Wheat cold resistance gene loci, primer combinations and their applications

Abstract

This invention belongs to the field of crop molecular marker technology, specifically relating to a wheat cold resistance gene locus, primer combinations, and their applications. This invention provides eight gene loci located on wheat chromosome 5A that are associated with cold resistance. KASP markers are provided based on these gene loci for easy verification. The application of these gene loci in identification kits and gene chip identification methods is also provided.

Description

Technical Field

[0001] This invention belongs to the field of crop molecular marker technology, specifically relating to a wheat cold resistance gene locus, primer combination, and its application. Background Art

[0002] Cold hardiness is an important agronomic trait in wheat, determining its growth and yield performance under cold conditions. Specifically, in cold weather, wheat with weak cold hardiness may experience severe impacts on its growth and development, including leaf damage and inhibited root growth, leading to slow or stagnant overall growth. Wheat with weak cold hardiness may fail to complete its normal growth cycle in cold environments, resulting in fewer grains per ear and lower thousand-grain weight, ultimately affecting yield. Conversely, wheat varieties with strong cold hardiness can maintain high yields even under cold conditions. Cold weather can also affect wheat quality, such as protein content and gluten quality. Wheat varieties with strong cold hardiness maintain good quality even under cold conditions. Cold hardiness also determines the geographical distribution range of wheat. Wheat varieties with strong cold hardiness can be planted in a wider range of areas, including some cold regions, thereby expanding the planting area and yield of wheat.

[0003] Therefore, improving the cold resistance of wheat is of great significance for ensuring wheat yield and quality. Breeding wheat varieties with strong cold resistance is an effective way to achieve this goal. While there are some reports on cold resistance gene loci located on wheat chromosome 5A, many loci remain to be explored. Summary of the Invention

[0004] This invention locates stable genetic loci significantly associated with wheat cold resistance through genome-wide association study (GWAS), and designs competitive allele-specific PCR (KASP) based on SNP differences between different alleles. It can be used for screening wheat cold-resistant germplasm materials and marker-assisted selection breeding of related genes.

[0005] To address the problems existing in the prior art, this invention provides a primer combination for wheat cold-resistance gene loci and its application, facilitating the screening and breeding of wheat germplasm resources. The discovery and development of more wheat cold-resistance loci facilitates researchers in breeding wheat varieties with stronger cold resistance, which is of great significance for improving wheat cold resistance and ensuring wheat yield and quality.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] Information on wheat cold resistance gene loci and SNPs significantly associated with wheat cold resistance is as follows:

[0008] SNP serial number QTL chromosome physical location SNP1 K5A482358 qCld-5A.1 5A 482358796 SNP2 K5A511491 qCld-5A.2 5A 511491562 SNP3 K5A521002 qCld-5A.3 5A 521002538 SNP4 K5A523148 qCld-5A.4 5A 523148083 SNP5 K5A526587 qCld-5A.5 5A 526587243 SNP6 K5A528271 qCld-5A.6 5A 528271904 SNP7 K5A537614 qCld-5A.7 5A 537614326 SNP8 K5A538995 qCld-5A.8 5A 538995933

[0009] The physical location is referenced from the sequence of Chinese Spring genome v1.0.

[0010] KASP primer combinations were designed based on wheat cold resistance gene loci. Forward primer F1 was fitted with a linker containing the FAM sequence “5'-GAAGGTGACCAAGTTCATGCT-3'”, and forward primer F2 was fitted with a linker containing the HEX sequence “5'-GAAGGTCGGAGTCAACGGATT-3'”. The primer combinations included the following eight types.

[0011]

[0012]

[0013] In the table above, solid lines underlined SNP sites.

[0014] Application of the gene loci in the identification kit.

[0015] The application of the gene loci in identifying gene chips.

[0016] The beneficial effects of the present invention are:

[0017] (1) This invention provides eight gene loci on wheat chromosome 5A that are related to cold resistance.

[0018] (2) KASP markers were provided based on gene loci to facilitate verification.

[0019] (3) It provides the application of gene loci in identification kits and identification gene chips. Attached Figure Description

[0020] Figure 1 Manhattan plot of cold resistance of 23SZR wheat in Example 1;

[0021] Figure 2 The image shows the QQ graph of the cold resistance of 23SZR wheat in Example 1;

[0022] Figure 3 Manhattan plot of cold resistance of 23YZR wheat in Example 1;

[0023] Figure 4 The image shows the QQ graph of the cold resistance of 23YZR wheat in Example 1;

[0024] Figure 5The KASP classification diagrams (ABCDEFGH represent the KASP classification diagrams of SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8, respectively);

[0025] Figure 6 Box plot of the data analysis results of frost damage severity of wheat with different allele types in Example 3; Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0027] Example 1: Genome-wide association analysis of wheat cold resistance

[0028] 1.1 Wheat material

[0029] The study used 203 natural populations for genome-wide association analysis. These materials mainly consist of wheat varieties and superior lines from the Huang-Huai wheat region and the northern winter wheat region of my country.

[0030] 1.2 Identification of cold resistance phenotype

[0031] Wheat varieties were planted in October 2022 at the Dishang Experimental Station (23SZR) of the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, and the Gaoyi Experimental Station (23YZR) of the Chinese Academy of Agricultural Sciences. The degree of frost damage was investigated in February 2023. Each variety was planted in one row with a row length of 1m and a row spacing of 25cm. Two replicates were set up for each planting site.

[0032] Frost damage is assessed using a 1-5 grade method:

[0033] Level 1 - No frost damage;

[0034] Grade 2 - Leaf tips are yellowed due to frost damage;

[0035] Level 3 - Half of the leaves are frozen to death;

[0036] Level 4 - All leaves withered;

[0037] Level 5 - The plant or most of the tillers have died from frost.

[0038] 1.3 Genome-wide association analysis

[0039] Based on the 660K microarray genotyping results, SNP markers with a minimum allele frequency (MAF) < 0.05 and missing data > 20% were removed using PLINK software. Quality control was performed on the 660K SNP microarray genotyping data. Genome-wide association analysis (GWAS) was conducted using a mixed linear model (MLM) in TASSEL 5.0 software, with a significant SNP threshold of P < 1.0e-3 (i.e., -log10P ≥ 3). A total of 198,762 SNPs were identified that could be used for subsequent GWAS analysis.

[0040] A GWAS study was conducted using phenotypic data of cold resistance levels under two different environmental conditions throughout the year and genotyping results from a 660K microarray. Eight significantly associated genetic loci were identified, as shown in Table 1 below, all located on chromosome 5A. For each locus, a suitable SNP was selected, resulting in eight cold resistance-related loci. The physical locations were determined using the sequences from the Chinese Spring genome v1.0.

[0041] Table 1. Information on SNP sites significantly associated with wheat cold resistance.

[0042] SNP serial number QTL Chromosome Positon SNP1 K5A482358 qCld-5A.1 5A 482358796 SNP2 K5A511491 qCld-5A.2 5A 511491562 SNP3 K5A521002 qCld-5A.3 5A 521002538 SNP4 K5A523148 qCld-5A.4 5A 523148083 SNP5 K5A526587 qCld-5A.5 5A 526587243 SNP6 K5A528271 qCld-5A.6 5A 528271904 SNP7 K5A537614 qCld-5A.7 5A 537614326 SNP8 K5A538995 qCld-5A.8 5A 538995933

[0043] Example 2: Development and validation of KASP markers for wheat cold resistance loci

[0044] 2.1 Wheat material

[0045] The study used 315 natural population materials for cold resistance site validation. These materials mainly consist of wheat varieties and superior lines from the Huang-Huai wheat region and the northern winter wheat region of my country.

[0046] 2.2 Research Methods

[0047] 2.2.1 Extraction of wheat genomic DNA

[0048] (1) Take an appropriate amount of young wheat leaves into a 2mL centrifuge tube, freeze them quickly with liquid nitrogen, and grind the leaves into powder on a grinder.

[0049] (2) Add 800 μL of TPS to the broken leaf tissue, incubate in a water bath at 80°C for 30 min, shake several times every 10 min, and centrifuge at 12000 r / min for 10 min at room temperature.

[0050] (3) Immediately after centrifugation, take 500 μL of the supernatant, add an equal volume of pre-cooled isopropanol, shake gently up and down a few times, and white flocculent matter will be produced. Centrifuge at 12000 r / min for 10 min.

[0051] (4) Discard the supernatant, and wash the precipitate with 300 μL of 75% ethanol until the precipitate becomes transparent;

[0052] (5) Let it stand in a fume hood to air dry. After the ethanol has evaporated, add 100 μL ddH2O to dissolve the DNA and store it at 20°C for later use.

[0053] 2.2.2 DNA quality testing and dilution

[0054] DNA was detected using 1% agarose gel electrophoresis to ensure the integrity of wheat genomic DNA.

[0055] DNA concentration was measured using NanoDrop 2000. The DNA was diluted to a working concentration of approximately 50 ng / μL and stored at -20°C for later use.

[0056] 2.2.3 KASP Primer Design

[0057] KASP primers were designed based on SNP sites. Forward primer F1 was fitted with a linker for the FAM sequence “5'-GAAGGTGACCAAGTTCATGCT-3'”, and forward primer F2 was fitted with a linker for the HEX sequence “5'-GAAGGTCGGAGTCAACGGATT-3'”. The primer sequences are shown in Table 2.

[0058] Table 2 KASP primer sequences

[0059]

[0060]

[0061] 2.2.4 KASP Reaction System and Procedure

[0062] 2.2.4.1 KASP Primer Preparation Method

[0063] Dilute the primers to 100 mM using ddH2O and prepare them as follows:

[0064] 12 μL of forward primer F1, 12 μL of forward primer F2, 30 μL of reverse primer R, and 46 μL of ddH2O, totaling 100 μL, were used as the working solution for KASP-labeled primers. Store at -20℃ for later use.

[0065] 2.2.4.2 KASP reaction system

[0066] The reaction system consists of 2.5 μL of HiGeno 2x Probe Mix (Beijing Jiacheng Biotechnology Co., Ltd.), 0.07 μL of primer working solution, 1 μL of template DNA, and 1.5 μL of ddH2O, for a total of 5 μL.

[0067] 2.2.4.3 KASP Amplification Program

[0068] (1) Pre-denaturate at 95℃ for 10 min;

[0069] (2) Denaturation at 95℃ for 20 seconds;

[0070] (3) Anneal at 61℃ for 40s (decreasing by 0.6℃ per cycle), repeat steps 2 and 3 10 times;

[0071] (4) Denaturation at 95℃ for 20 seconds;

[0072] (5) Anneal at 55℃ for 40s, repeat steps 4 and 5 35 times;

[0073] (6) Store at 25℃ and detect the signal.

[0074] 2.3 KASP genotyping results

[0075] After PCR amplification, information is read on a quantitative PCR instrument (BIO-RAD). Fluorescent signals with FAM tags (orange) cluster near the X-axis, fluorescent signals with HEX tags (blue) cluster near the Y-axis, and the rest (green) are heterozygous.

[0076] Example 3: Analysis of the detection results of KASP markers at wheat cold resistance loci

[0077] 3.1 Based on KASP marker detection, the genotyping of 315 wheat accessions is as follows:

[0078] Table 3 KASP classification results

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087] Note: NA represents missing data, H represents Heterozygote, and U represents Undetermined.

[0088] 3.2 Statistical Results Analysis

[0089] Table 3. Statistical analysis of the relationship between allele variation type and cold resistance.

[0090]

[0091]

[0092] Note: Statistical analysis was performed using the t-test (P < 0.05 indicates a significant difference; P < 0.01 indicates a highly significant difference).

Claims

1. A primer combination for a wheat cold resistance gene locus KASP, characterized in that: The forward primer F1 is coupled with a linker containing the FAM sequence "5'-GAAGGTGACCAAGTTCATGCT-3'", and the forward primer F2 is coupled with a linker containing the HEX sequence "5'-GAAGGTCGGAGTCAACGGATT-3'". The primer combination comprises: In the table above, solid lines underlined SNP sites.

2. The application of the KASP primer combination of claim 1 in the preparation of a wheat cold resistance identification kit.

3. The application of the KASP primer combination of claim 1 in the preparation of a gene chip for identifying wheat cold resistance.

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