CN110628935B - Molecular marking method and application of salt-tolerant gene LOC _ Os02g49700 of rice in adult stage - Google Patents
Molecular marking method and application of salt-tolerant gene LOC _ Os02g49700 of rice in adult stage Download PDFInfo
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Abstract
The invention discloses a molecular marking method of a salt-tolerant gene LOC _ Os02g49700 of rice in adult plant stage, wherein a pair of specific PCR primer pairs RM13924 is used for PCR amplification of genome DNA of a rice breeding material to be detected, if the primer pairs RM13924 can amplify a segment with the size of 195bp, the breeding material is provided with the salt-tolerant gene LOC _ Os02g49700, the RM13924 forward primer sequence is shown as SEQ ID No.1, and the reverse primer sequence is shown as SEQ ID No. 2. The molecular marking method can be used for rice salt-tolerant molecular marking auxiliary selective breeding.
Description
Technical Field
The invention relates to a molecular marking method of a salt-tolerant gene LOC _ Os02g49700 of rice in the adult plant stage, belonging to the field of stress-resistant breeding and molecular genetics of rice.
Background
Rice is one of the most important food crops in the world, is sensitive to salt damage, shows damage symptoms when the concentration of soluble salt in soil reaches 0.3 percent, and finally leads to remarkable yield reduction. The area of saline-alkali cultivated land in China is about 1 hundred million hectares, and the salt damage causes a great amount of yield reduction of crops. The cultivation of salt-tolerant rice varieties has important significance for solving the problem of grain safety.
Molecular genetics research shows that the salt tolerance of rice is quantitative character controlled by multiple genes. With the development of molecular marker technology, a large number of salt tolerance related Quantitative Trait Loci (QTL) are identified by utilizing marker technologies such as AFLP, RFLP, SSR and the like and genetic mapping populations, and the understanding of the salt tolerance inheritance mechanism of rice is promoted to a certain extent. However, in previous researches, double-parent genetic populations are mostly adopted to identify the QTL/gene of the target character, the genetic background is simple, the gene diversity is low, and the mining efficiency of favorable genes is low. The salt tolerance of different genotypes of rice is greatly different, and germplasm resources are natural population materials which are cultivated and reserved by people in long-term production practice, carry various genes which are favorable for variation, and are treasures for genetic improvement. With the development of biological technology, the high-density SNP markers obtained by the second-generation sequencing technology are utilized, and on the basis, the whole genome association analysis of the target traits can effectively promote the efficient excavation of target trait candidate genes. A large number of researches show that the whole genome association analysis is an effective method for excavating quantitative character sites obviously associated with target characters from germplasm resources, and is widely applied to genetic analysis research of important agronomic characters.
At present, salt tolerance related QTL at different periods such as rice germination period, seedling period, reproductive growth period and the like are reported, and part of candidate genes are finely positioned. However, many genes related to salt tolerance of rice are not discovered.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a brand-new novel salt-tolerant gene influencing the salt tolerance of the rice in the adult stage. And provides a molecular marking method, a detection method and application of the novel salt-tolerant gene.
The technical scheme of the invention is as follows:
the salt-tolerant gene LOC _ Os02g49700 of the invention at the adult stage of rice is positioned at 1 gene site of the 2 nd chromosome of rice related to salt tolerance of the adult stage of rice, and the gene can obviously improve the yield of rice under salt stress.
A molecular marking method of a salt-tolerant gene LOC _ Os02g49700 of rice in the adult stage comprises the steps of carrying out PCR amplification on a genomic DNA of a rice breeding material to be detected by using a pair of specific PCR primer pairs RM13924, wherein if the primer pairs RM13924 can amplify a 195bp fragment, the breeding material is provided with the salt-tolerant gene LOC _ Os02g49700, RM13924, a forward primer sequence is shown as SEQ ID No.1 (TAGGAGGGAGAAATCATTGTGG), and a reverse primer sequence is shown as SEQ ID No.2 (TCGTAGTACAACCACAACATGG).
A method for detecting whether rice contains a salt-tolerant gene LOC _ Os02g49700 at adult stage of rice is to use a pair of specific PCR primer pairs RM13924 to PCR amplify the genomic DNA of a rice breeding material to be detected, if the primer pairs RM13924 can amplify a 195bp fragment, the breeding material contains the salt-tolerant gene LOC _ Os02g49700, RM13924 forward primer sequences are shown as SEQ ID No.1 (TAGGAGGGAGAAATCATTGTGG), and reverse primer sequences are shown as SEQ ID No.2 (TCGTAGTACAACCACAACATGG).
The invention relates to application of a molecular marking method of a rice salt-tolerant gene LOC _ Os02g49700 or a method for detecting whether rice contains the rice salt-tolerant gene LOC _ Os02g49700 in adult-plant stage in rice salt-tolerant auxiliary selective breeding.
The invention utilizes 708 water rice core germplasm resources which are widely different from 77 countries including China, India, America, Philippines and the like, systematically analyzes the salt tolerance performance of the water rice core germplasm resources under the salt stress condition in the adult plant stage, and screens a salt tolerance gene LOC _ Os02g49700 which influences the rice yield under the salt stress condition in the adult plant stage by combining high-density SNP (single nucleotide polymorphism) markers and haploid analysis. Meanwhile, 1 pair of PCR primers RM13924 is screened out according to SNP variation near the target gene locus, and an effective molecular marker is further provided for molecular marker-assisted selective breeding. The new salt-tolerant gene LOC _ Os02g49700 and the closely linked molecular marker RM13924 thereof are expected to be applied to the auxiliary selection breeding of the rice salt-tolerant marker.
Compared with the prior art, the invention has the following beneficial effects:
1. the gene LOC _ Os02g49700 is a new gene detected by salt tolerant phenotype and SNP association analysis by using more than 700 germplasm resources with wide genetic differences from different countries around the world. The gene is obviously related to the yield of the rice under the salt stress condition in the adult plant period.
2. The molecular marker closely associated with the salt-tolerant gene provides effective information for the molecular marker-assisted selection of the salt-tolerant rice, and can obtain rice materials with high yield under the condition of salt stress. The molecular marker can be used for screening favorable genotypes of a breeding population under the salt stress of the rice in the adult plant period, effectively identifying salt-tolerant individuals with the gene and accelerating the breeding process.
Drawings
FIG. 1 salt-sensitive variety IR29 (P)1) With salt-tolerant variety Taipei 167 (P)2) Hybridization F2The salt tolerance performance of individuals in the adult stage of the population is mapped with RM13924 marker genotype. F for hybridizing salt-tolerant variety IR29 with salt-tolerant variety Taipei 1672Population, banding pattern of PCR amplification product of SSR marker RM13924 on 5% polyacrylamide gel electrophoresis and corresponding phenotype (1-30 is F randomly selected)2Generating single plants; m is DNA Ladder; p1Is 150bp, P 2195 bp).
Detailed Description
The present invention is further illustrated below with reference to specific examples, wherein the methods used are conventional methods unless otherwise specified.
Excavation of salt-tolerant new gene
1. Test material
The experimental materials were 708 parts of rice core germplasm resources (including 400 parts of indica, 247 parts of japonica, 39 parts of indica-japonica, 16 parts of Aus, and 6 parts of Basmati) from 77 countries including china, india, the united states, philippines, etc., respectively.
2. Salt tolerance phenotype identification of test materials
The experiment carries out continuous two-year salt-tolerant phenotype identification on 708 parts of rice materials, and ensures the reliability of phenotype data. The specific operation method comprises the following steps: 708 portions of rice material are directly sown in the rice field, and after the rice material grows to 2 leaves, the rice material is irrigated by 0.5 percent saline water which is prepared by fresh water and seawater according to a certain proportion, and the concentration of the saline water is kept until the rice is mature. Counting the number of seedlings after 1 month of sowing, recording heading period, examining the yield and the effective spike number of the cell after maturity, randomly selecting 10 spikes from each cell, and examining yield-related index characters such as total spike number, solid spike number, seed setting rate, thousand kernel weight and the like.
3. Genotype data processing and genome wide association analysis (GWAS)
The 32M SNP original genotype data in Rice SNP-Seek Database (http:// SNP-Seek. irri. org /) constructed by 3K Rice re-sequencing project is processed according to the following principle: 1) the same SNP locus has more than 2 alleles, only two alleles with the highest frequency are reserved, and the rest are processed according to deletion; 2) when MAF (minimum allele frequency) is less than or equal to 0.05 or the deletion rate is more than 20 percent, deleting the SNP data according to deletion processing to obtain 3,455,952 high-quality SNP markers in total; 3) randomly selecting partial SNP data, carrying out Kinship and principal component analysis by using an R software package GAPIT program, and analyzing a group structure; 4) extracting 1,101,404 SNP genotypes positioned on 44,332 annotated genes according to functional annotations of Nipponbare Genome gene of IRGSP-1.0 version produced by Rice Genome Annotation Project (http:// Rice. plant biology. msu. edu. /), performing whole Genome association analysis (GWAS) of candidate genes by utilizing a GAPIT program in a software package according to cell yield and test seed yield related traits under a salt stress condition, classifying the phenotype such as total panicle number, fructification rate, thousand seed weight and the like, and performing whole Genome association analysis (GWAS) on the candidate genes by utilizing a GAPIT program in the software package according to the Rice Genome Annotation Project
The horizontal SNP is defined as the SNP significantly associated with the trait of interest, resulting in SNP markers significantly associated with each trait as shown in table 1.
TABLE 1 summary of SNPs associated with investigational traits
SN is the number of seedlings, HD is the heading stage, GY is the yield of a cell, PN is the number of ears of the cell, TSN is the total number of ears of the ear, FGN is the number of solid grains of the ear, SSR is the seed setting rate, TGW is the weight of thousand grains
4. Identification of salt-tolerant candidate genes
230 known salt response-associated genes were screened from the funRicegenes database (https:// funRiceGenes. githuo. io /) and the QTARO database (http:// QTARO. abr. afrrc. go. jp). And comparing 903 genes detected to be associated with the salt tolerance related traits with 230 hypochondriac response genes, and selecting 43 associated genes with the same functional annotation and metabolic pathway for further haplotype analysis. If the phenotype corresponding to the main haplotype of the target gene has significant difference (P <0.001), the gene is defined as a salt-tolerant candidate gene. The study identifies 15 candidate genes (table 2) affecting the salt tolerance related traits of adult plants, wherein LOC _ Os02g49700 significantly affects the rice yield under the salt stress condition, and can explain the 32.7% variation of the rice yield in japonica rice subgroup. Further analysis found that LOC _ Os02g49700 encodes the homeobox of the leucine chain. In japonica rice groups, 1 SNP polymorphic site is identified to exist in the 5' UTR end S1_30381323 site of the gene, and the yield of rice under salt stress can be improved by changing A into C. By identifying PCR primer RM13924 (wherein the forward primer sequence is TAGGAGGGAGAAATCATTGTGG, and the reverse primer sequence is TCGTAGTACAACCACAACATGG) closely linked with LOC _ Os02g49700, an effective molecular marker can be further provided for molecular marker-assisted selective breeding. The new salt-tolerant gene LOC _ Os02g49700 and the closely linked molecular marker RM13924 thereof are expected to be applied to the auxiliary selection breeding of the rice salt-tolerant marker.
Table 2 identifies 15 salt-tolerant candidate genes associated with salt-tolerance-related traits in adult plant stage
aGenes obtained from the MSU Rice Genome Annotation Project database;banalyzing a statistic value corresponding to the peak SNP in the intra-gene association;canalyzing the variance of the gene haplotype;dthe character names are as in Table 1;e 2016(Y16)and 2017(Y17).
II, salt tolerance F2Marker validation analysis of segregating population salt-tolerant gene LOC _ Os02g49700
1. Salt stress treatment of test materials
F is constructed by utilizing salt-sensitive variety IR29 and variety Taipei 167 pairing with salt-tolerant candidate gene LOC _ Os02g497002Separating the colony, wherein 360 seedlings are cultured in a normal paddy field, the single seedling is transplanted in a 0.5% saline environment, and the yield-related characters are examined after the seedling is mature.
2. DNA extraction, PCR amplification and gel electrophoresis
Genomic DNA was extracted from each individual plant by the DNA extraction method of Temnykh et al (2000). The RM13924 marker was subjected to Polymerase Chain Reaction (PCR) using the genomic DNA of each individual strain as a template. Products of the PCR reaction were separated by polyacrylamide gel electrophoresis, stained with ethidium bromide, and imaged in a gel imaging system. And (5) judging and recording the band type of the progeny individual plant by referring to the amplified bands of the parents.
3. Marker assisted selection effect analysis and t-test analysis
F is divided according to the genotype represented by the RM13924 marker amplification band of the offspring individual plant2The salt tolerance and the corresponding situation of the individual with salt sensitivity to the genotype of the population are shown in figure 1, and the individual with salt sensitivity parent IR29 (P)1) The homozygous genotype individual has lower single plant yield and carries the salt-tolerant parent Taipei 167 (P)2) The single plant yield of the homozygous genotype individual is generally higher, while the single plant yield of the heterozygous genotype individual is centered on the salt-tolerant parent, which shows that RM13924 has a relatively ideal auxiliary selection effect on the salt-tolerant gene LOC _ Os02g 49700. F is to be2Individual division in a populationTwo groups, wherein one group is an individual with RM13924 locus genotype of IR29 homozygous genotype (called salt susceptible group), and 88 strains are counted; the other group is individuals (called salt-tolerant group) with RM13924 locus genotype being Taipei 167 homozygous genotype, and the total number is 85 strains. The average single plant yield obtained by the investigation of the two groups of individuals is subjected to t test (table 3), and the result shows that the single plant yield between the two groups of individuals reaches a very significant difference level, which indicates that a major gene LOC _ Os02g49700 influencing the yield under salt stress does exist near the RM13924 marker and is closely linked with the RM13924 marker.
TABLE 3 hybridization of salt-sensitive parent IR29 with salt-tolerant parent Taipei 167F2Individual yield expression of two groups of individuals with salt tolerance and salt-tolerance parent homozygous genotypes at RM13924 marker locus of population
***Indicating that the difference reached a significant level of 0.001.
Sequence listing
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<120> molecular marking method of salt-tolerant gene LOC _ Os02g49700 of rice in adult stage and application
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Claims (2)
1. Detecting whether the rice containsThe method for detecting the salt-tolerant gene LOC _ Os02g49700 of the rice in the adult stage comprises the steps of carrying out PCR amplification on the genomic DNA of the rice to be detected by using a pair of specific PCR primer pairs, wherein if the primer pairs can amplify a segment with the size of 195bp, the rice is provided with the salt-tolerant gene LOC _ Os02g49700, the forward primer sequence of the primer pairs is shown as SEQ ID No.1, and the reverse primer sequence is shown as SEQ ID No. 2; the rice to be detected is a salt-sensitive variety IR29 hybridized with a salt-tolerant variety Taipei 167F2And (4) a group.
2. The method for detecting whether the rice contains the salt tolerance gene LOC _ Os02g49700 of adult rice as claimed in claim 1 is applied to salt tolerance auxiliary selection breeding of rice.
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