InDel9b molecular marker of rice seedling-stage cold-resistant gene LTSS9b and application of InDel9b molecular marker

Abstract

The invention discloses an InDel9b molecular marker of a rice seedling stage cold-resistant gene LTSS9b and application, and relates to the field of rice breeding. The molecular marker is located in a promoter region (-392--1541) of a rice seedling stage cold-resistant gene LTSS9b (LOCOs09g23540), and the invention provides a primer group for detecting the InDel9b molecular marker and a kit comprising the primer group. The invention also provides application of the InDel9b molecular marker in identifying the cold resistance of the rice in the seedling stage and a method for identifying the cold resistance of the rice in the seedling stage. The molecular marker provided by the invention can be used for screening out the rice cold-resistant variety in the seedling stage, only conventional PCR (Polymerase Chain Reaction) is combined with electrophoresis in practical application, the cost is low, the flux is high, the specificity is good, the molecular marker is suitable for breeding and production practice, and the problem that the period is long in a conventional breeding method of the rice cold-resistant variety is effectively solved.

Description

Technical Field

[0001] The invention relates to the field of rice breeding, and in particular to an InDel9b molecular marker of a rice seedling cold-tolerant gene LTSS9b and an application thereof. Background Art

[0002] Rice chilling injury refers to the phenomenon that rice grows slowly, or even withers and dies, when it is exposed to temperatures below the temperature required for normal growth for a long time. Rice (Oryza sativa L.) originates from 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 and chilling injury can seriously inhibit germination and early growth of seedlings. Major rice-producing areas in my country often suffer from low temperature and chilling injury, resulting in reduced production. According to statistics, low temperature stress causes a loss of about 3-5 million tons of rice production in my country each year. Therefore, breeding cold-resistant varieties is an effective strategy to reduce the harm of low temperature to rice production.

[0003] Conventional breeding methods have a long cycle, and developing cold-tolerant molecular markers and using molecular marker-assisted selection (MAS) is simpler and more effective. Therefore, developing molecular markers related to rice cold tolerance, identifying rice cold tolerance through molecular markers or assisting in identifying rice cold tolerance, and accelerating the cold tolerance breeding process of rice are issues that need to be urgently addressed by those skilled in the art. Summary of the invention

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

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve its technical problem is: providing the InDel9b molecular marker of the rice seedling cold-tolerant gene LTSS9b, which is located at -392 to -1541 in the promoter region of the rice seedling cold-tolerant gene LTSS9b.

[0006] Furthermore, the identifier of the rice seedling cold-tolerance gene LTSS9b is LOC_Os09g23540.

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

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

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

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

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

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

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

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

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

[0016] Further, in step S3, 1% agarose gel electrophoresis was performed at 250-260V for 7-8min.

[0017] Further, in step S3, 1% agarose gel electrophoresis was performed at 250 V for 7 min.

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

[0019] Furthermore, the nucleotide sequence of the cold-tolerant rice variety is 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 shown in SEQ ID NO. 4, with a total length of 2771 bp.

[0021] The method for identifying cold tolerance of rice at the seedling stage is used in screening or auxiliary screening of cold-tolerant rice varieties.

[0022] A molecular marker breeding method for screening cold-resistant rice comprises the following steps: using the above-mentioned cold-resistant rice variety as a parent for breeding, using the above-mentioned method for identifying cold resistance of rice seedlings to identify the cold resistance of hybrids obtained by breeding, and screening hybrids with cold resistance.

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

[0024] 1. Insertion-deletion (InDel) molecular marker technology is based on the difference between the two parents in the whole genome, in which a certain number of nucleotides are inserted or deleted in the genome of one of the parents. According to the insertion or deletion sites in the genome, some primers are designed to amplify these insertion and deletion sites. The corresponding product fragments obtained after PCR amplification are subjected to electrophoresis, and finally the polymorphism of the sample is judged by observing different bands. The InDel detection cycle only takes 2-3 hours, and does not rely on the subjective experience of people in traditional detection, and accurate detection and identification results can be obtained in a short time.

[0025] 2. The present invention develops the promoter region (-392 to -1541) site of the cold-tolerant gene LTSS9b into an InDel molecular marker, and develops primers based on the molecular marker, which are then used to identify the cold tolerance of rice seedlings. The primers provided by the present invention were used to identify rice germplasm resources. The results showed that the PCR product of the cold-tolerant rice variety in the seedling stage had two bands (product size: 1169 bp and 2771 bp), while the cold-sensitive rice variety had only one band (product size: 2771 bp), indicating that the primers designed by the present invention can amplify the specific gene fragments of rice and distinguish them by electrophoresis.

[0026] 3. In practical applications, the InDel molecular marker provided by the present invention only requires PCR combined with 1% agarose gel electrophoresis, which has low cost, high throughput, and high specificity, and is suitable for cold-resistant breeding and production practice of rice seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the electrophoresis result of PCR amplification products;

[0028] Figure 2 This is the electrophoresis result of PCR amplification products;

[0029] Figure 3 This is the cold tolerance phenotype of the rice germplasm to be tested at the seedling stage. DETAILED DESCRIPTION

[0030] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0031] Example 1 Development of InDel9b Molecular Marker and Primers for Identifying Rice Seedling Cold Tolerance Gene LTSS9b

[0032] From MBKBASE( https: / / www.mbkbase.org / rice / blastOL ) website to download the LTSS9b genome sequences of the japonica rice variety Nipponbare and the indica rice variety R498. Through the genome sequences of LTSS9b in Nipponbare and R498, it was found that there was a deletion of about 1.6kb in Nipponbare. Therefore, primers were designed according to the sequence (forward primer: 5'-TGTAATCGTGGTGCACTGTA-3' (SEQ ID NO.1); reverse primer: 5'-CTTGTCGATGGCATTTCA-3' (SEQ ID NO.2)). The amplified product of Nipponbare had two bands, one of which was about 2.7kb in size and the other was about 1.2kb in size. Further, the genomic DNA of the cold-tolerant introgression line DG121 and the cold-sensitive variety Guichao 2 was extracted, amplified with the above primer set, and the PCR product was subjected to 1% agarose gel electrophoresis. The results are as follows: Figure 1 shown. Figure 1 In the figure, “+” represents an insertion of 1.6 kb, and “-” represents a deletion of 1.6 kb, R498: indica rice variety R498, Nip: japonica rice variety Nipponbare, GC2: cold-sensitive indica rice variety Guichao 2, DG121: cold-tolerant Jiangxi Dongxiang common wild rice introgression line.

[0033] Depend on Figure 1 It can be seen that the cold-tolerant introgression line DG121 has the same band as Nipponbare, with the same 1.6kb deletion, and the band of Guichao 2 is consistent with R498. Sanger sequencing of the amplified product revealed that the cold-tolerant introgression line DG121 has the nucleotide sequence (1169bp) shown in SEQ ID NO.3, and the cold-sensitive Guichao 2 has the nucleotide sequence (2771bp) shown in SEQ ID NO.4. The specific bands 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, the rice germplasm genome is amplified according to the above primer set, and the band pattern is detected by electrophoresis to determine the cold tolerance of rice.

[0038] Example 2 Identification of cold tolerance of rice seedlings using the above-mentioned InDel9b molecular marker

[0039] 1. Extraction of rice genomic DNA

[0040] The leaves of rice germplasm were taken and their genomic DNA was extracted using the CTAB method.

[0041] 2. PCR Amplification

[0042] Using the rice genomic DNA to be tested as a template, PCR amplification was performed using the following primer pairs to obtain the amplified product.

[0043] 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 was: 2×KOD One PCR Mix (purchased from TOYOBO, catalog 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 was as follows: preheating at 94°C for 5 min; 30 cycles: 98°C for 10 s, 55°C for 10 s, and 68°C for 1 min; and finally extension at 68°C for 5 min.

[0048] 3. Electrophoresis Identification

[0049] The PCR product obtained by the above amplification was subjected to electrophoresis using 1% agarose gel and the results were detected using a gel imaging system. The electrophoresis results were as follows: Figure 2 shown. Figure 2 In the figure, “+” represents an insertion of 1.6 kb, “-” represents a deletion of 1.6 kb, and the numbers are the numbers of the rice germplasm to be tested.

[0050] Now we select some test results from the test results of various samples to be tested for illustration. The electrophoresis test results are shown in Figure 2 The sample description corresponding to the electrophoresis result is shown in Table 1.

[0051] Table 1 Material information

[0052]

[0053]

[0054] Depend on Figure 2 As shown in Table 1, the electrophoresis bands of the cold-tolerant introgression line DG121 are two bands (1169 bp and 2771 bp), while the electrophoresis band of the cold-sensitive Guichao 2 is one band (2771 bp). The electrophoresis 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 electrophoresis 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, 174 are one band, so these varieties should be cold-sensitive rice varieties.

[0055] Example 3 Verification

[0056] In order to verify the accuracy of the identification results in Example 2, the above-mentioned rice varieties 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 treated with cold tolerance at the seedling stage. The selected healthy and plump seeds were placed in paper bags and placed in a 42°C oven for 2 days to break dormancy. The dormant seeds were then immersed in a 10% sodium hypochlorite solution for disinfection for 30 minutes. The disinfected seeds were rinsed with deionized water 3-4 times, placed in a 37°C incubator, soaked for 2 days, and the deionized water was changed once a day. After the seeds turned white, they were placed in a 96-well PCR plate with the bottom removed and cultured in a 28°C artificial climate chamber (14 hours of light and 10 hours of darkness) with deionized water, and the deionized water was changed every 2 days. When the plants grew to the 1-leaf 1-heart stage, they were replaced with a 1 / 3 concentration of nutrient solution (the volume ratio of nutrient solution: water = 1:2). When the plants grow to the 2-leaf 1-heart stage, the solution is replaced with complete nutrient solution. After taking photos, the plants are placed in a low-temperature incubator for a certain period of time (the treatment conditions for japonica rice are: 6°C treatment for 10 days, 28°C recovery for 10 days; the treatment conditions for indica rice are: 10°C treatment for 10 days, 28°C recovery for 7 days), and the phenotypes are counted (survival seedling rate % = number of surviving seedlings after recovery / total number of treated seedlings × 100%). Plant survival criteria: plants with green third leaves or new leaves growing after recovery are considered to be surviving plants, otherwise they are considered to be dead plants. The results of phenotypic identification are shown in Tables 2 and Figure 3 shown. Figure 3 In the figure, from top to bottom are the phenotype of temperate japonica rice before treatment, the phenotype of temperate japonica rice after treatment at 6℃ for 10 days and recovery at 28℃ for 10 days, the phenotype of tropical japonica rice before treatment, the phenotype of tropical japonica rice after treatment at 6℃ for 10 days and recovery at 28℃ for 10 days, the phenotype of indica rice before treatment, and the phenotype of indica rice after treatment at 10℃ for 10 days and recovery at 28℃ for 7 days. The scale is 1.5 cm.

[0058] Table 2 Genotypes and cold-tolerant phenotypes of materials

[0059]

[0060]

[0061] As shown in Table 2, rice varieties 82, 107, 164, 156, 1, 71, 48, 421, 396, 256, 359, 116, and 393 have a survival rate of more than 50% after cold treatment, and are cold-resistant rice varieties. 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 have a survival rate of less than 30% after cold treatment, and are cold-sensitive rice varieties. This result is consistent with the result of molecular marker identification in Example 2, indicating that the molecular markers, primers, and identification methods provided by the present invention can specifically identify the cold resistance of rice seedlings, and provide practical value for cold-resistant rice breeding.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The InDel9b molecular marker of the rice seedling cold-tolerant gene LTSS9b is characterized by: The InDel9b molecular marker is located at -392 to -1541 of the promoter region of the rice seedling cold-tolerant gene LTSS9b.

2. A primer set for detecting the InDel9b molecular marker described in claim 1, characterized in that: It comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.

2.

3. Use of the InDel9b molecular marker described in claim 1 or the primer set described in claim 2 in identifying cold tolerance of rice at the seedling stage.

4. A kit for identifying cold tolerance of rice seedlings, characterized in that: The method comprises the primer set as claimed in claim 2.

5. A method for identifying cold tolerance of rice seedlings, characterized in that: The following steps are involved: S1. Extract genomic DNA of the sample to be tested; S2. performing PCR amplification on the genomic DNA using the primer set described in claim 2 to obtain a PCR product; S3. Performing agarose gel electrophoresis on the PCR product obtained in step S2, and determining the cold tolerance of the rice variety according to the electrophoresis result.

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

7. The method for identifying cold tolerance of rice seedlings according to claim 5, characterized in that: In step S3, 1% agarose gel electrophoresis was performed at 250-260 V for 7-8 min.

8. The method for identifying cold tolerance of rice seedlings according to claim 5, characterized in that: In step S3, if two bands with lengths of 1169 bp and 2771 bp are obtained, the rice variety is determined to be cold-tolerant; if a band with a length of 2771 bp is obtained, the rice variety is determined to be cold-sensitive.

9. Use of the method for identifying cold tolerance of rice at the seedling stage according to any one of claims 5 to 8 in screening or assisting in screening of cold-tolerant rice varieties.

10. A molecular marker breeding method for screening cold-tolerant rice, characterized in that: The method comprises the following steps: using the cold-resistant rice variety described in claim 9 as a parent for breeding, using the method for identifying cold resistance of rice seedlings described in claim 5 to identify the cold resistance of hybrids obtained by breeding, and screening hybrids with cold resistance.

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