CN115896328A - Haplotype SNP molecular marker of functional gene for detecting rice alkali elimination value and detection method - Google Patents
Haplotype SNP molecular marker of functional gene for detecting rice alkali elimination value and detection method Download PDFInfo
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Abstract
The invention discloses a haplotype SNP molecular marker and a detection method for detecting functional genes of rice alkali digestion values, wherein the haplotype SNP molecular marker consists of SNP1 and SNP2, wherein the SNP1 is a nucleotide sequence shown as SEQ ID NO.1, and the 100bp position of the sequence is G or A; the SNP2 is a nucleotide sequence shown as SEQ ID NO.1, and the 2575bp position of the sequence is T or C. The haplotype SNP molecular marker related to the rice alkali elimination value can realize the rapid screening of rice high alkali elimination value germplasm resources, can also be used for the molecular marker-assisted selective breeding and the directional improvement of the rice variety alkali elimination value, and has great commercial value.
Description
Technical Field
The invention belongs to the field of agricultural molecular biology, and particularly relates to a haplotype SNP molecular marker of a functional gene for detecting the alkali digestion value of rice and a detection method.
Background
The relationship between the cooking and taste quality of rice and consumers is the closest in the rice quality character. About 90% of the dry matter in rice is starch, including amylose and amylopectin, the majority of which is amylopectin. The cooking taste quality of rice is mainly measured by 3 indexes of Amylose Content (AC), gel Consistency (GC) and Gelatinization Temperature (GT). Amylose is mainly synthesized by granular starch synthase I (GBSSI), and generally, the amylose content of rice is positively correlated with the consistency of gum and negatively correlated with the gelatinization temperature. The gelatinization temperature of rice is controlled by the ALK site located on chromosome 6, which encodes a starch synthase SSIIa (or SSII-3) which is responsible for elongating the short chains in amylopectin to long chains and causing an increase in gelatinization temperature. Japonica and indica rice carry different ALK alleles (SSIIa) J And SSIIa I ) Japonica rice type SSIIa J Contains 3 SNP variations and has low activity, so that the amylopectin content of short chain type in japonica rice is higher, and the gelatinization temperature of the japonica rice is generally lower than that of indica rice.
The measurement of gelatinization temperature has higher requirements on equipment and technology; therefore, in the rice quality analysis and identification, the alkali extinction value (ASV) of rice is used as a criterion for determining the gelatinization temperature, and the higher the alkali extinction value is, the lower the gelatinization temperature is. Generally, the alkali elimination value is divided into 7 grades, wherein the low alkali elimination value of 1-2 grades corresponds to the high gelatinization temperature (GT >74 ℃), the medium alkali elimination value of 3-5 grades corresponds to the medium gelatinization temperature (GT is in the range of 70-74 ℃), and the high alkali elimination value of 6-7 grades corresponds to the low gelatinization temperature (GT < 70 ℃). The industrial standard of the rice variety in China (NYT 593-2021 edible rice variety quality) requires that the alkali elimination value of a high-quality edible rice variety (divided into 3 grades) is not less than 5.0, and the alkali elimination values of excellent I and excellent II are more than 6.0. In order to breed a high-quality variety meeting the requirement, the alkali elimination value needs to be selected. The traditional breeding method mainly selects and fixes favorable target characters in a trend manner, and has great blindness and unpredictability; the MAS (marker-assisted selection) technology is based on the sequence difference of DNA (deoxyribonucleic acid), converts the phenotype difference in conventional breeding into genotype difference, and has strong reliability and high efficiency. However, there are few molecular markers that can be directly applied to breeding by selection based on the alkali elimination value, and particularly, there are fewer markers developed based on a functional gene (ALK) directly related to the alkali elimination value, so that molecular breeding for this trait cannot be always performed, and the state of conventional breeding is maintained.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a haplotype SNP molecular marker of a functional gene for detecting the alkali digestion value of rice.
The invention also provides a screening method of the haplotype SNP molecular marker.
The invention also provides application of the haplotype SNP molecular marker.
In the first aspect of the invention, a haplotype SNP molecular marker of a functional gene for detecting the rice alkali digestion value is provided, and the haplotype SNP molecular marker consists of SNP1 and SNP2, wherein the SNP1 is a nucleotide sequence shown as SEQ ID NO.1, and the 100bp position of the sequence is G or A; the SNP2 is a nucleotide sequence shown as SEQ ID NO.1, and the 2575bp position of the sequence is T or C.
In some embodiments of the invention, when the haplotype molecular marker is GT genotype according to SNP1 and SNP2, the alkali elimination value ASV is more than or equal to 6; when the haplotype molecule is marked as an AC genotype, the alkali elimination value ASV is less than 6; when the haplotype molecule is marked as GC genotype, the alkali digestion value ASV is less than 6.
In a second aspect of the present invention, a method for screening the molecular marker of the haplotype SNP molecular marker is provided, which comprises the following steps: performing whole genome sequencing on rice, obtaining alkali elimination value character associated sites by adopting a whole genome association analysis and phenotype analysis method according to a sequencing result, selecting SNP markers related to the alkali elimination value character of the rice from the SNPs with the allele frequency of more than 5 percent to obtain SNP1 positioned on the No. 6 chromosome 6750474bp of the rice genome, wherein the polymorphism is G or A and SNP2 positioned on the No. 6 chromosome 6752888bp of the rice genome, and the polymorphism is C or T.
In a third aspect of the present invention, an application of the above haplotype SNP molecular marker is provided, and the application is an application in rice breeding.
In some embodiments of the invention, the application is application in improving the rice alkali elimination value to cultivate high-quality rice varieties.
In some embodiments of the invention, the use is in assisting in the identification of rice having different rice gelatinization temperatures.
In some embodiments of the invention, the application is an application in detecting the alkalinity digestion value of rice.
A method for detecting the alkali extinction value of rice, comprising the steps of: and (3) carrying out sequence determination on the rice to be detected to obtain the genotype of the haplotype SNP molecular marker, and judging the alkali elimination value of the rice according to the genotype.
In some embodiments of the invention, when the haplotype molecular marker is GT genotype according to SNP1 and SNP2, the alkali elimination value ASV is more than or equal to 6; when the haplotype molecule is marked as an AC genotype, the alkali elimination value ASV is less than 6; when the haplotype molecular marker is GC genotype, the alkali elimination value ASV is less than 6.
In some embodiments of the invention, the sequencing employs one of whole genome re-sequencing, targeted sequencing and multiplex PCR sequencing.
A method of breeding rice, the method comprising the steps of: and (3) carrying out genotype detection on the haplotype SNP molecular markers in the rice sample, and selecting a GT genotype rice sample for breeding.
The haplotype SNP molecular marker and the detection method for detecting the functional gene of the rice alkali elimination value according to the embodiment of the invention have at least the following beneficial effects: the haplotype SNP molecular markers SNP1 and SNP2 related to the rice alkali elimination value can realize the rapid screening of the germplasm resources with high alkali elimination value (low gelatinization temperature) of rice, can also be used for the molecular marker-assisted selective breeding and the directional improvement of the alkali elimination value (gelatinization temperature) of rice varieties, and have great commercial value.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a Manhattan map of genome wide association analysis in example 1 of the present invention;
FIG. 2 is a graph showing the results of the phenotypic distributions corresponding to individuals having different genotypes at the chr6at6750474 (G/A) locus in example 1 of the present invention;
FIG. 3 is a graph showing the results of the phenotypic distribution of individuals with different genotypes at chr6at6752888 (T/C) sites in example 1 according to the present invention;
FIG. 4 is a graph showing the results of the phenotype distributions of individuals having different haplotypes in example 1 of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
Example 1 acquisition of haplotype SNP molecular markers of functional genes for detecting Rice alkali digestion values
1. Selecting materials
The rice material used in the invention comprises a natural population consisting of 322 parts of rice germplasm resources, and contains indica rice and japonica rice, and 25 parts of cultivated rice varieties, wherein the natural population is derived from 3K resources of the International Rice research institute. All materials were planted in Huazhi biological test base located in Changsha, hunan, and each material was replicated twice. Sowing in the last ten days of 5 months, transplanting in the middle ten days of 6 months, airing the harvested paddy, storing at room temperature for 3 months, and then using for experiments.
2. Sample processing and phenotypic analysis
And performing phenotype analysis on alkali elimination value characters of 347 samples obtained by final harvest by referring to agricultural industry standards (NY/T83-2017). Among them, 322 samples were used for the subsequent genome-wide association analysis, and 25 samples were used for the verification of the marker.
3. Genotype data collection and analysis
Genotype data for all samples were obtained by resequencing. The corresponding genotype of the germplasm resource natural population for the whole genome association analysis is from a public data set (3K RG 1M GWAS SNP dataset, https:// SNP-seek.irri.org/_ download.zul), the data set is SNP information of 3K resources of international rice, and about 1M SNP sites are uniformly distributed on 12 chromosomes of rice. The genotype dataset obtained by filtering the public dataset by using the sample name in the invention is directly used for the subsequent whole genome correlation analysis; after the other 25 cultivars are subjected to whole genome sequencing (Illumina, sequencing depth of 10), the obtained reads are subjected to quality control according to a standard method, and are subjected to SNP calling after being returned to a Nipponbare reference genome (MSU 7.0), so that genotypes of the cultivars are obtained.
4. Whole genome association analysis and site mining
The R software package rMVP and a mixed linear model are used for carrying out genome-wide association analysis to mine the association sites of the alkali elimination character, only SNP with allele frequency (MAF) >5% is reserved during association analysis, and as shown in figure 1, a region with significant association is found in the 6 th chromosome and covers an interval of 3.1M. After the segment is annotated according to a rice reference genome (Oryza _ sativa. IRGSP-1.0.47.Chr. Gfff 3), the segment is found to contain a reported main effective gene ALK (LOC _ Os06g12450, chr6: 6748358.6753338) related to gelatinization temperature, and all SNPs in the functional gene are extracted and used as candidate SNPs for subsequent analysis.
5. Identification of SNP markers and haplotypes associated with alkali digestion values
Dividing the alkali extinction table types of the natural population (322 parts) for correlation analysis into two types, namely ASV (American society for instrumentation) is more than or equal to 6 and ASV is less than 6; then, the genotypes of the population are also divided into two types according to the single candidate SNP, and the distribution frequency of the phenotypes in each genotype type is calculated, thereby determining the target SNP (e.g., the natural population is divided into population G (212 copies) and population a (110 copies) according to SNP1 (G/a), and the frequency of individuals having ASV ≧ 6 or ASV <6 in population G is not less than 85%, the SNP is determined as the target SNP for subsequent marker verification). Combining the target SNPs to obtain different genotype haplotypes, analyzing the relationship between each haplotype and the phenotype in the population by using the method, determining the genetic haplotypes which can be classified as the alkali elimination values, and using the genetic haplotypes for subsequent verification.
And 4 SNPs (chr 6at6750474G/A, chr6at6752357G/A, chr6at6752888T/C, chr6at 6753241G/A) are selected in the gene region, wherein chr6at6750474 is SNP1 of the present invention, chr6at6752888T/C is SNP2 of the present invention (the genotype sequence is the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2, SNP1 is the nucleotide sequence shown in SEQ ID NO.1, the 100bp position of the sequence is G or A, SNP2 is the nucleotide sequence shown in SEQ ID NO.1, and the 2575bp position of the sequence is T or C).
The invention obtains 2 SNPs (chr 6at6750474G/A, chr6at 6752888T/C) for identification of markers by screening. Dividing the alkali extinction phenotype of the natural population (322 parts) for correlation analysis into two categories, namely ASV more than or equal to 6 and ASV less than 6; then, the genotypes of the population are divided into two categories according to the 2 candidate SNPs, and the distribution frequency of the phenotypes in each genotype category is calculated, thereby determining the target SNP.
TABLE 1
As a result, as shown in Table 1 and FIGS. 2 to 4, the present invention was designed to combine phenotypes, and it was found that the 2 SNPs (SNP 1 chr6at6750474, SNP2 chr6at 6752888) have a significant linkage relationship with the rice alkali elimination trait by analyzing a haplotype obtained by combining the 2 SNP markers. By comparing 322 the relationship between rice alkali elimination values and haplotypes in rice samples, as shown in FIG. 2-4 and Table 1, it can be seen from FIG. 2 and Table 1 that when SNP1 chr6at6750474 is G, the alkali elimination value of 116 (53.9%) of the individuals (215 in total) containing the genotype in the natural population is greater than 6.0, and when SNP1 chr6at6750474 is A, the alkali elimination value of 104 (100%) of the individuals (104 in total) containing the genotype is less than 6.0, indicating that chr6at6750474 (G/A) is a molecular marker that can be used for screening low alkali elimination phenotype (ASV < 6); as can be seen from FIG. 3 and Table 1, when chr6at6752888 is T, the proportion of high alkali extinction phenotype (ASV ≧ 6) in the population reaches 92.3% (number of high alkali extinction individuals/total: 96/104), when chr6at6752888 is C, the proportion of low alkali extinction phenotype (ASV < 6) in the population reaches 90.7% (number of low alkali extinction individuals/total: 196/216), indicating that chr6at6752888 (T/C) can be used as a molecular marker for alkali extinction phenotype ranking (ASV ≧ 6 or ASV < 6); as can be seen from FIG. 4 and Table 1, 3 haplotypes (Haplotype) were formed from chr6at6750474 and chr6at6752888, and when Haplotype was G-T, i.e., the genotype sequence was SEQ ID NO.1 (position 100 in the sequence was chr6at6750474, position 2575 in the sequence was chr6at 6752888), the proportion of the high alkali extinction phenotype (ASV ≧ 6) in the population reached 93.2% (number of individuals with high alkali extinction/total number: 96/103); when Haplotype is G-C, the proportion of low alkalinity extinction phenotype (ASV < 6) reaches 82.1% (number of individuals with low alkalinity/total number: 92/112); when Haplotype is A-C, that is, when the genotype sequence is SEQ ID NO.2 (in the sequence, the 100 th site is chr6at6750474, the 2575 th site is chr6at 6752888), the proportion of the low-alkali elimination value phenotype (ASV < 6) reaches 100% (number of individuals with low-alkali elimination value/total number: 103/103), it is shown that the 2 haplotypes of G-T and A-C can be used as Haplotype markers of alkali elimination value grades (ASV is more than or equal to 6 or ASV < 6), respectively.
6. Mark verification
After 25 rice cultivars to be tested are typed according to chr6at6750474, chr6at6752888 and haplotype markers thereof, the linkage relationship between each type and the alkali elimination value table type is verified, and if the linkage relationship is consistent with the (5, identification of SNP markers and haplotypes related to the alkali elimination value), the genotype is considered to be a molecular marker with different alkali elimination value levels (ASV is more than or equal to 6 or ASV is less than 6).
The results are shown in Table 2, and it can be seen from the table that 18 varieties with G at the chr6at6750474 site in the 25 rice cultivars to be tested exist, wherein the ASV of 16 varieties is more than or equal to 6 and accounts for 88.8%; 7 varieties with the chr6at6750474 site A are provided, wherein the ASV of 6 varieties is less than 6, and the ratio is 85.7%; 18 varieties with the chr6at6752888 locus T exist, wherein the ASV of 16 varieties is more than or equal to 6, and the proportion is 88.8%; the site of chr6at6750474 is 7 varieties A, wherein the ASV of 6 varieties is less than 6, and the proportion is 85.7%; the haplotypes are 18G-T varieties, wherein the ASV of 16 varieties is more than or equal to 6 and accounts for 88.8 percent; 7 varieties with haplotypes of A-C exist, wherein the ASV of 6 varieties is less than 6, and the proportion is 85.7%; no haplotype was found to be a G-C haplotype. The result shows that each haplotype and the rice alkali elimination value show significant association, and the haplotype SNP molecular marker provided by the scheme can accurately identify the alkali elimination value of the rice.
TABLE 2 genotype and phenotype validation of 25 Rice varieties
In conclusion, the invention utilizes haplotype SNP molecular markers SNP1 and SNP2 for detecting the alkali elimination value of rice, can rapidly and accurately identify the alkali elimination value of the rice, accurately selects the rice sample with the alkali elimination value ASV more than or equal to 6, and can rapidly realize the breeding of the rice variety with high alkali elimination value.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.
Claims (9)
1. A haplotype SNP molecular marker for detecting functional genes of rice alkali elimination values is characterized in that the haplotype SNP molecular marker consists of SNP1 and SNP2, wherein the SNP1 is a nucleotide sequence shown as SEQ ID NO.1, and the 100bp position of the sequence is G or A; the SNP2 is a nucleotide sequence shown as SEQ ID NO.1, and the 2575bp position of the sequence is T or C.
2. The method for screening the haplotype SNP molecular markers according to claim 1, which comprises the following steps: performing whole genome sequencing on rice, obtaining alkali elimination value character associated sites by adopting a whole genome association analysis and phenotype analysis method according to a sequencing result, selecting SNP markers related to the alkali elimination value character of the rice from the SNPs with allele frequency of more than 5%, obtaining SNP1 at the position of 6750474bp of a rice genome, wherein the polymorphism is G or A and SNP2 at the position of 6752888bp of the rice genome, and the polymorphism is T or C.
3. The haplotype SNP molecular marker of claim 1 is applied to rice breeding.
4. The use of the haplotype SNP molecular markers according to claim 1 for assisting in identifying rice with different rice gelatinization temperatures.
5. The haplotype SNP molecular marker of claim 1 is applied to the detection of the rice alkali digestion value.
6. A method for detecting the alkali extinction value of rice is characterized by comprising the following steps: performing sequence determination on rice to be detected to obtain the genotype of the haplotype SNP molecular marker according to claim 1, and judging the alkali-eliminating value of rice according to the genotype.
7. The method of claim 6, wherein the haplotype molecular markers have an ASV value of 6 or more when SNP1 and SNP2 are GT genotypes; when the haplotype molecule is marked as an AC genotype, the alkali elimination value ASV is less than 6; when the haplotype molecule is marked as GC genotype, the alkali digestion value ASV is less than 6.
8. The method of claim 6, wherein sequencing is performed using one of whole genome re-sequencing, targeted sequencing, and multiplex PCR sequencing.
9. A method of breeding rice, comprising the steps of: genotyping the haplotype SNP molecular markers of claim 1 in a rice sample, selecting a rice sample with a GT genotype for breeding.
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| CN111154906A (en) * | 2020-01-20 | 2020-05-15 | 安徽省农业科学院水稻研究所 | SNP functional molecular marker suitable for rice screening special for rice flour and application thereof |
| CN116790801A (en) * | 2021-01-05 | 2023-09-22 | 中国科学院分子植物科学卓越创新中心 | Gene marker for distinguishing rice starch quality |
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| CN111154906A (en) * | 2020-01-20 | 2020-05-15 | 安徽省农业科学院水稻研究所 | SNP functional molecular marker suitable for rice screening special for rice flour and application thereof |
| CN116790801A (en) * | 2021-01-05 | 2023-09-22 | 中国科学院分子植物科学卓越创新中心 | Gene marker for distinguishing rice starch quality |
Non-Patent Citations (1)
| Title |
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| J. S. BAO等: "Nucleotide diversity in starch synthase IIa and validation of single nucleotide polymorphisms in relation to starch gelatinization temperature and other physicochemical properties in rice (Oryza sativa L.)", THEORETICAL AND APPLIED GENETICS, vol. 113, pages 1171 - 1183, XP019440333, DOI: 10.1007/s00122-006-0355-6 * |
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