Screening of DNA markers for high-grain-weight alleles in rice grain-weight QTL qTGW10-21.9 and its application

Abstract

The present invention provides a DNA marker Ten21945 for screening high-grain weight alleles on rice grain weight QTL qTGW10-21.9 and its application, and provides a DNA marker Ten21945 for screening high-grain weight alleles on rice thousand-grain weight QTL qTGW10-21.9 and its application in rice thousand-grain weight improvement breeding. The application of Ten21945 can accurately identify whether the rice variety to be identified carries the IR24 high thousand-grain weight allele on the qTGW10-21.9 locus. Examples show that the thousand-grain weight of rice materials carrying the IR24 high thousand-grain weight allele on qTGW10-21.9 is significantly increased, indicating that the DNA marker Ten21945 designed by the present invention can be effectively applied to the improvement breeding of rice thousand-grain weight.

Description

Technical Field

[0001] The invention belongs to the field of genetic breeding for improving rice grain weight, and relates to the identification of QTL controlling grain weight, the detection of high grain weight alleles and breeding applications. Background Art

[0002] The three elements of rice yield include: number of effective panicles, number of grains per panicle and grain weight. Discovering favorable alleles that control rice grain weight is the basis for improving rice grain weight. At present, 24 quantitative trait loci (QTLs) that control grain weight and grain shape have been cloned from natural rice variation populations, distributed on the remaining 9 chromosomes except chromosomes 4, 11 and 12. Most of these favorable alleles can be detected in modern improved rice varieties, indicating that these genes have been widely used in breeding practice and have played an important role in improving rice grain weight and grain shape.

[0003] Introducing the target favorable alleles into excellent rice varieties through molecular biological technology is an important means of targeted improvement. In the past nearly two decades, molecular marker-assisted selection has been a key technology for targeted improvement, especially in disease and insect resistance. Mining the QTL that controls grain weight and developing DNA markers closely linked to it is the basis for improving rice grain weight using molecular marker-assisted selection. The present invention finely locates a new QTL qTGW10-21.9 that controls grain weight from the backbone parent Teqing and IR24 derivative populations, and the IR24 allele increases grain weight. On this basis, a DNA marker for detecting the IR24 high grain weight allele on qTGW10-21.9 was developed for rice grain weight improvement. Summary of the invention

[0004] The technical problem solved by the present invention is as follows: a DNA marker which can accurately identify the IR24 high-grain weight allele at the rice grain weight QTL qTGW10-21.9 locus is developed, and examples are used to prove that the marker can be effectively applied to the breeding of high-grain weight rice varieties.

[0005] The present invention adopts the following technical solutions:

[0006] According to the chromosomal interval where qTGW10-21.9 is precisely located, the sequence differences between Teqing and IR24 in this interval are found. A DNA marker that can accurately identify the IR24 high grain weight allele at the qTGW10-21.9 locus is designed using the software Oligo 7.0 and is named Ten21945, where Ten represents chromosome 10 and the number represents the specific position on the chromosome of 21.945 Mb. The characteristics of the primers are:

[0007] Ten21945-F: 5'-TCAATCAATTCTGTTGGTATAACTATGATTG-3', as shown in SEQ ID No: 1 in the sequence listing;

[0008] Ten21945-R: 5'-GATGGAGAATTAACGAACGAACGG-3', as shown in SEQ ID NO. 2 in the sequence listing.

[0009] The brief steps for identifying the high grain weight allele at the qTGW10-21.9 locus using this specific DNA marker are as follows:

[0010] (1) DNA extraction: The genomic DNA of the control IR24 and the rice materials to be tested were extracted using conventional methods;

[0011] (2) PCR amplification: Using IR24 and DNA of the material to be tested as templates, Ten21945 was used for amplification. The PCR reaction program was set according to the instructions of the purchased Taq DNA polymerase, and the primer annealing temperature was 55°C;

[0012] (3) Gel electrophoresis: PCR products were separated by 6.0% non-denaturing polyacrylamide gel electrophoresis and visualized by silver staining;

[0013] (4) Allele identification: The amplified product band of the IR24 allele is used as a control. The material to be tested that has the same amplified product band as the control is the carrier of the IR24 high-grain-weight allele. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Electrophoresis results of Ten21945 amplified products

[0015] M: DNA molecular weight control; P1: IR24 (high grain weight allele); P2: Teqing (low grain weight allele); 1-32: strains to be tested DETAILED DESCRIPTION

[0016] Example: Using Ten21945 to identify the IR24 high grain weight allele at the qTGW10-21.9 locus

[0017] qTGW10-21.9 is a new QTL controlling grain weight. Based on its location in the Teqing and IR24 genomes and the previously obtained Teqing and IR24 whole genome sequence information, the sequence differences between the two parents within the interval were determined. The application software Oligo 7.0 was designed to accurately identify the specific DNA marker Ten21945 for the IR24 high grain weight allele at the qTGW10-21.9 locus.

[0018] Conventional methods such as DNA extraction, PCR amplification and electrophoresis were used to identify whether the rice material carried the IR24 high grain weight allele at the qTGW10-21.9 locus. Combined with the results of grain weight identification, it was confirmed that the strains carrying the IR24 high grain weight allele at the qTGW10-21.9 locus showed higher grain weight. The specific implementation steps are as follows:

[0019] (1) Rice materials and field planting: In May 2021, 32 near-isogenic lines selected from the Teqing and IR24 combinations and isolated at the qTGW10-21.9 locus were planted in the experimental field of the China National Rice Research Institute. Each line was planted with 8 plants in 1 row and 2 replicates were set up.

[0020] (2) DNA extraction and marker analysis: DNA was extracted from the leaves of the middle 6 plants in each line, and Ten21945 was used to amplify the DNA of Teqing, IR24 and the near-isogenic line to be tested. The PCR reaction program was set as follows: pre-denaturation for 2 minutes (94°C), denaturation for 45 seconds (94°C), annealing for 45 seconds (55°C), extension for 45 seconds (72°C), 30 cycles, final extension for 2 minutes (72°C), and storage at 10°C. The PCR reaction products were separated by non-denaturing polyacrylamide gel electrophoresis at a concentration of 6.0% and displayed by silver staining. The bands of the amplified products of Teqing and IR24 were used as controls. When the amplified product bands of the material to be tested were consistent with those of IR24, it carried the IR24 high-grain weight allele.

[0021] (3) Grain weight determination: After all strains mature, the seeds of the single plants that grow normally in the middle of each strain are collected and threshed after natural drying. Two portions of 300 full grains are selected from each strain for grain weight determination. Each portion is used as a technical replicate. If the difference between technical replicates exceeds 2.0%, the weight is re-determined.

[0022] (4) Data analysis: One-way ANOVA was used to compare the difference in grain weight between strains carrying the IR24 high-grain-weight allele and those not carrying the allele, with a significance level of p < 0.05.

[0023] The following results were obtained through the above experiments:

[0024] Among the tested rice materials, 15 strains carrying the IR24 high-grain-weight allele were detected, and the remaining strains did not carry the allele ( Figure 1 ). The results of variance analysis showed that there was a significant difference in grain weight between the two strains carrying different alleles (p=0.0007), and the strain carrying the IR24 high grain weight allele increased grain weight by 0.40 g. This result proves that the DNA marker Ten21945 developed by the present invention can accurately identify the IR24 high grain weight allele at the qTGW10-21.9 locus, and can be effectively used in molecular breeding for the IR24 high grain weight allele at the qTGW10-21.9 locus. Sequence Listing <110> China National Rice Research Institute <120> Screening of DNA markers for high-grain-weight alleles in rice grain-weight QTL qTGW10-21.9 and its application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 31 <212> DNA <213> Rice (Oryza sativa) <400> 1 tcaatcaatt ctgttggtat aactatgatt g 31 <210> 2 <211> twenty four <212> DNA <213> Rice (Oryza sativa) <400> 2 gatggagaat taacgaacga acgg 24

Claims

1. Application of DNA marker Ten21945 in screening high grain weight alleles in rice grain weight QTL qTGW10-21.9, characterized in that: The DNA marker Ten21945 was amplified by its amplification primers Ten21945-F and Ten21945-R, Ten21945-F: 5'-TCAATCAATTCTGTTGGTATAACTATGATTG-3', and the nucleotide sequence was the nucleotide sequence shown in SEQ ID NO: 1; Ten21945-R: 5'-GATGGAGAATTAACGAACGAACGG-3', and the nucleotide sequence was the nucleotide sequence shown in SEQ ID NO: 2; Ten in Ten21945 represents chromosome 10, and the number represents the specific position on the chromosome, which is 21.945 Mb.

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