of the disease grade results and the molecular marker detection results were used for verification.
[0054] The results showed that among the 137 RILs, the SNP genotypes of 72 RILs were consistent with AhELSR1, indicating that they contained the major resistance gene AhELSR1 for early spot disease; the SNP genotypes of 65 RILs were different from AhELSR1, indicating that they did not contain the major resistance gene AhELSR1. The T test found that the incidence level of RIL families containing the early spot disease resistance gene AhELSR1 in 2019-2021 was significantly lower than that of the 65 RIL families without AhELSR1 at the 0.01 level.
[0055] Table 1 Differences in disease grade among RIL families selected using the SNP markers of the present invention.
[0056]
[0057] The designed AhELSR1 gene-specific SNP detection primers were used to perform genotyping on 10 early spot disease resistance resources and 50 early spot disease susceptible varieties. Using genomic DNA as a template and the sequence shown in SEQ ID NO. 2-3 as a primer, PCR amplification was performed according to the aforementioned PCR reaction system and reaction procedure. The amplified product was sequenced, the sequence was aligned to the reference sequence, and the bases at the SNP site were detected. In 2021, a unified field resistance identification of early spot disease resistance of 60 peanut varieties was carried out at the Zhanjiang Experimental Station of the Chinese Academy of Tropical Agricultural Sciences.
[0058] The results showed that the average resistance grade of 50 susceptible peanut varieties was 6.82±1.36, and the genotype detection did not contain the major resistance gene AhELSR1 for early spot disease; the average resistance grade of 10 resistant resources was 3.66±1.42, and the genotype detection all contained the major resistance gene AhELSR1 for early spot disease.
[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.
Claims
1. Use of a reagent for detecting the major gene AhELSR1 for peanut early spot resistance in identifying or assisting in identifying peanut early spot resistance, characterized in that: The CDS sequence of AhELSR1 is shown in SEQ ID NO.1; when the 415th, 417th, 445th, 454th and 469th nucleotides of the CDS sequence of AhELSR1 are G, C, A, C and C, respectively, the corresponding peanut has strong resistance to early spot disease; when the 415th, 417th, 445th, 454th and 469th nucleotides of the CDS sequence of AhELSR1 are A, G, G, A and G, respectively, the corresponding peanut has weak resistance to early spot disease.
2. Application of a reagent for detecting the major gene AhELSR1 for peanut early spot resistance in early prediction of peanut early spot resistance, characterized in that: The CDS sequence of AhELSR1 is shown in SEQ ID NO.1; when the 415th, 417th, 445th, 454th and 469th nucleotides of the CDS sequence of AhELSR1 are G, C, A, C and C, respectively, the corresponding peanut has strong resistance to early spot disease; when the 415th, 417th, 445th, 454th and 469th nucleotides of the CDS sequence of AhELSR1 are A, G, G, A and G, respectively, the corresponding peanut has weak resistance to early spot disease.
3. Use of a reagent for detecting the major gene AhELSR1 for resistance to early spot disease in peanuts in screening for resistance to early spot disease, characterized in that: The CDS sequence of AhELSR1 is shown in SEQ ID NO.1; when the 415th, 417th, 445th, 454th and 469th nucleotides of the CDS sequence of AhELSR1 are G, C, A, C and C, respectively, the corresponding peanut has strong resistance to early spot disease; when the 415th, 417th, 445th, 454th and 469th nucleotides of the CDS sequence of AhELSR1 are A, G, G, A and G, respectively, the corresponding peanut has weak resistance to early spot disease.
4. The use according to any one of claims 1 to 3, characterized in that: The reagent is a primer pair for detecting the AhELSR1, and the sequence of the primer pair is shown in SEQ ID NO. 2-3.
5. A method for identifying resistance to peanut early spot disease, characterized in that: include: (1) using the primer pairs shown in SEQ ID NOs. 2-3, PCR amplification of the DNA of the peanut to be identified; (2) sequencing the PCR amplification products, analyzing the sequences of the PCR amplification products, and determining the resistance of the peanut to be identified to early spot disease based on the sequences of the PCR amplification products; (3) If the 415th, 417th, 445th, 454th and 469th nucleotides of the PCR amplification product sequence corresponding to the CDS sequence shown in SEQ ID NO.1 are G, C, A, C and C, the peanut to be identified has strong resistance to early blight; if the 415th, 417th, 445th, 454th and 469th nucleotides of the PCR amplification product sequence corresponding to the CDS sequence shown in SEQ ID NO.1 are A, G, G, A and G, the peanut to be identified has weak resistance to early blight.