SNP markers associated with dollar spot resistance in Paspalum marinum and their application

Abstract

The present invention discloses a SNP marker related to the dollar spot disease resistance trait of sea paspalum and an application thereof. The SNP marker is located at the 401bp of the sequence shown in SEQ ID NO.1 and has an A / G polymorphism. The SNP marker provided by the present invention has a key regulatory effect on the dollar spot disease resistance of sea paspalum. Based on the SNP marker, the dollar spot disease resistance of sea paspalum can be predicted or screened and identified, and can also be used for map-based cloning and molecular marker-assisted selection to accelerate the breeding process of dollar spot disease-resistant materials of sea paspalum.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular breeding, and particularly relates to a SNP marker related to the dollar spot disease resistance trait of sea paspalum and an application thereof. Background Art

[0002] Paspalum vaginatum Sw., also known as seaside paspalum or Hawaiian grass, is a perennial herbaceous plant of the genus Paspalum, family Poaceae, tribe Panicum, angiosperms, monocots, class Poaceae, mainly distributed in tropical and subtropical coastal areas. It has well-developed stolons and rhizomes, linear or narrow lanceolate leaves, and the lawns it forms are uniformly dense and dark green in color. It is an excellent grass species for green lawns and sports field lawns. Dollar spot disease occurs more frequently on sea paspalum and is one of the important diseases affecting lawn grass. Dollar spot disease seriously damages the beauty and functionality of the lawn, and causes weed erosion and plant death. Dollar spot disease often occurs at night when the temperature is low and there is more dew. After being infected with dollar spot disease, the lawn will have round, sunken, straw-colored patches the size of a coin. In severe cases, larger irregular patches will appear. When a single plant is infected, water-soaked spots will appear on the leaves, with reddish-brown stripes around the spots. The leaves will quickly die due to the rapid spread of the spots.

[0003] Dollar spot is becoming increasingly destructive to lawn grasses around the world, and the management problems it brings are becoming more and more prominent. The extensive use of chemical fungicides not only costs huge amounts of money in lawn grass management, but also seriously damages the ecological environment. Currently, dollar spot pathogens around the world have been reported to have developed resistance to fungicides, forming a vicious circle. Therefore, breeding varieties of sea paspalum that are resistant to dollar spot is the most environmentally friendly and effective means.

[0004] Genome-wide association analysis (GWAS) is based on linkage disequilibrium (LD), using single nucleotide polymorphism (SNP) of a population as a molecular genetic marker, and searching for genetic markers and candidate genes associated with target traits based on the association between phenotype and the whole genome. GWAS has become an effective means to analyze the genetic variation of complex traits. Summary of the invention

[0005] In view of this, the present invention aims to discover molecular markers related to the dollar spot disease resistance trait of sea paspalum based on whole genome association analysis, in order to assist in the effective screening of sea paspalum varieties with strong dollar spot disease resistance and accelerate the breeding process of sea paspalum.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides a SNP marker associated with the dollar spot disease resistance trait of sea paspalum, the SNP marker is located at the 401bp from the 5' end of the sequence shown in SEQ ID NO.1, and has an A / G polymorphism; specifically, the SNP marker is located at the 4618003 nucleotide position of chromosome 6 of the sea paspalum genome, so the marker is named Pv.chr06:p4618003.

[0008] The primers used to detect the above-mentioned SNP markers also fall within the scope of protection of the present invention. It is understandable that the primers can be primers designed based on primer amplification blocked mutation technology to directly detect the base type at the SNP site, or primers for amplifying the target sequence including the SNP marker.

[0009] The second aspect of the present invention provides the use of the above-mentioned SNP marker or primer, including A1) or A2)

[0010] A1) Application in detecting or identifying the trait of resistance to dollar spot disease in Paspalum marinum;

[0011] A2) Application in Paspalum breeding.

[0012] The third aspect of the present invention provides a method for detecting the dollar spot disease resistance trait of sea paspalum, specifically: detecting the genotype of the test material at the SNP marker provided by the present invention, and the relationship between the dollar spot disease resistance ability and the genotype is GG>AG>AA; specifically: when the genotype of the sea paspalum at the SNP marker to be tested is GG, the sea paspalum is a variety with strong resistance to dollar spot disease; when the genotype of the sea paspalum at the SNP marker to be tested is AG, the sea paspalum is a variety with moderate resistance to dollar spot disease; when the genotype of the sea paspalum at the SNP marker to be tested is AA, the sea paspalum is a variety that is not resistant to dollar spot disease.

[0013] In the above detection method, the genotype detection process is: using the genome of the tested sea paspalum material as a template, amplifying the target fragment containing the SNP marker and sequencing it, or directly resequencing the genomic DNA.

[0014] In a specific embodiment of the present invention, the primers used for amplification are shown in SEQ ID NOs. 2-3.

[0015] The fourth aspect of the present invention provides a method for breeding Paspalum marinum, specifically: by detecting the SNP marker provided by the present invention, selecting Paspalum marinum material with a genotype of GG for auxiliary breeding.

[0016] A fifth aspect of the present invention provides a detection kit, which includes primers for detecting the above-mentioned SNP markers.

[0017] The above detection kit includes primers with sequences as shown in SEQ ID NOs. 2-3.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention uses resequencing data of 82 sea paspalum germplasms and combines them with phenotypic data of dollar spot disease resistance of sea paspalum to perform whole genome association analysis, locates for the first time the QTL site affecting the dollar spot disease resistance trait in sea paspalum, and develops corresponding SNP markers. The study found that the SNP molecular marker Pv.chr06:p4618003 located at the 4618003bp position of chromosome 6 has a high contribution rate to the dollar spot disease resistance trait of sea paspalum, and its data contribution rate to the disease index (DI) of the dollar spot disease resistance trait of sea paspalum reaches 91.35%. It can be seen that the SNP marker plays a key role in the regulation of the dollar spot disease resistance trait of sea paspalum.

[0020] Primers designed based on the SNP molecular marker can be used for map-based cloning and molecular marker-assisted selection, thereby effectively screening for sea paspalum varieties with dollar spot disease resistance. The detection is convenient and rapid, not affected by the environment, and has high breeding efficiency. It accelerates the breeding process of sea paspalum and overcomes the shortcomings of traditional breeding methods such as specific experiments, many operational restrictions, long time periods, and many uncertain factors. It is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a statistical graph showing the incidence of dollar spot disease in 82 species of Paspalum in DI in Example 1.

[0022] Figure 2 This is a result diagram of the DI resistance phenotype of dollar spot disease corresponding to different genotypes of the SNP marker Pv.chr06:p4618003 for the dollar spot disease resistance trait of Paspalum provided by the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0024] Unless otherwise specified, the following examples are based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions.

[0025] Example 1 Location of the major QTL for resistance to dollar spot disease in Paspalum marinum

[0026] (1) Phenotypic determination of resistance of Paspalum marinum to dollar spot disease.

[0027] A total of 82 samples of Paspalum marinum germplasms were used in this example, which were from China (19 samples), the United States (49 samples), Argentina (6 samples) and other countries (8 samples). The specific germplasm names and sources are shown in Table 1.

[0028] All test materials were hydroponically grown, with a capacity of 3L in the hydroponic pot, Hoagland's complete nutrient solution added once a week, and pruned once every two weeks at a height of 5cm. By adopting the method of live inoculation, the disease phenotype of the detached leaves of Paspalum marinum was recorded by taking photos every 6 hours, and the disease index of the leaves in each time period was calculated, and the comprehensive score membership function value DI of the total disease index was calculated based on the disease index.

[0029] Table 1 Information on the germplasm resources of Paspalum marinum

[0030]

[0031]

[0032] (2) Resequencing of the Paspalum population.

[0033] Sampling was done in the germplasm resource garden. About 2g of tender leaves were taken from each germplasm. They were quickly frozen with liquid nitrogen and sent to Myvi Metabolism for sequencing. The sequencing strategy was Illumina PE150. Quality control was performed on the raw data after sequencing. The filtering criteria were as follows: ① Filter sequences containing adapters; ② Filter a pair of sequences with an N content of more than 10% in the single-end sequence; ③ Filter a pair of sequences with a low-quality (Q≤5) base number of more than 50%. After filtering the adapter sequence, inaccurate sequence, and low-quality sequence, clean data was obtained for the next sequence alignment. After constructing the index file for the SeaIsle2000 reference genome, the clean data was aligned to the reference genome using BWA software; the alignment results were sorted using SAMtools software; the repeated markers generated during library preparation were processed using PicardTools software; and the population variation information was analyzed using GATK software. VCFtools software was used to further screen and filter SNPs. The command was “vcftools --vcf [vcf_file] --max --missing0.8 --maf 0.05 --mac 3 --minQ 30 --minDP 3 --min --alleles 2 --max-alleles2 --recode --recode-INFO-all --out [out_prefix]”.

[0034] (3) Genome-wide association analysis.

[0035] The vcf file was converted using plink software. The Balding-Nichols kinship matrix was generated using EMMAX software using the command "emmax-kin-intel64-vd 10-o[out_prefix][tped_prefix]". The association analysis was performed using the command "emmax-intel64-vd 10-t[tped_prefix]-p[pheno_file]-k[kin_file]-o[out_prefix]". The P value of each locus of Paspalum marinum was obtained from the association analysis. When P<1E-06, the SNP was a significant SNP. The SNP with the smallest P value was the peak SNP. The materials were grouped according to the different allele types of the peak SNP in the population, and variance analysis was performed. The percentage of the ratio of the variance between groups to the total variance was the contribution rate of the peak SNP.

[0036] Through analysis, the interval of the major QTL site for dollar spot disease resistance in sea paspalum was limited to the 4618003rd and 4618252th bases of chromosome chr06 of sea paspalum. The corresponding SNPs were chr06_4618003 (A / G) and chr06_4618252 (G / A), and the peak SNP was chr06_4618003 (A / G). The contribution rate of this QTL to the overall disease index of sea paspalum was 91.35%. It can be seen that this QTL plays a key role in regulating the dollar spot disease resistance trait of sea paspalum.

[0037] Example 2 Development of SNP molecular markers for resistance to dollar spot disease in Paspalum marinum

[0038] According to the above-identified QTL and peak SNP for resistance to dollar spot disease of Paspalum chinensis, SNP markers were developed, and the 400 bp sequences before and after the SNP were extracted as the characteristic sequence of the SNP marker Pv.chr06:p4618003 (as shown in SEQ ID NO.1, where the 401 bp is the SNP site and there is an A / G mutation), and the primers for the SNP molecular marker Pv.chr06:p4618003 were designed as follows:

[0039] Forward primer: 5'-ATGGTTACTTCAAACAGAACGAGCT-3' (SEQ ID NO. 2);

[0040] Reverse primer: 5'-TCAGAGGATATGATGACCCTTTCC-3' (SEQ ID NO. 3).

[0041] Through conventional PCR amplification and sequencing detection, the bases of SNP molecular markers are divided into three types. When the base type (i.e., genotype) of Pv.chr06:p4618003 is GG, the average membership function value of the comprehensive score of the average disease index of Paspalum is 20.17, which is a variety with strong resistance to Paspalum dollar spot disease; when the base type is AG, the average score of Paspalum is 28.42, which is a variety with medium resistance to Paspalum dollar spot disease; when the base type is AA, the average score of Paspalum is 54.94, which is a variety with weak resistance to Paspalum dollar spot disease.

[0042] The membership function values ​​of the comprehensive score of the disease index of Paspalum marinum corresponding to different genotypes are as follows: Figure 2 The results showed that the resistance traits of the sea paspalum dollar spot disease corresponding to different genotypes of the SNP marker Pv.chr06:p4618003 were significantly different. Therefore, the resistance of the sea paspalum dollar spot disease to be tested can be quickly and simply evaluated by identifying the type of the SNP marker, so as to quickly screen the sea paspalum materials with high resistance to dollar spot disease.

[0043] In addition to using the above primers to amplify the sequence, the material can also be subjected to genome resequencing, and the SNP molecular marker Pv.chr06:p4618003 published by the present invention can be subjected to SNP genotyping according to the method in Example 1 to determine the genotype type of the SNP, so as to quickly predict the material's resistance to dollar spot disease.

[0044] Example 3 Application of SNP markers in identification of resistance to dollar spot disease in Paspalum marinum

[0045] DNA of young leaves of three materials known to have strong resistance to dollar spot disease, Qiong2, 17HN-39, and 17HN-25, were extracted and sent to a sequencing company for genome resequencing. The SNP marker genotypes of the three materials were identified respectively, and the disease index of the leaves of the three materials was identified according to the method of Example 1, and the membership function value of the comprehensive score of the disease index was calculated.

[0046] DNA of young leaves of three materials known to have moderate resistance to dollar spot disease, namely PI 647893 (HI 10), Utah1 and TYB 2, were extracted and sent to a sequencing company for genome resequencing. The SNP marker genotypes of the three materials were identified respectively, and the disease index of the leaves of the three materials was identified according to the method of Example 1, and the membership function value of the comprehensive score of the disease index was calculated.

[0047] DNA from young leaves of three materials, grin_UPG144, grin_UPG145, and Kai Luna, which were previously known to have poor resistance to dollar spot disease, were extracted and sent to a sequencing company for genome resequencing. The SNP marker genotypes of the three materials were identified respectively, and the disease index of the leaves of the three materials was identified according to the method of Example 1, and the membership function value of the comprehensive score of the disease index was calculated.

[0048] The test results of the above three groups are shown in Table 2:

[0049] Table 2 Identification of genotypes and dollar spot resistance of different materials at Pv.chr06:p4618003

[0050]

[0051] The results showed that: ① For the three materials grin_UPG144, grin_UPG145 and Kai Luna with poor resistance to dollar spot disease known in the early stage, the genotype at the 4618003bp position of chromosome chr06 (i.e., the 401bp position of the sequence shown in SEQ ID NO.1) was AA, which was basically consistent with the membership function value of the disease index comprehensive score corresponding to the AA genotype of the SNP marker in Example 2; ② For the three materials PI 647893 (HI 10), Utah1 and TYB2, the genotype at the 4618003bp position of chromosome chr06 is AG, which is basically consistent with the membership function value corresponding to the AG genotype of the SNP marker in Example 2; ③ For the three materials Qiong2, 17HN-39 and 17HN-25 with strong resistance to dollar spot disease known in the early stage, the genotype at the 4618003bp position of chromosome chr06 is GG, which is basically consistent with the membership function value corresponding to the GG genotype of the SNP marker in Example 2.

[0052] It can be seen that the genotype type of the SNP molecular marker Pv.chr06:p4618003 can be used to effectively identify the dollar spot disease resistance of different sea paspalum materials.

[0053] In summary, the SNP marker Pv.chr06:p4618003 provided by the present invention plays a key regulatory role in the resistance of sea paspalum to dollar spot disease. Based on this SNP marker, the resistance of sea paspalum to dollar spot disease can be predicted or screened and identified. The identification method is simple and the selection efficiency is high. It can be used for map-based cloning and molecular marker-assisted selection to accelerate the breeding process of sea paspalum resistant to dollar spot disease materials.

[0054] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. Use of a SNP marker associated with the dollar spot disease resistance trait of Paspalum marinum or a primer for detecting the SNP marker in detecting or identifying the dollar spot disease resistance trait of Paspalum marinum, characterized in that: The SNP marker is located at the 401 bp of the sequence shown in SEQ ID NO. 1 and has an A / G polymorphism.

2. Use of a SNP marker associated with dollar spot disease resistance in Paspalum marinum or a primer for detecting the SNP marker in breeding for dollar spot disease resistance in Paspalum marinum, characterized in that: The SNP marker is located at the 401 bp of the sequence shown in SEQ ID NO. 1 and has an A / G polymorphism.

3. A method for detecting the dollar spot disease resistance of Paspalum marinum, characterized in that: The genotype of the test material at the SNP marker related to the dollar spot disease resistance trait of Paspalum marinum is detected. The SNP marker is located at the 401bp of the sequence shown in SEQ ID NO.1, has an A / G polymorphism, and the relationship between the dollar spot disease resistance ability and the genotype is GG>AG>AA.

4. The method for detecting the dollar spot disease resistance trait of Paspalum marinum according to claim 3, characterized in that: The genotype detection process is: using the genome of the tested sea paspalum material as a template, amplifying the target fragment containing the SNP marker and sequencing it, or directly resequencing the genomic DNA.

5. The method for detecting the dollar spot disease resistance trait of Paspalum marinum according to claim 4, characterized in that: The primers used for the amplification are shown in SEQ ID NOs. 2-3.

6. A method for breeding Paspalum marinum, characterized in that: The SNP marker associated with the dollar spot disease resistance trait of sea paspalum was detected. The SNP marker was located at the 401bp of the sequence shown in SEQ ID NO.1 and had A / G polymorphism. Materials with genotype GG were selected for assisted breeding of the dollar spot disease resistance trait of sea paspalum.

7. A detection kit, characterized in that: The invention comprises primers for detecting a SNP marker related to the trait of dollar spot disease resistance of sea paspalum, wherein the SNP marker is located at the 401 bp position of the sequence shown in SEQ ID NO.1 and has an A / G polymorphism.

8. The detection kit according to claim 7, characterized in that It includes primers whose sequences are shown in SEQ ID NO. 2-3.

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