Primer group for detecting purity of two-line hybrid rice and application of primer group

Abstract

The invention provides the primer group for detecting the purity of the rice two-line hybrid, the primer group is high in specificity and free of impurity peaks, the method for detecting the purity of the rice two-line hybrid by utilizing the primer group can be used for detecting a plurality of samples at high throughput, the detection efficiency and accuracy are greatly improved, and in the breeding process of the rice two-line sterile line, the purity of the rice two-line hybrid is greatly improved. Therefore, the breeding period can be effectively shortened, and the high-quality development of the seed industry in China is promoted.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and genetic breeding, and in particular to a primer set for detecting the purity of two-line hybrid rice and an application thereof. Background Art

[0002] Thermo-sensitive Genic Male Sterile 5 (TMS5) gene in rice is a core gene in the two-line hybrid rice breeding system. Its temperature-dependent male sterility provides a crucial genetic foundation for efficient hybrid rice seed production. The discovery and functional elucidation of this gene have driven innovation in hybrid rice technology, enabling the transition from the traditional "three-line method" to the more convenient "two-line method," significantly reducing breeding costs and improving seed production efficiency. The TMS5 gene, located on rice chromosome 2 (LOC_Os02g12130), encodes a protein with ribonuclease (RNase Z) activity. RNase Z is ubiquitous in eukaryotes and primarily cleaves the 3'-end precursor sequence of tRNA, participating in tRNA maturation. Under normal conditions, TMS5 maintains the steady-state expression of genes involved in pollen development by cleaving specific mRNAs in the cytoplasm, such as UbL40 mRNA. However, when the ambient temperature rises to a critical value (usually above 24°C), the stability of the TMS5 protein decreases significantly, resulting in its inability to effectively degrade the target mRNA. These undegraded mRNAs (such as UbL40) accumulate abnormally in pollen mother cells, triggering disordered programmed cell death (PCD) of the tapetum, ultimately leading to pollen abortion. In a low-temperature environment (≤23°C), the function of the TMS5 protein is restored, pollen develops normally, and the plants can self-pollinate to reproduce sterile seeds. This "high-temperature sterility, low-temperature fertility" phenotypic switching mechanism makes it possible for two-line hybrid rice to switch between sterile line reproduction and hybrid seed production by simply controlling temperature without the need for a maintainer line, greatly simplifying the breeding process.

[0003] With the continued expansion of the two-line hybrid rice seed production industry, and the attendant risks of extreme low temperatures inducing sterile maternal line conversion, market requirements for two-line hybrid purity are becoming increasingly stringent. Currently, hybrid purity testing is based on PCR amplification of genomic DNA using specifically designed primers. Currently, various markers are used to identify the temperature-sensitive gene TMS5, each with its own advantages and disadvantages. Polyacrylamide gel electrophoresis-based markers, linked upstream of the TMS5 gene, require less demanding equipment but are inefficient and carry the risk of genetic segregation. Agarose gel electrophoresis-based markers are suitable for testing small sample volumes but have low throughput. KASP markers, designed using SNP typing instruments, offer high throughput but are expensive. Therefore, developing new markers and methods that are low-cost, high-throughput, rapid, efficient, and provide reliable results is crucial for current two-line hybrid rice purity testing. Summary of the Invention

[0004] The present invention aims to provide a primer set for detecting the purity of two-line hybrid rice and its application.

[0005] On the one hand, the present invention provides a primer set for detecting the purity of rice two-line hybrids or a primer set for detecting the genotype of the rice thermo-sensitive genic male sterility gene (TMS5), the primer set comprising a universal primer and two specific primers, the nucleotide sequence of the universal primer being shown in SEQ ID No. 2; the nucleotide sequences of the two specific primers being shown in SEQ ID No. 5-6.

[0006] In one embodiment, the rice thermo-sensitive genic male sterility gene (TMS5) is LOC_Os02g12130.

[0007] In one embodiment, the two specific primers are modified with different fluorescent signals. Preferably, the two specific primers are modified with carboxyfluorescein FAM fluorescent signal at the 5' end and hexachlorofluorescein phosphoramidate HEX fluorescent signal at the 5' end, respectively. More preferably, the two specific primers include a first specific primer and a second specific primer; the nucleotide sequence of the first specific primer is shown in SEQ ID No.5, and the 5' end of the first specific primer is modified with carboxyfluorescein FAM fluorescent signal; the nucleotide sequence of the second specific primer is shown in SEQ ID No.6, and the 5' end of the second specific primer is modified with hexachlorofluorescein phosphoramidate HEX fluorescent signal.

[0008] In one embodiment, the primer set is used to detect the rice TMS5 gene, and the two specific primers detect the A / A type and the A / C type of the TMS5 gene, respectively. Preferably, the two specific primers include a first specific primer and a second specific primer; the nucleotide sequence of the first specific primer is shown in SEQ ID No. 5, and the first specific primer detects the A / A type of the TMS5 gene; the nucleotide sequence of the second specific primer is shown in SEQ ID No. 6, and the second specific primer detects the A / C type of the TMS5 gene.

[0009] On the other hand, the present invention provides a method for detecting the purity of two-line hybrid rice or a method for detecting the genotype of the thermo-sensitive genic male sterility gene (TMS5) of rice, comprising the step of amplifying using the primer set.

[0010] In one embodiment, the method comprises the steps of:

[0011] (1) Extracting genomic DNA from the rice to be tested;

[0012] (2) using the genomic DNA of the rice to be tested as a template and performing PCR amplification using the primer set;

[0013] (3) Detect the amplified product in a capillary electrophoresis machine.

[0014] In one embodiment, the PCR reaction procedure is: pre-denaturation at 94°C to 98°C for 2 to 10 minutes; denaturation at 94°C to 98°C for 30s to 60s, annealing at 42°C to 65°C for 30s to 60s, extension at 72°C for 45s to 60s, 28 to 35 cycles; extension at 72°C for 3 to 10 minutes; and cooling at 4°C for 5 to 20 minutes.

[0015] In one embodiment, the PCR reaction program is: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 5 min; cooling at 4°C for 10 min.

[0016] In one embodiment, the PCR reaction system includes the following components: DNA polymerase buffer 4-10 μL, MgCl2 solution (25 mM) 0.8-3.2 μL, dNTPS 0.4-2.0 μL, Taq DNA polymerase 0.1-1.0 μL, TMS5-F-2 primer 0.1-0.5 μL, TMS5-AR primer 0.1-0.5 μL, TMS5-CR primer 0.1-0.5 μL, genomic DNA 1.6-4.0 μL, and ddH2O filling.

[0017] In one embodiment, the PCR reaction system is: DNA polymerase buffer 4 μL, MgCl2 solution (25 mM) 2 μL, dNTP 0.4 μL, Taq DNA polymerase 0.1 μL, TMS5-F-2 primer 0.4 μL, TMS5-AR primer 0.2 μL, TMS5-CR primer 0.2 μL, genomic DNA 1.6 μL, ddH2O filling.

[0018] In one embodiment, the TMS5-F-2 is a universal primer, and the nucleotide sequence of the universal primer is shown in SEQ ID No. 2; the TMS5-AR and TMS5-CR are specific primers, the TMS5-AR is the first specific primer, and the TMS5-CR is the second specific primer. The nucleotide sequence of the specific primer TMS5-AR is shown in SEQ ID No. 5, and the nucleotide sequence of the specific primer TTMS5-CR is shown in SEQ ID No. 6.

[0019] In one embodiment, the universal primer is TMS5-F-2; the two specific primers are TMS5-AR and TMS5-CR, the first specific primer is TMS5-AR, the second specific primer is TMS5-CR, the nucleotide sequence of the specific primer TMS5-AR is shown in SEQ ID No. 5, and the nucleotide sequence of the specific primer TTMS5-CR is shown in SEQ ID No. 6.

[0020] In one embodiment, the method for determining the TMS5 genotype of the rice to be tested is: if the amplified product shows a single main peak at 153 bp or 159 bp, the TMS5 genotype of the rice to be tested is C / C type, and the rice to be tested is a two-line male parent; if the amplified product shows a single main peak at 159 bp, the TMS5 genotype of the rice to be tested is A / A type, and the rice to be tested is a two-line female parent; if the amplified product shows a single main peak at 153 bp or 159 bp, and a single main peak appears at 159 bp at the same time, the TMS5 genotype of the rice to be tested is A / C type, and the rice to be tested is a two-line hybrid.

[0021] Preferably, the two specific primers include a first specific primer and a second specific primer; the nucleotide sequence of the first specific primer is as shown in SEQ ID No. 5, and the 5' end of the first specific primer is modified by adding carboxyfluorescein FAM fluorescent signal; the nucleotide sequence of the second specific primer is as shown in SEQ ID As shown in No. 6, the 5' end of the second specific primer is modified with a hexachlorofluorescein phosphoramidate (HEX) fluorescence signal; the method for determining the TMS5 genotype of the rice to be tested is as follows: if a single main peak with green fluorescence (HEX fluorescence) appears at 153 bp or 159 bp of the amplified product, the TMS5 genotype of the rice to be tested is C / C, and the rice to be tested is a two-line male parent; if a single main peak with blue fluorescence (FAM fluorescence) appears at 159 bp of the amplified product, the TMS5 genotype of the rice to be tested is A / A, and the rice to be tested is a two-line female parent; if a single main peak with green fluorescence (HEX fluorescence) appears at 153 bp or 159 bp of the amplified product, and a single main peak with blue fluorescence (FAM fluorescence) appears at 159 bp, the TMS5 genotype of the rice to be tested is A / C, and the rice to be tested is a two-line hybrid.

[0022] In one embodiment, the rice sample to be tested is derived from a plant part of rice, such as leaves, fruits, seeds or rhizomes.

[0023] On the other hand, the present invention provides a kit for detecting the purity of two-line hybrid rice or a kit for detecting the genotype of the thermo-sensitive genic male sterility gene (TMS5) of rice, the kit comprising the primer set.

[0024] On the other hand, the present invention provides the use of the primer set or the kit in the purity detection of two-line thermosensitive hybrid rice, or in the molecular marker-assisted breeding of two-line thermosensitive male sterile rice line materials.

[0025] Advantageous Effects of the Invention

[0026] The present invention provides a primer set and method for detecting the purity of two-line hybrid rice. The primer set of the present invention has strong specificity and no impurity peaks. The detection method has low cost, high throughput, high efficiency, and stable and reliable results. The method is applicable to the purity detection of two-line hybrid rice and the breeding of two-line sterile lines.

[0027] The following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the drawings and the following detailed description of the preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Amplification results of different primer combinations.

[0029] Figure 2 .The results of primer combination TMS5-F-2+TMS5-AR / TMS5-CR verification of 112 rice materials. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0031] The sources of the primers in the primer sets described herein are not particularly limited. All raw materials required for the PCR amplification system are commercially available products and can be selected from sources known in the art. The rice materials used can be obtained from commercial sources or national germplasm banks, or are widely used and promoted by those skilled in the art.

[0032] Example 1: Design of specific fluorescence detection primers for the two-line thermosensitive male sterility gene TMS5 in rice

[0033] 1. SNP differential site analysis

[0034] The mutation of the TMS5 gene is a gain-of-function mutation, which makes rice more sensitive to temperature. The pollen is completely sterile under high temperatures and completely fertile under low temperatures. Currently, the main mutation type used for this gene is the mutation of the 71st base of the TMS5 genome from C to A. The homozygous A / A type is temperature-sensitive.

[0035] 2. Design and screening of fluorescent primers for capillary electrophoresis

[0036] This example designs specific detection primers for the TMS5 gene based on capillary electrophoresis and fluorescent PCR techniques, and in accordance with the principle that Taq DNA polymerase can only perform effective polymerization when the 3' terminal base of the primer is completely complementary to the template.

[0037] The primer set design is based on the A / C polymorphism at base 71 of the TMS5 genome. The homozygous A / A type represents a temperature-sensitive genotype. Based on this, four universal forward primers, TMS5-F-1, TMS5-F-2, TMS5-F-3, and TMS5-F-4, were designed, as well as two back primers, TMS5-AR and TMS5-CR, containing differential bases at their 3' ends. The last base at the 3' end of TMS5-AR is the complementary base T of the A type at position 71 of the TMS5 gene, and the last base at the 3' end of TMS5-CR is the complementary base G of the C type at position 71 of the TMS5 gene. Sequence information is shown in Table 1.

[0038] Table 1 Detection primer sequence information

[0039]

[0040] The 5' end of primer TMS5-AR was modified with carboxyfluorescein (FAM), a fluorescent signal, while the 5' end of primer TMS5-CR was modified with hexachlorofluorescein phosphoramidate (HEX). Four universal forward primers were mixed with TMS5-AR and TMS5-CR, respectively, and amplified using DNA from two different rice lines as templates. The universal forward primer combined with primer TMS5-AR specifically amplified the A-type bases of TMS5, producing a FAM signal; the universal forward primer combined with primer TMS5-CR specifically amplified the C-type bases of TMS5, producing a HEX signal.

[0041] PCR reaction conditions: pre-denaturation at 95°C for 2 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 40 s; extension at 72°C for 5 min; and cooling at 4°C for 10 min.

[0042] PCR reaction system: A 20 μL reaction system includes the following components: DNA polymerase buffer 4 μL, MgCl2 solution (25 mM) 2 μL, dNTP 0.4 μL, Taq DNA polymerase 0.1 μL, TMS5-F-2 primer 0.4 μL, TMS5-AR primer 0.2 μL, TMS5-CR primer 0.2 μL, genomic DNA 1.6 μL, and ddH2O filling.

[0043] PCR amplification products were detected in a capillary electrophoresis machine.

[0044] 3. Result judgment

[0045] The method for determining the type of two-line rice accession is as follows: If a single main peak with green fluorescence (HEX fluorescence) appears at 153bp or 159bp in the amplified product, the rice sample is determined to contain homozygous C / C bases, indicating that the accession is a two-line male parent. If a single main peak with blue fluorescence (FAM fluorescence) appears at 159bp in the amplified product, the rice sample is determined to contain homozygous A / A bases, indicating that the accession is a two-line female parent. If a single main peak with green fluorescence (HEX fluorescence) appears at 153bp or 159bp in the amplified product, and a single main peak with blue fluorescence (FAM fluorescence) appears at 159bp, the rice sample is determined to contain heterozygous A / C bases, indicating that the accession is a two-line hybrid. Note: Some two-line male accessions have a 6bp deletion upstream of base 71, resulting in two fragment sizes for the male parent.

[0046] The results are as follows Figure 1 As shown, Figure 1 Amplification plots A and B show the amplification patterns of four universal forward primers (TMS5-F-1, TMS5-F-2, TMS5-F-3, and TMS5-F-4) and two back primers, respectively. The amplification products of primers TMS5-F-1, TMS5-F-3, and TMS5-F-4 exhibited spurious peaks in the paternal lineages and hybrid lineages of the two lines. Due to competitive binding, no peaks were detected in the maternal lineages of the two lines. Primer TMS5-F-2 consistently detected the TMS5 genotypes of the paternal, maternal, and hybrid lineages of the two lines, and the amplified products were consistent with expectations. In other words, the combination of primer TMS5-F-2 with TMS5-AR and TMS5-CR can be used to detect TMS5 gene mutations.

[0047] Example 2: Screening and verification of fluorescence detection primers in 112 rice materials

[0048] In order to verify the practicality of the fluorescence detection primers TMS5-F-2+TMS5-AR / TMS5-CR described in Example 1, 112 rice materials with known two-line temperature-sensitive phenotypes and genotypes were used for verification. The names of the rice materials are shown in Table 2. The verification results are shown in Table 2. Figure 2 As shown, the fluorescent detection primer combination TMS5-F-2 + TMS5-AR / TMS5-CR can clearly distinguish the TMS5 gene genotypes of these 112 rice materials, and the detection results are completely consistent with the expected results. This shows that the fluorescent detection marker of the TMS5 gene of the present invention is fully suitable for purity testing of two-line rice hybrids and breeding of thermosensitive male sterile lines.

[0049] Table 2 Names and types of 112 two-line rice materials

[0050]

[0051]

[0052]

[0053]

[0054] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A primer set for detecting the purity of a two-line hybrid rice or a primer set for detecting the genotype of the thermo-sensitive genic male sterility gene (TMS5) in rice, characterized in that: The primer set includes a universal primer and two specific primers. The nucleotide sequence of the universal primer is shown in SEQ ID No. 2; the nucleotide sequences of the two specific primers are shown in SEQ ID No. 5-6.

2. The primer set according to claim 1, characterized in that The two specific primers are modified with different fluorescent signals respectively.

3. The primer set according to claim 1, characterized in that The primer set is used to detect the rice TMS5 gene, and the two specific primers respectively detect the A / A type and the A / C type of the TMS5 gene.

4. A method for detecting the purity of two-line hybrid rice or a method for detecting the genotype of the thermo-sensitive genic male sterility gene (TMS5) in rice, characterized in that: The method comprises the step of amplifying using the primer set described in any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The method comprises the following steps: (1) Extracting genomic DNA from the rice to be tested; (2) using the genomic DNA of the rice to be tested as a template and performing PCR amplification using the primer set described in any one of claims 1 to 3; (3) Detect the amplified product in a capillary electrophoresis machine.

6. The method according to claim 5, characterized in that The PCR reaction procedure is as follows: pre-denaturation at 94°C to 98°C for 2 to 10 min; denaturation at 94°C to 98°C for 30s to 60s, annealing at 42°C to 65°C for 30s to 60s, extension at 72°C for 45s to 60s, 28 to 35 cycles; extension at 72°C for 3 to 10 min; and cooling at 4°C for 5 to 20 min.

7. The method according to claim 5, characterized in that The PCR reaction system is as follows: DNA polymerase buffer 4-10 μL, MgCl2 solution (25 mM) 0.8-3.2 μL, dNTPS 0.4-2.0 μL, Taq DNA polymerase 0.1-1.0 μL, TMS5-F-2 primer 0.1-0.5 μL, TMS5-AR primer 0.1-0.5 μL, TMS5-CR primer 0.1-0.5 μL, genomic DNA 1.6-4.0 μL, ddH2O filler; The TMS5-F-2 is a universal primer; the TMS5-AR and TMS5-CR are specific primers.

8. The method according to claim 5, characterized in that The method for determining the TMS5 genotype of the rice to be tested is as follows: if a single main peak appears at 153 bp or 159 bp in the amplified product, the TMS5 genotype of the rice to be tested is C / C type, and the rice to be tested is a two-line male parent; if a single main peak appears at 159 bp in the amplified product, the TMS5 genotype of the rice to be tested is A / A type, and the rice to be tested is a two-line female parent; If the amplified product shows a single main peak at 153 bp or 159 bp, and a single main peak appears at 159 bp at the same time, the TMS5 genotype of the tested rice is A / C type, and the tested rice is a two-line hybrid.

9. A kit for detecting the purity of two-line hybrid rice or a kit for detecting the genotype of the thermo-sensitive genic male sterility gene (TMS5) in rice, characterized in that: The kit comprises the primer set according to any one of claims 1 to 3.

10. Use of the primer set according to any one of claims 1 to 3 or the kit according to claim 9 in purity detection of two-line thermo-sensitive hybrid rice, or in molecular marker-assisted breeding of two-line thermo-sensitive male sterile rice lines.

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