A haplotype molecular marker for the rice yield-regulating gene GWY10 and its application

Abstract

The application discloses a haplotype molecular marker of a rice yield regulation gene GWY10 and application thereof, and belongs to the technical field of plant molecular breeding. The application obtains the haplotype molecular marker of the rice yield regulation gene GWY10 by screening a core sequence of the rice yield regulation gene GWY10 and SNP sites of upstream and downstream sequences of the gene, the haplotype molecular marker can quickly identify the function of the rice yield regulation gene GWY10, thereby more efficiently and directly judging the phenotype of a rice variety, in addition, the haplotype molecular marker can be used for development of a rice yield regulation gene GWY10 chip or for design of a detection marker based on PCR, and is very practical, and therefore, the haplotype molecular marker has a good application prospect in rice breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a haplotype molecular marker of the rice yield-regulating gene GWY10 and its application. Background Technology

[0002] Molecular markers are polymorphic markers based on the genetic material DNA. Based on the association between markers and traits, functional or closely linked markers can be used to rapidly identify functional genes in a variety. Single nucleotide polymorphisms (SNPs) are the most numerous and widely distributed type of molecular marker. The development of SNP markers is based on DNA sequencing. Over the past two decades, rapid advancements in genome sequencing and a significant decrease in sequencing costs have enabled scientists worldwide to discover a large number of SNP markers through sequencing thousands of rice germplasm resources. Simultaneously, the completion of large-scale, high-quality rice genome sequencing has laid a solid foundation for screening functional gene haplotypes.

[0003] SNPs reflect DNA genetic variations at the level of single base variations, thus compensating to some extent for the shortcomings of first-generation molecular markers (such as restriction fragment length polymorphism, RFLP) and second-generation molecular markers (such as microsatellite DNA polymorphism, SSR). Therefore, they are called third-generation genetic markers. The main advantages of SNPs are: high density and wide distribution (one SNP appears every 232 bp in rice gene sequences); and rapid, high-throughput detection and automated analysis.

[0004] Rice is one of the world's most important food crops. Research on rice yield-regulating genes helps to increase yield, improve quality, and enhance resource utilization efficiency, providing crucial support for global food security and sustainable agricultural development. Existing research indicates that the rice yield-regulating gene GWY10 regulates grain width through direct regulation by the transcription factor OsbZIP47, interaction with CDKB2, and expression differences caused by natural variations. However, there are currently few reports on haplotype molecular markers closely linked to the rice yield-regulating gene GWY10. Summary of the Invention

[0005] The purpose of this invention is to provide a haplotype molecular marker for the rice yield-regulating gene GWY10 and its application. The haplotype molecular marker provided by this invention can rapidly identify the function of the rice yield-regulating gene GWY10, thereby more efficiently and directly determining the phenotype of rice varieties. Therefore, it has good application prospects in rice breeding.

[0006] In a first aspect, the present invention provides a haplotype molecular marker for the rice yield-regulating gene GWY10, including SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 loci; wherein, SNP1 is located at 19396150 bp on chromosome 10, with polymorphism A or G; SNP2 is located at 19396170 bp on chromosome 10, with polymorphism A or G; SNP3 is located at 19396202 bp on chromosome 10, with polymorphism A or G; and SNP4 is located at 19396223 bp on chromosome 10, with polymorphism A or G. The polymorphisms of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 are C or A, respectively. SNP5 is located at 19401650 bp on chromosome 10, with a polymorphism of A or G. SNP7 is located at 19509221 bp on chromosome 10, with a polymorphism of G or C. SNP8 is located at 19607019 bp on chromosome 10, with a polymorphism of A or C. The physical locations of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 were determined based on the whole genome sequence of Nipponbare, MSU7.0.

[0007] In this invention, by screening the SNP sites of the core sequence of the rice yield-regulating gene GWY10 and its upstream and downstream sequences, a haplotype molecular marker of the rice yield-regulating gene GWY10 is obtained. This haplotype molecular marker can rapidly identify the function of the rice yield-regulating gene GWY10, thereby more efficiently and directly determining the phenotype of rice varieties. In addition, this haplotype molecular marker can be used for the development of rice yield-regulating gene GWY10 chips or for designing PCR-based detection markers, which is very practical.

[0008] In a second aspect, the present invention provides the application of the haplotype molecular markers described above in the identification of the rice yield-regulating gene GWY10.

[0009] In some implementation schemes, when the haplotype formed by SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 in sequence is AAAACCAGA, the rice yield-regulating gene GWY10 reduces grain width and / or thousand-grain weight.

[0010] In some implementation schemes, when the haplotype formed by SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 in sequence is GGGAAGCC, the rice yield-regulating gene GWY10 increases grain width and / or thousand-grain weight.

[0011] In a third aspect, the present invention provides the application of haplotype molecular markers as described above in rice breeding.

[0012] In a fourth aspect, the present invention provides the application of haplotype molecular markers as described above in screening and / or identifying high-yielding rice varieties.

[0013] In a fifth aspect, the present invention provides a method for detecting a haplotype molecular marker of the rice yield-regulating gene GWY10, comprising the following steps: detecting the rice genome sequence to obtain the rice genome sequence result; and detecting the genotype of the haplotype molecular marker of the rice yield-regulating gene GWY10 based on the rice genome sequence result.

[0014] In some implementation schemes, methods for detecting the genome sequence of rice include at least one of whole-genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

[0015] It is understood that the method for detecting the genome sequence of rice can be conventionally selected according to the actual situation. In this invention, the preferred method for detecting the genome sequence of rice includes at least one of whole genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

[0016] In a sixth aspect, the present invention provides a method for rice breeding, comprising the following steps: detecting the genotype of the haplotype molecular marker of the rice yield regulating gene GWY10 as described above in a rice sample, and selecting rice samples with the haplotype GGGAAGCC formed by the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites in sequence for breeding.

[0017] The rice breeding method provided by this invention is simple and efficient, and it has the advantage of high accuracy by using haplotype molecular markers of the rice yield regulating gene GWY10 for assisted breeding.

[0018] In some implementations, the phenotype of rice samples with haplotype GGGAAGCC is analyzed, and rice samples with high grain width and / or thousand-grain weight are selected for breeding.

[0019] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention obtains a haplotype molecular marker for the rice yield-regulating gene GWY10 by screening the core sequence and SNP sites of its upstream and downstream sequences. This haplotype molecular marker can rapidly identify the function of the rice yield-regulating gene GWY10, thereby more efficiently and directly determining the phenotype of rice varieties. In addition, this haplotype molecular marker can be used for the development of GWY10 microarrays or for designing PCR-based detection markers, which is very practical. Therefore, this haplotype molecular marker has good application prospects in rice (especially high-yield rice) breeding. Attached Figure Description

[0020] Figure 1 The present invention provides 16 haplotype marker combinations of the rice yield regulating gene GWY10 and its neighboring sequences in Example 1 of this invention. Among them, the 8th and 9th sites are haplotype sites of the rice grain width and thousand-grain weight gene GWY10. The genotype "CA" at the 8th and 9th sites is a genotype that reduces grain width and thousand-grain weight, and "TC" is a genotype that increases grain width and thousand-grain weight.

[0021] Figure 2 This is a graph showing the results of the analysis of differences in grain width and thousand-grain weight between haplotype rice varieties Hap1 and Hap2 in Example 2 of this invention; where "***" indicates that the results are statistically significant at the significance level of P<0.001; the error bars in the graph are "mean ± 2 × standard error", and the small range of the error bars indicates that the sample mean is close to the population mean, and the data is highly reliable. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0024] Example 1: Obtaining the haplotype molecular marker of the rice yield-regulating gene GWY10

[0025] In this embodiment, haplotype markers for the rice yield-regulating gene GWY10 were designed based on information from 4726 rice varieties and 14,541,446 SNP sites provided by the website RiceVarMap. Specifically, the steps include:

[0026] 1) Based on the article published by Professor Li Yibo's team at Huazhong Agricultural University (Waseem A, Qi S, Yana C, et al. The quantitative trait locus GWY10 controls rice grain width and yield[J]. Plant Physiology, 2024.), the haplotype markers consisting of 13 sites in the promoter region of the rice yield-regulating gene GWY10 were obtained. The specific information is shown in Table 1 below:

[0027] Table 1. Two haplotypes H1 and H2 in the promoter region of the rice yield-regulating gene GWY10.

[0028] Haplotype H1 H2 vg1019604947 A G vg1019604999 G A vg1019605309 CTTCGGGCATA C vg1019605386 C T vg1019605453 G A vg1019606113 A ATGG vg1019606178 TTC T vg1019606384 T C vg1019607019 C A vg1019607219 G A vg1019607584 A G vg1019607712 ACC A vg1019607715 A AG

[0029] Two SNP sites, “vg1019607019” and “vg1019606384”, were selected.

[0030] 2) Download SNP variation information of 4726 rice varieties from the RiceVarMap v2.0 website, and then extract SNP genotype information of 250kb upstream and downstream regions of the functional site of the rice yield regulating gene GWY10, totaling 12407 SNP variation sites from 4726 rice varieties.

[0031] 3) The vcf files containing the 12,407 SNP gene information from 4,726 varieties were converted to plink format using PLINK software. Then, the correlation coefficients (r) with the two SNP loci (vg1019606384 and vg1019607019) were obtained using PLINK software analysis. 2 (0-1 indicates the degree of linkage between two SNPs, and 1 indicates that the two SNPs are completely linked.) For SNPs with a correlation value greater than 0.64, SNPs associated with both SNPs were extracted, resulting in a total of 7 SNPs that were highly associated with both vg1019606384 and vg1019607019 sites in the promoter region of the rice grain width and thousand-grain weight gene GWY10.

[0032] 4) Genotypes of the above 9 SNP loci were extracted from 4726 rice varieties, and haplotype analysis was performed using Haploview software. The "Examine haplotypes above" parameter was set to 0.4, resulting in 16 haplotype groups, Haplotype 1 to 16. The results are as follows: Figure 1 As shown.

[0033] 5) Extraction Figure 1 The eight loci (01-07 and 09) of Haplotype1 and Haplotype2 form two haplotype combinations, Hap1 and Hap2 (details are shown in Table 2 below). Rice varieties with the Hap1 haplotype have a genotype that reduces grain width and thousand-grain weight by regulating the rice yield gene GWY10, while rice varieties with the Hap2 haplotype have a genotype that increases grain width and thousand-grain weight by regulating the rice yield gene GWY10.

[0034] Table 2 Haplotype markers of the rice yield-regulating gene GWY10

[0035] Serial Number chromosome Location Hap1 Hap2 1 chr10 19396150 A G 2 chr10 19396170 A G 3 chr10 19396202 A G 4 chr10 19396223 C A 5 chr10 19401650 C A 6 chr10 19402015 A G 7 chr10 19509221 G C 8 chr10 19607019 A C

[0036] As shown in Table 2, the two haplotype molecular markers are highly linked to the functional site of the rice yield-regulating gene GWY10, and can be used to predict rice grain width and thousand-grain weight.

[0037] Example 2: Application of haplotype molecular markers for the rice yield-regulating gene GWY10

[0038] Phenotypic data of publicly available rice varieties were downloaded from the RFGB (Rice Functional Genomics Database), including 1171 varieties with phenotypic records for both grain width and thousand-grain weight. Genotyping of these varieties was performed on the RiceVarMap v2.0 website. The results showed that 467 rice varieties belonged to the "Hap1" haplotype in Example 1, 212 rice varieties belonged to the "Hap2" haplotype, and the remaining 492 varieties...

[0039] The rice varieties are of other haplotypes, as detailed in Table 3 below.

[0040] Table 3. Phenotypic data and GWY10 haplotype markers of 1171 rice varieties.

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[0067] Comparing the grain width and thousand-grain weight phenotypes of the above 467 Hap1 rice varieties and 212 Hap2 rice varieties, the results are as follows: Figure 2 As shown.

[0068] from Figure 2The results show that varieties carrying the GWY10 Hap1 haplotype have smaller grain width and thousand-grain weight, while varieties carrying the GWY10 Hap2 haplotype have larger grain width and thousand-grain weight. These results indicate that haplotype molecular markers of the rice yield-regulating gene GWY10 can accurately identify rice grain width and thousand-grain weight.

[0069] In summary, the haplotype molecular markers provided by this invention can rapidly identify the function of the rice yield-regulating gene GWY10, thereby more efficiently and directly determining the phenotype of rice varieties. Therefore, they have good application prospects in rice breeding.

[0070] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0071] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of haplotype molecular marker in identifying yield-controlling gene GWY10 in rice, characterized in that, The haplotype molecular marker comprises SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites; The SNP1 is located at the genomic position of 10# chromosome 19396150bp, and the polymorphism is A or G; the SNP2 is located at the genomic position of 10# chromosome 19396170bp, and the polymorphic site is A or G; the SNP3 is located at the genomic position of 10# chromosome 19396202bp, and the polymorphism is A or G; the SNP4 is located at the genomic position of 10# chromosome 19396223bp, and the polymorphic site is C or A; the SNP5 is located at the genomic position of 10# chromosome 19401650bp, and the polymorphism is C or A; the SNP6 is located at the genomic position of 10# chromosome 19402015bp, and the polymorphism is A or G; the SNP7 is located at the genomic position of 10# chromosome 19509221bp, and the polymorphism is G or C; and the SNP8 is located at the genomic position of 10# chromosome 19607019bp, and the polymorphism is A or C; The physical positions of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites are determined based on the whole genome sequence MSU7.0 version of Nipponbare; When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites in turn is AAACCAGA, the rice yield control gene GWY10 reduces the grain width and / or the thousand-grain weight; When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites in turn is GGGAAGCC, the rice yield control gene GWY10 increases the grain width and / or the thousand-grain weight.

2. Use of haplotype molecular markers in rice breeding, characterized in that, The haplotype molecular marker comprises SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites; The haplotype molecular marker comprises SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites; The genomic position of the SNP1 is 10# chromosome 19396150bp, and the polymorphism is A or G; the genomic position of the SNP2 is 10# chromosome 19396170bp, and the polymorphic site is A or G; the genomic position of the SNP3 is 10# chromosome 19396202bp, and the polymorphism is A or G; the genomic position of the SNP4 is 10# chromosome 19396223bp, and the polymorphic site is C or A; the genomic position of the SNP5 is 10# chromosome 19401650bp, and the polymorphism is C or A; the genomic position of the SNP6 is 10# chromosome 19402015bp, and the polymorphism is A or G; the genomic position of the SNP7 is 10# chromosome 19509221bp, and the polymorphism is G or C; and the genomic position of the SNP8 is 10# chromosome 19607019bp, and the polymorphism is A or C; The physical positions of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites are determined based on the whole genome sequence of Nipponbare MSU7.0 version; When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites in turn is AAACCAGA, the rice yield control gene GWY10 reduces the grain width and / or the thousand-grain weight; When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites in turn is GGGAAGCC, the rice yield control gene GWY10 increases the grain width and / or the thousand-grain weight.

3. Use of haplotype molecular markers for screening and / or identifying high yielding rice varieties, characterized in that, The haplotype molecular marker comprises the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 sites; The genomic position of the SNP1 is 10# chromosome 19396150bp, and the polymorphism is A or G; the genomic position of the SNP2 is 10# chromosome 19396170bp, and the polymorphic site is A or G; the genomic position of the SNP3 is 10# chromosome 19396202bp, and the polymorphism is A or G; the genomic position of the SNP4 is 10# chromosome 19396223bp, and the polymorphic site is C or A; the genomic position of the SNP5 is 10# chromosome 19401650bp, and the polymorphism is C or A; the genomic position of the SNP6 is 10# chromosome 19402015bp, and the polymorphism is A or G; the genomic position of the SNP7 is 10# chromosome 19509221bp, and the polymorphism is G or C; and the genomic position of the SNP8 is 10# chromosome 19607019bp, and the polymorphism is A or C; The physical positions of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites are determined based on the whole genome sequence of Nipponbare, MSU7.0 version; When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites in sequence is AAACCAGA, the rice yield regulatory gene GWY10 reduces the grain width and / or the thousand-grain weight; When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites in sequence is GGGAAGCC, the rice yield regulatory gene GWY10 increases the grain width and / or the thousand-grain weight.

4. A method for detecting a haplotype molecular marker of a rice yield regulatory gene GWY10, characterized in that, The method comprises the following steps: detecting the genomic sequence of rice to obtain a genomic sequence result of the rice; and detecting the genotype of a haplotype molecular marker of a rice yield regulatory gene GWY10 according to the genomic sequence result of the rice; The haplotype molecular marker comprises SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites; The SNP1 is located at the genomic position of 10th chromosome 19396150bp, and the polymorphism is A or G; the SNP2 is located at the genomic position of 10th chromosome 19396170bp, and the polymorphic site is A or G; the SNP3 is located at the genomic position of 10th chromosome 19396202bp, and the polymorphism is A or G; the SNP4 is located at the genomic position of 10th chromosome 19396223bp, and the polymorphic site is C or A; the SNP5 is located at the genomic position of 10th chromosome 19401650bp, and the polymorphism is C or A; the SNP6 is located at the genomic position of 10th chromosome 19402015bp, and the polymorphism is A or G; the SNP7 is located at the genomic position of 10th chromosome 19509221bp, and the polymorphism is G or C; and the SNP8 is located at the genomic position of 10th chromosome 19607019bp, and the polymorphism is A or C; The physical positions of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites are determined based on the whole genome sequence of Nipponbare, MSU7.0 version; When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites in sequence is AAACCAGA, the rice yield regulatory gene GWY10 reduces the grain width and / or the thousand-grain weight; When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites in sequence is GGGAAGCC, the rice yield regulatory gene GWY10 increases the grain width and / or the thousand-grain weight.

5. The detection method according to claim 4, characterized in that, The method for detecting the genome sequence of rice comprises at least one of whole genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

6. A method of breeding rice, characterized by, The method comprises the following steps: detecting the genotype of a haplotype molecular marker of a yield-regulating gene GWY10 in a rice sample, and selecting a rice sample with a haplotype of GGGAAGCC formed by SNPs at SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 in sequence for breeding. The haplotype molecular marker comprises SNPs at SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8. The SNP1 is located at 19396150bp on chromosome 10, and the polymorphism is A or G; the SNP2 is located at 19396170bp on chromosome 10, and the polymorphic site is A or G; the SNP3 is located at 19396202bp on chromosome 10, and the polymorphism is A or G; the SNP4 is located at 19396223bp on chromosome 10, and the polymorphic site is C or A; the SNP5 is located at 19401650bp on chromosome 10, and the polymorphism is C or A; the SNP6 is located at 19402015bp on chromosome 10, and the polymorphism is A or G; the SNP7 is located at 19509221bp on chromosome 10, and the polymorphism is G or C; and the SNP8 is located at 19607019bp on chromosome 10, and the polymorphism is A or C. The physical positions of the SNPs at SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 are determined based on the MSU7.0 version of the whole genome sequence of Nipponbare.

7. The method of claim 6, wherein, The phenotype of the rice sample with the haplotype of GGGAAGCC is analyzed, and a rice sample with high grain width and / or 1000-grain weight is selected for breeding.

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