An InDel molecular marker for identifying cold resistance of wheat and its application

Abstract

The invention discloses an InDel molecular marker for identifying wheat cold resistance and its application, belonging to the field of molecular biology. An InDel molecular marker for identifying wheat cold resistance traits, the InDel molecular marker is located on chromosome 5A of wheat, and the nucleotide sequence is shown in SEQ ID NO.1. The invention utilizes PCR and electrophoresis technology to perform genotype selection on wheat cold resistance regulating genes to achieve the purpose of providing markers for wheat cold resistance molecular marker assisted breeding. The invention is simple to operate and has high accuracy.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to an InDel molecular marker for identifying cold resistance of wheat and an application thereof. Background Art

[0002] Wheat is one of the three major grain crops in the world and is the staple food for more than 35% of the world's population. Its continued increase in production and stable yield are related to world food security. Most of the varieties promoted in the wheat-growing areas in the middle and lower reaches of the Yangtze River are weak winter or spring varieties. In recent years, extreme weather events have occurred frequently due to abnormal climate, among which low temperature and cold damage have become one of the most important natural disasters affecting wheat growth in this wheat-growing area. Wheat varieties with different cold resistance show large genetic differences in their response to adverse low temperatures. The promotion and application of cold-resistant and cold-damage-resistant wheat varieties is the key to stable production. In traditional breeding, physiological and biochemical or artificially created adverse stress conditions are often used to evaluate the resistance of varieties and individuals of cold-resistant breeding targets, which has the disadvantages of complicated processing, poor accuracy and high workload.

[0003] With the development of molecular biology, molecular marker-assisted selection can improve breeding efficiency and accelerate the breeding process. Finding molecular markers that are closely linked to agronomic traits is the prerequisite for molecular marker-assisted selection breeding. Insertion / deletion polymorphism (InDel) molecular markers are based on the design of specific primers on the sequences on both sides of the insertion / deletion site, and are a type of molecular marker that reflects sequence length polymorphism through polymerase chain reaction (PCR) amplification. InDel markers are developed based on sequence differences, with stable variations and high accuracy, avoiding problems such as ambiguity in subsequent analysis due to nonspecificity and complexity. In the entire genome, the polymorphism frequency of InDel markers is second only to SNP markers. Compared with SNP markers, InDel markers are simple and easy to operate, and can be directly used on an electrophoresis platform to achieve the purpose of genotyping. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of existing phenotypic identification technology, provide an InDel molecular marker for wheat cold resistance identification, and use PCR and electrophoresis technology to perform genotype selection on wheat cold resistance regulatory genes to achieve the purpose of providing markers for wheat cold resistance molecular marker-assisted breeding.

[0005] The technical solution adopted by the present invention is: an InDel molecular marker for identifying the cold resistance trait of wheat, the InDel molecular marker is located on chromosome 5A of wheat, and the nucleotide sequence is shown in SEQ ID NO.1.

[0006] The present invention also discloses a primer pair for amplifying the above-mentioned InDel molecular marker, comprising:

[0007] Ind025-F: 5'-TGGGTTGCTTTCATCATTCA-3' (SEQ ID NO.2)

[0008] Ind025-R: 5'-AAGGATCCCCCTGTTTATGG-3' (SEQ ID NO. 3).

[0009] The invention also discloses a method for identifying the cold resistance trait of wheat, comprising: taking the genome DNA of the wheat to be tested as a template and using the above primer pair to perform PCR amplification.

[0010] The present invention also discloses a method for identifying the cold resistance of wheat. The PCR amplification system is as follows: 3 μl of template DNA, 0.4 μl of 10 μmol of upstream and downstream primers, 0.4 μl of 10 mM dNTPs, 2 μl of 10×PCR buffer, and ddH 2 O supplemented the reaction system to 20 μl.

[0011] Furthermore, the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, for 40 cycles; extension at 72°C for 10 minutes, storage at 16°C, and separation of amplified products by 3.5% agarose gel electrophoresis.

[0012] Furthermore, after PCR amplification and electrophoresis, the InDel marker showed a 200 bp fragment in the cold-resistant variety, while it showed a 218 bp fragment in the non-cold-resistant variety, with a difference of 18 bp.

[0013] The invention also discloses a kit, which is used for detecting the cold resistance trait of wheat and comprises the above primer pair.

[0014] The invention also discloses the application of the above-mentioned InDel molecular marker or primer pair in identifying the cold resistance trait of wheat.

[0015] The invention also discloses the application of the InDel molecular marker or primer pair in cold-resistant wheat auxiliary breeding.

[0016] Beneficial effects achieved by the present invention: The present invention provides an InDel molecular marker for wheat cold resistance identification, and uses PCR and electrophoresis technology to perform genotype selection on wheat cold resistance genes to achieve the purpose of providing markers for wheat cold resistance molecular marker-assisted breeding. The present invention is simple to operate and has a high accuracy rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The genotype detection results of the molecular marker Ind025 on wheat varieties;

[0018] Figure 2 It is the association analysis between some cold-resistant genotypes and non-cold-resistant genotypes and phenotypes;

[0019] Figure 3 It is an InDel-marked sequence for identifying the cold resistance trait of wheat. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with the examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention.

[0021] Example 1 Development of InDel molecular markers for wheat cold resistance

[0022] There were two obvious low temperature periods (lowest temperature < minus 5°C) between December 20, 2020 and January 27, 2021. The lowest temperature reached minus 8 to minus 9°C for four consecutive days from December 29, 2020 to January 1, 2021. The lowest temperature reached minus 10°C on January 6, 2021. A week after the low temperature ended, the field phenotype of spring wheat varieties YD16 and Wheaton was investigated and the frost damage level was identified. It was found that the frost damage level of YD16 was 0.46±0.50, which was significantly (p=3.22e-37) lower than that of wheaton (3.49±0.61). These results show that Wheaton is a cold-intolerant wheat variety, while spring wheat YD16 is a cold-tolerant wheat variety that can tolerate temperatures as low as minus 10°C.

[0023] 40 F clones constructed by crossing YD16 and Wheaton 2-3 The freezing injury levels of the families were identified and the freezing death rates of YD16 and Wheaton were 0% and 54.1%, respectively. 3 Among the strains, 9 strains had a freezing death rate lower than 5%; 7 strains had a freezing death rate higher than 50%; and 24 strains had a freezing death rate between 5% and 50%. Using the 55K SNP chip analysis, it was found that the SNP marker on chromosome 5A was closely linked to the freezing resistance phenotype. Subsequently, we randomly sequenced a region and found that the amplified sequence of the sequencing primer Seq025 primer had an 18bp Indel variation between YD16 and Wheaton. Using the amplified sequence of the Seq025 primer as the template sequence, the molecular marker Ind025 (Ind025-F: 5'-TGGGTTGCTTTCATCATTCA-3'; Ind025-R: 5'-AAGGATCCCCCTGTTTATGG-3') was designed. Ind025 was used to identify 8 F 2 - 3A total of 3260 plants in the family were subjected to genotypic analysis and phenotypic identification. The results showed that the marker genotype of Ind025 was closely associated with the frost resistance phenotype; the average frost damage grade of plants carrying the 200bp fragment frost resistance genotype was 0.9, and the average freezing death rate was 2.4%; the average frost damage grade of plants carrying the 218bp fragment non-freezing genotype was 3.8, and the average freezing death rate was 49.4%. These results show that the molecular marker Ind025 can accurately identify the frost resistance of wheat.

[0024] Example 2 Application of InDel molecular markers for wheat cold resistance

[0025] The InDel molecular marker Ind025 was used to identify the cold resistance of 96 wheat varieties and statistically analyze the phenotypes. The specific steps are as follows:

[0026] 1. Extraction of leaf genomic DNA

[0027] ① Take a plant leaf of about 4 cm and place it in a 2 mL centrifuge tube.

[0028] ② Add a grinding steel ball, cover the lid tightly, put it into liquid nitrogen for 60 seconds, and then use a grinding machine to grind the sample for 90 seconds at a grinding frequency of 60 Hz.

[0029] ③ Add 1000 μl of 1.5% CTAB extract to the ground sample, place it in a 70°C oven, and shake it every 20 minutes.

[0030] ④ Cool to room temperature, add 500 μL of chloroform, mix by inversion, and centrifuge at 12000 r / min for 5 min.

[0031] ⑤ Pipette 600μL of supernatant into a new 1.5ml centrifuge tube, add an equal amount of isopropanol, mix by inverting 50 times, and let stand at -20℃ for 1h; centrifuge at 12000r / min for 5min and discard the supernatant.

[0032] ⑥ Add 1 mL of 75% alcohol to wash and let stand for 1 hour.

[0033] ⑦ Balance and centrifuge at 12000r / min for 5min, discard the alcohol, absorb the excess liquid, and blow dry at room temperature;

[0034] ⑧ Add 200 μL of ddHO 2 O, dissolve at room temperature for 30 min, and store at -20℃ for later use.

[0035] 2. PCR amplification and detection

[0036] The 20 μL PCR reaction system is as follows: 3 μL template DNA, 0.4 μL each of 10 μmol upstream and downstream primers, 0.4 μL of 10 mM dNTPs, 2 μL of 10× PCR buffer, and ddH 2 HO supplemented the reaction system to 20 μL.

[0037] The PCR reaction procedure was: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, 40 cycles; 72°C extension for 10 min, 16°C storage. The amplified products were separated by 3.5% agarose gel electrophoresis, and the test results were as follows: Figure 1 As shown, 200 bp is the freeze-resistant genotype, and 218 bp is the non-freeze-resistant genotype.

[0038] The InDel marker genotypes of some varieties were counted and statistically analyzed in combination with the phenotypic freezing damage level (refer to the Shen Zongtan identification method). There were 40 varieties with cold-resistant genotypes and 56 varieties with non-cold-resistant genotypes. Figure 2 As shown, the cold-resistant genotype and the non-cold-resistant genotype plants were significantly correlated with the phenotype. Figure 3 InDel-marked cold-resistant and non-cold-resistant sequences for identifying wheat cold-resistant traits.

[0039] The above experiments have confirmed that the cold resistance of wheat varieties can be identified by detecting the deletion sequence related to the cold resistance of wheat varieties using the InDel primer Ind025.

[0040] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Yangzhou University Yangzhou Yangtze River Seed Co., Ltd. <120> An InDel molecular marker for identifying cold resistance of wheat and its application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 gagtggagtg ccaccctg 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 tgggttgctt tcatcattca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 aaggatcccc ctgtttatgg 20

Claims

1. A method for identifying the cold resistance of wheat, characterized in that: include: Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed on the InDel molecular markers for identifying the cold resistance traits of wheat; The InDel molecular marker is located on chromosome 5A of wheat, and the nucleotide sequence is shown in SEQ ID NO.1; The primer pairs used for amplification included: Ind025-F: 5'-TGGGTTGCTTTCATCATTCA-3'; Ind025-R: 5'-AAGGATCCCCCTGTTTATGG-3'; After PCR amplification and electrophoresis, the InDel marker showed a 200bp fragment in the cold-resistant variety, while it showed a 218bp fragment in the non-cold-resistant variety, with a difference of 18bp.

2. A method for identifying the cold resistance of wheat according to claim 1, characterized in that: The PCR amplification system is: 3 μl of template DNA, 0.4 μl of 10 μmol of upstream and downstream primers, 0.4 μl of 10 mM dNTPs, 2 μl of 10×PCR buffer, and 20 μl of the reaction system supplemented with ddH2O.

3. The method for identifying the cold resistance of wheat according to claim 1, characterized in that: The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, and 40 cycles; extension at 72°C for 10 minutes, storage at 16°C, and separation of amplified products by 3.5% agarose gel electrophoresis.

4. Use of the primer pair described in claim 1 in identifying the cold resistance trait of wheat.

5. Use of the primer pair according to claim 1 in cold-resistant wheat assisted breeding.

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