Application of haplotype molecular marker of rice lodging-resistant gene STRONG2 in rice breeding

Abstract

The invention discloses application of a haplotype molecular marker of a rice lodging-resistant gene STRONG2 in rice breeding, and belongs to the technical field of plant molecular breeding. The haplotype molecular marker of the rice lodging-resistant gene STRONG2 is obtained by screening a core sequence of the rice lodging-resistant gene STRONG2 and SNP sites of upstream and downstream sequences of the gene, and the haplotype molecular marker can quickly identify the function of the rice lodging-resistant gene STRONG2, so that the lodging resistance of a rice variety is more efficiently and directly judged; therefore, the haplotype molecular marker has a good application prospect in rice breeding, and when the haplotype molecular marker is applied to rice breeding, the breeding period can be remarkably shortened, the breeding efficiency can be improved, and manpower and material resources can be saved; besides, the haplotype molecular marker can be used for developing a rice lodging-resistant gene STRONG2 chip or designing a detection marker based on PCR (Polymerase Chain Reaction), and is very practical.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding, and particularly relates to the application of a haplotype molecular marker of a rice lodging-resistant gene STRONG2 in rice breeding. Background Art

[0002] Molecular markers are polymorphism markers based on genetic material DNA. According to the association relationship between markers and traits, functional markers or tightly linked markers of genes can be used to quickly identify the functional genes contained in varieties. Single Nucleotide Polymorphisms (SNP) markers are the most numerous and widely distributed type of molecular marker. The development of SNP markers is based on DNA sequencing. In the past two decades, genomic sequencing has developed rapidly, and the sequencing cost has decreased significantly. Scientists around the world have sequenced thousands of rice germplasm resources and discovered a large number of SNP markers. At the same time, the completion of large-scale and high-quality rice genome sequencing has also laid a solid foundation for screening functional gene haplotypes.

[0003] The DNA genetic variations reflected by SNPs are more reflected at the level of single-base variations, which to a certain extent make up for the deficiencies in the methods of the first-generation molecular markers (such as Restriction Fragment Length Polymorphism, RFLP) and the second-generation molecular markers (such as Microsatellite DNA Polymorphism, SSR), so they are called the third-generation gene genetic markers. The main advantages of SNPs are: high density, wide distribution, and in the rice gene sequence, one SNP appears every 232 bp; and they can be quickly detected by high-throughput methods and automated analysis can be achieved.

[0004] Rice is one of the most important food crops in the world. Studying rice lodging-resistant genes helps to improve lodging resistance, improve quality, and enhance resource utilization efficiency, providing important support for global food security and sustainable agricultural development. The rice lodging-resistant gene STRONG2 positively regulates the stem diameter by increasing the content of mannose and cellulose in rice. Existing studies have shown that the natural variation of the SNP14931253 functional site in its promoter region regulates the transcription level of STRONG2 by affecting the binding of the transcription factor MYB61 to the STRONG2 gene promoter, and then regulates the stem diameter of rice, thereby affecting the lodging resistance of rice varieties. However, there are few research reports on the haplotype molecular markers closely linked to the rice lodging-resistant gene STRONG2. Summary of the Invention

[0005] The object of the present invention is to provide the application of the haplotype molecular marker of the rice lodging resistance gene STRONG2 in rice breeding, so as to solve the problem in the prior art that there is a lack of haplotype molecular markers that can quickly identify the function of the rice lodging resistance gene STRONG2 and then efficiently and directly judge the lodging resistance of rice varieties.

[0006] In the first aspect, the present invention provides the application of the haplotype molecular marker of the rice lodging resistance gene STRONG2 in rice breeding. The haplotype molecular marker includes SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci. Among them, the genomic position where SNP1 is located is at 14921508 bp on chromosome 3, and the polymorphism is A or G; the genomic position where SNP2 is located is at 14921839 bp on chromosome 3, and the polymorphic site is T or C; the genomic position where SNP3 is located is at 14922417 bp on chromosome 3, and the polymorphism is C or T; the genomic position where SNP4 is located is at 14924358 bp on chromosome 3, and the polymorphic site is C or T; the genomic position where SNP5 is located is at 14925325 bp on chromosome 3, and the polymorphism is A or G; the genomic position where SNP6 is located is at 14925960 bp on chromosome 3, and the polymorphism is A or C; the genomic position where SNP7 is located is at 14928230 bp on chromosome 3, and the polymorphism is G or T; the genomic position where SNP8 is located is at 14928766 bp on chromosome 3, and the polymorphism is C or G; the genomic position where SNP9 is located is at 14928962 bp on chromosome 3, and the polymorphism is T or C; the genomic position where SNP10 is located is at 14929229 bp on chromosome 3, and the polymorphism is T or C; the genomic position where SNP11 is located is at 14931253 bp on chromosome 3, and the polymorphism is C or T; the genomic position where SNP12 is located is at 14931647 bp on chromosome 3, and the polymorphism is C or T. The physical positions of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci are determined based on the whole-genome sequence MSU7.0 version of Nipponbare.

[0007] In the present invention, the inventor screened the core sequence of the rice lodging resistance gene STRONG2 and the SNP sites of the upstream and downstream sequences of this gene, and obtained a haplotype molecular marker of the rice lodging resistance gene STRONG2. This haplotype molecular marker can quickly identify the function of the rice lodging resistance gene STRONG2, thereby more efficiently and directly judging the lodging resistance of rice varieties. Therefore, it has good application prospects in rice breeding. When this haplotype molecular marker is applied to rice breeding, it can significantly shorten the breeding cycle, improve the breeding efficiency, and save manpower and material resources. In addition, this haplotype molecular marker can be used for the development of a rice lodging resistance gene STRONG2 chip, or for designing PCR-based detection markers, which is very practical.

[0008] In some embodiments, the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 sites in sequence is ATCCAAGCTTCC or GCTTGCTGCCTT.

[0009] In some embodiments, when the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 sites in sequence is ATCCAAGCTTCC, the rice lodging resistance gene STRONG2 reduces the rice stem diameter; or when the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 sites in sequence is GCTTGCTGCCTT, the rice lodging resistance gene STRONG2 increases the rice stem diameter.

[0010] In some embodiments, the rice includes lodging-resistant rice.

[0011] In a second aspect, the present invention provides a method for detecting a haplotype molecular marker of the rice lodging resistance gene STRONG2, including the following steps: detecting the genomic sequence of rice to obtain the genomic sequence result of the rice; and detecting the genotype of the haplotype molecular marker of the above-mentioned rice lodging resistance gene STRONG2 according to the genomic sequence result of the rice.

[0012] In some embodiments, the method for detecting the genomic sequence of rice includes at least one of whole-genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

[0013] Understandably, the method for detecting the genomic sequence of rice can be routinely selected according to the actual situation, as long as the genomic sequence of rice can be obtained; in the present invention, the method for detecting the genomic sequence of rice preferably includes at least one of whole-genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

[0014] In a third 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 lodging resistance gene STRONG2 in a rice sample, and selecting a rice sample with the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci in sequence being GCTTGCTGCCTT for breeding.

[0015] The method for rice breeding provided by the present invention is simple and efficient, and uses the haplotype molecular marker of the rice lodging resistance gene STRONG2 for assisted breeding, having the advantage of high accuracy.

[0016] In some embodiments, the phenotype of the rice sample with the haplotype GCTTGCTGCCTT is analyzed, and a rice sample with a large culm diameter is selected for breeding.

[0017] In a fourth aspect, the present invention provides the application of a substance for detecting the haplotype molecular marker of the rice lodging resistance gene STRONG2 in rice breeding.

[0018] In some embodiments, the substance includes at least one of a primer set and a kit.

[0019] Understandably, the substance can be routinely selected according to the actual usage needs, as long as the genotype of the haplotype molecular marker of the rice lodging resistance gene STRONG2 can be detected; in the present invention, the substance preferably includes at least one of a primer set and a kit.

[0020] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention screens the SNP sites of the core sequence of the rice lodging resistance gene STRONG2 and the upstream and downstream sequences of this gene, and obtains the haplotype molecular marker of the rice lodging resistance gene STRONG2. This haplotype molecular marker can quickly identify the function of the rice lodging resistance gene STRONG2, thereby more efficiently and directly judging the lodging resistance of rice varieties. Therefore, it has good application prospects in rice breeding. When this haplotype molecular marker is applied to rice breeding, it can significantly shorten the breeding cycle, improve the breeding efficiency, and save manpower and material resources. In addition, this haplotype molecular marker can be used for the development of a rice lodging resistance gene STRONG2 chip, or for designing PCR-based detection markers, which is very practical. Description of the Drawings

[0021] Figure 1 This is a combination of two haplotype markers of the rice lodging resistance gene STRONG2 and its adjacent sequences in Example 1 of the present invention. Among them, the 11th site is the functional site of the rice lodging resistance gene STRONG2. "C" means that reducing the rice stem diameter results in weak lodging resistance, and "T" means that increasing the rice stem diameter results in strong lodging resistance.

[0022] Figure 2 This is a result diagram of the analysis of the stem diameter differences between Hap1 and Hap2 haplotype rice varieties in Example 2 of the present invention. Among them, "***" indicates that the results are extremely significantly statistically significant at a significance level of P < 0.001. The error bars in the figure are "mean ± 2 × standard error". The range of the error bars is small, indicating that the sample mean is close to the population mean and the data reliability is high. Detailed Embodiments

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0024] The experimental methods without specific conditions indicated in the embodiments are usually carried out according to the conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in "Molecular Cloning: A Laboratory Manual" written by M.R. Green, "Molecular Biology" written by Robert·F·Weaver, etc., or according to the experimental methods recommended by the reagent and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial channels without special instructions.

[0025] Example 1 Obtaining Haplotype Molecular Markers for the Rice Lodging Resistance Gene STRONG2

[0026] In this example, haplotype markers for the rice lodging resistance gene STRONG2 were screened and designed based on the information of 4,726 rice varieties and 14,541,446 SNP loci provided by the website RiceVarMap. Specifically, the following steps were included:

[0027] 1) The functional gene locus of the rice lodging resistance gene STRONG2 was found according to the article published by the team of Zhang Zhanying / Li Zichao from the College of Agronomy, China Agricultural University (Zhao, Y., Wang, X., Gao, J., Rehman Rashid, M.A., Wu, H., Hu, Q., Sun, X, Li, J, Zhang, H., Xu, P., et al. (2025). The MYB61-STRONG2 module regulates culm diameter and lodging resistance in rice. J. Integr. Plant Biol. 67:243 - 257.). The specific information is shown in Table 1 below.

[0028] Table 1 Information on the Functional Gene Locus of the Rice Lodging Resistance Gene STRONG2

[0029]

[0030]

[0031] 2) The SNP variation information of 4,726 rice varieties was downloaded from the RiceVarMap v2.0 website, and then the SNP genotype information of the regions 10 kb upstream and downstream of the functional locus of the rice lodging resistance gene STRONG2 was extracted. A total of 368 SNP variation sites were from 4,726 rice varieties.

[0032] 3) The vcf file of the 368 SNP gene information from the above 4,726 varieties was converted into the plink format using the software PLINK, and then the correlation coefficient r 2 (between 0 and 1, indicating the degree of linkage between two SNPs, equal to 1 when the two SNPs are completely linked) greater than 0.64 with the SNP locus (vg0314931253) of the functional gene of the rice lodging resistance gene STRONG2 was obtained through software PLINK analysis, and 11 SNP loci highly associated with the vg0314931253 locus in the promoter region of the rice lodging resistance gene STRONG2 were obtained.

[0033] 4) Genotypes of the above 12 SNP loci in 4,726 rice varieties were extracted, and haplotype analysis was performed using Haploview software with the "Examine haplotypes above" parameter set to 0.6, obtaining two groups of haplotypes, Hap1 and Hap2. The results are as Figure 1 shown.

[0034] 5) Organize the genotypes of the two groups of haplotypes, Hap1 and Hap2, in Figure 1 . The specific information is shown in Table 2 below. Among them, for rice varieties with the Hap1 haplotype, the rice lodging resistance gene STRONG2 has a genotype that reduces the culm diameter of rice and results in weak lodging resistance. For rice varieties with the Hap2 haplotype, the rice lodging resistance gene STRONG2 has a genotype that increases the culm diameter of rice and results in strong lodging resistance.

[0035] Table 2 Haplotype markers of the rice lodging resistance gene STRONG2

[0036]

[0037]

[0038] As can be seen from Table 2, the two groups of haplotype molecular markers are highly linked to the functional locus (chr03:14931253) of the rice lodging resistance gene STRONG2 and can be used to predict the lodging resistance of rice.

[0039] Example 2 Application of haplotype molecular markers of the rice lodging resistance gene STRONG2

[0040] According to the tabular data file provided in the article published by the team of Zhang Zhanying / Li Zichao from the College of Agronomy, China Agricultural University (Zhao, Y., Wang, X., Gao, J., Rehman Rashid, M.A., Wu, H., Hu, Q., Sun, X, Li, J, Zhang, H., Xu, P., et al. (2025). The MYB61 - STRONG2 module regulates culm diameter and lodging resistance in rice. J. Integr. Plant Biol. 67:243 - 257.), 340 rice varieties contain phenotypic records of culm diameter. Query the genotypes of 244 rice varieties on the RiceVarMap v2.0 website. The results show that 76 rice varieties are the "Hap1" haplotype in Example 1, and 168 rice varieties are the "Hap2" haplotype. The specific situation is shown in Table 3 below.

[0041] Table 3 Phenotypes of 244 rice varieties and haplotype markers of STRONG2

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049] Comparing the stem diameter phenotypes of the above 76 Hap1 rice varieties and 168 Hap2 rice varieties, the results are as Figure 2 shown.

[0050] From Figure 2 it can be seen that the varieties carrying the STRONG2 Hap1 haplotype all have smaller rice stem diameters, while the varieties carrying the STRONG2 Hap2 haplotype have larger rice stem diameters. The above results indicate that the haplotype molecular marker of the rice lodging resistance gene STRONG2 can accurately identify the thickness of the rice stem and thus determine the lodging resistance of the sample.

[0051] In summary, the haplotype molecular marker provided by the present invention can quickly identify the function of the rice lodging resistance gene STRONG2. When this haplotype molecular marker is applied to rice breeding, it can significantly shorten the breeding cycle, improve the breeding efficiency, and save manpower and material resources. In addition, this haplotype molecular marker can be used for the development of a rice lodging resistance gene STRONG2 chip, or for designing PCR-based detection markers, which is very practical.

[0052] It should be noted that the above embodiments all belong to the same inventive concept. The descriptions of the embodiments have their own emphases. For the parts not described in detail in individual embodiments, reference can be made to the descriptions in other embodiments.

[0053] The above embodiments only represent the implementation manners of the present invention. The descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. Application of the haplotype molecular marker of the rice lodging resistance gene STRONG2 in rice breeding, characterized in that, The haplotype molecular markers include SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci; Among them, the genomic location of SNP1 is at 14,921,508 bp on chromosome 3, and the polymorphism is A or G; the genomic location of SNP2 is at 14,921,839 bp on chromosome 3, and the polymorphic site is T or C; the genomic location of SNP3 is at 14,922,417 bp on chromosome 3, and the polymorphism is C or T; the genomic location of SNP4 is at 14,924,358 bp on chromosome 3, and the polymorphic site is C or T; the genomic location of SNP5 is at 14,925,325 bp on chromosome 3, and the polymorphism is A or G; the genomic location of SNP6 is at 14,925,960 bp on chromosome 3, and the polymorphism is A or C; the genomic location of SNP7 is at 14,928,230 bp on chromosome 3, and the polymorphism is G or T; the genomic location of SNP8 is at 14,928,766 bp on chromosome 3, and the polymorphism is C or G; the genomic location of SNP9 is at 14,928,962 bp on chromosome 3, and the polymorphism is T or C; the genomic location of SNP10 is at 14,929,229 bp on chromosome 3, and the polymorphism is T or C; the genomic location of SNP11 is at 14,931,253 bp on chromosome 3, and the polymorphism is C or T; the genomic location of SNP12 is at 14,931,647 bp on chromosome 3, and the polymorphism is C or T; The physical locations of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci are determined based on the whole-genome sequence of Nipponbare version MSU7.

0.

2. The application according to claim 1, wherein The haplotypes composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci in sequence are ATCCAAGCTTCC or GCTTGCTGCCTT.

3. The application according to claim 2, characterized in that, When the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci in sequence is ATCCAAGCTTCC, the rice lodging resistance gene STRONG2 reduces the rice stem diameter; or when the haplotype composed of the SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci in sequence is GCTTGCTGCCTT, the rice lodging resistance gene STRONG2 increases the rice stem diameter.

4. The application according to claim 1, characterized in that The rice includes lodging-resistant rice.

5. A detection method for haplotype molecular markers of the rice lodging resistance gene STRONG2, characterized in that, It includes the following steps: detecting the genomic sequence of rice to obtain the genomic sequence result of rice; detecting the genotype of the haplotype molecular marker of the rice lodging resistance gene STRONG2 as claimed in claim 1 according to the genomic sequence result of the rice.

6. The detection method according to claim 5, wherein The method for detecting the genomic sequence of rice includes at least one of whole genome resequencing, targeted sequencing, multiplex PCR sequencing, and gene chip detection.

7. A method for rice breeding, characterized in that, It includes the following steps: detecting the genotype of the haplotype molecular marker of the rice lodging resistance gene STRONG2 as claimed in claim 1 in a rice sample, and selecting a rice sample with the haplotype composed of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, and SNP12 loci in sequence being GCTTGCTGCCTT for breeding.

8. The method according to claim 7, wherein Analyze the phenotype of the rice sample with the haplotype of GCTTGCTGCCTT, and select a rice sample with a large stem diameter for breeding.

9. Application of a substance for detecting the haplotype molecular marker of the rice lodging resistance gene STRONG2 in rice breeding.

10. The application according to claim 9, characterized in that, The substance includes at least one of a primer set and a kit.

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