Detection of specific DNA amplification primers for large fragment chromosome translocation in three rice varieties and their application

Abstract

The application discloses specific DNA amplification primers for detecting large fragment translocation of three rice varieties and application of the primers. The three varieties are BANDI, a indica rice variety, LUDI GOCHYA, another indica rice variety, and Kasalath, an Aus variety. The specific DNA amplification primers include four primers, namely, TG7-CHR12, TG58-CHR8-1, TG58-CHR8-2 and TG87-CHR5. In the specific primers, 2251bp, 4207bp, 3813bp and 4113bp bands can be detected in sequence, and the bands are chromosome translocation genotypes. The four specific primers are used to amplify the translocation type and non-translocation type varieties, and the results show that the markers can accurately identify whether the rice chromosomes have translocation or not. The DNA markers designed in the application have high accuracy and strong specificity, and have strong practicability in molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of rice breeding technology and relates to specific DNA amplification primers for detecting large-segment chromosomal translocations in three rice varieties and their applications. Background Technology

[0002] Large-segment translocations of rice chromosomes are a key factor leading to gamete abortion in the F1 generation of hybrids, significantly affecting the full utilization of heterosis in rice. Significant progress has been made in rice pan-genomes, providing more information on chromosomal structural variations (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 chromosomal translocations are mainly concentrated in sequence repetitive regions, making it difficult to develop specific DNA amplification primers at the translocation breakpoints. Developing specific DNA amplification primers for large-segment chromosomal translocation fragments would enable rapid and accurate identification of the genotype of varieties originating from these translocations, thus improving breeding efficiency.

[0003] Three large translocation events occurred at the midpoint of rice chromosome 9, translocating to the tail ends of chromosomes 12, 8, and 5, respectively. The corresponding rice varieties are the indica rice variety BANDI, the indica rice variety LUDI GOCHYA, and the Aus rice variety Kasalath. Specific detection primers developed targeting the breakpoints of these large translocations at the tail ends can directly identify translocated and non-translocated types. Based on the sequence characteristics at these three translocation sites, the patent holder developed four specific DNA amplification primers; testing on translocated and non-translocated varieties confirmed that these four markers can accurately distinguish between large-segment chromosomal translocations and non-translocations. The specific DNA amplification primers provided by this invention have universal application value for identifying large-segment translocation events in these three rice varieties. In breeding research, the detection of large-segment translocation fragments can be used to adjust the pairing of hybrid rice varieties. Summary of the Invention

[0004] The technical problem solved by this invention is to provide four specific DNA amplification markers for detecting large-fragment translocations in three rice varieties, and their detection methods. The technical solution adopted by this invention is as follows.

[0005] A method for detecting large fragment translocations in 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 existence of large-segment chromosomal translocations based on the amplification product results;

[0017] The 2251bp band detected on the TG7-CHR12 primers indicates the chromosomal translocation genotype of the indica rice variety BANDI.

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

[0019] The 3813bp band detected on the TG58-CHR8-2 primers indicates the chromosome translocation genotype of the indica rice variety LUDI GOCHYA.

[0020] The 4113bp band detected on the TG87-CHR5 primers indicates the chromosomal translocation genotype of the Aus rice variety Kasalath.

[0021] The genotypes that do not show bands of 2251bp, 4207bp, 3813bp, and 4113bp in sequence on the above four primer pairs are those without chromosomal translocation.

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

[0023] The remaining 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, for a total of 33 cycles, and a final extension at 68℃ for 10 minutes.

[0024] In a specific implementation, the PCR amplification products are identified by electrophoresis or sequencing.

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

[0026] This invention provides primers or primer combinations for detecting large-fragment translocations in 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: BANDI (indica rice), LUDI GOCHYA (indica rice), and Kasalath (Australian rice).

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

[0038] It also provides the application of primers or primer combinations for detecting large-segment translocations in three rice varieties in the detection of large-segment chromosome translocations in 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 this invention were used to amplify translocation and non-translocation varieties. The results showed that these markers can accurately identify the presence or absence of chromosome translocation in rice. The DNA markers designed in this invention have high accuracy and strong specificity, and are highly practical in molecular breeding. Attached Figure Description

[0040] Figure 1 The detection results of four specific DNA amplification markers for large-segment chromosomal translocations in three rice varieties in typical rice varieties. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0042] Example 1: Development of four specific DNA amplification markers for large-segment chromosomal translocations in three rice varieties

[0043] Large chromosomal translocations are a serious obstacle to the utilization of heterosis in rice, significantly affecting grain filling. Therefore, rapid and accurate identification of large chromosomal translocation breakpoints in cultivars is one of the most crucial steps in the selection of hybrid parents. We used high-quality rice pan-genome sequences to discover several large chromosomal translocation events. First, we downloaded the pan-genome sequence from the rice pan-genome website (https: / / cgm.sjtu.edu.cn / TGSrice / index.html). Analysis revealed chromosomal translocation events exceeding 5 Mb in rice varieties TG7, TG58, and TG87, all originating from the same source. The reference genome for these translocations was the Nipponbare T2T reference genome downloaded from the RiceSuperPIRdb website (http: / / www.ricesuperpir.com / web / download). Then, whole-genome collinearity analysis was performed using the NGenomeSyn program (https: / / github.com / hewm2008 / NGenomeSyn) to obtain specific sequence information of large-segment chromosomal translocations present in the three varieties. Chromosomal translocations often occur in regions rich in repetitive sequences, which poses a significant challenge to accurately identifying chromosomal translocation breakpoints. To overcome the interference of repetitive sequences, molecular markers targeting translocation-specific sequences need to be developed. After extensive screening and testing, we finally developed four pairs of specific DNA molecular markers for the three varieties. For chromosome 12 of the indica rice variety BANDI, amplification primers TG7-CHR12 were designed. The forward primer TG7-CHR12-F sequence is 5'-CAATGTAGTATGAATGAGGGCT-3', and the reverse primer TG7-CHR12-R sequence is 5'-TGGGACTAAAACTCTTGCTCAA-3'. Primers TG58-CHR8-1 were designed for chromosome 8 of the indica rice variety LUDI GOCHYA. The forward primer TG58-CHR8-1-F sequence is 5'-CATACCTTGCCTCACGACTA-3', and the reverse primer TG58-CHR8-1-R sequence is 5'-CTTCCCTCTCCGCCTTCC-3'. Primers TG58-CHR8-2 were also designed for chromosome 8 of the indica rice variety LUDI GOCHYA. The forward primer TG58-CHR8-2-F sequence is 5'-CGAACCTAGGGTTGCTCGTA-3', and the reverse primer TG58-CHR8-2-R sequence is 5'-AACCGGAGAAGTGGCGTTTC-3'.Primers TG87-CHR5 were designed for chromosome 5 of the Aus cultivar Kasalath. The forward primer TG87-CHR5-F sequence is 5'-CAACTGCCCAACACATGAG-3', and the reverse primer TG87-CHR5-R sequence is 5'-CCGCTTCTGCTGGTAGTAGG-3'.

[0044] Genomic DNA was extracted from the samples using the CTAB method. Three common rice varieties, Nipponbare (NIP), 9311 (Indica), and the local Indica variety Digu (DG), were used as amplification controls. PCR amplification was performed using the five DNA amplification markers mentioned above: annealing temperatures were 55.0℃, 57.0℃, 59.0℃, and 59.0℃ respectively; all other PCR reaction conditions were consistent except for the annealing temperature: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 s, annealing for 30 s, 68℃ extension for 4 min, for a total of 33 cycles, a final extension at 68℃ for 10 min, and storage at 16℃. Results showed that the TG7-CHR12 marker detected a band of approximately 2251 bp, TG58-CHR8-1 detected a band of 4207 bp, TG58-CHR8-2 detected a band of 3813 bp, and the TG87-CHR5 PCR product detected a band of 4113 bp. The product sizes were all consistent with theoretical values. Figure 1 Furthermore, non-translocation varieties could not amplify the corresponding specific bands. These specific molecular markers offer advantages of specificity, speed, and convenience, providing a powerful tool for identifying three large-segment chromosomal translocation events under various hybridization combinations and offering important reference for breeding practices.

Claims

1. A primer combination for detecting large-segment translocations in three rice varieties, characterized in that, The primer combination is as follows: 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: BANDI (indica rice), LUDI GOCHYA (indica rice), and Kasalath (Australian rice).

2. Application of primer combinations for detecting large-segment translocations in three rice varieties in the detection of large-segment chromosome translocations in three rice varieties; the three rice varieties are: indica rice variety BANDI, indica rice variety LUDI GOCHYA, and Aus variety Kasalath; The primer combination is as follows: 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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