Haplotype molecular marker related to thousand-grain weight of rice and application thereof

Abstract

This invention discloses a haplotype molecular marker associated with thousand-grain weight in rice and its application, belonging to the field of plant molecular breeding technology. The haplotype molecular marker provided by this invention consists of multiple tightly linked SNPs, and its association with the "significantly increased thousand-grain weight" phenotype is extremely significant. P The value is <0.01), and it has good stability. By detecting this haplotype molecular marker, the thousand-grain weight performance of rice can be predicted quickly and accurately at the seedling stage without waiting for the grains to mature, which greatly improves the breeding efficiency. In addition, this haplotype molecular marker can be directly used for molecular marker-assisted breeding of high-yield or large-grain rice varieties, or it can be integrated into rice functional gene chips as a core marker, or used to develop low-cost PCR detection kits, which is highly practical. It provides a direct molecular tool for the genetic improvement of rice thousand-grain weight, which helps to achieve the targeted breeding of high-yield, large-grain rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a haplotype molecular marker related to the thousand-grain weight of rice 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 they can be detected rapidly with high throughput and automated analysis.

[0004] As one of the world's most important food crops, rice has seen a surge in genetic information in recent years, thanks to the in-depth advancement of functional genomics research. Large-scale, high-quality genotypic and agronomic phenotypic data have been continuously produced, forming a rich genetic information resource library. Among these, the thousand-grain weight is a key agronomic trait, directly reflecting the fullness and size of the grains. Breeding rice varieties with high thousand-grain weight is one of the effective strategies for increasing yield per unit area.

[0005] Although current rice functional genomics research has accumulated a large amount of genotype and phenotypic data, the mining of precise genetic markers for thousand-grain weight is still insufficient, and there is a lack of efficient molecular markers that can be directly used in breeding practices, which restricts the efficiency of thousand-grain retargeting breeding. Summary of the Invention

[0006] The purpose of this invention is to provide a haplotype molecular marker related to the thousand-grain weight of rice and its application. In this invention, by detecting the genotype of this haplotype molecular marker, the thousand-grain weight phenotype of rice can be predicted rapidly and accurately, greatly improving breeding efficiency; therefore, it has good application prospects in rice breeding.

[0007] In a first aspect, the present invention provides a haplotype molecular marker related to the thousand-grain weight of rice, comprising at least one of a first group of haplotype molecular markers and a second group of haplotype molecular markers; wherein, the first group of haplotype molecular markers includes SNP1 and SNP2 loci, SNP1 locus is located at 25026065 bp on chromosome 2, and the polymorphic locus is G or A; SNP2 locus is located at 25026078 bp on chromosome 2, and the polymorphic locus is A or G; when the dominant haplotype formed by SNP1 and SNP2 loci is GA, it indicates an increase in the thousand-grain weight of rice varieties; the second group of haplotype molecular markers includes SNP3, SNP4, SNP5, SNP6, and SNP7 loci, SNP3 locus is located at 5888189 bp on chromosome 9, and the polymorphic locus is... The loci are G or T; SNP4 is located at 5888211 bp on chromosome 9, and the polymorphic locus is T or C; SNP5 is located at 5888261 bp on chromosome 9, and the polymorphic locus is T or G; SNP6 is located at 5888315 bp on chromosome 9, and the polymorphic locus is A or G; SNP7 is located at 5888336 bp on chromosome 9, and the polymorphic locus is G or A; when the dominant haplotype formed by SNP3, SNP4, SNP5, SNP6, and SNP7 in sequence is GTTAG, it indicates an increase in the thousand-grain weight of rice varieties; the physical locations of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7 loci were determined based on the whole genome sequence of Nipponbare, MSU7.0 version.

[0008] In this invention, the inventors obtained haplotype molecular markers related to the thousand-grain weight of rice through association analysis and haplotype analysis of rice genomic and phenotypic data; these haplotype molecular markers consist of multiple tightly linked SNPs and exhibit a highly significant association with the "significantly increased thousand-grain weight" phenotype. P The value is <0.01), and it has good stability. By detecting this haplotype molecular marker, the thousand-grain weight performance of rice can be predicted quickly and accurately at the seedling stage without waiting for the grains to mature, which greatly improves the breeding efficiency. In addition, this haplotype molecular marker can be directly used for molecular marker-assisted breeding of high-yield or large-grain rice varieties, or it can be integrated into rice functional gene chips as a core marker, or used to develop low-cost PCR detection kits, which is highly practical. It provides a direct molecular tool for the genetic improvement of rice thousand-grain weight, which helps to achieve the targeted breeding of high-yield, large-grain rice varieties.

[0009] In some implementations, haplotype molecular markers include a first group of haplotype molecular markers and a second group of haplotype molecular markers.

[0010] In this invention, when rice carries the dominant haplotype GA or the dominant haplotype GTTAG, its thousand-grain weight is significantly increased; when rice carries both the dominant haplotype GA and the dominant haplotype GTTAG, its thousand-grain weight is further significantly increased; the dominant haplotype GA and the dominant haplotype GTTAG have a synergistic effect.

[0011] In a second aspect, the present invention provides the application of any of the haplotype molecular markers described above in the identification or auxiliary identification of the thousand-grain weight phenotype of rice.

[0012] In a third aspect, the present invention provides the application of substances that detect any of the above-mentioned haplotype molecular markers in the identification or auxiliary identification of the thousand-grain weight phenotype of rice.

[0013] Understandably, the substance can be a reagent or kit, as long as it can efficiently obtain the genotype of the haplotype molecular marker.

[0014] In a fourth aspect, the present invention provides a gene chip for detecting the thousand-grain weight phenotype of rice, including probes for detecting any of the above-mentioned SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7 sites in rice.

[0015] In this invention, the genotype of haplotype molecular markers is detected by probes, thereby enabling accurate and rapid acquisition of the thousand-grain weight phenotype of rice.

[0016] In a fifth aspect, the present invention provides the application of any of the haplotype molecular markers or gene chips described above in rice breeding.

[0017] In a sixth aspect, the present invention provides the application of any of the haplotype molecular markers or gene chips described above in screening and / or identifying high-yielding and / or large-grained rice varieties.

[0018] In a seventh aspect, the present invention provides a method for detecting haplotype molecular markers related to the thousand-grain weight of rice, comprising the following steps: extracting genomic DNA from the rice sample to be tested; detecting the genotype of any of the haplotype molecular markers mentioned above in the genomic DNA to obtain the genotype of the haplotype molecular marker.

[0019] In some implementations, the step of detecting the genotype of any of the haplotype molecular markers mentioned above in genomic DNA includes at least one of whole-genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

[0020] It is understandable that the detection method can be conventionally selected based on the actual situation, as long as the genotype of the haplotype molecular marker can be obtained. For example, in this invention, the detection method preferably includes at least one of whole-genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

[0021] In an eighth aspect, the present invention provides a method for breeding high-yield and / or large-grain rice varieties, comprising the following steps: detecting the genotype of any of the haplotype molecular markers mentioned above in rice samples, and selecting rice samples with dominant haplotype GA and / or dominant haplotype GTTAG for breeding.

[0022] This invention provides a simple breeding method that can quickly and accurately predict the thousand-grain weight of rice during the seedling stage without waiting for the grains to mature, thus greatly improving breeding efficiency.

[0023] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention obtains haplotype molecular markers related to the thousand-grain weight of rice through association analysis and haplotype analysis of rice genomic and phenotypic data; these haplotype molecular markers consist of multiple tightly linked SNPs, and their association with the "significantly increased thousand-grain weight" phenotype is extremely significant. P The value is <0.01), and it has good stability. By detecting this haplotype molecular marker, the thousand-grain weight performance of rice can be predicted quickly and accurately at the seedling stage without waiting for the grains to mature, which greatly improves the breeding efficiency. In addition, this haplotype molecular marker can be directly used for molecular marker-assisted breeding of high-yield or large-grain rice varieties, or it can be integrated into rice functional gene chips as a core marker, or used to develop low-cost PCR detection kits, which is highly practical. It provides a direct molecular tool for the genetic improvement of rice thousand-grain weight, which helps to achieve the targeted breeding of high-yield, large-grain rice varieties. Attached Figure Description

[0024] Figure 1 This is a comparison chart of the thousand-grain weight phenotype differences between varieties carrying the dominant haplotype (GA) on chromosome 2 and those not carrying it (other haplotypes) in Example 2 of the present invention. "Indicates the result at the significance level" P <0.01 indicates extremely significant statistical significance; the error bars in the figure represent "mean ± 2 × standard error". The small range of the error bars indicates that the sample mean is close to the population mean, and the data is highly reliable. Figure 2 This is a comparison chart of the thousand-grain weight phenotype differences between varieties carrying the dominant haplotype on chromosome 9 (GTTAG) and those not carrying it (other haplotypes) in Example 2 of the present invention. "Indicates the result at the significance level" P<0.01 indicates extremely significant statistical significance; the error bars in the figure represent "mean ± 2 × standard error". The small range of the error bars indicates that the sample mean is close to the population mean, and the data is highly reliable. Figure 3 This is a comparison chart of the thousand-grain weight phenotypes in Example 2 of the present invention, showing the differences between varieties carrying both dominant haplotypes of chromosomes 2 and 9 (double dominance) and varieties carrying only a single dominant haplotype (single dominance) or non-carrying varieties (double disadvantage). "Indicates the result at the significance level" P <0.001, which is statistically significant. "Indicates the result at the significance level" P <0.01, which is statistically significant. "Indicates the result at the significance level" P <0.05 indicates extremely significant statistical significance; the error bars in the figure represent “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

[0025] 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.

[0026] 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.

[0027] Example 1: Obtaining molecular markers for dominant haplotypes in rice thousand-grain weight In this embodiment, the aim is to screen and discover dominant haplotype molecular markers in rice thousand-grain weight.

[0028] Specifically, it includes the following steps: 1) Data Sources and Processing 3,605 rice varietal resources with complete thousand-grain weight phenotype records and whole-genome SNP genotype data were obtained from the publicly available rice genome database RiceAtlas. The thousand-grain weight was recorded in grams (g).

[0029] 2) Determination of dominant haplotype PCA (principal component analysis) was performed on the above genotype data using PLINK software, with 10 principal components. GWAS analysis was performed on the rice genotype and phenotypic data using Fast3VmrMLM (version 1.0) software, with the software covariate file being the results of the PCA analysis above, and the remaining parameters set to default values.

[0030] Haplotype analysis was performed on SNP sites in the GWAS results: The haplotype block estimation function (--blocks) of PLINK software was used to search for strongly associated SNPs (correlation coefficient r) within a 200kb range upstream and downstream of significant SNP sites. 2 Haplotype blocks with a value greater than 0.9.

[0031] SNP genotype information was extracted from the haplotype blocks, and statistical analysis was performed on haplotype blocks of different phenotypes in combination with sample phenotypic values. Haplotypes with "sample quantity ≥ 20 and corresponding phenotype as dominant" were defined as dominant haplotypes.

[0032] Association analysis and haplotype analysis of large-scale genomic and phenotypic data revealed a haplotype block closely related to thousand-grain weight in the 25026065-25026078 bp region of chromosome 2. Two SNP loci within this region (located in Table 1, SNP1 and SNP2) exhibited strong linkage disequilibrium in the population. Statistical analysis showed that when the genotype combination of these two SNP loci was "GA", varieties carrying this haplotype showed a significantly increased thousand-grain weight.

[0033] Meanwhile, another haplotype block associated with thousand-grain weight was found in chromosome 9, from 5888189 to 5888336 bp, containing five closely linked SNP loci (locations shown in Table 1, SNP3-7). When the genotype combination of these five loci was "GTTAG", it was also significantly associated with an increased thousand-grain weight.

[0034] The physical locations of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7 were determined based on the whole genome sequence MSU7.0 version of Nipponbare.

[0035] Table 1. Information on dominant haplotypes per thousand grains of rice

[0036] Example 2: Validation of molecular markers for dominant haplotypes in rice thousand-grain weight In this embodiment, the validation data came from self-developed sequencing data, including 500 rice samples of different varieties collected from fields in Ezhou and Gong'an, Hubei Province. The thousand-grain weight phenotypic values ​​of these rice samples were measured, and genotypic data were obtained using next-generation sequencing technology.

[0037] 1) Verification of chromosome 2 haplotype (group I haplotype molecular markers) Statistical results of the above sample dataset show that among the 500 varieties, 48 ​​varieties carry the dominant haplotype "GA", and the remaining 452 varieties carry other haplotypes, as shown in Table 2.

[0038] Comparing the thousand-grain weight data of 48 dominant haplotype varieties with 452 other haplotype varieties, the results are as follows: Figure 1 As shown.

[0039] from Figure 1 It can be seen that the average thousand-grain weight of the variety population carrying the dominant haplotype was significantly higher than that of the non-carrying variety population, and the difference reached a highly significant level. P <0.01). This fully demonstrates that the haplotype "GA" can serve as a dominant molecular marker for predicting the thousand-grain weight trait in rice.

[0040] 2) Validation of chromosome 9 haplotype (second group haplotype molecular markers) Of the 500 varieties, 252 varieties carried the dominant haplotype "GTTAG", while the remaining 248 varieties carried other haplotypes, as shown in Table 2.

[0041] Comparing the thousand-grain weight data of 252 dominant haplotype varieties with 248 other haplotype varieties, the results are as follows: Figure 2 As shown.

[0042] from Figure 2 It can be seen that the average thousand-grain weight of the variety population carrying the dominant haplotype was significantly higher than that of the non-carrying variety population, and the difference reached a highly significant level. P <0.01). This fully demonstrates that the haplotype "GTTAG" can serve as a dominant molecular marker for predicting the thousand-grain weight trait in rice.

[0043] 3) Validation of the dominant haplotype superposition effect (simultaneous presence of both group 1 and group 2 haplotype molecular markers). Among the 500 varieties mentioned above, 13 varieties carried dominant haplotypes on both chromosomes 2 and 9, 274 varieties carried dominant haplotypes in only one haplotype patch, and the remaining 213 varieties did not carry dominant haplotypes. The details are shown in Table 2.

[0044] Comparing the thousand-grain weight data of the above varieties, the results are as follows: Figure 3 As shown, from Figure 3 It can be seen that the average thousand-grain weight of the variety population carrying the double-dominant haplotype is significantly higher than that of the single-dominant variety population. P <0.05, while the average thousand-grain weight of the single dominant group was significantly higher than that of the double inferior group ( P The difference was <0.001), and the difference reached a highly significant level, which fully verified the superposition effect of the two dominant haplotypes.

[0045] Table 2. Phenotypes and haplotypes of 1000-grain weight of 500 rice varieties

[0046] 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.

[0047] 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. A haplotype molecular marker associated with 1000-grain weight in rice, characterized in that, Including at least one of the first group of haplotype molecular markers and the second group of haplotype molecular markers; The first group of haplotype molecular markers includes SNP1 and SNP2 sites. The SNP1 site is located at 25026065 bp on chromosome 2, and the polymorphic site is G or A. The SNP2 site is located at 25026078 bp on chromosome 2, and the polymorphic site is A or G. When the dominant haplotype formed by the SNP1 and SNP2 sites is GA, it indicates an increase in the thousand-grain weight of rice varieties. The second group of haplotype molecular markers includes SNP3, SNP4, SNP5, SNP6, and SNP7 loci. SNP3 is located at 5888189 bp on chromosome 9, with a polymorphic site of G or T; SNP4 is located at 5888211 bp on chromosome 9, with a polymorphic site of T or C; SNP5 is located at 5888261 bp on chromosome 9, with a polymorphic site of T or G; SNP6 is located at 5888315 bp on chromosome 9, with a polymorphic site of A or G; and SNP7 is located at 5888336 bp on chromosome 9, with a polymorphic site of G or A. When the dominant haplotype formed by the SNP3, SNP4, SNP5, SNP6, and SNP7 loci is GTTAG, it indicates an increase in the thousand-grain weight of rice varieties. The physical locations of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7 loci were determined based on the whole genome sequence MSU7.0 version from Nipponbare.

2. The haplotype molecular marker of claim 1, wherein The haplotype molecular markers include a first group of haplotype molecular markers and a second group of haplotype molecular markers.

3. The application of haplotype molecular markers as described in any one of claims 1-2 in identifying or assisting in the identification of the thousand-grain weight phenotype of rice.

4. The use of the haplotype molecular marker of any one of claims 1-2 in identifying or assisting in the identification of the thousand-grain weight phenotype of rice.

5. A gene chip for detecting the phenotype of 1000-grain weight of rice, characterized by, Including probes for detecting SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, and SNP7 sites in rice as described in any one of claims 1-2.

6. The application of haplotype molecular markers as described in any one of claims 1-2 or the gene chip as described in claim 5 in rice breeding.

7. The application of the haplotype molecular marker as described in any one of claims 1-2 or the gene chip as described in claim 5 in screening and / or identifying high-yielding and / or large-grained rice varieties.

8. A method for detecting haplotype molecular markers related to thousand-grain weight in rice, characterized in that, Includes the following steps: Genomic DNA was extracted from the rice samples to be tested; The genotype of the haplotype molecular marker in the genomic DNA as described in any one of claims 1-2 is detected to obtain the genotype of the haplotype molecular marker.

9. The detection method according to claim 8, characterized in that, In the step of detecting the genotype of the haplotype molecular marker in the genomic DNA as described in any one of claims 1-2, the detection method includes at least one of whole genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

10. A method for breeding high-yield and / or large-grain rice varieties, characterized in that, The method includes the following steps: detecting the genotype of the haplotype molecular marker in rice samples as described in any one of claims 1-2, and selecting rice samples with the dominant haplotype GA and / or the dominant haplotype GTTAG for breeding.

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