A KASP marker related to melon powdery mildew resistance and its application

Abstract

The invention discloses a KASP marker related to melon powdery mildew resistance and its application, belonging to the technical field of plant disease resistance breeding. The primer sequence of the KASP marker is as follows: the upstream primer for the susceptible site is shown in SEQ ID NO.2, the upstream primer for the disease resistance site is shown in SEQ ID NO.3, and the shared downstream primer is shown in SEQ ID NO.4. The application of the KASP marker related to melon powdery mildew resistance of the invention can be applied to the screening of melons resistant to powdery mildew, the identification of melons resistant to powdery mildew, and the breeding of melons resistant to powdery mildew, and has the characteristics of high efficiency, high accuracy and no pollution.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease resistance breeding, and in particular relates to a KASP marker related to melon powdery mildew resistance and an application thereof. Background Art

[0002] Muskmelon (Cucumis melo L.) is an annual diploid plant belonging to the Cucurbitaceae family and the genus Cucumis. It is widely planted worldwide for its flavor and taste, and has become a high-end fruit and a fruit that is popular among consumers. Powdery mildew is an important threat to melon cultivation at present, which seriously affects the yield and quality of melon and is a global disease in melon production. Powdery mildew prevention and control has become an urgent problem to be solved in production. At present, the prevention and control of powdery mildew in production is mainly through the use of chemical agents such as pesticides, time and space isolation such as isolation facilities, and the application of disease-resistant varieties. Among them, chemical agents are prone to cause environmental pollution and pose a risk to human health. Time and space isolation is costly and is not suitable for the current situation in my country where arable land is insufficient and the scale is small and scattered. The application of disease-resistant varieties can overcome the above risks, adapt to my country's national conditions, and be economical, environmentally friendly and effective.

[0003] At present, melon varieties mainly include conventional varieties purified by traditional hybridization of conventional materials, biparental hybrid varieties, physical and chemical mutagenesis breeding such as radiation, and biotechnology breeding such as molecular marker-assisted selection. Traditional breeding technology has a long cycle, time-consuming and labor-intensive disease resistance identification, high cost, and unstable results. Especially for disease-resistant breeding, pathogens mutate quickly, and traditional breeding has the risk of not adapting to the market after successful variety selection. Although mutation breeding can obtain new genetic resources, it has high uncertainty, high screening costs, and the offspring may have reversion mutations and unstable traits. Molecular marker-assisted selection breeding can greatly shorten the breeding cycle, improve selection efficiency, and reduce costs by exploring mutation sites closely linked to powdery mildew resistance genes, designing and developing related molecular markers for breeding selection, and the selected varieties can be quickly put on the market to solve production problems. Summary of the invention

[0004] The purpose of the embodiment of the present invention is to provide a KASP marker related to melon powdery mildew resistance and its application, aiming to solve the defects of low efficiency, high cost and insufficient related genes in the existing breeding of melon powdery mildew resistant varieties, through genotyping and phenotypic identification, to explore a site that can cause functional non-synonymous mutations that is different from the retrieved and published disease resistance site, and develop related molecular markers and applications.

[0005] The purpose of the embodiment of the present invention is achieved through the following technical solutions:

[0006] On one hand, the present invention provides a mutation site related to melon powdery mildew resistance, wherein the mutation site is located at the 7901th nucleotide G→A mutation of the MELO3C015361.2 gene on melon chromosome 2, and the nucleotide sequence of the MELO3C015361.2 gene is shown in SEQ ID NO.1.

[0007] Another aspect of the present invention provides a KASP marker related to melon powdery mildew resistance, using the above mutation site, the primer sequence of the KASP marker is as follows:

[0008] Susceptibility site binding upstream primer (as shown in SEQ ID NO.2):

[0009] 5'-GAAGGTCGGAGTCAACGGATTCAGGAAGCTTATATAAAATGCTTTATT-3';

[0010] Disease resistance site binding upstream primer (as shown in SEQ ID NO.3):

[0011] 5'-GAAGGTGACCAAGTTCATGCTCAGGAAGCTTATATAAAATGCTTTATC-3';

[0012] Common downstream primer (as shown in SEQ ID NO.4):

[0013] 5'-AGTTCAAGTTCTAGTTTGCTGTGGA-3'.

[0014] The invention also discloses an application of a KASP marker related to melon powdery mildew resistance, which is used for screening melons resistant to powdery mildew.

[0015] The invention also discloses an application of a KASP marker related to melon powdery mildew resistance, which is used for identification of melon resistance to powdery mildew.

[0016] Furthermore, the application of the KASP marker related to melon powdery mildew resistance is used for breeding of melons resistant to powdery mildew.

[0017] Furthermore, the powdery mildew resistant melon breeding is preferably melon powdery mildew resistance marker-assisted breeding.

[0018] The specific identification method steps for the identification of melon resistance to powdery mildew are as follows:

[0019] (1) extracting genomic DNA of the melon material to be screened;

[0020] (2) using the above-mentioned KASP-labeled primer sequences, a competitive allele-specific PCR method was used to amplify and detect melon genomic DNA;

[0021] (3) Determination of amplification test results:

[0022] (3.1) If the amplification result is C, it is a melon homozygous disease-resistant material;

[0023] (3.2) If the amplification result is T, it is a homozygous susceptible material of melon;

[0024] (3.3) If the amplification result is C / T, it is a melon hybrid disease-resistant material.

[0025] Furthermore, the melon material is the part of the melon to be identified, such as melon leaves or melon fruits.

[0026] The beneficial effects of the present invention are:

[0027] 1. The KASP primers of the present invention are designed by performing powdery mildew inoculation identification on two parents (resistant and susceptible) and their reciprocal cross F1 at the seedling stage, finding that the disease resistance is dominantly inherited, then resequencing the two parents to screen the differential sites, performing differential site genotype analysis on two extreme separation pools of the F2 generation, combining bioinformatics analysis to screen functional mutation sites, and verifying through natural populations to obtain primers designed for gene sites related to melon powdery mildew resistance.

[0028] 2. After searching the mutation sites of the present invention, no identical melon powdery mildew resistance sites were found in the public.

[0029] 3. The KASP primers developed by the present invention are used for screening melon powdery mildew resistant materials and variety breeding, and have the characteristics of high efficiency, high accuracy and no pollution. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] At present, the selection of melon powdery mildew resistant varieties in melon breeding has the disadvantages of low efficiency, high cost and insufficient related genes. In order to solve the above technical problems, the present invention proposes a KASP marker related to melon powdery mildew resistance and its application. Through genotyping and phenotypic identification, a site that can cause functional non-synonymous mutations different from the retrieved and published disease resistance sites is discovered, and related molecular markers and applications are developed.

[0032] The KASP described in the present invention is competitive allele-specific PCR (kompetitive allele-specific PCR).

[0033] The test materials used in the examples of the present invention include: a melon material with high resistance to powdery mildew, P143, and a melon material with high susceptibility to powdery mildew, P01, both of which are high-generation inbred lines bred by Ningbo Academy of Agricultural Sciences. P01 and P143 were crossed with each other as parents to obtain reciprocal cross F1, and the F1 with P01 as the female parent and P143 as the male parent was self-pollinated to obtain an F2 population.

[0034] Among them, the melon variety P01 is a thick-skinned melon material, which is recorded in the article "Gao Tianyi, Hao Fangmin, Zang Quanyu, Ma Erlei, Huang Yunping, Wang Yuhong. QTL positioning analysis of melon vine blight resistance based on SLAF-seq and BSA technology [J]. Acta Agriculturae Boreali-Sinica, 2021, 36(2): 40-45". The public can obtain this melon variety from the applicant within twenty years from the date of application, only for repeating experiments related to the present invention.

[0035] Melon variety P143 is recorded in the article "Song Hui, Huang Yunping, Gu Binquan, Fu Yujing, Zang Quanyu, Xing Nailin, Zhang Xiangqin. Data processing of physiological indicators of melon tolerance to low temperature and weak light and screening of extreme materials [J]. Acta Agriculturae Boreali-Sinica, 2020, 35(S1): 183-188". The public can obtain this melon variety from the applicant within twenty years from the date of application, only for repeating experiments related to the present invention.

[0036] Example 1

[0037] Experimental steps:

[0038] 1) Two parents (P143, P01) and reciprocal cross F1 and F2 were sown in plug trays for seedling raising, with 10 plants each for the parents and F1 and 200 plants for F2. Powdery mildew was inoculated and identified at the 1-leaf 1-heart stage, and the incidence of powdery mildew was observed 15 days later. Results P143 and reciprocal cross F1 had no powdery mildew, but P01 had severe powdery mildew, indicating that the powdery mildew resistance gene of P143 was dominant.

[0039] 2) Sampling of leaves of parents and F2 individual plants, 20 plants each of disease-free and diseased materials in the F2 population were selected and mixed as disease-resistant pools and disease-susceptible pools. The genomic DNA of the above samples was extracted using the Quanshijin Plant Genomic DNA Extraction Kit. The DNA samples were randomly broken into fragments of 350 bp in length by a Covaris crusher. The TruSeq Library Construction Kit was used to build the library, and the reagents and consumables recommended in the instructions were strictly used. The DNA fragments were end-repaired, ployA-tailed, sequencing adapter-added, purified, PCR amplified, and other steps to complete the entire library preparation. After the library construction was completed, Qubit2.0 was used for preliminary quantification, and the library was diluted to 1 ng / μl, and then the insert size of the library was detected using Agilent 2100. After the insert size met expectations, the effective concentration of the library was accurately quantified using the Q-PCR method (library effective concentration>2nM) to ensure the quality of the library. After the library inspection is qualified, different libraries are pooled according to the effective concentration and the target data volume requirements for Illumina HiSeq TM PE150 sequencing. The Raw data obtained from the machine is quality controlled to obtain Clean data, and the Clean data is aligned to the reference genome (http: / / cucurbitgenomics.org / organism / 18). The alignment results are deduplicated by SAMTOOLS (parameter: rmdup), and the average coverage depth of the reference genome (excluding N region) is between 15.66X and 19.49X. The UnifiedGenotyper module of GATK3.8 software is used to detect SNPs and InDels of multiple samples, and VariantFiltration is used for filtering. ANNOVAR is used to annotate the SNP detection results. The window greater than the threshold at a 95% confidence level is selected as the candidate interval. In order not to ignore the influence of minor QTLs, candidate SNPs and InDels were selected across the whole genome. If the phenotypes of the reference parent and the offspring were the same, sites with All-index close to 0 in the offspring pool were selected. If the phenotypes of the reference parent and the offspring were opposite, sites with All-index close to 1 in the offspring pool were selected as candidate sites. The annotation results of ANNOVAR were extracted for the candidate sites, and genes at sites causing stop loss or stop gain or non-synonymous mutations or alternative splicing were preferentially selected as candidate genes.

[0040] 3) According to the candidate gene mutation site, KASP and SSR primers were designed to perform genotyping on the remaining individual plants in the F2 population. Results The 7901st nucleotide G→A mutation of the MELO3C015361.2 gene on chromosome 2 (the sequence of the MELO3C015361.2 is shown in SEQ ID NO.1) had the highest phenotypic consistency of more than 95%. At the same time, this gene is related to plant disease resistance. Therefore, this site can be used as a melon powdery mildew resistance site.

[0041] Example 2

[0042] 1. Experimental materials: 30 high-generation melon inbred lines, with P143 and P01 as controls. All 32 materials were bred by Ningbo Academy of Agricultural Sciences.

[0043] 2. Test steps:

[0044] 1) 32 materials were used for seedling cultivation in plug trays, with 3 replicates for each material, 10 plants each. At the 1-leaf 1-heart stage, melon powdery mildew was inoculated by spraying spore suspension in an artificial climate chamber, and the incidence of powdery mildew was observed 15 days later.

[0045] 2) For each material, 5 leaves were selected for mixed sampling, and the genomic DNA of the samples was extracted using the Quanshijin Plant Genomic DNA Extraction Kit. The concentration was measured using NanoDrop 2000, and each DNA stock solution was diluted to 20 ng / ul.

[0046] 3) A KASP molecular marker was designed based on the antisense sequence of the 7901st nucleotide G→A mutation of the MELO3C015361.2 gene on chromosome 2 (the sequence of the MELO3C015361.2 is shown in SEQ ID NO.1), and its sequence is as follows:

[0047] Susceptibility site binding upstream primer (as shown in SEQ ID NO.2):

[0048] 5'-GAAGGTCGGAGTCAACGGATTCAGGAAGCTTATATAAAATGCTTTATT-3';

[0049] Disease resistance site binding upstream primer (as shown in SEQ ID NO.3):

[0050] 5'-GAAGGTGACCAAGTTCATGCTCAGGAAGCTTATATAAAATGCTTTATC-3';

[0051] Common downstream primer (as shown in SEQ ID NO.4):

[0052] 5'-AGTTCAAGTTCTAGTTTGCTGTGGA-3'.

[0053] 4) Prepare the PCR reaction system as follows: 2 μL of 2xTaq DNA Polymerase Mix, 1 μL of SNP PrimerMix (4x), and 2 μL of DNA sample.

[0054] 5) PCR steps are shown in Table 1.

[0055] Table 1 PCR steps

[0056]

[0057] 6) SNP site detection uses the fluorophores FAM and VIC to distinguish two isogenic sites. Passive reference dye ROX is used to correct the signal difference between wells due to reaction volume errors. The relevant excitation and emission wavelengths are shown in Table 2. The reading software is OEM equipment from LGC.

[0058] Table 2 Related excitation and emission wavelengths

[0059]

[0060] 7) The genotype reading software (Kluster Caller) of LGC_OMEGA was used to analyze the result data of PCR amplification. The relative fluorescence values ​​corresponding to VIC and FAM of each PCR reaction well were obtained. According to the relative fluorescence value, the samples were clustered, and the genotype was further determined according to the sample cluster and the fluorescence type. The genotype and phenotype results of the measured samples are shown in Table 3. The results were consistent with the genotype and phenotype results except 21A-5, 21A-14, 21A-18 and 21A-29. These 4 materials were detected using the disclosed melon powdery mildew resistance site, and the results contained known powdery mildew resistance sites. Among the 12 materials with genotype C / C, only 21A-16 and 21A-21 contained known resistance sites. The powdery mildew resistance levels of these two materials were higher than those containing only the sites of the present invention and the known sites. The resistance of the material containing only the sites of the present invention was slightly higher than that of the material containing only the known resistance sites. The genotype and phenotype of this site are generally consistent.

[0061] Table 3 Sample genotypes and phenotypes

[0062] Sample No. genotype Phenotype Sample No. genotype Phenotype Sample No. genotype Phenotype 21A-1 C / C anti- 21A-12 T / T feel 21A-23 T / T feel 21A-2 C / C anti- 21A-13 C / C anti- 21A-24 T / T feel 21A-3 T / T feel 21A-14 T / T anti- 21A-25 T / T feel 21A-4 C / C anti- 21A-15 T / T feel 21A-26 C / C anti- 21A-5 T / T anti- 21A-16 C / C anti- 21A-27 T / T feel 21A-6 T / T feel 21A-17 C / C anti- 21A-28 T / T feel 21A-7 T / T feel 21A-18 T / T anti- 21A-29 T / T anti- 21A-8 C / C anti- 21A-19 T / T feel 21A-30 T / T feel 21A-9 T / T feel 21A-20 C / C anti- P143 C / C anti- 21A-10 C / C anti- 21A-21 C / C anti- P01 T / T feel 21A-11 T / T feel 21A-22 C / C anti-

[0063] 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 and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A KASP marker related to melon powdery mildew resistance, characterized in that: The primer sequences of the KASP markers are as follows: The upstream primer for the susceptible site binding is shown in SEQ ID NO.2; The disease resistance site binding upstream primer is shown in SEQ ID NO.3; The common downstream primer is shown as SEQ ID NO.

4.

2. Use of the KASP marker associated with melon powdery mildew resistance according to claim 1, characterized in that: Used for screening melons resistant to powdery mildew.

3. Use of the KASP marker associated with melon powdery mildew resistance according to claim 1, characterized in that: Used for identification of melon resistance to powdery mildew.

4. Use of the KASP marker associated with melon powdery mildew resistance according to claim 1, characterized in that: Used for powdery mildew resistant melon breeding.

5. The use of KASP markers related to melon powdery mildew resistance according to claim 4, characterized in that: The powdery mildew resistant melon breeding is melon powdery mildew resistance marker assisted breeding.

6. The use of KASP markers related to melon powdery mildew resistance according to claim 3, characterized in that: The specific identification method steps for the identification of melon resistance to powdery mildew are as follows: (1) Extracting genomic DNA from the melon material to be screened; (2) using the primer sequence of the KASP marker described in claim 1, a competitive allele-specific PCR method is used to amplify and detect melon genomic DNA; (3) Based on the mutation sites related to melon powdery mildew resistance, the amplification test results were judged: (3.1) If the amplification result is C, it is a melon homozygous disease-resistant material; (3.2) If the amplification result is T, it is a homozygous susceptible material of melon; (3.3) If the amplification result is C / T, it is a melon heterozygous disease-resistant material; The mutation site is located at the 7901th nucleotide G→A mutation of the MELO3C015361.2 gene on melon chromosome 2, and the sequence of the MELO3C015361.2 is shown in SEQ ID NO.

1.

7. The use of KASP markers related to melon powdery mildew resistance according to claim 6, characterized in that: The melon material is the part of the melon to be identified, such as melon leaves and melon fruits.

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