InDel9b Molecular Marker of Cold Tolerance Gene LTSS9b at Rice Seedling Stage and Its Application

Abstract

The present invention discloses an InDel9b molecular marker of the cold tolerance gene LTSS9b at the rice seedling stage and its application, which relates to the field of rice breeding. This molecular marker is located in the promoter region (-392 to -1541) of the cold tolerance gene LTSS9b (LOC_Os09g23540) at the rice seedling stage. The present invention provides a primer set for detecting the above InDel9b molecular marker, and a kit including the above primer set. The present invention also provides the application of the above InDel9b molecular marker in identifying the cold tolerance at the rice seedling stage and a method for identifying the cold tolerance at the rice seedling stage. The molecular marker provided by the present invention can screen out cold tolerance varieties at the rice seedling stage. In practical applications, only conventional PCR combined with electrophoresis is required, which has low cost, high throughput and good specificity, and is suitable for breeding and production practices, effectively solving the problem of long cycle existing in the conventional breeding methods of cold tolerance rice varieties.

Description

Technical Field

[0001] The present invention relates to the field of rice breeding, and specifically relates to an InDel9b molecular marker of the cold tolerance gene LTSS9b at the seedling stage of rice and its application. Background Art

[0002] Rice cold damage refers to the phenomenon that rice is in a temperature lower than its normal growth requirement for a long time, resulting in slow growth, and even withering and death. Rice (Oryza sativa L.) originated in tropical and subtropical regions and is more sensitive to cold stress than other cereal crops such as barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). Long-term low-temperature cold damage will severely inhibit germination and early seedling growth. Therefore, breeding cold-tolerant varieties is an effective strategy to reduce the harm of low temperature to rice production.

[0003] The conventional breeding method has a long cycle. Developing cold tolerance molecular markers and using molecular marker-assisted selection breeding (MAS) is simpler and more effective. Therefore, developing molecular markers related to rice cold tolerance, identifying or assisting in identifying rice cold tolerance through molecular markers, and accelerating the process of rice cold tolerance breeding are problems that need to be solved urgently by those skilled in the art. Summary of the Invention

[0004] Aiming at the above deficiencies in the prior art, the present invention provides an InDel9b molecular marker of the cold tolerance gene LTSS9b at the seedling stage of rice and its application. The molecular marker provided by the present invention can screen out cold-tolerant rice varieties at the seedling stage, effectively solving the problem of long cycle existing in the conventional breeding method of cold-tolerant rice varieties.

[0005] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is: providing an InDel9b molecular marker of the cold tolerance gene LTSS9b at the seedling stage of rice, and the InDel9b molecular marker is located at -392 to -1541 in the promoter region of the cold tolerance gene LTSS9b at the seedling stage of rice.

[0006] Further, the identifier of the cold tolerance gene LTSS9b at the seedling stage of rice is LOC_Os09g23540.

[0007] A primer set for detecting the above InDel9b molecular marker, including a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.2.

[0008] The application of the above InDel9b molecular marker or the above primer set in identifying the cold tolerance at the seedling stage of rice.

[0009] A kit for identifying cold tolerance at the seedling stage of rice, comprising the above primer set.

[0010] A method for identifying cold tolerance at the seedling stage of rice, comprising the following steps:

[0011] S1. Extract genomic DNA of the sample to be tested;

[0012] S2. Perform PCR amplification on the genomic DNA using the primer set described in claim 2 to obtain a PCR product;

[0013] S3. Perform agarose gel electrophoresis on the PCR product obtained in step S2, and determine the cold tolerance of the rice variety according to the electrophoresis result.

[0014] Furthermore, in step S2, the PCR amplification system comprises 5 μL of 2×KOD One PCR Mix, 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 μL of template DNA, and 3 μL of water.

[0015] Furthermore, in step S2, the PCR amplification program is preheating at 94°C for 5 min; 30 cycles: 98°C for 10 s, 55°C for 10 s, 68°C for 1 min; and finally extending at 68°C for 5 min.

[0016] Furthermore, in step S3, perform 1% agarose gel electrophoresis at 250 - 260 v for 7 - 8 min.

[0017] Furthermore, in step S3, perform 1% agarose gel electrophoresis at 250 v for 7 min.

[0018] Furthermore, in step S3, if two bands with lengths of 1169 bp and 2771 bp are obtained, it is determined as a cold-tolerant rice variety; if one band with a length of 2771 bp is obtained, it is determined as a cold-sensitive rice variety.

[0019] Furthermore, the nucleotide sequence of the cold-tolerant rice variety is as shown in SEQ ID NO.3, with a total length of 1169 bp.

[0020] Furthermore, the nucleotide sequence of the cold-sensitive rice variety is as shown in SEQ ID NO.4, with a total length of 2771 bp.

[0021] Use of the above method for identifying cold tolerance at the seedling stage of rice in screening or assisting in screening rice varieties with cold tolerance.

[0022] A molecular marker breeding method for screening cold-tolerant rice, comprising the following steps: Using the above cold-tolerant rice variety as a parent for breeding, using the above method for identifying cold tolerance at the seedling stage of rice to identify the cold tolerance of the hybrid offspring obtained from the breeding, and screening the hybrid offspring with cold tolerance.

[0023] The present invention has the following beneficial effects:

[0024] 1. The insertion-deletion (InDel) molecular marker technology is based on the differences between two parents in the whole genome, where there are a certain number of nucleotide insertions or deletions in the genome of one parent. According to the insertion or deletion sites in the genome, some primers are designed to amplify these insertion-deletion sites. The corresponding product fragments obtained after PCR amplification are electrophoresed, and finally the polymorphism of the sample is judged by observing different bands. The InDel detection cycle only takes 2-3 hours, does not rely on the subjective experience of humans in traditional detection, and can obtain accurate detection and identification results in a short time.

[0025] 2. The present invention develops the InDel molecular marker at the (-392~-1541) site of the promoter region of the cold-tolerant gene LTSS9b, and develops primers based on this molecular marker, and then uses them to identify the cold tolerance of rice seedlings. Using the primers provided by the present invention to identify rice germplasm resources, the results show that the PCR products of rice varieties with cold tolerance at the seedling stage will have two bands (product sizes: 1169bp and 2771bp), while cold-sensitive rice varieties will have only one band (product size: 2771bp), indicating that the primers designed by the present invention can amplify the specific gene fragments of rice and distinguish them through electrophoresis.

[0026] 3. In practical applications, the InDel molecular marker provided by the present invention only requires PCR combined with 1% agarose gel electrophoresis, with low cost, high throughput, and high specificity, and is suitable for cold tolerance breeding and production practice of rice seedlings. Description of the Drawings

[0027] Figure 1 It is the electrophoresis result diagram of the PCR amplification product;

[0028] Figure 2 It is the electrophoresis result diagram of the PCR amplification product;

[0029] Figure 3 It is the cold tolerance phenotype diagram of the rice germplasm to be detected at the seedling stage. Detailed Embodiments

[0030] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. Those not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0031] Example 1 Development of the InDel9b Molecular Marker and Primers for Identifying the Cold-Tolerant Gene LTSS9b of Rice Seedlings

[0032] The LTSS9b genomic sequences of the japonica rice cultivar Nipponbare and the indica rice cultivar R498 were downloaded from the website of MBKBASE (https: / / www.mbkbase.org / rice / blastOL). Through the genomic sequences of LTSS9b in Nipponbare and R498, a deletion of approximately 1.6 kb was found in Nipponbare. Then, primers were designed according to the sequences (forward primer: 5’-TGTAATCGTGGTGCACTGTA-3’ (SEQ ID NO.1); reverse primer: 5’-CTTGTCGATGGCATTTCA-3’ (SEQ ID NO.2)). There were two bands in the amplification product of Nipponbare, one band was approximately 2.7 kb in size, and the other band was approximately 1.2 kb in size. Further, the genomic DNAs of the cold-tolerant introgression line DG121 and the cold-sensitive cultivar Guichao 2 were extracted and amplified with the above primer set. The PCR products were subjected to 1% agarose gel electrophoresis, and the results were as Figure 1 shown. Figure 1 In the figure, “+” represents the insertion of 1.6 kb, “-” represents the deletion of 1.6 kb, R498: indica rice cultivar R498, Nip: japonica rice cultivar Nipponbare, GC2: cold-sensitive indica rice cultivar Guichao 2, DG121: cold-tolerant introgression line of Dongxiang common wild rice in Jiangxi.

[0033] It can be Figure 1 seen from the figure that the band of the cold-tolerant introgression line DG121 was consistent with that of Nipponbare, with the same 1.6 kb deletion, and the band of Guichao 2 was consistent with that of R498. Sanger sequencing was performed on the amplification product, and it was found that the cold-tolerant introgression line DG121 had the nucleotide sequence shown in SEQ ID NO.3 (1169 bp), and the cold-sensitive Guichao 2 had the nucleotide sequence shown in SEQ ID NO.4 (2771 bp). The specific band of the cold-tolerant gene can be distinguished by electrophoresis.

[0034] The primer sequences developed for the above molecular markers are as follows:

[0035] Forward primer: 5’-TGTAATCGTGGTGCACTGTA-3’ (SEQ ID NO.1);

[0036] Reverse primer: 5’-CTTGTCGATGGCATTTCA-3’ (SEQ ID NO.2)

[0037] Therefore, by amplifying the genomic DNA of rice germplasm with the above primer set and detecting the band pattern by electrophoresis, the cold tolerance of rice can be judged.

[0038] Example 2 Identification of cold tolerance at the seedling stage of rice using the above InDel9b molecular marker

[0039] 1. Extraction of genomic DNA of rice to be tested

[0040] Take the leaves of rice germplasm and extract its genomic DNA by CTAB method.

[0041] 2. PCR amplification

[0042] Using the genomic DNA of rice to be tested as a template, perform PCR amplification with the following primer pairs to obtain amplification products.

[0043] Among them, the primer sequences are as follows:

[0044] Forward primer: 5’-TGTAATCGTGGTGCACTGTA-3’ (SEQ ID NO.1);

[0045] Reverse primer: 5’-CTTGTCGATGGCATTTCA-3’ (SEQ ID NO.2)

[0046] The PCR amplification system is: 2×KOD One PCR Mix (purchased from TOYOBO, product number: 365700) 5 μL, F primer 0.5 μL, R primer 0.5 μL, template DNA 1 μL, water 3 μL;

[0047] The PCR amplification program is: preheat at 94°C for 5 min; 30 cycles: 98°C for 10 s, 55°C for 10 s, 68°C for 1 min; finally extend at 68°C for 5 min.

[0048] 3. Electrophoresis identification

[0049] Electrophorese the above-mentioned PCR products obtained by amplification on a 1% agarose gel, detect the results with a gel imaging system, and the electrophoresis results are as Figure 2 shown. Figure 2 Among them, “+” represents an insertion of 1.6 kb, “-” represents a deletion of 1.6 kb, and the numbers are the numbers of rice germplasm to be detected.

[0050] Now, select some detection results from the detection results of multiple samples to be tested for illustration. The electrophoresis detection results are shown in Figure 2 , and the sample description corresponding to this electrophoresis result is shown in Table 1.

[0051] Table 1 Material information

[0052]

[0053]

[0054] From Figure 2As can be seen from Table 1, the electrophoretic bands of the cold-tolerant introgression line DG121 are two bands (1169 bp and 2771 bp), while the electrophoretic band of the cold-sensitive Guichao 2 is one band (2771 bp). The electrophoretic bands of rice varieties 82, 107, 164, 156, 1, 71, 48, 421, 396, 256, 359, 116, and 393 are two bands, so these varieties should be cold-tolerant rice varieties. The electrophoretic bands of rice varieties 91, 316, 74, 104, 134, 172, 154, 137, 186, 222, 224, 290, 355, 366, 236, 148, 3, 407, 90, 280, 97, 124, and 174 are one band, so these varieties should be cold-sensitive rice varieties.

[0055] Verified in Example 3

[0056] To verify the accuracy of the identification results in Example 2, the above-mentioned rice varieties to be tested, namely 91, 82, 107, 164, 316, 156, 74, 1, 71, 104, 48, 421, 134, 396, 172, 154, 256, 137, 186, 222, 224, 290, 355, 366, 359, 236, 148, 3, 116, 407, 90, 280, 393, 97, 124, and 174, were subjected to cold tolerance treatment at the seedling stage, as follows:

[0057] Rice varieties from 20 different countries were subjected to cold tolerance treatment at the seedling stage. The selected healthy and plump seeds were put into paper bags and placed in an oven at 42 °C for 2 days to break dormancy. The seeds after breaking dormancy were immersed in 10% sodium hypochlorite solution for 30 minutes for disinfection. The disinfected seeds were rinsed 3 - 4 times with deionized water, and then placed in an incubator at 37 °C for 2 days, with the deionized water changed once a day. After the seeds showed white tips, they were placed in a 96-well PCR plate without a bottom and cultured with deionized water in an artificial climate chamber at 28 °C (14 hours of light and 10 hours of darkness), and the deionized water was changed once every 2 days. When the plants grew to the one-leaf and one-heart stage, they were changed to a 1 / 3 concentration of nutrient solution (volume ratio of nutrient solution: water = 1:2). When the plants grew to the two-leaf and one-heart stage, they were changed to full nutrient solution. After taking pictures and saving them, they were subjected to low-temperature treatment in a low-temperature incubator for a certain period of time (the treatment conditions for japonica rice were: treated at 6 °C for 10 days and recovered at 28 °C for 10 days; the treatment conditions for indica rice were: treated at 10 °C for 10 days and recovered at 28 °C for 7 days), and the phenotypes were counted (survival rate of live seedlings % = number of live seedlings after recovery / total number of seedlings treated × 100%). The judgment standard for plant survival: plants with the third leaf being green or having new leaves growing after recovery were considered surviving plants, and vice versa. The phenotypic identification results are shown in Table 2 and Figure 3 as shown. Figure 3Among them, from top to bottom are the phenotypic diagrams of temperate japonica rice before treatment, the phenotypic diagrams of temperate japonica rice after 10 days of treatment at 6°C and 10 days of recovery at 28°C, the phenotypic diagrams of tropical japonica rice before treatment, the phenotypic diagrams of tropical japonica rice after 10 days of treatment at 6°C and 10 days of recovery at 28°C, the phenotypic diagrams of indica rice before treatment, and the phenotypic diagrams of indica rice after 10 days of treatment at 10°C and 7 days of recovery at 28°C. The scale bar is 1.5 cm.

[0058] Table 2 Genotypes and Cold Tolerance Phenotypes of Materials

[0059]

[0060]

[0061] As can be seen from Table 2, after cold treatment, the survival rates of rice varieties 82, 107, 164, 156, 1, 71, 48, 421, 396, 256, 359, 116, 393 are all above 50%, and they are cold-tolerant rice varieties. After cold treatment, the survival rates of rice varieties 91, 316, 74, 104, 134, 172, 154, 137, 186, 222, 224, 290, 355, 366, 236, 148, 3, 407, 90, 280, 97, 124, 174 are all below 30%, and they are cold-sensitive rice varieties. This result is consistent with the molecular marker identification results in Example 2, indicating that the molecular markers, primers and identification methods provided by the present invention can specifically identify the cold tolerance of rice seedlings at the seedling stage, providing practical value for rice cold tolerance breeding.

[0062] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of a primer set for detecting InDel9b molecular marker in identifying cold tolerance at seedling stage of rice, characterized in that, The primer set includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.

2. The rice varieties include Nipponbare, R498, introgression line DG121, Gui-chao 2, and rice subspecies Tej, Trj, and Ind.

2. A method for identifying cold tolerance at the seedling stage of rice, characterized in that, It includes the following steps: S1. Extract the genomic DNA of the sample to be tested. S2. Perform PCR amplification on the genomic DNA using the primer set described in claim 1 to obtain a PCR product. The primer set includes a forward primer and a reverse primer. The nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.

2. S3. Perform agarose gel electrophoresis on the PCR product obtained in step S2, and determine the cold tolerance of the rice variety according to the electrophoresis results. If two bands with lengths of 1169 bp and 2771 bp are obtained, it is determined as a cold-tolerant rice variety; if only one band with a length of 2771 bp is obtained, it is determined as a cold-sensitive rice variety. The rice varieties include Nipponbare, R498, introgression line DG121, Gui-chao 2, and rice subspecies Tej, Trj, and Ind.

3. The method for identifying cold tolerance at the seedling stage of rice according to claim 2, characterized in that In step S2, the PCR amplification system includes 5 μL of 2×KOD One PCR Mix, 0.5 μL of the forward primer, 0.5 μL of the reverse primer, 1 μL of template DNA, and 3 μL of water. The PCR amplification program is preheating at 94°C for 5 min; 30 cycles: 98°C for 10 s, 55°C for 10 s, 68°C for 1 min; and finally extending at 68°C for 5 min.

4. The method for identifying cold tolerance at the seedling stage of rice according to claim 2, wherein, In step S3, perform 1% agarose gel electrophoresis at 250 - 260 v for 7 - 8 min.

5. Use of the method for identifying cold tolerance at the seedling stage of rice according to any one of claims 2 - 4 in screening rice varieties with cold tolerance. The rice varieties include Nipponbare, R498, introgression line DG121, Gui-chao 2, and rice subspecies Tej, Trj, and Ind.

6. A molecular marker breeding method for screening cold-tolerant rice, characterized in that It includes the following steps: Use the cold-tolerant rice variety described in claim 5 as a parent for breeding, and use the method for identifying cold tolerance at the seedling stage of rice described in claim 2 to identify the cold tolerance of the hybrid offspring obtained from the breeding, and screen the hybrid offspring with cold tolerance. The rice varieties include Nipponbare, R498, introgression line DG121, Gui-chao 2, and rice subspecies Tej, Trj, and Ind.

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