Molecular Marker of Cold Tolerance Gene CTES11b at Rice Young Bud Stage and Its Application

Abstract

The present invention discloses an InDel marker closely linked to the cold tolerance gene CTES11b during the rice young shoot stage. The sequence of the InDel marker is AGGCTACGCACCAATCT, which is located between the 203rd and 221st bases of the nucleotide sequence shown in SEQ ID No. 2. When the sequence between the 203rd and 221st bases is deleted, it has the cold tolerance gene CTES11b during the rice young shoot stage and shows cold tolerance. The present invention identifies the cold tolerance gene CTES11b on chromosome 11 during the young shoot stage and a co-dominant molecular marker for genotype identification thereof. Through the assisted screening of the cold tolerance gene CTES11b marker during the young shoot stage, rice varieties or materials with significantly improved cold tolerance during the young shoot stage can be obtained.

Description

Technical Field

[0001] The present invention belongs to the field of cold tolerance genetic improvement and cold tolerance molecular assisted breeding of rice, and particularly relates to a molecular marker of a cold tolerance gene CTES11b at the rice young shoot stage and its application in cold tolerance genetic improvement of rice. Background Art

[0002] In recent years, great changes have taken place in rice production in China. On the one hand, the production goal has changed from pursuing yield to reducing costs and improving economic benefits; on the other hand, with the rapid development of China's economy, the main rural labor force has transferred to cities and towns, and the shortage of rural labor has become the main limiting factor for the development of rice production. Under such circumstances, direct seeding, due to its time-saving, labor-saving and cost-saving features, is deeply welcomed by farmers and has been rapidly popularized. According to incomplete statistics, the direct seeding area of rice has reached 567,000 hectares in Anhui Province, 490,000 hectares in Hubei Province, 433,000 hectares in Jiangxi Province, 693,000 hectares in Jiangsu Province, 373,000 hectares in Zhejiang Province, 100,000 hectares in Guangdong Province, and 400,000 hectares in Heilongjiang Province (Chen Pin et al., 2013; Zhang Xijuan et al., 2016). Early rice direct seeding in the double cropping rice area in South China and early japonica rice direct seeding in the cold highland rice area in Northeast China often encounter low temperature weather in spring, resulting in missing seedlings and ridges, and serious yield reduction. This requires that rice seeds for direct seeding have strong cold tolerance at the young shoot stage. However, in the above-mentioned rice-growing areas, transplanting seedlings has always been the main method, and cold tolerance at the young shoot stage has not been used as a breeding goal for a long time. As a result, many high-yield and high-quality rice varieties cannot be used as direct seeding rice due to their weak cold tolerance at the bud stage, which restricts the production of direct seeding rice.

[0003] Cultivating cold-tolerant varieties during the young shoot stage is an important goal in rice breeding, especially in the selection of direct-seeded rice varieties. Traditional breeding methods are time-consuming, laborious, difficult in phenotypic identification, and have low breeding efficiency. Since cold tolerance at the young shoot stage is a typical quantitative trait, involving multiple gene loci and having an interaction effect with the environment, it makes the genetic improvement of cold tolerance at the young shoot stage difficult. Utilizing cold-tolerant genes at the young shoot stage and molecular markers tightly linked to them to carry out marker-assisted selection breeding can effectively solve this problem. So far, some gene loci controlling cold tolerance at the young shoot stage have been mapped (Yan Changjie et al., 1999; Zhang et al., 2005; Takeuchi et al., 2007; Qiao Yongli et al., 2005; Chen Wei et al., 2005; Zhang Luxia et al., 2007; Gong Yingjun et al., 2009; Lin Jing et al., 2010; Zhou Yong et al., 2013; Yang Luomiao et al., 2014; Zhu Jinyan et al., 2015; Yang et al., 2016; Jiang Shukun et al., 2020). Some linked marker information has also been reported, but the recombination rate is relatively high and the selection effect is poor. At the same time, a small number of tightly linked markers belong to SNP information obtained by sequencing and have not been converted into tightly linked markers. Therefore, there are few reports and patents on the auxiliary molecular markers for cold-tolerant gene loci at the young shoot stage and the application of the markers in rice molecular breeding. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a molecular marker for the cold-tolerant gene CTES11b at the young shoot stage of rice and its application in the genetic improvement of cold tolerance in rice.

[0005] The technical solution of the present invention is: an InDel marker tightly linked to the cold-tolerant gene CTES11b at the young shoot stage of rice, and the sequence of the InDel marker is AGGCTACGCACCAATCT; it is located between the 203rd and 221st bases of the nucleotide sequence shown in SEQ ID No.2. When the sequence between the 203rd and 221st bases is deleted, it has the cold-tolerant gene CTES11b at the young shoot stage of rice and shows cold tolerance.

[0006] A primer pair for detecting the above InDel marker.

[0007] Furthermore, the nucleotide sequence of the upstream primer of the primer pair is as shown in SEQ ID No.3, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No.4.

[0008] A kit containing the above-mentioned primer pair.

[0009] The application of the above-mentioned InDel marker or primer pair or kit in the auxiliary breeding of cold-tolerant traits at the young shoot stage of rice.

[0010] A method for detecting whether rice contains the cold tolerance gene CTES11b at the young shoot stage, comprising the following steps:

[0011] (1) Extract the genomic DNA of the rice to be detected;

[0012] (2) Using the genomic DNA as a template, design a primer pair according to the upstream and downstream sequences of the InDel molecular marker for PCR amplification;

[0013] (3) Detect and analyze the amplification product. If the sequence AGGCTACGCACCAATCT is deleted at the corresponding InDel locus in the amplification product, the cold tolerance gene CTES11b at the young shoot stage of rice exists and shows cold tolerance.

[0014] Furthermore, the nucleotide sequence of the upstream primer of the primer pair is as shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No. 4; if a 291bp fragment can be amplified, the test material carries the cold tolerance gene CTES11b at the young shoot stage of rice; if a 308bp fragment can be amplified, the test material does not carry the cold tolerance gene CTES11b at the young shoot stage of rice.

[0015] A molecular marker for the cold tolerance gene CTES11b at the young shoot stage of rice, whose nucleotide sequence is as shown in SEQ ID No. 1.

[0016] The application of the above-mentioned molecular marker in the assisted breeding of the cold tolerance gene CTES11b at the young shoot stage of rice.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention identifies the cold tolerance gene CTES11b at the young shoot stage on chromosome 11 and a co-dominant molecular marker that can genotype it.

[0019] 2. Through the assisted screening of the cold tolerance gene CTES11b marker at the young shoot stage, rice varieties or materials with significantly improved cold tolerance at the young shoot stage can be obtained.

[0020] 3. The molecular marker of the present invention can be used for the selection of cold tolerance genotypes at the young shoot stage in the breeding populations of the hybrid offspring and related derivative materials of "Lijiangxintuanheigu", "Kongyu 131" and their hybrid offspring with either of them as one of the parents, and can effectively identify cold tolerance individuals carrying this gene, facilitating timely hybridization and transfer, improving breeding efficiency, and accelerating the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic diagram of the detection result of the CTES11b molecular marker in the embodiment of the present invention;

[0022] Legend of the marks in the figure: M: Molecular marker standard; 1: Shennong 265; 2: Kongyu 131; 3: Jiudao 35; 4 - 12: F8 generation rice lines with weak cold tolerance at the bud stage; 13: Lijiangxintuanheigu; 14: Longdao 345; 15 - 22: F8 generation rice lines with strong cold tolerance at the bud stage. Detailed implementation mode

[0023] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the test materials used in the following examples are all obtained from commercial channels.

[0024] 1. Discovery and molecular marker of cold tolerance gene CTES11b at the rice young bud stage

[0025] "Lijiangxintuanheigu" and "Kongyu 131" are representative rice germplasm resources with cold tolerance at the young bud stage. Using "Lijiangxintuanheigu" with strong cold tolerance at the young bud stage as the male parent and the high - yield and disease - resistant variety "Shennong 265" as the female parent for hybridization, a recombinant inbred line population of F8 generation containing 144 lines was obtained by the single - seed descent method. From 2021 to 2022, QTL analysis of cold tolerance traits at the young bud stage was continuously carried out using this population, and a stable - inheritance major QTL - CTES11b ( Figure 1 ) was detected between 22174756bp and 22459727bp on chromosome 11 of rice. Its increasing - effect allele comes from the cold - tolerant resource "Lijiangxintuanheigu". A 17 - bp InDel (AGGCTACGCACCAATCT) closely linked to the cold tolerance gene CTES11b at the young bud stage was detected in this interval. Introducing the CTES11b gene can increase the cold tolerance level of rice at the bud stage by an average of 2 - 3 levels. In the F8 generation population, the cold tolerance level at the bud stage of the F8 generation lines carrying this gene is 1 - 3 levels, and the cold tolerance level at the bud stage of the lines without this gene is 5 - 7 levels.

[0026] This InDel is located between the 203rd and 221st bases of the nucleotide sequence shown in SEQ ID No.2; when the sequence AGGCTACGCACCAATCT between the 203rd and 221st bases is deleted (as shown in SEQ ID No.1), it has the cold tolerance gene CTES11b of rice at the young bud stage and shows cold tolerance. According to the information upstream and downstream of this InDel, various different primer pairs can be designed to detect this InDel.

[0027] 2. Development and verification of co - dominant molecular markers for identifying the CTES11b genotype of rice

[0028] According to the previous positioning results and sequence differences, the primer of InDel molecular marker CTB11-1 (the upstream primer sequence is shown in SEQ ID No. 3, and the downstream primer sequence is shown in SEQ ID No. 4) closely linked to the cold tolerance gene CTES11b at the young shoot stage was designed. This marker was applied to the breeding offspring population of "Lijiangxintuanheigu" and "Shennong 265" for verification:

[0029] (1) Genomic DNA was extracted using the conventional CTAB method.

[0030] (2) Using genomic DNA as a template and InDel molecular marker CTB11-1 as primers for PCR amplification; the PCR amplification system was 15 μL, containing 50 mM KCl, 10 mM Tris-HCl (pH 8.8), 0.1% Triton-X, 1.5 mM MgCl2, 200 μM each of dNTPs, 0.2 μM of each primer, and 0.5 U Taq DNA polymerase. The reaction program was pre-denaturation at 94 °C for 5 min; each cycle included denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 2 min, for 30 cycles; finally, extension at 72 °C for 8 min.

[0031] (3) The PCR amplification products were separated by 3.5% agarose gel electrophoresis.

[0032] (4) The results were observed and recorded using a gel imaging system.

[0033] The offspring materials carrying the cold tolerance gene CTES11b at the young shoot stage (cold tolerance grade 1-3) could amplify a 291 bp band, while the offspring without the cold tolerance gene CTES11b at the young shoot stage (cold tolerance grade 5-7) could amplify a 308 bp band. It can be seen that this molecular marker primer can be used to identify whether the test material contains the cold tolerance gene CTES11b at the young shoot stage. And this 291 bp amplification product (nucleotide sequence is shown in SEQ ID NO. 1) can be used as a molecular marker for the cold tolerance gene CTES11b at the young shoot stage of rice.

[0034] 3. Molecular breeding application of the cold tolerance gene CTES11b at the young shoot stage of rice

[0035] Through sequencing, identification analysis, etc., it was determined that the cold-tolerant germplasm "Kongyu 131" also contains the cold-tolerant gene CTES11b at the young shoot stage, and a 291bp band can be amplified by using the molecular marker CTB11-1 for detection. Therefore, this patent also includes using "Kongyu 131" or its derived offspring as parents to hybridize with the variety to be improved that does not contain this gene, then performing hybridization or backcrossing, and then using the molecular marker of the present invention for assisted selection to transfer the cold-tolerant gene CTES11b at the young shoot stage into the breeding material to be improved.

[0036] Specific steps: In the hybrid offspring of "Kongyu 131" or the hybrid offspring of its derived materials, use the molecular marker of the present invention for tracking selection or perform assisted final selection in stable materials until the breeding materials are stable.

[0037] The present invention will be further described below in conjunction with examples:

[0038] Using "Kongyu 131" as the male parent and "Jiudao 35" as the female parent for hybridization, use the molecular marker of the present invention for assisted selection of the F6 generation materials. Three stable lines containing the CTES11b gene were selected in the F6 generation. When these lines were detected using the marker CTB11-1, a 291bp band could be amplified instead of a 308bp band. After measurement, the cold tolerance equivalence of these three materials at the bud stage was between 1 and 3 levels. And a new rice variety "Longdao 345" with cold tolerance at the young shoot stage was selected from them.

Claims

1. An InDel marker closely linked to the cold tolerance gene in the rice young shoot stage. The sequence of the InDel marker is AGGCTACGCACCAATCT, which is located at the 204th to 220th bases of the nucleotide sequence shown in SEQ ID No.

2. When there is a deletion, it shows cold tolerance. CTES11b 2. The application of the primer pair or kit for detecting the InDel marker according to claim 1 in the assistant breeding of the cold tolerance trait in the rice young shoot stage.

3. A method for detecting whether rice contains the cold tolerance gene in the rice young shoot stage CTES11b , characterized in that comprising the following steps: (1) Extracting genomic DNA of the rice to be detected; (2) Using the genomic DNA as a template, designing a primer pair according to the upstream and downstream sequences of the InDel marker described in claim 1 for PCR amplification; (3)Detect and analyze the amplification products. If the sequence of AGGCTACGCACCAATCT is deleted at the corresponding InDel locus in the amplification products, the cold tolerance gene in rice young shoot stage exists. CTES11b It shows cold tolerance. The nucleotide sequence of the cold tolerance gene CTES11b in rice young shoot stage is shown in SEQ ID No.

1.

4. The method according to claim 3, characterized in that The nucleotide sequence of the upstream primer of the primer pair is as shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No. 4; when the amplification product is 291 bp, the test material carries the cold tolerance gene at the rice young shoot stage CTES11b ; when the amplification product is 308 bp, the test material does not carry the cold tolerance gene at the rice young shoot stage CTES11b .

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