Functional marker primer group of rice restorer gene Rf4 and application of functional marker primer group

Abstract

The invention provides a functional marker primer group of a rice restorer gene Rf4. The functional marker primer group comprises a primer pair ZcDI-Rf4-MH63-F and a primer pair ZcDI-Rf4-MH63-R for detecting the haplotype of the Rf4 gene H1, a primer pair ZcDI-Rf4-ZS97-F and a primer pair ZcDI-Rf4-ZS97-R for detecting the haplotype of the Rf4 gene H4, and a primer pair ZcDI-Rf4-Nip-F and a primer pair ZcDI-Rf4-Nip-R for detecting the haplotype of the Rf4 gene H5. The invention also provides an application of the functional marker primer group. The functional marker primer group comprises more than one of the following applications: detection of rice restorer gene Rf4 haplotype; the method is used for predicting the restorability of the rice restorer; the method is used for rice restorer line breeding. The functional marker primer group is designed by taking the characteristic recombinant sequences of the H1 haplotype with the strongest restoring force on the locus of the restoring gene Rf4 and the H4 and H5 haplotypes with the highest genotype frequency in the maintainer line as targets, has the characteristics of high specificity and tight linkage, and avoids the defect that primers are directly designed in functional genes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of crop molecular genetic breeding, and particularly relates to a functional marker primer group of rice restorer gene Rf4 and application thereof. BACKGROUND

[0002] The wild abortive type sterile cell gene of rice is the gene WA352 located in mitochondria, Rf3 on chromosome 1 and Rf4 on chromosome 10, which can restore the wild abortive type sterile line. Rf3 controls before transcription of the gene WA352, that is, reduces the expression amount of WA352; Rf4 controls from after transcription to before translation of the gene WA352, that is, degrades WA352 and inhibits the translation of WA352. Both of the two genes can restore the wild abortive type sterile line, but the restoring power is different, the pollen conversion rate of the sterile line carrying Rf4 is 9% higher than that of the sterile line carrying Rf3. Meanwhile, the restoring effect of Rf3 and Rf4 genes is a typical additive effect, and the H1 haplotype represented by the Rf4 haplotype carried by Minghui 63 on the Rf4 locus has two functional copies, and the restoring power is stronger. Therefore, the identification of the Rf4 locus haplotype of the parent by using the molecular marker assisted breeding technology has an important role in production in terms of selecting the restoring power and selective breeding with a clear genetic background.

[0003] In the process of improving the restorer line, generally, the restorer line is crossed with the restorer line, and the advantage is that the restoring genes do not separate, and the offspring strain to be bred does not need to be identified in terms of the restoring power. However, in some conventional rice or maintainer lines not carrying the restoring gene, other advantageous traits need to be introduced into the specific restorer line. SUMMARY

[0004] The technical problem to be solved by the application is to provide a functional marker primer group of rice restorer gene Rf4 and application thereof in view of the deficiencies of the prior art, the functional marker primer group is designed according to the recombination sequence of the H1 haplotype with the strongest restoring power of rice and the H4 and H5 haplotypes with the highest genotype frequency in the maintainer line as the target, and has the characteristics of high specificity and close linkage.

[0005] To solve the above technical problem, the technical scheme adopted by the application is as follows: a functional marker primer group of rice restorer gene Rf4, characterized in that the functional marker primer group comprises a primer pair for detecting the H1 haplotype on the Rf4 gene locus of rice, a primer pair for detecting the H4 haplotype on the Rf4 gene locus of rice and a primer pair for detecting the H5 haplotype on the Rf4 gene locus of rice. The primer pair for detecting the H1 haplotype on the Rf4 gene locus of rice comprises ZcDI-Rf4-MH63-F and ZcDI-Rf4-MH63-R, and the nucleotide sequences are shown in SEQ ID No. 1-2. The primer pair for detecting the H4 haplotype at the rice restorer gene Rf4 locus comprises ZcDI-Rf4-ZS97-F and ZcDI-Rf4-ZS97-R, and the nucleotide sequences are shown in SEQ ID Nos. 3-4, respectively. The primer pair for detecting the H5 haplotype at the rice restorer gene Rf4 locus comprises ZcDI-Rf4-Nip-F and ZcDI-Rf4-Nip-R, and the nucleotide sequences are shown in SEQ ID Nos. 5-6, respectively.

[0006] The application further provides an application of the functional marker primer set, and the application comprises one or more of the following applications. The functional marker primer set is used for detecting the haplotype of the rice restorer gene Rf4 locus. The functional marker primer set is used for predicting the restorability of a rice restorer line. The functional marker primer set is used for breeding a rice restorer line.

[0007] Preferably, when the functional marker primer set is used for detecting the haplotype of the rice restorer gene Rf4 locus, the detection method comprises the following steps: extracting the genomic DNA of a leaf of a rice to be detected as a template, performing PCR amplification on the template by using the functional marker primer set, and judging the haplotype of the rice restorer gene Rf4 according to the PCR amplification product band.

[0008] Preferably, the reaction system of the PCR amplification comprises the following components: 7.5 μL of 2x PCR Master Mix, 0.7 μL of 10 μM ZcDI-Rf4-MH63-F, 0.7 μL of 10 μM ZcDI-Rf4-MH63-R, 0.7 μL of 10 μM ZcDI-Rf4-ZS97-F, 0.7 μL of 10 μM ZcDI-Rf4-ZS97-R, 0.7 μL of 10 μM ZcDI-Rf4-Nip-F, 0.7 μL of 10 μM ZcDI-Rf4-Nip-R, 1 μL of a DNA template, and ddH2O, which is added to 15 μL.

[0009] Preferably, the judgment method of the haplotype of the rice restorer gene Rf4 locus comprises the following steps: if the PCR amplification product band is 109 bp, the rice restorer gene Rf4 locus to be detected is the H4 haplotype; if the PCR amplification product band is 213 bp, the rice restorer gene Rf4 locus to be detected is the H1 haplotype; and if the PCR amplification product band is 152 bp, the rice restorer gene Rf4 locus to be detected is the H5 haplotype.

[0010] Compared with the prior art, the present application has the following advantages: 1、 The present application analyzes the collinearity between the sequences of four haplotype gene clusters on the functional gene Rf4 recovery locus, screens characteristic recombination sequences for the H1 haplotype with the strongest recovery and the H4 and H5 haplotypes with the highest genotype frequency in the maintainer line, and designs functional markers with the characteristic recombination sequences as the target, which has the characteristics of high specificity and close linkage. This strategy avoids the drawbacks of directly designing primers in the functional gene, i.e. the drawbacks of non-functional gene matching amplification after primer off-target and the inability to distinguish the H1 and H7 haplotypes with strong recovery (the double-copy functional gene sequence of H1 and the single-copy functional gene sequence of H7 are completely identical, and cannot be directly distinguished).

[0011] 2、 The present application designs functional marker primer sets for different haplotypes on the recovery gene Rf4 locus, which can quickly predict the recovery of the offspring of the hybrid population, serve as a simple preliminary preparation, and reduce the overall workload of determining the recovery.

[0012] The present application will be further described in detail below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 FIG. 1 is a schematic diagram of the collinearity relationship of the homologous gene cluster G1 on the recovery gene Rf4 locus between different rice varieties in Example 1 of the present application.

[0014] Figure 2 FIG. 2 is a schematic diagram of the collinearity relationship of the homologous gene cluster G2 on the recovery gene Rf4 locus between different rice varieties in Example 1 of the present application.

[0015] Figure 3 FIG. 3 is a comparison result diagram of the functional marker primer set designed in Example 1 of the present application with the reference genome sequence of Minghui 63 and Zhen Shan 97 rice.

[0016] Figure 4 FIG. 4 is an electropherogram of the detection of the genotypes of rice recovery lines and maintainer lines by the functional marker primer set in Example 2 of the present application. DETAILED DESCRIPTION

[0017] Example 1 This example is the design of the functional marker primer set of the rice recovery gene Rf4.

[0018] The genomic sequences of 3 rice varieties Minghui 63 (MH63): CP054686.1, Zhen Shan 97 (ZS97): CP056062.1, Nip: AP014967.1 were obtained from NCBI website; the genomic sequences of 2 rice varieties II 32: NH277 and IR24: NH186 were obtained from RiceSuperPIRdb database (http: / / www.ricesuperpir.com / ) and subjected to multiple sequence collinearity alignment, as shown in Figure 1 FIG. 1, the gene fragments carried by linkage group G1 (group 1) are all on the negative strand of chromosome 10 of rice, Rf-a and Rf-b are functional genes, the red segments are non-functional gene fragments highly homologous to the functional genes, the gene clusters where the Rf genes are located have undergone multiple transverse amplification events (including inversion, duplication, insertion and deletion), and the green prisms are the segments where the molecular markers are located; as shown in Figure 2As shown, the gene fragments carried by linkage group G2 (group 2) are all on the positive strand of chromosome 10 of rice except the last fragment; Tables 1-5 are the physical positions of the linkage groups of each rice variety on the reference genome. By analyzing the collinearity between the sequences of the four haplotype gene clusters at the functional gene Rf4 recovery locus, the characteristic recombination sequences were screened for the H1 haplotype with the strongest recovery and the H4 and H5 haplotypes with the highest genotype frequency in the maintainer line, and the functional markers were designed by targeting the characteristic recombination sequences. The primer pair ZcDI-Rf4-MH63-F and ZcDI-Rf4-MH63-R for detecting the H1 haplotype at the Rf4 recovery locus was designed, with the nucleotide sequences being 5'-CCAATCATGTTCACCTGGCAC-3' (SEQ ID No. 1) and 5'-GCACTAATCCCGAGAACGCA-3' (SEQ ID No. 2), which targeted a part of the sequence in the H1 marker target, and the nucleotide sequence of the target sequence was shown in SEQ ID No. 7, with a nucleotide sequence length of 213 bp; the primer pair ZcDI-Rf4-ZS97-F and ZcDI-Rf4-ZS97-R for detecting the H4 haplotype at the Rf4 recovery locus was designed, with the nucleotide sequences being 5'-ACGTCCATCCTGTTATAAACAAGCA-3' (SEQ ID No. 3) and 5'-TACGAAACGGCATCCACCC-3' (SEQ ID No. 4), which targeted a part of the sequence in the H4 marker target, and the nucleotide sequence of the target sequence was shown in SEQ ID No. 8, with a nucleotide sequence length of 109 bp; the primer pair ZcDI-Rf4-Nip-F and ZcDI-Rf4-Nip-R for detecting the H5 haplotype at the Rf4 recovery locus was designed, with the nucleotide sequences being 5'-AGCCGTCCACAGGAGTAGTC-3' (SEQ ID No. 5) and 5'-GCCGACCGTCCGACCCAA-3' (SEQ ID No. 6), which targeted a part of the sequence in the H5 marker target, and the nucleotide sequence of the target sequence was shown in SEQ ID No. 9, with a nucleotide sequence length of 152 bp.

[0019] The target sequences of the designed functional marker primer sets were subjected to sequence alignment with the MH63 and ZS97 reference genomes on the RIGW (http: / / rice.hzau.edu.cn / rice_rs3 / ) website, and the results were shown in FIGS. 1-4. Figure 3 As shown, the selected target sequences have high specificity and will not be off-target to other homologous sequences.

[0020] Table 1 Physical positions of linkage groups of rice MH63 on the reference genome Table 2 Physical location of linkage groups on the reference genome of rice ZS97 Table 3 Physical location of linkage groups on the reference genome of rice Nip Table 4 Physical location of linkage groups on the reference genome of rice II32 Table 5 Physical location of linkage groups on the reference genome of rice IR24 Example 2 This example is the application of the functional marker primer set designed in Example 1 for the detection of rice restorer gene Rf4 haplotype and the prediction of the restorability of rice restorer lines.

[0021] Genomic DNA of rice leaves to be tested was extracted; the extracted genomic DNA of rice leaf DNA was used as a template to perform PCR amplification with the designed functional primer marker set ZcDI-Rf4-MH63, ZcDI-Rf4-ZS97, ZcDI-Rf4-Nip, to obtain PCR products; the reaction system of the PCR amplification was as follows: 2x PCR Master Mix 7.5 μL, 10 μM of ZcDI-Rf4-MH63-F 0.7 μL, 10 μM of ZcDI-Rf4-MH63-R 0.7 μL, 10 μM of ZcDI-Rf4-ZS97-F 0.7 μL, 10 μM of ZcDI-Rf4-ZS97-R 0.7 μL, 10 μM of ZcDI-Rf4-Nip-F 0.7 μL, 10 μM of ZcDI-Rf4-Nip-R 0.7 μL, DNA template 1 μL, and ddH2O was added to 15 μL; the program of the PCR amplification was as follows: 94℃ for 5 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 10 min; the obtained PCR products were separated on a polyacrylamide gel and then silver-stained for color development, the amplified PCR products were photographed and saved, and the results were as shown in Figure 4As shown, the Rf4 gene H4 haplotype with a length of 109bp is amplified in the 1-10 maintaining rice varieties Mingnuo 208A, Mingyuan A, Mingrui A, Ming 218A, Mingtuo A, Mingshang A, Guangkang A, Mingtao A, Minghe A and Mingsao A; the Rf4 gene H1 haplotype with a length of 213bp is amplified in the 11-17 restoring rice varieties Minghuo 86, Minghuo 63, Minghuo 3009, Minghuo 1831, Minghuo 6559, Minghuo 1259 and Minghuo 510; and the Rf4 gene H5 haplotype with a length of 152bp is amplified in the 18 maintaining rice variety Nipponbare.

[0022] Example 3 In this example, the functional marker primer set designed in Example 1 is used to detect the seed setting rate of hybrid rice varieties in which the Rf4 restoring gene-carrying H1 haplotype restoring line is the male parent in variety approval.

[0023] As shown in Table 6, the seed setting rate of hybrid rice varieties in variety approval is basically around 80%.

[0024] Table 6 Seed setting rate of hybrid rice varieties in which the Rf4 gene-carrying H1 haplotype restoring line is the male parent Example 4 In this example, the functional marker primer set designed in Example 1 is used in the breeding of rice restoring lines.

[0025] Using the functional marker primer set, the excellent restoring line Minghuo 1831 carrying the H1 haplotype is successfully identified, and based on this material, it is combined with Tianxiang 2A to successfully breed a new hybrid rice variety Tianxiangyou 1831; in 2024, Tianxiangyou 1831 successfully passed the national crop variety approval (Ministry of Agriculture and Rural Affairs of the People's Republic of China Announcement No. 867), and the field test data confirmed that its actual seed setting rate reached 82.6%, reaching the expected level.

[0026] The above is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change and equivalent change made according to the technical essence of the present application to the above embodiment are still within the protection scope of the technical solution of the present application.

Claims

1. A functional marker primer set of rice restorer gene Rf4, characterized in that, The functional marker primer set includes primer pairs for detecting the H1 haplotype at the Rf4 locus of the rice restorer gene, primer pairs for detecting the H4 haplotype at the Rf4 locus of the rice restorer gene, and primer pairs for detecting the H5 haplotype at the Rf4 locus of the rice restorer gene. The primer pairs for detecting the H1 haplotype at the Rf4 locus of the rice restorer gene include ZcDI-Rf4-MH63-F and ZcDI-Rf4-MH63-R, with nucleotide sequences shown in SEQ ID No. 1 to 2, respectively. The primer pairs for detecting the H4 haplotype at the Rf4 locus of the rice restorer gene include ZcDI-Rf4-ZS97-F and ZcDI-Rf4-ZS97-R, with nucleotide sequences as shown in SEQ ID No. 3 to 4, respectively. The primer pairs for detecting the H5 haplotype at the Rf4 locus of the rice restorer gene include ZcDI-Rf4-Nip-F and ZcDI-Rf4-Nip-R, with nucleotide sequences shown in SEQ ID No. 5 to 6, respectively.

2. Use of a functional marker primer set according to claim 1, characterized in that The application includes one or more of the following: The functional marker primer set was used for the detection of haplotypes at the rice restorer gene Rf4 locus; The functional marker primer set is used for predicting the resilience of rice restorer lines; The functional marker primer set is used for rice restorer line breeding.

3. The application according to claim 2, characterized in that, When the functional marker primer set is used to detect the haplotype of the rice restorer gene Rf4 locus, the detection method is as follows: extract genomic DNA from the rice leaf to be tested as a template, perform PCR amplification using the functional marker primer pair, and determine the haplotype of the rice restorer gene Rf4 based on the PCR amplification product band.

4. The application according to claim 3, characterized in that, The PCR amplification reaction system consisted of: 7.5 μL of 2×PCRMaster Mix, 0.7 μL of 10 μM ZcDI-Rf4-MH63-F, 0.7 μL of 10 μM ZcDI-Rf4-MH63-R, 0.7 μL of 10 μM ZcDI-Rf4-ZS97-F, 0.7 μL of 10 μM ZcDI-Rf4-ZS97-R, 0.7 μL of 10 μM ZcDI-Rf4-Nip-F, 0.7 μL of 10 μM ZcDI-Rf4-Nip-R, 1 μL of DNA template, and ddH2O to a final volume of 15 μL. The PCR amplification program was as follows: 94℃ for 5 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 10 min.

5. The application according to claim 3, characterized in that, The method for determining the haplotype of the rice restorer gene Rf4 locus is as follows: if the PCR amplification product band is 109 bp, then the rice restorer gene Rf4 locus to be tested is haplotype H4; if the PCR amplification product band is 213 bp, then the rice restorer gene Rf4 locus to be tested is haplotype H1; if the PCR amplification product band is 152 bp, then the rice restorer gene Rf4 locus to be tested is haplotype H5.

Images