Specific DNA (deoxyribonucleic acid) amplification primer for detecting large-segment chromosome translocation of three rice varieties and application of specific DNA amplification primer

Abstract

The invention discloses specific DNA (deoxyribonucleic acid) amplification primers for detecting large-fragment translocation of three rice varieties and application of the specific DNA amplification primers. The three varieties are respectively an indica type rice variety BANDI, an indica type rice variety LUDI GOCHYA and an Aus variety Kasalath. The total number of the specific DNA amplification primers is four, and the specific DNA amplification primers comprise TG7-CHR12, TG58-CHR8-1, TG58-CHR8-2 and TG87-CHR5. On the specific primers, the 2251bp band, the 4207bp band, the 3813bp band and the 4113bp band can be detected in sequence, and the genotypes are chromosome translocation genotypes. The four specific primers are used for amplifying translocation type and non-translocation type varieties, and the result shows that the markers can accurately identify whether the rice chromosome translocation exists or not. The DNA marker designed by the invention is high in accuracy and strong in specificity, and has relatively strong practicability in molecular breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of rice breeding, and relates to specific DNA amplification primers for detecting large-segment chromosome translocation of three rice varieties and applications thereof. Background Art

[0002] Large translocation of rice chromosomes is a key factor leading to the abortion of male and female gametes in the hybrid F1 generation, which significantly affects the full utilization of rice hybrid vigor. The current rice pan-genome has made significant progress and provided more information on chromosome structural variation (Zhang Fetal. Long-read sequencing of 111 rice genomes reveals significantly larger pan-genomes. Genome Res. 2022 May; 32(5): 853-863. doi: 10.1101 / gr.276015.121.; Qin P et al. Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations. Cell. 2021 Jun 24; 184(13): 3542-3558.e16. doi: 10.1016 / j.cell.2021.04.046.). Large-segment chromosome translocations are mainly concentrated in sequence duplication regions, and it is difficult to develop specific DNA amplification primers at the chromosome translocation breakpoints. Developing specific DNA amplification primers for large-segment chromosome translocation fragments can quickly and accurately identify the genotype of the source of large-segment chromosome translocation varieties, which is conducive to improving breeding efficiency.

[0003] Three large-segment translocation events occurred in the middle of rice chromosome 9, which were translocated to the tail ends of chromosomes 12, 8 and 5, respectively. The varieties corresponding to the translocation are the indica rice variety BANDI, the indica rice variety LUDI GOCHYA and the Aus variety Kasalath. Developing specific detection primers at the breakpoints of the large-segment translocation at the tail end can directly identify the translocation type and the non-translocation type. The patentee developed four specific DNA amplification primers based on the sequence characteristics at the above three translocation sites; the translocation varieties and non-translocation varieties were tested, and it was confirmed that these four markers can accurately distinguish between large-segment chromosome translocation types and non-translocation types. The specific DNA amplification primers provided by the present invention have universal application value for the identification of large-segment translocation events of three rice varieties. In breeding research, the detection of large-segment translocation fragments can be used to adjust the hybrid rice variety grouping. Summary of the invention

[0004] The technical problem solved by the present invention is to provide four specific DNA amplification markers for detecting large translocation of three rice varieties and their detection methods. The present invention adopts the following technical solutions.

[0005] A method for detecting large translocation of rice varieties, characterized by:

[0006] (1) Using the genomic DNA of the sample to be tested as a template, PCR amplification was performed using chromosome translocation-specific DNA primers TG7-CHR12, TG58-CHR8-1, TG58-CHR8-2 or TG87-CHR5;

[0007] The primers are:

[0008] TG7-CHR12-F: 5'-CAATGTAGTATGAATGAGGGCT-3';

[0009] TG7-CHR12-R: 5'-TGGGACTAAAACTCTTGCTCAA-3';

[0010] TG58-CHR8-1-F: 5'-CATACCTTGCCTCACGACTA-3';

[0011] TG58-CHR8-1-R: 5'-CTTCCCTCTCCGCCTTCC-3';

[0012] TG58-CHR8-2-F: 5'-CGAACCTAGGGTTGCTCGTA-3';

[0013] TG58-CHR8-2-R: 5'-AACCGGAGAAGTGGCGTTTC-3';

[0014] TG87-CHR5-F: 5'-CAACTGCCCAACACATGAGC-3';

[0015] TG87-CHR5-R: 5'-CCGCTTCTGCTGGTAGTAGG-3';

[0016] (2) Determine the presence of large-segment chromosomal translocation based on the amplification product results;

[0017] On the primer TG7-CHR12, the 2251bp band was detected for the chromosome translocation genotype of the indica rice variety BANDI;

[0018] The 4207 bp band detected on primer TG58-CHR8-1 was the chromosome translocation genotype of the indica rice variety LUDI GOCHYA;

[0019] The 3813 bp band detected by primer TG58-CHR8-2 was the chromosome translocation genotype of the indica rice variety LUDI GOCHYA;

[0020] The 4113 bp band detected by primer TG87-CHR5 was the chromosome translocation genotype of Aus rice variety Kasalath;

[0021] The genotypes in which the 2251bp, 4207bp, 3813bp and 4113bp bands cannot be detected in sequence on the above four pairs of primers are those in which no chromosomal translocation has occurred.

[0022] Specifically, the annealing temperatures of the chromosome translocation-specific DNA primers TG7-CHR12, TG58-CHR8-1, TG58-CHR8-2, and TG87-CHR5 for PCR amplification were 55.0°C, 57.0°C, 59.0°C, and 59.0°C, respectively;

[0023] The remaining PCR reaction conditions were the same: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing for 30 s, extension at 68°C for 4 min, for a total of 33 cycles, and final extension at 68°C for 10 min.

[0024] In a specific embodiment, the PCR amplification product is determined by electrophoresis or sequencing.

[0025] More specifically, the electrophoresis is performed using 1% agarose electrophoresis.

[0026] The present invention provides primers or primer combinations for detecting large fragment translocations of three rice varieties, wherein the primers are:

[0027] TG7-CHR12-F: 5'-CAATGTAGTATGAATGAGGGCT-3';

[0028] TG7-CHR12-R: 5'-TGGGACTAAAACTCTTGCTCAA-3';

[0029] TG58-CHR8-1-F: 5'-CATACCTTGCCTCACGACTA-3';

[0030] TG58-CHR8-1-R: 5'-CTTCCCTCTCCGCCTTCC-3';

[0031] TG58-CHR8-2-F: 5'-CGAACCTAGGGTTGCTCGTA-3';

[0032] TG58-CHR8-2-R: 5'-AACCGGAGAAGTGGCGTTTC-3';

[0033] TG87-CHR5-F: 5'-CAACTGCCCAACACATGAGC-3';

[0034] TG87-CHR5-R: 5'-CCGCTTCTGCTGGTAGTAGG-3';

[0035] Each primer pair is packaged separately;

[0036] The three rice varieties are: Indica rice variety BANDI, Indica rice variety LUDI GOCHYA, and Aus rice variety Kasalath.

[0037] The present invention also provides application of the method in detecting large-segment chromosome translocation in three rice varieties.

[0038] Also provided is the use of primers or primer combinations for detecting large-segment translocations of three rice varieties in detecting large-segment chromosomal translocations of three rice varieties; the three rice varieties are: indica rice variety BANDI, indica rice variety LUDIGOCHYA, and Aus variety Kasalath.

[0039] The four specific primers of the present invention were used to amplify translocation and non-translocation varieties, and the results showed that these markers can accurately identify the presence or absence of rice chromosome translocation. The DNA markers designed by the present invention have high accuracy and strong specificity, and are highly practical in molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Detection results of four specific DNA amplification markers for large chromosomal translocation in three rice varieties in typical rice varieties. DETAILED DESCRIPTION

[0041] The present invention is further described below with reference to the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0042] Example 1. Development of four specific DNA amplification markers for large chromosome translocations in three rice varieties

[0043] Large chromosome translocation is one of the serious obstacles to the utilization of rice heterosis, and it significantly affects the fruiting of rice. Therefore, rapid and accurate identification of large chromosome translocation breakpoints of cultivated varieties is one of the most important links in the selection of hybrid parent groups. Therefore, we used the high-quality pan-genome sequence of rice to discover multiple large chromosome translocation events. First, the pan-genome sequence was downloaded from the rice pan-genome website (https: / / cgm.sjtu.edu.cn / TGSrice / index.html). After analysis, it was found that rice varieties TG7, TG58 and TG87 had chromosome translocation events exceeding the 5-Mb level, and the three were from the same source. The reference genome for chromosome translocation is the Nipponbare T2T reference genome downloaded from the RiceSuperPIRdb website (http: / / www.ricesuperpir.com / web / download). Then, the whole genome colinearity analysis was performed between the genomes using the NGenomeSyn program (https: / / github.com / hewm2008 / NGenomeSyn), and the specific sequence information of the large-segment chromosomal translocations in the three varieties was obtained. Chromosome translocations often occur in regions rich in repetitive sequences, which poses a great challenge to accurately identify the breakpoints of chromosomal translocations. In order to overcome the interference of repetitive sequences, it is necessary to develop molecular markers for the specific sequences of the translocation type. After a lot of screening and testing. Finally, we developed four pairs of specific DNA molecular markers for the three varieties. The amplification primer TG7-CHR12 was designed for chromosome 12 of the indica rice variety BANDI. The forward primer TG7-CHR12-F sequence is 5'-CAATGTAGTATGAATGAGGGCT-3', and the reverse primer TG7-CHR12-R sequence is 5'-TGGGACTAAAACTCTTGCTCAA-3'. Primer TG58-CHR8-1 was designed for chromosome 8 of the indica rice variety LUDI GOCHYA, the sequence of the forward primer TG58-CHR8-1-F was 5'-CATACCTTGCCTCACGACTA-3', and the sequence of the reverse primer TG58-CHR8-1-R was 5'-CTTCCCTCTCCGCCTTCC-3'. Primer TG58-CHR8-2 was designed for chromosome 8 of the indica rice variety LUDI GOCHYA, the sequence of the forward primer TG58-CHR8-2-F was 5'-CGAACCTAGGGTTGCTCGTA-3', and the sequence of the reverse primer TG58-CHR8-2-R was 5'-AACCGGAGAAGTGGCGTTTC-3'.Primer TG87-CHR5 was designed for chromosome 5 of the Aus variety Kasalath. The sequence of the forward primer TG87-CHR5-F was 5'-CAACTGCCCAACACATGAG-3', and the sequence of the reverse primer TG87-CHR5-R was 5'-CCGCTTCTGCTGGTAGTAGG-3'.

[0044] The CTAB method was used to extract the genomic DNA of the samples. Three conventional varieties of japonica rice, Nipponbare (NIP), indica rice 9311, and indica rice local variety Digu (DG) were used as amplification controls. The above five DNA amplification markers were used for PCR amplification: the annealing temperatures were 55.0℃, 57.0℃, 59.0℃, and 59.0℃, respectively; except for the annealing temperature, the other PCR reaction conditions were the same: 94℃ pre-denaturation for 2 minutes, 94℃ denaturation for 30 seconds, annealing for 30 seconds, 68℃ extension for 4 minutes, a total of 33 cycles, 68℃ final extension for 10 minutes, and storage at 16℃. The results showed that the marker TG7-CHR12 detected a band with a fragment size of about 2251bp, TG58-CHR8-1 detected a band of 4207bp, TG58-CHR8-2 detected a band of 3813bp, and the PCR product of TG87-CHR5 detected a band of 4113bp. The product sizes were consistent with the theory ( Figure 1 ), and the non-translocation varieties could not amplify the corresponding specific bands. The above specific molecular markers have the advantages of specificity, rapidity and convenience, providing a powerful tool for identifying three large-segment chromosome translocation events under various hybrid combinations, and providing an important reference for breeding practice.

Claims

1. A method for detecting large translocations in rice varieties, characterized in that: (1) Using the genomic DNA of the sample to be tested as a template, PCR amplification was performed using chromosome translocation-specific DNA primers TG7-CHR12, TG58-CHR8-1, TG58-CHR8-2, or TG87-CHR5; The primers are: TG7-CHR12-F: 5'-CAATGTAGTATGAATGAGGGCT-3'; TG7-CHR12-R:5'-TGGGACTAAAACTCTTGCTCAA-3'; TG58-CHR8-1-F: 5'-CATACCTTGCCTCACGACTA-3'; TG58-CHR8-1-R: 5'-CTTCCCTCTCCGCCTTCC-3'; TG58-CHR8-2-F: 5'-CGAACCTAGGGTTGCTCGTA-3'; TG58-CHR8-2-R:5'-AACCGGAGAAGTGGCGTTTC-3'; TG87-CHR5-F:5'-CAACTGCCCAACACATGAGC-3'; TG87-CHR5-R: 5'-CCGCTTCTGCTGGTAGTAGG-3'; (2) Determine the presence of large-segment chromosomal translocation based on the amplification product results; On the primer TG7-CHR12, the 2251bp band was detected for the chromosome translocation genotype of the indica rice variety BANDI; The 4207 bp band detected by primer TG58-CHR8-1 was the chromosome translocation genotype of the indica rice variety LUDI GOCHYA; The 3813 bp band detected by primer TG58-CHR8-2 was the chromosome translocation genotype of the indica rice variety LUDI GOCHYA; The 4113 bp band detected by primer TG87-CHR5 was the chromosome translocation genotype of Aus rice variety Kasalath; The genotypes in which the 2251bp, 4207bp, 3813bp and 4113bp bands cannot be detected in sequence on the above four pairs of primers are those in which no chromosomal translocation has occurred.

2. The method according to claim 1, characterized in that: The annealing temperatures of the chromosome translocation-specific DNA primers TG7-CHR12, TG58-CHR8-1, TG58-CHR8-2, and TG87-CHR5 for PCR amplification were 55.0°C, 57.0°C, 59.0°C, and 59.0°C, respectively; The remaining PCR reaction conditions were the same: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing for 30 s, extension at 68°C for 4 min, for a total of 33 cycles, and final extension at 68°C for 10 min.

3. The method according to claim 1, characterized in that The PCR amplification products were determined by electrophoresis or sequencing.

4. The method according to claim 3, characterized in that The electrophoresis was performed using 1% agarose electrophoresis.

5. Primers or primer combinations for detecting large translocations of three rice varieties, characterized in that: The primers are: TG7-CHR12-F: 5'-CAATGTAGTATGAATGAGGGCT-3'; TG7-CHR12-R:5'-TGGGACTAAAACTCTTGCTCAA-3'; TG58-CHR8-1-F: 5'-CATACCTTGCCTCACGACTA-3'; TG58-CHR8-1-R: 5'-CTTCCCTCTCCGCCTTCC-3'; TG58-CHR8-2-F: 5'-CGAACCTAGGGTTGCTCGTA-3'; TG58-CHR8-2-R:5'-AACCGGAGAAGTGGCGTTTC-3'; TG87-CHR5-F:5'-CAACTGCCCAACACATGAGC-3'; TG87-CHR5-R: 5'-CCGCTTCTGCTGGTAGTAGG-3'; Each primer pair is packaged separately; The three rice varieties are: Indica rice variety BANDI, Indica rice variety LUDI GOCHYA, and Aus rice variety Kasalath.

6. Use of the method according to any one of claims 1 to 4 in detecting large chromosome translocations in three rice varieties.

7. Use of primers or primer combinations for detecting large chromosome translocations in three rice varieties in detecting large chromosome translocations in three rice varieties; the three rice varieties are: indica rice variety BANDI, indica rice variety LUDI GOCHYA, Aus variety Kasalath; The primers are: TG7-CHR12-F: 5'-CAATGTAGTATGAATGAGGGCT-3'; TG7-CHR12-R:5'-TGGGACTAAAACTCTTGCTCAA-3'; TG58-CHR8-1-F: 5'-CATACCTTGCCTCACGACTA-3'; TG58-CHR8-1-R: 5'-CTTCCCTCTCCGCCTTCC-3'; TG58-CHR8-2-F: 5'-CGAACCTAGGGTTGCTCGTA-3'; TG58-CHR8-2-R:5'-AACCGGAGAAGTGGCGTTTC-3'; TG87-CHR5-F:5'-CAACTGCCCAACACATGAGC-3'; TG87-CHR5-R: 5'-CCGCTTCTGCTGGTAGTAGG-3'.

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