Haplotype identification marker of fertility restorer gene rf4 in rice

Abstract

The application belongs to the field of plant molecular breeding, and specifically discloses haplotype identification markers of a rice fertility restoration gene Rf4 Based on the high-quality rice reference genome sequences of 33 different varieties published by Professor Qin Peng's team of Sichuan Agricultural University, the published gene sequences or functional gene sites are compared to distinguish the varieties containing target genes, and then the bioinformatics method is used to quickly develop a haplotype identification marker combination of a rice fertility restoration gene Rf4 .

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular breeding, and in particular relates to a group of haplotype markers for identifying a rice fertility restorer gene Rf4. Background Art

[0002] Rice is one of the most important food crops in the world, and how to increase the yield of rice varieties has always been the main goal of scientists. The success of the three-line system has solved the problem of large-scale hybrid rice seed production. The recovery ability of the restorer line directly affects the yield of seed production. In addition to the major fertility restorer genes Rf3, Rf4, Rf1a, Rf1b, and Rf2, there are also some minor fertility restorer genes that have been located and cloned (Tomohiko et al., A fertility restorer gene, Rf4, widely used for hybrid rice breeding encodes a pentatricopeptide repeat protein. Rice (NY) 2014; 7:28.). The combined effect of the major and minor fertility restorer genes in the restorer line determines the recovery ability of the restorer line.

[0003] Molecular markers are polymorphic markers based on the genetic material DNA. Based on the correlation between markers and traits, functional markers or tightly linked markers can be used to rapidly identify functional genes within a variety. Single-nucleotide polymorphism (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 decade since the advent of the 454 sequencer in 2005, next-generation sequencing technology has been continuously refined, significantly increasing genome sequencing efficiency and reducing sequencing costs. Whole genome sequences have been completed for numerous species, greatly advancing functional genomic research. Over the past few decades, scientists worldwide have cloned multiple rice genes and sequenced thousands of rice germplasm resources, discovering a large number of SNP markers.

[0004] Rice is a model plant for monocot research and is also the most important food crop in the world. Molecular biology researchers prioritize resequencing different varieties, using the SNP markers discovered through resequencing to construct a high-density rice haplotype map (HapMap), and using genome-wide association mapping (GWAS) to conduct association analysis on important agronomic traits, identify candidate gene loci associated with these traits, and establish a set of efficient, rapid, mature, stable, low-cost, and high-throughput genotyping methods.

[0005] In rice, 4726 cultivated rice varieties have been resequenced. Of these, 3243 were completed by Li Zhikang's group and published in GigaScience (The 3,000 Rice Genomes Project, 2014), 950 were completed by Han Bin's group and published in Nat Genet (Huang et al., 2010) (Huang et al., 2012), and 533 were completed by a rice group at Huazhong Agricultural University and published in Nucleic Acids Res (Zhao et al., 2014). In 2021, Professor Qin Peng's group at Sichuan Agricultural University completed the pan-genome sequencing and assembly of 33 rice varieties, obtaining reference genome-level sequences, which were published in Cell (Qin et al., 2021). The completion of large-scale, high-quality rice genome sequencing has laid a solid foundation for screening haplotypes of functional genes.

[0006] The cytoplasmic male sterility (CMS) system is the genetic basis for exploiting heterosis in crops. Currently, the production and application of "three-line" hybrid rice (Oryza sativa L.) primarily relies on wild-abortive cytoplasmic male sterility and its restorer system (CMS-WA / Rf). Research has shown that the restorer gene Rf4, encoding a mitochondrially localized PPR (pentatricopeptide repeat) protein, can restore fertility in wild-abortive cytoplasmic male sterile rice lines. This restorer gene, Rf4, encodes a mitochondrially localized PPR (pentatricopeptide repeat) protein that restores fertility by degrading the mRNA transcript of the sterility gene WA352. Currently, the restoration ability of restorer lines is primarily verified by examining fertility after hybridization. If the presence of fertility restorer genes in rice varieties could be accurately identified, the restoration ability of the variety could be rapidly assessed without hybridization.

[0007] The differences between rice varieties are essentially differences in genotype, and DNA molecular markers can directly reflect differences at the genetic level. The DNA genetic variation reflected by the single nucleotide polymorphisms (SNPs) used in the present invention is more reflected in the variation level of a single base, which to a certain extent makes up for the shortcomings of first-generation molecular markers (such as restriction fragment length polymorphisms, RFLP) and second-generation molecular markers (such as microsatellite DNA polymorphisms, SSR) methods, so it is called the third generation of genetic marker methods. The main advantages of SNPs are: high density and wide distribution. In rice, one SNP appears every 232bp; it can quickly achieve high-throughput detection and is easy to implement automated analysis.

[0008] The rich diversity and complex mechanisms of action of rice genes form the genetic foundation for rice breeding and improvement. Global scientists have significantly enriched rice germplasm resources and databases of high-quality rice gene diversity. This provides sufficient gene sources and genetic information for precise selection of breeding parents for whole-genome molecular breeding, laying the foundation for the development of high-yield, high-quality, and multi-resistant rice varieties. This present invention develops precise identification markers based on functional genes and applies these findings to rice genetic improvement. Summary of the Invention

[0009] The purpose of this invention is to develop a set of haplotype marker combinations for identifying the rice fertility restorer gene Rf4. Based on the high-quality rice reference genome sequences of 33 different varieties recently published by Professor Qin Peng's team at Sichuan Agricultural University, the authors compared the published gene sequences or functional gene loci to distinguish varieties containing the target gene. Subsequently, bioinformatics methods were used to rapidly develop a set of haplotype marker combinations for identifying the rice fertility restorer gene Rf4. Simultaneously, using the already developed rice gene chip GSR40K, the researchers screened a set of marker combinations on the GSR40K gene chip that can accurately identify the function of the fertility restorer gene Rf4.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] Based on the reference genome sequences of 33 different varieties of rice published in Cell by Professor Qin Peng's team at Sichuan Agricultural University, a haplotype marker combination of the fertility restorer gene Rf4 consisting of 196 SNP sites was screened out. The SNP site information is shown in Table 3 in Example 1.

[0012] Furthermore, using the developed rice gene chip GSR40K, we screened all high-quality markers in the fertility restorer gene Rf4 region of the rice GSR40K gene chip, and identified seven SNP sites that intersected with the above markers. The specific information is as follows:

[0013] The genomic location of SNP1 is 18819461bp on chromosome 10, where the base is C;

[0014] The genomic location of SNP2 is 18820606bp on chromosome 10, where the base is T;

[0015] The genomic location of SNP3 is 18837209bp on chromosome 10, where the base is G;

[0016] The genomic location of SNP4 is 18844245bp on chromosome 10, where the base is T;

[0017] The genomic location of SNP5 is 18844789bp on chromosome 10, where the base is T;

[0018] The genomic location of SNP6 is 18852356bp on chromosome 10, where the base is T;

[0019] The genomic location of SNP7 is 18852860bp on chromosome 10, where the base is C;

[0020] The physical locations of the above SNP sites were determined based on the MSU7.0 version of the whole genome sequence of Nipponbare.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] Using molecular markers to identify the functional genes of rice varieties and determine the phenotype of rice varieties is more efficient and direct.

[0023] By using the gene core sequence and upstream and downstream sequences, sufficient haplotype markers can be developed to identify the function of the gene. The haplotype markers of the functional genes screened by the present invention can be used to develop a rice fertility restorer gene Rf4 chip.

[0024] In the process of screening and optimizing markers, the present invention fully retains the polymorphic differential SNP typing markers, and also takes into account the spacing between markers and the specific sequence characteristics near the markers. The optimized marker combination can be directly used for chip design or for designing PCR-based detection markers, which is very practical.

[0025] This method uses a gene chip to accurately detect a relatively small number of SNP haplotype markers, enabling gene function identification. In practical applications, functional genes can be identified using a limited set of markers. Newly cloned genes can also be quickly optimized using similar methods to generate haplotype marker sets, eliminating the need to redesign markers for all new genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The 7 markers screened in Example 2 were used to identify the genotypes of the fertility restorer gene Rf4 in 33 rice varieties. DETAILED DESCRIPTION

[0027] Example 1:

[0028] Based on the pan-genome sequences of 33 rice genetically diverse materials published in Cell by Professor Qin Peng's team at Sichuan Agricultural University (Qin et al., 2021), we screened and designed haplotype markers for the cloned fertility restorer gene Rf4:

[0029] 1. Search for the functional site of the rice fertility restorer gene Rf4 (as shown in Table 1 below); screen all differential SNP markers within the 20 Kb region upstream and downstream of the Rf4 functional site in 33 high-quality genomic sequences, totaling 1,102,307 SNP markers.

[0030] Table 1: Function of the rice fertility restorer gene Rf4 (http: / / www.xhhuanglab.cn / tool / RiceNavi.html)

[0031]

[0032] 2. Based on the functional site of the rice fertility restorer gene Rf4 found in step 1, the varieties containing the Rf4 functional gene in 33 high-quality reference genomes were identified through sequence alignment, as shown in Table 2.

[0033] Table 2: Rice varieties containing the fertility restorer gene Rf4

[0034]

[0035] 3. From the SNP sites screened in step 1, select the haplotype markers for the optimized fertility restorer gene R f4 according to the following criteria:

[0036] 1) 94 SNP markers shared by rice varieties CG14, Tumba, and Y3551 that are inconsistent with the reference genome Nipponbare and different from 30 other varieties (Qin et al., 2021);

[0037] 2) There are 655 SNPs that are consistent between rice varieties CG14, Tumba, and Y3551 and the reference genome Nipponbare;

[0038] 3) The above two steps yielded a total of 749 marker combinations that can be used to identify the fertility restorer gene Rf4;

[0039] 4) 79 InDel markers were filtered out, leaving 670 markers;

[0040] 5) Retain uniformly distributed SNP markers: retain markers with adjacent distances greater than 100 bp; repeat the iterations to retain 196 markers with distances greater than 100 bp from the optimized markers.

[0041] 4. The haplotype markers of the fertility restorer gene Rf4 are as follows (the physical location of the SNP site is determined based on the MSU7.0 version of the Nipponbare whole genome sequence):

[0042] Table 3 Haplotype markers of fertility restorer gene Rf4

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] Example 2:

[0049] Based on the rice high-density whole-genome SNP chip GSR40K independently developed by Wuhan Shuanglvyuan Creative Technology Research Institute Co., Ltd., we further screened and optimized the haplotype markers of the fertility restorer gene Rf4:

[0050] 1. All high-quality markers in the fertility restorer gene Rf4 region of the rice GSR40K gene chip were screened, and a total of 11 high-quality SNP markers were found, as shown in Table 4. These 11 SNP sites were detected in all 33 high-quality genomes. Compared with the haplotype marker set screened in Example 1, only 7 markers were common intersection marker combinations (F1018748017CT, R1018749162AC, R1018765765CT, R1018772801CA, R1018773345GA, F1018780912TC, F1018781416TC). The remaining 4 markers (R1018744204AG, F1018766728TC, R1018769460AG, and R1018783722GA) were different from all the markers in Example 1.

[0051] Table 4 Marker combinations in the fertility restorer gene Rf4 gene segment in the GSR40K gene chip

[0052]

[0053] 2. Using the four markers R1018744204AG, F1018766728TC, R1018769460AG, and R1018783722GA from the above markers, we tested the CG14, Tumba, and Y3551 varieties. Their genotypes were inconsistent. The remaining seven markers overlapped with the haplotype marker set selected in Example 1. We used these seven marker loci to genotype 33 high-quality genomes. The results are as follows: Figure 1 shown.

[0054] 3. Based on the specific phenotypes of the 33 pan-genomic materials in Example 1, the screened common marker combinations were optimized to screen out a group of marker combinations that can accurately identify the function of the fertility restorer gene Rf4 in the gene chip GSR40K, as shown in Table 5.

[0055] Table 5: Haplotype markers in the gene chip GSR40K that can accurately identify the fertility restorer gene Rf4

[0056]

Claims

1. A reagent for detecting haplotype molecular markers of the rice fertility restorer gene Rf4, characterized in that: The haplotype molecular marker consists of 7 SNP sites, and the SNP site information is as follows: The genomic location of SNP1 is 18819461 bp on chromosome 10; The genomic location of SNP2 is 18820606bp on chromosome 10; The genomic location of SNP3 is 18,837,209 bp on chromosome 10; The genomic location of SNP4 is 18,844,245 bp on chromosome 10; The genomic location of SNP5 is 18,844,789 bp on chromosome 10; The genomic location of SNP6 is 18,852,356 bp on chromosome 10; The genomic location of SNP7 is 18,852,860 bp on chromosome 10; The physical locations of the above SNP sites were determined based on the MSU7.0 version of the whole genome sequence of Nipponbare.

2. Use of the reagent according to claim 1 in identifying the rice fertility restorer gene Rf4, characterized in that: The genotype of the SNP1 is C, the genotype of the SNP2 is T, the genotype of the SNP3 is G, the genotype of the SNP4 is T, the genotype of the SNP5 is T, the genotype of the SNP6 is T, and the genotype of the SNP7 is C, and the rice contains the restorer gene Rf4.

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