Genome-wide SNP molecular markers associated with rice drought resistance and their applications

Abstract

The present invention relates to the technical field of molecular markers, and in particular to whole-genome SNP molecular markers related to rice drought resistance and their applications. The present invention uses "drought-resistant" and "sensitive" rice varieties to screen SNP molecular markers from the whole genome, and obtains 191 SNP molecular markers significantly associated with drought-resistant phenotypes, which are used to efficiently and quickly identify the drought resistance of rice varieties. The molecular markers of the present invention can be used to identify the drought resistance of rice variety resources, analyze and screen breeding materials and segregated populations, thereby facilitating the selection and breeding process of water-saving and drought-resistant rice varieties and improving the efficiency of drought-resistant molecular breeding.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, and in particular to whole-genome SNP molecular markers related to rice drought resistance and applications thereof. Background Art

[0002] Due to abnormal climate change and frequent extreme weather, drought stress has become one of the important obstacles to rice production, posing a serious threat to food security. Breeding and planting water-saving and drought-resistant rice varieties is the most economical and effective measure to reduce drought losses. Discovering genes related to rice drought resistance traits, developing molecular markers closely related to drought resistance traits, and using molecular marker-assisted selection are of great significance for breeding water-saving and drought-resistant rice varieties.

[0003] Single Nucleotide Polymorphisms (SNPs) mainly refer to DNA sequence polymorphisms at the genome level caused by the variation of a single nucleotide, including single base conversion, transversion, deletion and insertion, etc. SNPs are widely distributed in the rice genome. With the advancement of high-throughput sequencing technology, SNPs have become a new generation of molecular markers.

[0004] Wang Wensheng et al. designed PCR primers based on SNP sites significantly associated with the LOC_Os03g20680 gene that controls plant height under drought stress in rice in CN 108048597A. The marker can better distinguish drought-resistant and sensitive varieties. The drought resistance of rice varieties with A / A genotype is significantly better than that of rice varieties with T / T genotype. At the same time, his research team also provided SNP molecular markers related to rice seedling root length and their applications in CN 108034654 A. The SNP molecular marker is from the LOC_Os08g31580 gene. The SNP molecular marker is located at the 19,547,859bp position of rice chromosome 8, and the base is C or T. It can be used for rice root system improvement and drought-resistant breeding. However, although similar drought-resistant functional SNP markers can distinguish genotypes, they are limited to a specific gene and genetic background. Rice drought resistance is a complex quantitative trait controlled by multiple genes, with a rich genetic background and complex molecular mechanisms. A single SNP marker is difficult to meet the actual needs of production and also limits its scope of application. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides SNP molecular markers related to rice drought resistance and applicable to the whole genome and applications thereof.

[0006] Specifically, the present invention first provides a SNP molecular marker associated with rice drought resistance, which is one or more SNP molecular markers as shown in SEQ ID NO.1 to SEQ ID NO.191, wherein a mutation occurs at the 50 bp position, resulting in polymorphism in rice drought resistance; the mutation form of the SNP molecular marker is as follows:

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[0013] In order to facilitate the description of the location of SNP sites, the present invention defines them with reference to the genome sequence version Rice Genome Annotation Project-MSU Rice Genome Annotation (Osa1) Release 7. In fact, those skilled in the art can find corresponding SNP sites in other versions of rice genome sequences based on the above definition, and the SNP molecular markers formed therefrom are also within the protection scope of the present invention.

[0014] Specifically, the nucleotide sequences of each molecular marker are shown in Table 1 below; wherein a mutation occurs at the 50 bp position, resulting in polymorphism in the drought resistance phenotype of rice.

[0015] Table 1

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[0026] As a preferred embodiment of the present invention, the SNP molecular marker combination is a combination of 191 molecular markers provided by the present invention.

[0027] The invention also provides a gene chip, which comprises the SNP molecular marker.

[0028] The present invention also provides primers for amplifying the SNP molecular markers.

[0029] The present invention also provides a reagent or a kit containing the primer.

[0030] Furthermore, the present invention also provides the use of the SNP molecular marker, or gene chip, or primer, or reagent or kit in early prediction of rice drought resistance phenotype.

[0031] Furthermore, the present invention also provides the use of the SNP molecular marker, or gene chip, or primer, or reagent or kit in screening drought-resistant rice.

[0032] Furthermore, the present invention also provides the use of the SNP molecular marker, or gene chip, or primer, or reagent or kit in breeding drought-resistant rice.

[0033] Based on the above technical solution, the beneficial effects of the present invention are as follows:

[0034] The present invention uses "drought-resistant" and "sensitive" rice varieties to screen SNP molecular markers from whole-genome SNP markers, and obtains 191 SNP molecular markers significantly associated with drought-resistant phenotypes, which can well distinguish drought-resistant and sensitive materials under complex genetic background conditions, and can be used to efficiently and quickly identify the drought resistance of rice varieties. The molecular markers of the present invention can be used to identify the drought resistance of rice variety resources, analyze and screen breeding materials and segregated populations, thereby facilitating the breeding process of water-saving and drought-resistant rice varieties and improving the efficiency of drought-resistant molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Manhattan plot for drought resistance association analysis.

[0036] Figure 2 Cluster analysis for highly resistant materials and sensitive materials.

[0037] Figure 3 The clustering results of highly resistant and sensitive materials at 191 SNP sites.

[0038] Figure 4The clustering results are as follows: 200 nuclear sterile materials and 24 resistant and susceptible materials. DETAILED DESCRIPTION

[0039] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0040] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] Example 1

[0042] The present invention screened out typical drought-resistant and sensitive varieties from more than 220,000 rice materials in the low-temperature and low-humidity gene resource storage bank of the Shanghai Agricultural Biological Gene Center (Table 2), wherein the drought resistance index was obtained by planting for two consecutive years under relatively strict drought conditions and then testing the yield phenotype. The resequencing genotype information of drought-resistant and sensitive materials in the rice core germplasm was used to screen the SNP sites of these materials and the SNP sites common to the 56K chip (Li Zhikang et al., SNP molecular marker combination for rice genotyping and its application), and a total of 32,274 SNP sites were obtained.

[0043] Table 2

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[0045]

[0046] In order to further screen out SNP sites associated with drought resistance, case-control genome-wide association analysis was performed on all 32,274 SNP sites based on 13 highly resistant materials and 11 sensitive materials. The significance threshold was a P value less than 0.05. After Bonferroni correction, 191 significant SNP sites were obtained that were associated with drought resistance phenotypes, as shown in Table 3. Some of the SNP sites are not within the gene and may be closely linked to the adjacent drought resistance genes. Manhattan plot of association analysis results, see Figure 1 .

[0047] Based on the genotype frequencies of 191 SNP sites related to drought resistance phenotypes in high-resistant materials and sensitive materials, the present invention inferred the possible alleles of high-resistant materials at each SNP site, and obtained the genotypes of high-resistant materials at 191 SNP sites (Table 3). By calculating the similarity between 13 drought-resistant materials and 11 sensitive materials and the high-resistant genotype, it was found that the similarity between the materials with high-resistant phenotypes and the high-resistant genotypes was significantly higher than that of the sensitive materials, see Figure 2. 191 SNP loci related to drought resistance were used to cluster 24 materials, and it was found that these SNP loci can clearly distinguish highly resistant materials from sensitive materials, see Figure 3 .

[0048] Table 3

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[0059] Example 2

[0060] The 191 SNP loci obtained in Example 1 were used for genotype scanning, and 200 individual plants of a dominant nuclear sterile segregation population obtained by hybridization of African upland rice and excellent rice varieties were selected for screening to verify the genotyping effect of the drought-resistant SNP loci.

[0061] Among them, the pedigree information of 200 rice samples from the dominant nuclear sterile segregation group used for screening: 138 specific representative rice varieties were selected and hybridized with the dominant nuclear sterile in equal amounts and planted together, including rice micro-core germplasm, African upland rice, backbone parents and major resistance source varieties (drought-resistant, high-quality, high-yield, disease-resistant, and salt-tolerant), with rich diversity and a broad genetic basis.

[0062] The specific operation of screening drought-resistant rice is as follows:

[0063] 1) Extracting genomic DNA from 200 rice samples to be tested;

[0064] 2) Using the rice whole genome SNP chip system, the genomic DNA to be tested is hybridized to obtain the SNP genotype data of the material to be tested for subsequent analysis.

[0065] 3) Analyze the genotypes in the material, perform preliminary clustering, and combine the yield phenotypes of dryland varieties to determine the drought resistance of the material. The results are shown in Figure 4 .

[0066] From the drought resistance phenotype (Table 4), the yield of the drought-resistant group under water-planting and drought-management conditions was significantly higher than that of the sensitive group (Table 5), indicating that under complex genetic background conditions, this group of drought-resistant SNPs can still distinguish drought-resistant and sensitive materials well, and can be applied to water-saving and drought-resistant rice breeding practice.

[0067] Table 4

[0068] Drought-resistant plant H11 S29 D48 D32 Y22 H6 Y7 S39 S8 average Dryland yield (g) 53.68 55.17 56.29 56.66 69.87 76.69 80.23 88.97 100.45 70.89 Sensitive individual D15 H16 H45 H20 D2 H27 H13 Y32 H12 average Dryland yield (g) 8.36 13.11 13.58 20.54 21.78 21.96 27.08 30.81 33.66 22.71

[0069] Table 5

[0070]

[0071] 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. A combination of SNP molecular markers associated with rice drought resistance, characterized in that: It is all SNP molecular markers shown in SEQ ID NO.1 to SEQ ID NO.191, wherein a mutation occurs at the 50 bp position, resulting in polymorphism in rice drought resistance; the mutation form of the SNP molecular marker is as follows:

2. A gene chip, characterized in that: It comprises the combination of SNP molecular markers according to claim 1.

3. A primer set for amplifying the SNP molecular marker according to claim 1.

4. A reagent or kit comprising the primer set according to claim 3.

5. Use of the combination of SNP molecular markers according to claim 1, or the gene chip according to claim 2, or the primer set according to claim 3, or the reagent or kit according to claim 4 in early prediction of rice drought resistance phenotype.

6. Use of the combination of SNP molecular markers according to claim 1, or the gene chip according to claim 2, or the primer set according to claim 3, or the reagent or kit according to claim 4 in screening drought-resistant rice.

7. Use of the combination of SNP molecular markers according to claim 1, or the gene chip according to claim 2, or the primer set according to claim 3, or the reagent or kit according to claim 4 in breeding drought-resistant rice.

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