An SNP molecular marker for identifying the haplotype of the rice fertility restoration gene Rf4 and its application in restoring rice fertility

Abstract

The present invention discloses an SNP molecular marker for identifying the haplotype of the rice fertility restoration gene Rf4 and its application in restoring rice fertility. The present invention discovers that there are copy number variations and sequence base variations in the rice wild abortive cytoplasmic male sterility fertility restoration gene Rf4, and there are a total of 7 allelic genotype sequences and 8 haplotypes, including 5 single-copy haplotypes: rf4j, rf4i, rf4aus, Rf4a<supgt;I< / supgt;, and Rf4b<supgt;M< / supgt>, as well as 3 double-copy haplotypes: Rf4a<supgt;I< / supgt>-rf4b, rf4a-Rf4b<supgt;M< / supgt>, and Rf4a<supgt;M< / supgt>-Rf4b<supgt;M< / supgt>. Based on these allelic genotype sequences and SNP variations, the present invention develops a set of molecular markers to achieve the purpose of screening and identifying rice varieties, screening strong restoring rice containing the double-copy haplotype of Rf4a<supgt;M< / supgt>-Rf4b<supgt;M< / supgt>, and accelerating the breeding and identification of strong restoring lines by crossing with sterile lines.

Description

Technical Field

[0001] The present invention relates to the technical field of crop molecular genetic breeding, and particularly, to an SNP molecular marker for identifying the haplotype of rice fertility restoration gene Rf4 and its application in restoring rice fertility. Background Art

[0002] Rice (Oryza sativa) is the staple food crop for over half of the world's population and has made outstanding contributions to global food security. The discovery and application of rice cytoplasmic male sterility (CMS) and fertility restorer (Rf) germplasms are the core genetic materials for realizing the production of "three-line" hybrid rice, becoming a model for the utilization of crop heterosis and significantly increasing rice yield. The main types of CMS / Rf fertility control genetic systems used in "three-line" hybrid rice are three categories: Wild Abortive (CMS-WA / Rf3Rf4), BoroⅡ (CMS-BT / Rf1aRf1b), and Hong-Lian (CMS-HL / Rf5Rf6), among which 90% of "three-line" hybrid rice adopts the CMS-WA / Rf3Rf4 fertility control genetic system.

[0003] The CMS-WA / Rf3Rf4 fertility control genetic system consists of the WA352 sterile gene located in the mitochondrial genome and the major fertility restorer genes Rf3 and Rf4 located in the nuclear genome. Rf4 has been cloned (Tang et al., The rice restorer Rf4 for Wild abortive cytoplasmic male sterility encodes a mitochondrial-localized PPR protein that functions in reduction of WA352 transcripts. Molecular Plant, 2014, 7: 1497-1500.). It encodes a PPR (pentatricopeptide repeat) protein containing 782 amino acids. The Rf4 protein functions in restoring fertility by degrading the mRNA of WA352. It has been reported that there are multiple alleles of Rf4, including the dominant (functional) Rf4 in restorer lines, the recessive (non-functional) rf4i in indica sterile or maintainer lines, and the recessive rf4j in japonica rice (Tang et al., The rice restorer Rf4 for Wild abortive cytoplasmic male sterility encodes a mitochondrial-localized PPR protein that functions in reduction of WA352 transcripts. Molecular Plant, 2014, 7: 1497-1500.). In recent years, molecular marker-assisted selection breeding has been widely promoted and applied. The reported Rf4 molecular markers for marker-assisted selection breeding are all linked markers with a certain genetic distance from Rf4, which can only distinguish between the dominant Rf4 and recessive rf4 allelic genotypes, and cannot identify germplasm materials with different dominant Rf4 haplotypes, which is not conducive to the efficient selection of strong restorer lines. Currently, there is no report on effective molecular markers located within the Rf4 gene locus and its coding region that can identify different dominant Rf4 haplotypes. SUMMARY OF THE INVENTION

[0004] The object of the present invention is to overcome the above deficiencies of the prior art, and it is found that the fertility restoration gene Rf4 of cytoplasmic male sterile rice is a complex locus composed of two sub-loci a and b, with copy number variation and sequence base variation, and there are a total of 7 allelic genotype sequences. Based on these allelic genotype sequences and their single nucleotide polymorphism (SNP) variations, the present invention develops a set of differential PCR specific amplification molecular markers, and provides a SNP molecular marker for identifying the haplotype of the rice fertility restoration gene Rf4 and its application in restoring rice fertility.

[0005] The first object of the present invention is to provide the application of a reagent for detecting SNP sites in the rice genome.

[0006] The second object of the present invention is to provide a set of PCR reagents for differentiating or assisting in differentiating rice varieties, detecting the similarity between any two rices, and / or identifying rice fertility.

[0007] The third object of the present invention is to provide a kit containing the above-mentioned PCR reagents.

[0008] The fourth object of the present invention is to provide the application of the above-mentioned kit in differentiating or assisting in differentiating rice varieties, detecting the similarity between any two rices, and / or identifying rice fertility.

[0009] The fifth object of the present invention is to provide a method for improving the fertility restoration ability of wild abortive cytoplasmic male sterile rice.

[0010] The sixth object of the present invention is to provide a method for constructing rice with high fertility restoration ability for wild abortive cytoplasmic male sterility.

[0011] In order to achieve the above objects, the present invention is realized through the following solutions:

[0012] The application of a reagent for detecting SNP sites in the rice genome in any one of the following:

[0013] Differentiating or assisting in differentiating rice varieties;

[0014] Detecting the similarity between any two rices;

[0015] Identifying rice fertility;

[0016] The SNP sites are as follows:

[0017] The SNP site 1 is located at the 21058519th base of chromosome 10 of rice where the Rf4 gene is located, and is T or A;

[0018] The SNP site 2 is located at the 21058518th base of chromosome 10 of rice where the Rf4 gene is located, and is G or C;

[0019] The SNP locus 3 is located at the 21,058,193rd base of chromosome 10 of rice where the Rf4 gene is located, and is A or T;

[0020] The SNP locus 4 is located at the 21,058,190th base of chromosome 10 of rice where the Rf4 gene is located, and is C or T;

[0021] The SNP locus 5 is located at the 21,058,560th base of chromosome 10 of rice where the Rf4 gene is located, and is A or T;

[0022] The SNP locus 6 is located at the 21,058,559th base of chromosome 10 of rice where the Rf4 gene is located, and is T or C;

[0023] The SNP locus 7 is located at the 21,058,745th base of chromosome 10 of rice where the Rf4 gene is located, and is T or A;

[0024] The SNP locus 8 is located at the 21,058,435th base of chromosome 10 of rice where the Rf4 gene is located, and is A or G;

[0025] The SNP locus 9 is located at the 21,058,942nd base of chromosome 10 of rice where the Rf4 gene is located, and is A or G;

[0026] The SNP locus 10 is located at the 21,058,935th base of chromosome 10 of rice where the Rf4 gene is located, and is G or T;

[0027] The SNP locus 11 is located at the 21,058,931st base of chromosome 10 of rice where the Rf4 gene is located, and is G or A;

[0028] The SNP locus 12 is located at the 21,058,924th base of chromosome 10 of rice where the Rf4 gene is located, and is T or C;

[0029] The SNP locus 13 is located at the 21,059,126th base of chromosome 10 of rice where the Rf4 gene is located, and is A or C;

[0030] The SNP locus 14 is located at the 21,058,070th base of chromosome 10 of rice where the Rf4 gene is located, and is C or T;

[0031] The SNP locus 15 is located at the 20,978,195th base of chromosome 10 of rice where the Rf4 gene is located, and is A or G;

[0032] The SNP locus 16 is located at the 20,977,909th base of chromosome 10 of rice where the Rf4 gene is located, and is A or G;

[0033] The SNP locus 17 is located at the 20,977,907th base of chromosome 10 of rice where the Rf4 gene is located, and is T or G;

[0034] The SNP loci 18-23 are located at the 21,055,894-21,055,889th bases of chromosome 10 of rice where the Rf4 gene is located, and are GGAATA or TCGCAC;

[0035] The SNP loci 24-26 are located at the 21,055,647-21,055,645th bases of chromosome 10 of rice where the Rf4 gene is located, and are TGT or CGC.

[0036] The chromosome 10 of rice where the Rf4 gene is located has the GenBank accession number CP018166.1.

[0037] Preferably, the reagent for detecting SNP loci in the rice genome comprises any one or several combinations of primer pairs with nucleotide sequences shown in SEQ ID NO: 8-23.

[0038] Preferably, if the base of SNP locus 1 is T, the base of SNP locus 2 is G, the base of SNP locus 3 is A, and the base of SNP locus 4 is C, then the cytoplasmic male sterility restoration gene Rf4 locus of rice is the rf4j single-copy haplotype, and the rice variety is one or more of 9522, IRGC 125981, IRGC 128077, IRGC 128086, IRGC 128314, IRGC 128360, IRGC128467, IRGC 131964, IRGC 131968, IRGC 132274, IRGC 132278, IRGC 132307, IRGC132339, IRGC 132345, Zhonghua 11, Taichung 65, Nipponbare, and Xiushui;

[0039] If the base of SNP locus 5 is A and the base of SNP locus 6 is T, then the Rf4 locus is the rf4i single-copy haplotype, and the rice varieties are one or more of 1209A, 843A, 9311, D62A, D62B, E Nong 13, F32A, F32B, F59A, F59B, IR64, IRGC125858, IRGC 126249, IRGC 127056, IRGC 127268, IRGC 127698, IRGC 127725, IRGC128041, IRGC 128335, IRGC 132315, IRGC 132364, R211, R286, Zhonghui 7259, Zhonghui 7265, Gang 46A, Gang 46B, Hua A, Hua B, Shuanggui A, Yixiang 1A, Yixiang 1B, Chuannong 1B, Chuannong 3A, Chuannong 3B, Ju 2A, Guanghui 128, Guangtai A, Changhui 121, Changnong 1A, Guihuazhan, Huhan 1A, Huhan 1B, Huhan 7A, Huhan 7B, Taifeng A, Zhenshan 97A, Zhenshan 97B, Rong 7A, Shu 6A, Shu 8B, Shu 9A, Xinuo 1A, Xinuo 1B, Xida 2A, Xida 2B, Xida 5A, Xida 5B, Ganxiang 73A, Ganxiang 73B, Ganxiang A, Ganxiang B, Jin 23A, Jin 23B, Changjing 1B and Liangzhan;

[0040] If the base of SNP locus 7 is T and the base of SNP locus 8 is A, then the Rf4 locus is the rf4aus single-copy haplotype, and the rice varieties are one or more of Albania, CISOKAN, IRGC 128344, IRGC 128317, Baxiang and Weed Rice 13;

[0041] If the base of SNP locus 14 is C, the base of SNP locus 15 is A, the base of SNP locus 16 is A and the base of SNP locus 17 is T, then the Rf4 locus is Rf4a I single-copy haplotype, and the rice varieties are Jalmagna and / or Guanghui 102;

[0042] If the base of SNP locus 14 is C, the bases of SNP loci 18-23 are GGAATA and the bases of SNP loci 24-26 are TGT, then the Rf4 locus is Rf4b M single-copy haplotype, and the rice varieties are Mianhui 725 and / or R60;

[0043] If the base of SNP locus 14 is C, the base of SNP locus 15 is A, and the base of SNP locus 16 is A, and the base of SNP locus 17 is T; the bases of SNP loci 18-23 are GGAATA, and the bases of SNP loci 24-26 are TGT, then the Rf4 locus is Rf4a M -Rf4bM The double-copy haplotype, and the rice variety is one or more of CDR22, IR30, IRGC 117425, IRGC 127105, IRGC 127340, IRGC 128109, R88, Zhonghui 7203, Zhonghui 7248, Lehui 188, Yihui 3551, Hanhui 3, Changhui 851, Changhui 881, Minghui 63, Minghui 86, Ce 64, Suhui 5, Shuhui 498, Shuhui 5040, Shuhui 548, Xihui 16, Xihui 22, Xihui 28, Xihui 952, Ganhu 8130 and Fuhui 838;

[0044] The base of SNP locus 13 is A, the base of SNP locus 14 is C, the base of SNP locus 15 is A, the base of SNP locus 16 is A, and the base of SNP locus 17 is T; the bases of SNP loci 18-23 are GGAATA, and the bases of SNP loci 24-26 are TGT, then the Rf4 locus is Rf4a I -rf4b double-copy haplotype, and the rice variety is one or more of IR24, IR8, IRGC127742, L6B, OM052, ZMB1, Zhonghui 7206, Zhonghui 7208, Zhonghui 7217, Zhonghui 7258, Zhonghui 7270, Zhonghui 7271, Zhonghui 7272, Zhonghui 7273, Zhonghui 81, Fengxinzhan, Jiuxiangzhan, Huazhan, Pinghui 661, Pinghui 718, Miyang 46, Guanghui 998, Chenghui 727, Xinhuzhan, Changhui 871, Changxianghui 1, Gui 99, Xiangqing, Zhenshan Rf4I, Shuhui 3034, Shuhui 527, Changhui 1, Minhui 3301, Yahui 2115, Huanghuazhan and Heihui 1;

[0045] The base of SNP locus 9 is A, the base of SNP locus 10 is G, the base of SNP locus 11 is G, the base of SNP locus 12 is T, the base of SNP locus 13 is A, the base of SNP locus 14 is C, the base of SNP locus 15 is A, the base of SNP locus 16 is A, and the base of SNP locus 17 is T; the bases of SNP loci 18-23 are GGAATA, and the bases of SNP loci 24-26 are TGT, then the Rf4 locus is rf4a-Rf4b M Double-copy haplotype, and the rice variety is one or more of IRGC 121441, IRGC 125883, IRGC 126154, IRGC 126159, IRGC127199, IRGC 132424, Xianhui 207 and Ganhu 993;

[0046] The rice with the Rf4 locus being a single-copy haplotype of rf4j, rf4i or rf4aus is a non-restoring line;

[0047] The Rf4 locus is Rf4a I or Rf4b M Single-copy haplotype, Rf4a M -Rf4b M 、Rf4a I -rf4b or rf4a-Rf4b M Rice with a double-copy haplotype is a restorer line.

[0048] A rice plant with a cytoplasmic male sterility restoring gene Rf4 locus of the rf4j single-copy haplotype, wherein the base at SNP locus 1 is T, the base at SNP locus 2 is G, the base at SNP locus 3 is A, and the base at SNP locus 4 is C.

[0049] A rice plant with a cytoplasmic male sterility restoring gene Rf4 locus of the rf4i single-copy haplotype, wherein the base at SNP locus 5 is A and the base at SNP locus 6 is T.

[0050] A rice plant with a cytoplasmic male sterility restoring gene Rf4 locus of the rf4aus single-copy haplotype, wherein the base at SNP locus 7 is T and the base at SNP locus 8 is A.

[0051] A rice plant with a cytoplasmic male sterility restoring gene Rf4 locus of Rf4a I Single-copy haplotype, wherein the base at SNP locus 14 is C, the base at SNP locus 15 is A, the base at SNP locus 16 is A, and the base at SNP locus 17 is T.

[0052] A rice plant with a cytoplasmic male sterility restoring gene Rf4 locus of Rf4b M Single-copy haplotype, wherein the base at SNP locus 14 is C, the bases at SNP loci 18-23 are GGAATA, and the bases at SNP loci 24-26 are TGT.

[0053] A rice plant with a cytoplasmic male sterility restoring gene Rf4 locus of Rf4a M -Rf4b M Double-copy haplotype, wherein the base at SNP locus 14 is C, the base at SNP locus 15 is A, the base at SNP locus 16 is A, and the base at SNP locus 17 is T; the bases at SNP loci 18-23 are GGAATA, and the bases at SNP loci 24-26 are TGT.

[0054] A rice plant with a cytoplasmic male sterility restoring gene Rf4 locus of Rf4a IRice with a double-copy haplotype of -rf4b, where the base at SNP locus 13 is A, the base at SNP locus 14 is C, the base at SNP locus 15 is A, the base at SNP locus 16 is A, and the base at SNP locus 17 is T; the bases at SNP loci 18-23 are GGAATA, and the bases at SNP loci 24-26 are TGT.

[0055] A cytoplasmic male sterility restoring gene Rf4 locus is rf4a-Rf4b M Rice with a double-copy haplotype, where the base at SNP locus 9 is A, the base at SNP locus 10 is G, the base at SNP locus 11 is G, the base at SNP locus 12 is T, the base at SNP locus 13 is A, the base at SNP locus 14 is C, the base at SNP locus 15 is A, the base at SNP locus 16 is A, and the base at SNP locus 17 is T; the bases at SNP loci 18-23 are GGAATA, and the bases at SNP loci 24-26 are TGT.

[0056] A set of PCR reagents for identifying or assisting in the identification of rice varieties, detecting the similarity between any two rice plants, and / or identifying the fertility of rice, consisting of PCR reagent 1, PCR reagent 2, PCR reagent 3, PCR reagent 4, PCR reagent 5, PCR reagent 6, PCR reagent 7, and PCR reagent 8;

[0057] The PCR reagent 1 includes a primer combination with the nucleotide sequences shown in SEQ ID NO: 8-9;

[0058] The PCR reagent 2 includes a primer combination with the nucleotide sequences shown in SEQ ID NO: 10-11;

[0059] The PCR reagent 3 includes a primer combination with the nucleotide sequences shown in SEQ ID NO: 12-13;

[0060] The PCR reagent 4 includes a primer combination with the nucleotide sequences shown in SEQ ID NO: 14-15;

[0061] The PCR reagent 5 includes a primer combination with the nucleotide sequences shown in SEQ ID NO: 16-17;

[0062] The PCR reagent 6 includes a primer combination with the nucleotide sequences shown in SEQ ID NO: 18-19;

[0063] The PCR reagent 7 includes a primer combination with the nucleotide sequences shown in SEQ ID NO: 20-21;

[0064] The PCR reagent 8 includes a primer combination with nucleotide sequences shown in SEQ ID NO: 22 to 23.

[0065] A kit containing the above-mentioned PCR reagent.

[0066] Use of the above-mentioned kit in identifying or assisting in the identification of rice varieties, detecting the similarity between any two rice plants, and / or identifying rice fertility.

[0067] A method for improving the fertility restoration ability of wild abortive cytoplasmic male sterility in rice, enabling the rice restorer gene Rf4 locus to carry Rf4a M -Rf4b M double-copy haplotype, where the Rf4a M -Rf4b M The double-copy haplotype is: the base at SNP locus 14 is C, the base at SNP locus 15 is A, the base at SNP locus 16 is A, and the base at SNP locus 17 is T; the bases at SNP loci 18 to 23 are GGAATA, and the bases at SNP loci 24 to 26 are TGT.

[0068] Preferably, use the above-mentioned kit to identify that the Rf4 locus is the Rf4a M -Rf4b M double-copy haplotype rice, and hybridize it with the sterile line;

[0069] The sterile line is the rice with the Rf4 locus being the rf4i single-copy haplotype identified by the above-mentioned kit;

[0070] The rf4i single-copy haplotype is: the base at SNP locus 5 is A and the base at SNP locus 6 is T.

[0071] A method for constructing rice with high fertility restoration ability for wild abortive cytoplasmic male sterility, enabling the cytoplasmic male sterility restorer gene Rf4 locus of rice to be Rf4a M -Rf4b M double-copy haplotype, where the Rf4a M -Rf4b M The double-copy haplotype is: the base at SNP locus 14 is C, the base at SNP locus 15 is A, the base at SNP locus 16 is A, and the base at SNP locus 17 is T; the bases at SNP loci 18 to 23 are GGAATA, and the bases at SNP loci 24 to 26 are TGT.

[0072] Preferably, use the above-mentioned kit to identify that the Rf4 locus is the Rf4a M -Rf4b M double-copy haplotype rice, and hybridize it with the sterile line to construct rice with high fertility restoration ability;

[0073] The sterile line is such that the Rf4 locus of rice is identified as the rf4i single-copy haplotype by the described kit;

[0074] The rf4i single-copy haplotype is: the base at SNP locus 5 is A and the base at SNP locus 6 is T.

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] The present invention discloses an SNP molecular marker for identifying the haplotype of the rice fertility restoration gene Rf4 and its application in restoring rice fertility. By aligning the sequences of the Rf4 locus and the gene coding region of 165 rice varieties, the present invention discovers that the rice wild abortive cytoplasmic male sterility fertility restoration gene Rf4 is a complex locus composed of two sub-loci, a and b, with copy number variation and sequence base variation. There are 7 allelic genotype sequences in cultivated rice, a total of 8 haplotypes, including 5 single-copy haplotypes: rf4j, rf4i, rf4aus, Rf4a I , and Rf4b M , and 3 double-copy haplotypes: Rf4a I -rf4b, rf4a-Rf4b M and Rf4a M -Rf4b M . Based on these allelic genotype sequences and their single nucleotide polymorphism (SNP) variations, the present invention develops a set of differential PCR specific amplification molecular markers and establishes a method for accurately and rapidly identifying the haplotype of the Rf4 gene locus, which can accurately distinguish different haplotypes of the Rf4 complex locus to achieve the purpose of screening and identifying rice varieties. At the same time, using this set of molecular markers to screen strong fertility-restoring rice germplasm resources containing the Rf4a M -Rf4b M double-copy haplotype, and by crossing with the sterile line, it accelerates the breeding and identification of strong fertility-restoring lines and plays a key role in the cultivation of excellent restoring lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 It is a schematic diagram of the genomic structure variation of the Rf4 locus (located on chromosome 10) in 3 rice varieties. The gray background indicates other PPR genes in this interval, the black background indicates the functional Rf4 genotype, and the white background indicates the non-functional rf4 genotype.

[0078] Figure 2 For A, it is the functional Rf4 (including Rf4a M , Rf4b M , Rf4a I) and partial differential amino acid regions of non-functional rf4-encoded proteins. The bold white background indicates the differential amino acids in three PPR motifs (PPR13, PPR14, and PPR15) between Rf4 and rf4; Figure 2 B is the genotype composition and haplotype of the Rf4 locus detected in cultivated rice.

[0079] Figure 3 Schematic diagram for functional verification of different genotypes of Rf4. The CMS-WA sterile line J23A was used as the transformation receptor, and the pollen phenotype was completely sterile. Among them, the transformants with different rf4 genotypes (t represents transgenic) all showed pollen abortion, and the transformants with the Rf4 (Rf4a M ) genotype could restore pollen fertility and showed pollen fertility. The scale bar in the figure is 50 μm.

[0080] Figure 4 Schematic diagram for identifying genotypes and haplotypes in different cultivated rice materials using allele-specific molecular markers of the Rf4 locus. Figure 4 The rice materials in A contain a single copy of the recessive rf4 allele (rf4j, rf4i, or rf4aus). Figure 4 The rice materials in B contain a single copy of the dominant Rf4, and the haplotypes they form are rf4a-Rf4b M , Rf4a I -rf4b, Rf4a I , or Rf4a M . Figure 4 The rice varieties in C contain two copies of Rf4, both of which are the Rf4a M -Rf4b M haplotype.

[0081] Figure 5 To verify the dosage effect of Rf4 on fertility restoration using different Rf4 near-isogenic lines. Among them Figure 5 A shows the pollen staining rate (upper figure) and spikelet seed setting rate (lower figure) of J23A×ZSRf4I (WA352 / rf4iRf4a I rf4b) and J23A×ZSRf4M (WA352 / rf4iRf4a M Rf4b M ). The scale bar in the figure: 50 μm for the upper figure and 5 cm for the lower figure. Figure 5 B shows the expression level of Rf4 in different lines. Figure 5 C shows the expression level of WA352 in different lines. The data are the mean ± standard deviation of three biological replicates, n = 3. Different letters indicate significant differences between groups (p < 0.05). Specific implementation methods

[0082] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the test methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0083] Example 1 Genomic Structural Variation Analysis, Haplotype Identification and Functional Analysis of the Rf4 Locus

[0084] I. Experimental Methods

[0085] CMS-WA is the wild abortive cytoplasmic male sterility in rice, which is based on the CMS-WA / Rf3Rf4 fertility control genetic system. The CMS-WA / Rf3Rf4 fertility control genetic system consists of the sterility gene WA352 located in the mitochondrial genome and the major fertility restoring genes Rf3 and Rf4 located in the nuclear genome. Rf4 encodes a PPR (pentatricopeptide repeat) protein containing 782 amino acids. There are multiple alleles of Rf4, including the dominant (functional) Rf4 in the restorer line, the recessive (non-functional) rf4i in indica rice sterile lines or maintainer lines, and the recessive rf4j in japonica rice.

[0086] 1. Genomic Structural Variation Analysis of the Rf4 Locus

[0087] Based on the alignment and analysis of the sequences of the fertility restoring gene Rf4 (restorer line), its alleles rf4j (japonica rice) and rf4i (sterile line and maintainer line, translation terminated prematurely), and the Rf4 locus and its flanking sequences of rice varieties Minghui 63 (MH63), Nipponbare (Nip) and Zhenshan 97 (ZS97), it was found that there are structural variations in the Rf4 locus.

[0088] Minghui 63 (MH63) is an indica rice restorer line; Nipponbare (Nip) is a japonica rice variety; Zhenshan 97 (ZS97) is an indica rice sterile line or maintainer line.

[0089] MH63 contains two copies of Rf4, named a copy and b copy (or called Rf4a and Rf4b sub-loci), whose coding region sequences are the same but the upstream and downstream sequences at the distal ends are different; Nip contains a single copy of rf4j located at the a locus; ZS97 contains a single copy of rf4i located at the a locus, where rf4i is a truncated pseudogene formed by sequence insertion (premature stop codon) ( Figure 1 )

[0090] 2. Allelic Variation, Haplotype Identification and Functional Analysis of the Rf4 Locus

[0091] Based on the SNP differences in the a and b copy sequences of the Rf4 gene, primers STI-Rf4-F, STI-Rf4a-R (abbreviated as F and a-R respectively), STI-Rf4b-R (abbreviated as b-R), rf4i-F1, rf4i-R1 (abbreviated as i-F1 and i-R1 respectively), R / rf4-seq-F1 to F4, rf4i-seq-F1, and rf4i-seq-F2 were designed (Table 1).

[0092] Table 1 Primer sequences for PCR amplification and sequencing of cultivated rice germplasm resources

[0093]

[0094] Genomic DNA (SDS method) was extracted from 165 domestic and foreign cultivated rice germplasm resources collected from the Genetic Engineering Laboratory of South China Agricultural University as templates, and PCR amplification and sequencing analysis were carried out using the primer sets described in Table 1. Among them, primers F and a-R were used for specific amplification of the a copy, F and b-R were used for specific amplification of the b copy, and i-F1 and i-R1 were used for specific amplification of the rf4i copy of indica rice male sterile lines or non-restoring materials. R / rf4-seq-F1 to F4 are sequencing primers for Rf4, rf4j, rf4aus, rf4a, and rf4b, and rf4i-seq-F1 and rf4i-seq-F2 are sequencing primers for rf4i.

[0095] The 20 μL PCR reaction system used for amplification is shown in Table 2 below:

[0096] Table 2 PCR reaction system of Example 1

[0097]

[0098] The PCR reaction conditions were the STI PCR method (Zhao et al., STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences. Molecular Plant, 2022, 4: 620 - 629..), specifically as follows:

[0099]

[0100] Among them, "*" represents the nested inner cycle, and applicable PCR instruments include brands such as Jena, LongGene, and Dongshenglong.

[0101] II. Experimental results

[0102] j represents japonica rice; i represents indica rice; aus represents the aus type variety of indica rice; I corresponds to the Rf4 genotype of the indica rice restorer line IR24, and M corresponds to the Rf4 genotype of MH63.

[0103] Through sequencing and amino acid sequence alignment analysis, it was found that there are 7 genotypes at the Rf4 locus in cultivated rice. These 7 allelic genotypes were named rf4j, rf4i, rf4aus, rf4a, rf4b, Rf4 M and Rf4 I , respectively. The nucleotide sequences of rf4j, rf4i, rf4aus, rf4a, rf4b, Rf4 M and Rf4 I are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

[0104] Rf4 includes two genotypes, Rf4 M and Rf4 I . In the tested rice materials, Rf4 M exists at either the a or b locus, so they are named Rf4a M and Rf4b M , respectively. Rf4 I only exists at the a locus, so it is named Rf4a I , and there is no Rf4b I .

[0105] According to the rice variety source and the sub-locus where the gene is located, the recessive alleles include rf4j, rf4i, rf4aus, rf4a, and rf4b.

[0106] The proteins encoded by the recessive rf4 and the functional Rf4 (Rf4a M , Rf4b M , Rf4a I ) have 14 amino acid differences within three PPR motifs (amino acids at positions 519 - 623) of PPR13, PPR14, and PPR15, respectively ( Figure 2 A).

[0107] The proteins encoded by Rf4 M and Rf4 I (i.e., Rf4a I ) have glutamic acid at position 303 (Rf4 M ) and lysine at position 303 (Rf4 I ), and alanine at position 529 (Rf4 M ) and Rf4 IThere are 2 amino acid differences at the amino acid position (glycine).

[0108] Among the 165 cultivated rice germplasm resources tested, a total of 8 haplotypes were found, including 5 single-copy haplotypes: rf4j, rf4i, rf4aus, Rf4a I , and Rf4b M , as well as 3 double-copy haplotypes: Rf4a I -rf4b, rf4a-Rf4b M and Rf4a M -Rf4b M ( Figure 2 B).

[0109] To verify the functions of different genotypes at the Rf4 locus, the present invention transformed rf4a, rf4b, rf4aus, rf4j, and Rf4 into the sterile line J23A respectively. All transformants with rf4 genotypes (rf4a, rf4b, rf4aus, and rf4j) showed pollen abortion, while the Rf4 transformants could restore fertility and showed pollen fertility ( Figure 3 ).

[0110] Example 2 Development of haplotype-specific molecular markers for the Rf4 locus

[0111] According to the upstream and downstream sequences of the Rf4 locus and the SNP differences of different haplotypes of Rf4, a molecular marker method for accurately and rapidly identifying and screening different Rf4 haplotypes of rice germplasm was developed.

[0112] 1. Analyze the SNP differences in the 7 allelic genotype sequences of the a and b copies of the Rf4 gene according to the sequencing results of Example 1, and design molecular markers specific for different haplotypes, including rf4j-F, rf4j-R, rf4i-F2, rf4i-R2, rf4aus-F, rf4aus-R, rf4a-F, rf4a-R, rf4a / b-F, rf4a / b-R, Rf4-F, Rf4-R, a-locus-F, a-locus-R, b-locus-F, and b-locus-R. Actin1-F and Actin1-R are positive controls (Table 3).

[0113] Table 3 Primer sequences with nucleotide sequences shown in SEQ ID NO: 8-25

[0114]

[0115]

[0116] 2. Using the genomic DNA of 165 cultivated rices in Example 1 as templates, PCR amplifications were carried out respectively with 8 pairs of molecular markers in Table 3.

[0117] The 20 μL PCR reaction system used for amplification is shown in Table 4 below:

[0118] Table 4 PCR reaction system of Example 2

[0119]

[0120] The PCR reaction conditions were as follows:

[0121]

[0122] The PCR products were detected by 1.5% agarose gel electrophoresis.

[0123] II. Experimental results

[0124] The gene number of the following Rf4 gene in the MBKbase database (http: / / www.mbkbase.org / riice) is: OsR498G1018978200.01. The number of the 10th chromosome of rice where the Rf4 gene is located in GenBank is CP018166.1.

[0125] The amplification product of the rf4j-F and rf4j-R primers is 371 bp. The nucleotide sequence of the amplification product is as shown in SEQ ID NO: 26. At the 23rd, 24th, 349th, and 352nd bases at the 5' end of the amplification product (i.e., the 1237th, 1238th, 1563rd, and 1566th bases of the Rf4 gene; the 21058519th, 21058518th, 21058193rd, and 21058190th bases of CP018166.1), there are respectively one SNP site of T / A, G / C, A / T, and C / T.

[0126] The amplification product of the rf4i-F2 and rf4i-R2 primers is 358 bp. The nucleotide sequence of the amplification product is as shown in SEQ ID NO: 27. At the 341st and 342nd bases at the 5' end of the amplification product (i.e., the 1196th and 1197th bases of the Rf4 gene; the 21058560th and 21058559th bases of CP018166.1), there are respectively one SNP site of A / T and T / C.

[0127] The amplification product of the rf4aus-F and rf4aus-R primers is 351 bp. The nucleotide sequence of the amplification product is as shown in SEQ ID NO: 28. At the 20th and 330th bases at the 5' end of the amplification product (i.e., the 1011th and 1321st bases of the Rf4 gene; the 21058745th and 21058435th bases of CP018166.1), there are respectively one SNP site of T / A and A / G.

[0128] The amplification products of the rf4a-F and rf4a-R primers are 446 bp. The nucleotide sequence of the amplification product is as shown in SEQ ID NO: 29. At the 10th, 17th, 21st, and 428th bases at the 5' end of the amplification product (i.e., the 814th, 821st, 825th, and 1232nd bases of the Rf4 gene; the 21058942nd, 21058935th, 21058931st, and 21058924th bases of CP018166.1), there are respectively one SNP site of A / G, G / T, G / A, and T / C.

[0129] The amplification products of the rf4a / b-F and rf4a / b-R primers are 197 bp. The nucleotide sequence of the amplification product is as shown in SEQ ID NO: 30. At the 176th base at the 5' end of the amplification product (i.e., the 630th base of the Rf4 gene; the 21059126th base of CP018166.1), there is one SNP site of A / C.

[0130] The amplification products of the Rf4-F and Rf4-R primers are 262 bp. The nucleotide sequence of the amplification product is as shown in SEQ ID NO: 31. At the 238th base at the 5' end of the amplification product (i.e., the 1686th base of the Rf4 gene; the 21058070th base of CP018166.1), there is one SNP site of C / T.

[0131] The amplification products of the a-locus-F and a-locus-R primers are 331 bp. The nucleotide sequence of the amplification product is as shown in SEQ ID NO: 32. At the 21st, 307th, and 309th bases at the 5' end of the amplification product (i.e., the 2030th, 2316th, and 2318th bases after the stop codon of the Rf4 gene; the 20978195th, 20977909th, and 20977907th bases of CP018166.1), there are respectively one SNP site of A / G, A / G, and T / G.

[0132] The amplification product of b-locus-F and b-locus-R primers is 282 bp, and the nucleotide sequence of the amplification product is shown in SEQ ID NO: 33. SNP sites exist at the 15th to 20th and 262th to 264th consecutive bases of the 5' end of the amplification product, respectively. There are continuous SNP sites of GGAATA / TCGCAC at the 15th to 20th positions of the 5' end of the amplification product, and there are continuous SNP sites of TGT / CGC at the 262nd to 264th positions of the 5' end of the amplification product (i.e., the 1513th to 1518th and 1760th to 1762th bases after the stop codon of the Rf4 gene; the 21055894th to 21055889th and 21055647th to 21055645th bases of CP018166.1).

[0133] Nipponbare, Taichung 65, Zhonghua 11 and 9522 are japonica rice varieties. Jin 23A, Jin 23B, Zhenshan 97A, Zhenshan 97B, IR64 and 9311, Ganhui 993, Xianhui 207, Zhenshan Rf4I, IR8, Jalmagna, Guanghui 102, Mianhui 725, R60, Minghui 63, Shuhui 498, Fuhui 838 and IR30 are indica rice varieties. CISOKAN, Baxiang, Zaweidao 13 and Albania are indica rice aus varieties. Actin1 was used as a control.

[0134] The results show that if Figure 4 As shown in A, the rf4j-F and rf4j-R primer pairs specifically amplify a 371 bp fragment in japonica rice (such as Nipponbare, Taichung 65, Zhonghua 11 and 9522); the rf4i-F2 and rf4i-R2 primer pairs specifically amplify a 358 bp fragment in wild-type cytoplasmic male sterile lines, maintainer lines and some non-restoring indica rice varieties (such as Jin 23A, Rujin 23B, Zhenshan 97A, Zhenshan 97B, IR64 and 9311); the rf4aus-F and rf4aus-R primer pairs specifically amplify a 351 bp fragment in indica rice aus varieties (such as CISOKAN, Baxiang, Weedy Rice 13 and Albania), and the rf4j, rf4i and rf4aus genes are all located at the a locus, which is a single copy rf4.

[0135] As shown in Table 5, the rf4j gene is located at locus a. The rice varieties with the Rf4 locus being the rf4j single-copy haplotype are 9522, IRGC125981, IRGC 128077, IRGC 128086, IRGC 128314, IRGC 128360, IRGC 128467, IRGC 131964, IRGC 131968, IRGC 132274, IRGC 132278, IRGC 132307, IRGC 132339, IRGC132345, Zhonghua 11, Taichung 65, Nipponbare, and Xiushui.

[0136] The rf4i gene is located at locus a. The rice varieties with the Rf4 locus being the rf4i single-copy haplotype are 1209A, 843A, 9311, D62A, D62B, Enong 13, F32A, F32B, F59A, F59B, IR64, IRGC 125858, IRGC 126249, IRGC127056, IRGC 127268, IRGC 127698, IRGC 127725, IRGC 128041, IRGC 128335, IRGC132315, IRGC 132364, R211, R286, Zhonghui 7259, Zhonghui 7265, Gang 46A, Gang 46B, Hua A, Hua B, Shuanggui A, Yixiang 1A, Yixiang 1B, Chuannong 1B, Chuannong 3A, Chuannong 3B, Ju 2A, Guanghui 128, Guangtai A, Changhui 121, Changnong 1A, Guihuazhan, Huhan 1A, Huhan 1B, Huhan 7A, Huhan 7B, Taifeng A, Zhenshan 97A, Zhenshan 97B, Rong 7A, Shu 6A, Shu 8B, Shu 9A, Xinuo 1A, Xinuo 1B, Xida 2A, Xida 2B, Xida 5A, Xida 5B, Ganxiang 73A, Ganxiang 73B, Ganxiang A, Ganxiang B, Jin 23A, Jin 23B, Changjing 1B, Liangzhan.

[0137] The rf4aus gene is located at locus a. The rice varieties with the Rf4 locus being the rf4aus single-copy haplotype are Albania, CISOKAN, IRGC 128344, IRGC 128317, Baxiang, and Weedy Rice 13.

[0138] Figure 4B shows a rice variety carrying a single copy of dominant Rf4, in which the amplified fragments of the rf4a-F and rf4a-R primer pair (446bp), the amplified fragments of the rf4a / bF and rf4a / bR primer pair (197bp), the amplified fragments of the Rf4-F and Rf4-R primer pair (262bp), the amplified fragments of the a-locus-F and a-locus-R primer pair (331bp) and the amplified fragments of the b-locus-F and b-locus-R primer pair (282bp) were amplified simultaneously, a total of five pairs of molecular markers for rice varieties (such as Ganhui 993 and Xianhui 207), and the corresponding haplotype of the Rf4 locus is rf4a-Rf4b M .

[0139] As shown in Table 5, the rf4a gene is located at locus a, and Rf4b M The gene is located at the b locus, and the Rf4 locus is rf4a-Rf4b M The rice varieties with double copy haplotypes are IRGC 121441, IRGC 125883, IRGC 126154, IRGC 126159, IRGC127199, IRGC 132424, Xianhui 207 and Ganhui 993.

[0140] like Figure 4 As shown in B, the amplified fragments of the rf4a / bF and rf4a / bR primer pairs, the amplified fragments of the Rf4-F and Rf4-R primer pairs, the amplified fragments of the a-locus-F and a-locus-R primer pairs, and the amplified fragments of the b-locus-F and b-locus-R primer pairs were amplified simultaneously, for a total of four pairs of molecular markers of rice varieties (such as Zhenshan Rf4I and IR8), and the corresponding haplotype of the Rf4 locus is Rf4a I -rf4b.

[0141] As shown in Table 5, Rf4a I The gene is located at locus a, the rf4b gene is located at locus b, and the Rf4 locus is Rf4a I The rice varieties with -rf4b double copy haplotype are IR24, IR8, IRGC 127742, L6B, OM052, ZMB1, Zhonghui 7206, Zhonghui 7208, Zhonghui 7217, Zhonghui 7258, Zhonghui 7270, Zhonghui 7271, Zhonghui 7272, Zhonghui 7273, Zhonghui 81, Fengxinzhan, Jiuxiangnian, Huazhan, Pinghui 661, Pinghui 718, Miyang 46, Guanghui 998, Chenghui 727, Xinhuazhan, Changhui 871, Changxianghui 1, Gui 99, Xiangqing, Zhenshan Rf4I, Shuhui 3034, Shuhui 527, Changhui 1, Minhui 3301, Yahui 2115, Huanghuazhan and Heihui 1.

[0142] likeFigure 4 As shown in Figure B, the amplified fragments of the primer pair Rf4-F and Rf4-R and the amplified fragments of the primer pair a-locus-F and a-locus-R are simultaneously amplified, and the rice varieties with two pairs of molecular markers (such as Jalmagna and Guanghui 102) have the haplotype of Rf4a at the corresponding Rf4 locus I .

[0143] As shown in Table 5, Rf4a I gene is located at the a locus, and the Rf4 locus is Rf4a I The rice varieties with single-copy haplotype are Jalmagna and Guanghui 102

[0144] As Figure 4 shown in Figure B, the amplified fragments of the primer pair Rf4-F and Rf4-R and the amplified fragments of the primer pair b-locus-F and b-locus-R are simultaneously amplified, and the rice varieties with two pairs of molecular markers (such as Mianhui 725 and R60) have the haplotype of Rf4b at the corresponding Rf4 locus M .

[0145] As shown in Table 5, Rf4b M gene is located at the b locus, and the Rf4 locus is Rf4b M The rice varieties with single-copy haplotype are R60 and Mianhui 725

[0146] As Figure 4 shown in Figure C, the amplified fragments of the primer pair Rf4-F and Rf4-R, the amplified fragments of the primer pair a-locus-F and a-locus-R, and the amplified fragments of the primer pair b-locus-F and b-locus-R are simultaneously amplified, indicating that the rice varieties (such as Minghui 63, Shuhui 498, Fuhui 838, and IR30) carry double-copy dominant Rf4 (Rf4a M -Rf4b M ).

[0147] As shown in Table 5, Rf4a M gene is located at the a locus, Rf4b M gene is located at the b locus, and the Rf4 locus is Rf4a M -Rf4b MThe rice varieties with double-copy haplotypes are CDR22, IR30, IRGC 117425, IRGC 127105, IRGC 127340, IRGC128109, R88, Zhonghui 7203, Zhonghui 7248, Lehui 188, Yihui 3551, Hanhui 3, Changhui 851, Changhui 881, Minghui 63, Minghui 86, Ce 64, Suhui 5, Shuhui 498, Shuhui 5040, Shuhui 548, Xihui 16, Xihui 22, Xihui 28, Xihui 952, Ganhu 8130, Fuhui 838.

[0148] Table 5 Information on rice varieties and Rf4 locus haplotypes

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] Example 3 Verification of the restoring ability with different copy numbers of Rf4

[0157] I. Experimental method

[0158] Verify the dosage effect of double-copy Rf4 on the fertility restoration of CMS-WA, and screen for restorer lines with strong restoring ability (rice carrying double-copy Rf4).

[0159] Using the sterile line J23A(rf4i) as the female parent, and respectively using the Rf4 single-gene restorer lines ZSRf4I(Rf4a I rf4b) and ZSRf4M(Rf4a M Rf4a M ) with a recessive rf3 background as the male parents for hybridization. Obtain the hybrid F1 generations J23A×ZSRf4I(WA352 / rf4iRf4a I rf4b) and J23A×ZSRf4M(WA352 / rf4iRf4a M Rf4b M ).

[0160] To confirm the dosage effect of the restorer gene Rf4 on the fertility restoration of CMS-WA, the present invention also developed specific primers Rf4-qF, Rf4-qR and WA352-qF, WA352-qR for detecting the expression levels of Rf4 and the sterile gene WA352, and the primers for the internal reference genes were UFC1-qF, UFC1-qR and Atp6-qF, Atp6-qR (Table 6).

[0161] Table 6 Primer sequences with nucleotide sequences shown in SEQ ID NO: 24-31

[0162]

[0163]

[0164] II. Experimental results

[0165] The pollen stainability rate and spikelet setting rate of J23A×ZSRf4M (WA352 / rf4iRf4a M Rf4b M ) are higher than those of J23A×ZSRf4I (WA352 / rf4iRf4a I rf4b)( Figure 5 A), indicating that ZSRf4M carrying double-copy dominant Rf4 has stronger restoring ability.

[0166] The results of qRT-PCR detection (referring to the system and method of ChamQ Universal SYBR qPCR Master Mix of Novoprotein Co., Ltd.) showed that the expression level of Rf4 in the anthers of J23A×ZSRf4M at the rice microspore mother cell stage was about 2 times that of Rf4 in J23A×ZSRf4I( Figure 5 B), and the expression level of the corresponding WA352 was opposite (since the Rf4 protein has the effect of mediating the degradation of WA352 mRNA)( Figure 5 C).

[0167] The expression levels of Rf4 and WA352 further verified the dosage effect of double-copy Rf4 on the fertility restoration of CMS-WA, and also indicated that the Rf4 molecular marker in Example 2 can accurately identify and screen rice varieties carrying double-copy Rf4.

[0168] Example 4 A kit for identifying the haplotype of the cytoplasmic male sterility restorer gene Rf4 in rice

[0169] I. Composition

[0170] 1. Primer sequences with nucleotide sequences shown in SEQ ID NO: 8-25 (Table 3 of Example 1), Taq PCR Mix and ddH2O.

[0171] II. Usage Method

[0172] Using the genomic DNA of the rice to be tested as a template, the 20 μL PCR reaction system for PCR amplification is as follows: 10 μL of 2×Taq PCR Mix, 7.0 μL of ddH2O, 1.0 μL of 4 μM forward primer, 1.0 μL of 4 μM reverse primer, and 50 - 100 ng / μL DNA template.

[0173] The PCR reaction conditions are as follows: treated at 94°C for 4 min; treated at 94°C for 30 s, 59°C for 30 s, 72°C for 20 s, repeated for 28 - 29 nested inner cycles; treated at 72°C for 2 min.

[0174] Detect the PCR products by 1.5% agarose gel electrophoresis.

[0175] III. Result Interpretation

[0176] As Figure 4 shown in A, in a rice variety to be tested, if only the primer pair with the nucleotide sequence as shown in SEQ ID NO: 8 - 9 amplifies a specific fragment, then the cytoplasmic male sterility restoration gene Rf4 locus is the rf4j single - copy haplotype.

[0177] As Figure 4 shown in A, in a rice variety to be tested, if only the primer pair with the nucleotide sequence as shown in SEQ ID NO: 10 - 11 amplifies a specific fragment, then its Rf4 locus is the rf4i single - copy haplotype.

[0178] As Figure 4 shown in A, in a rice variety to be tested, if only the primer pair with the nucleotide sequence as shown in SEQ ID NO: 12 - 13 amplifies a specific fragment, then its Rf4 locus is the rf4aus single - copy haplotype.

[0179] As Figure 4 shown in B, in a rice variety to be tested, if the primer pairs with the nucleotide sequences as shown in SEQ ID NO: 18 - 19 and SEQ ID NO: 22 - 23 amplify 2 specific fragments simultaneously, then its Rf4 locus is Rf4a I single - copy haplotype.

[0180] As Figure 4 shown in B, in a rice variety to be tested, if the primer pairs with the nucleotide sequences as shown in SEQ ID NO: 18 - 19 and SEQ ID NO: 20 - 21 amplify 2 specific fragments simultaneously, then its Rf4 locus is Rf4b M single - copy haplotype.

[0181] AsFigure 4 As shown in Figure B, in a rice variety to be tested, if primer pairs with nucleotide sequences as shown in SEQ ID NO: 14-15, SEQ ID NO: 16-17, SEQ ID NO: 18-19, SEQ ID NO: 20-21, and SEQ ID NO: 22-23 simultaneously amplify 5 specific fragments, then its Rf4 locus is rf4a-Rf4b M Double-copy haplotype.

[0182] As Figure 4 As shown in Figure B, in a rice variety to be tested, if primer pairs with nucleotide sequences as shown in SEQ NO: 16-17, SEQ ID NO: 18-19, SEQ ID NO: 20-21, and SEQ ID NO: 22-23 simultaneously amplify 4 specific fragments, then its Rf4 locus is Rf4a I -rf4b double-copy haplotype.

[0183] As Figure 4 As shown in Figure C, in a rice variety, if primer pairs with nucleotide sequences as shown in SEQ ID NO: 18-19, SEQ ID NO: 20-21, and SEQ ID NO: 22-23 simultaneously amplify 3 specific fragments, then its Rf4 locus is Rf4a M -Rf4b M Double-copy haplotype.

[0184] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to list all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting SNP sites in the rice genome in any of the following: Identifying or assisting in identifying rice varieties; Identifying rice fertility; The SNP sites are as follows: The SNP site 1 is located at the 21058519th base of chromosome 10 of rice where the Rf4 gene is located, and is T or A; The SNP site 2 is located at the 21058518th base of chromosome 10 of rice where the Rf4 gene is located, and is G or C; The SNP site 3 is located at the 21058193rd base of chromosome 10 of rice where the Rf4 gene is located, and is A or T; The SNP site 4 is located at the 21058190th base of chromosome 10 of rice where the Rf4 gene is located, and is C or T; The SNP site 5 is located at the 21058560th base of chromosome 10 of rice where the Rf4 gene is located, and is A or T; The SNP site 6 is located at the 21058559th base of chromosome 10 of rice where the Rf4 gene is located, and is T or C; The SNP site 7 is located at the 21058745th base of chromosome 10 of rice where the Rf4 gene is located, and is T or A; The SNP site 8 is located at the 21058435th base of chromosome 10 of rice where the Rf4 gene is located, and is A or G; The SNP site 9 is located at the 21058942nd base of chromosome 10 of rice where the Rf4 gene is located, and is A or G; The SNP site 10 is located at the 21058935th base of chromosome 10 of rice where the Rf4 gene is located, and is G or T; The SNP site 11 is located at the 21058931st base of chromosome 10 of rice where the Rf4 gene is located, and is G or A; The SNP site 12 is located at the 21058924th base of chromosome 10 of rice where the Rf4 gene is located, and is T or C; The SNP site 13 is located at the 21059126th base of chromosome 10 of rice where the Rf4 gene is located, and is A or C; The SNP site 14 is located at the 21058070th base of chromosome 10 of rice where the Rf4 gene is located, and is C or T; The SNP site 15 is located at the 20978195th base of chromosome 10 of rice where the Rf4 gene is located, and is A or G; The SNP locus 16 is located at the 20,977,909th base of chromosome 10 of rice where the Rf4 gene is located, and is A or G; The SNP locus 17 is located at the 20,977,907th base of chromosome 10 of rice where the Rf4 gene is located, and is T or G; The SNP loci 18 - 23 are located at the 21,055,894 - 21,055,889th bases of chromosome 10 of rice where the Rf4 gene is located, and are GGAATA or TCGCAC; The SNP loci 24 - 26 are located at the 21,055,647 - 21,055,645th bases of chromosome 10 of rice where the Rf4 gene is located, and are TGT or CGC; The chromosome 10 of rice where the Rf4 gene is located has the GenBank accession number CP018166.

1.

2. The application according to claim 1, characterized in that, The reagent for detecting SNP sites in the rice genome comprises any one or several combinations of primer combinations with nucleotide sequences shown in SEQ ID NO: 8 to 23.

3. The application according to claim 1, characterized in that, If the base of SNP site 1 described in claim 1 is T, the base of SNP site 2 is G, the base of SNP site 3 is A, and the base of SNP site 4 is C, then the cytoplasmic male sterility restoration gene Rf4 locus of rice is the rf4j single-copy haplotype, and the rice variety is one or several of 9522, IRGC 125981, IRGC 128077, IRGC128086, IRGC 128314, IRGC 128360, IRGC 128467, IRGC 131964, IRGC 131968, IRGC132274, IRGC 132278, IRGC 132307, IRGC 132339, IRGC 132345, Zhonghua 11, Taichung 65, Nipponbare, and Xiushui; If the base of SNP site 5 is A and the base of SNP site 6 is T, then the Rf4 locus is the rf4i single-copy haplotype, and the rice variety is one or several of 1209A, 843A, 9311, D62A, D62B, Enong 13, F32A, F32B, F59A, F59B, IR64, IRGC 125858, IRGC 126249, IRGC 127056, IRGC 127268, IRGC 127698, IRGC 127725, IRGC 128041, IRGC128335, IRGC 132315, IRGC 132364, R211, R286, Zhonghui 7259, Zhonghui 7265, Gang 46A, Gang 46B, Hua A, Hua B, Shuanggui A, Yixiang 1A, Yixiang 1B, Chuannong 1B, Chuannong 3A, Chuannong 3B, Ju 2A, Guanghui 128, Guangtai A, Changhui 121, Changnong 1A, Guihuazhan, Huhan 1A, Huhan 1B, Huhan 7A, Huhan 7B, Taifeng A, Zhenshan 97A, Zhenshan 97B, Rong 7A, Shu 6A, Shu 8B, Shu 9A, Xinuo 1A, Xinuo 1B, Xida 2A, Xida 2B, Xida 5A, Xida 5B, Ganxiang 73A, Ganxiang 73B, Ganxiang A, Ganxiang B, Jin 23A, Jin 23B, Changjing 1B, and Liangzhan; If the base of SNP site 7 is T and the base of SNP site 8 is A, then the Rf4 locus is the rf4aus single-copy haplotype, and the rice variety is one or several of Albania, CISOKAN, IRGC 128344, IRGC 128317, Baxiang, and Weedy rice 13; If the base of SNP locus 14 is C, the base of SNP locus 15 is A, the base of SNP locus 16 is A and the base of SNP locus 17 is T, then the Rf4 locus is Rf4a I For the single-copy haplotype, the rice varieties are Jalmagna and / or Guanghui 102; The base of the SNP locus 14 is C, the bases of the SNP loci 18-23 are GGAATA, and the bases of the SNP loci 24-26 are TGT, then the Rf4 locus is Rf4b M Single-copy haplotype, and the rice varieties are Mianhui 725 and / or R60; The base of SNP locus 14 is C, the base of SNP locus 15 is A, the base of SNP locus 16 is A, and the base of SNP locus 17 is T; the bases of SNP loci 18-23 are GGAATA, and the bases of SNP loci 24-26 are TGT, then the Rf4 locus is Rf4a M -Rf4b M Double-copy haplotype, and the rice variety is one or more of CDR22, IR30, IRGC 117425, IRGC 127105, IRGC127340, IRGC 128109, R88, Zhonghui 7203, Zhonghui 7248, Lehui 188, Yihui 3551, Hanhui 3, Changhui 851, Changhui 881, Minghui 63, Minghui 86, Ce 64, Suhui 5, Shuhui 498, Shuhui 5040, Shuhui 548, Xihui 16, Xihui 22, Xihui 28, Xihui 952, Ganhu 8130, and Fuhui 838; The base of the SNP locus 13 is A, the base of the SNP locus 14 is C, the base of the SNP locus 15 is A, the base of the SNP locus 16 is A, and the base of the SNP locus 17 is T; the bases of the SNP loci 18-23 are GGAATA, and the bases of the SNP loci 24-26 are TGT, then the Rf4 locus is Rf4a I -rf4b double-copy haplotype, and the rice variety is one or more of IR24, IR8, IRGC127742, L6B, OM052, ZMB1, Zhonghui 7206, Zhonghui 7208, Zhonghui 7217, Zhonghui 7258, Zhonghui 7270, Zhonghui 7271, Zhonghui 7272, Zhonghui 7273, Zhonghui 81, Fengxinzhan, Jiuxiangzhan, Huazhan, Pinghui 661, Pinghui 718, Miyang 46, Guanghui 998, Chenghui 727, Xinhuzhan, Changhui 871, Changxianghui 1, Gui 99, Xiangqing, Zhenshan Rf4I, Shuhui 3034, Shuhui 527, Changhui 1, Minhui 3301, Yahui 2115, Huanghuazhan, and Heihui 1; The base of SNP site 9 is A, the base of SNP site 10 is G, the base of SNP site 11 is G, the base of SNP site 12 is T, the base of SNP site 13 is A, the base of SNP site 14 is C, the base of SNP site 15 is A, the base of SNP site 16 is A, and the base of SNP site 17 is T; the bases of SNP sites 18 to 23 are GGAATA, and the bases of SNP sites 24 to 26 are TGT, then the Rf4 locus is rf4a-Rf4b M Double copy haplotype, rice varieties are one or more of IRGC 121441, IRGC 125883, IRGC 126154, IRGC 126159, IRGC 127199, IRGC 132424, Xianhui 207 and Ganhui 993; The rice with the Rf4 locus being the rf4j, rf4i, or rf4aus single-copy haplotype is a non-restorer line; The Rf4 locus is Rf4a I or Rf4b M Single-copy haplotype, Rf4a M -Rf4b M 、Rf4a I -rf4b or rf4a-Rf4b M Rice with double-copy haplotype is a restorer line.

4. A set of PCR reagents for identifying or assisting in identifying rice varieties and / or determining rice fertility, characterized in that, It is composed of PCR reagent 1, PCR reagent 2, PCR reagent 3, PCR reagent 4, PCR reagent 5, PCR reagent 6, PCR reagent 7, and PCR reagent 8; The PCR reagent 1 includes the primer combination with the nucleotide sequences as shown in SEQ ID NOs: 8-9 described in claim 2; The PCR reagent 2 includes the primer combination with the nucleotide sequences as shown in SEQ ID NOs: 10-11 described in claim 2; The PCR reagent 3 includes the primer combination with the nucleotide sequences as shown in SEQ ID NOs: 12-13 described in claim 2; The PCR reagent 4 includes the primer combination with the nucleotide sequences as shown in SEQ ID NOs: 14-15 described in claim 2; The PCR reagent 5 includes the primer combination with the nucleotide sequences as shown in SEQ ID NOs: 16-17 described in claim 2; The PCR reagent 6 includes the primer combination with the nucleotide sequences as shown in SEQ ID NOs: 18-19 described in claim 2; The PCR reagent 7 includes the primer combination with the nucleotide sequences as shown in SEQ ID NOs: 20-21 described in claim 2; The PCR reagent 8 includes the primer combination with the nucleotide sequences as shown in SEQ ID NOs: 22-23 described in claim 2.

5. A kit containing the PCR reagents according to claim 4.

6. Use of the kit according to claim 5 in identifying or assisting in the identification of rice varieties and / or in identifying the fertility of rice.

7. A method for improving the fertility restoration ability of wild abortive cytoplasmic male sterility in rice, characterized in that The Rf4 locus of the rice restorer gene carries Rf4a M -Rf4b M A double-copy haplotype, wherein the Rf4a M -Rf4b M The double-copy haplotype is: the base at SNP locus 14 described in claim 1 is C, the base at SNP locus 15 is A, the base at SNP locus 16 is A, and the base at SNP locus 17 is T; the bases at SNP loci 18-23 are GGAATA, and the bases at SNP loci 24-26 are TGT.

8. The method according to claim 7, characterized in that Identify that the Rf4 locus is Rf4a using the kit according to claim 5 M -Rf4b M Rice with a double-copy haplotype is hybridized with a sterile line; The sterile line is that the Rf4 locus of rice is identified as the rf4i single-copy haplotype by the kit described in claim 5; The rf4i single-copy haplotype is: the base at SNP locus 5 described in claim 1 is A and the base at SNP locus 6 is T.

9. A method for constructing rice with high fertility restoration ability to wild abortive cytoplasmic male sterility, characterized in that Convert the rice cytoplasmic male sterility restoring gene Rf4 locus to Rf4a M -Rf4b M double-copy haplotype, wherein the Rf4a M -Rf4b M The double-copy haplotype is: the base at SNP locus 14 described in claim 1 is C, the base at SNP locus 15 is A, the base at SNP locus 16 is A, and the base at SNP locus 17 is T; the bases at SNP loci 18-23 are GGAATA, and the bases at SNP loci 24-26 are TGT.

10. The method according to claim 9, characterized in that Identify that the Rf4 locus is Rf4a using the kit according to claim 5 M -Rf4b M Rice with a double-copy haplotype is hybridized with a sterile line to construct rice with high fertility restoration ability; The sterile line is that the Rf4 locus of rice is identified as the rf4i single-copy haplotype by the kit described in claim 5; The rf4i single-copy haplotype is: the base at SNP locus 5 described in claim 1 is A and the base at SNP locus 6 is T.

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