SNP (Single Nucleotide Polymorphism) site closely linked with maximum relative root length character of wheat after salt stress, CAPS (Cyclic Activated Polymorphism) molecular marker and application

Abstract

The invention discloses an SNP site closely linked with the maximum relative root length character of wheat after salt stress, a CAPS molecular marker and application, and belongs to the technical field of wheat genetic molecular breeding. The SNP (Single Nucleotide Polymorphism) site is at least one of the 274th site, the 689th site, the 725th site, the 767th site, the 811th site and the 1038th site of a TaHSFA2-2A gene sequence. The six SNPs have two haplotypes, namely, the haplotype Hap-2A-1 and the haplotype Hap-2A-2, and the two haplotypes of the six SNPs are linked with each other. Correlation analysis proves and natural population verifies that the maximum relative root length of Hap-2A-2 after salt stress in the wheat germination period is obviously larger than that of Hap-2A-1. Therefore, the method has important application value in predicting the maximum relative root length of the wheat after salt stress or preparing a reagent for predicting the maximum relative root length of the wheat after salt stress.

Description

Technical Field

[0001] The invention belongs to the technical field of wheat genetic molecular breeding, and particularly relates to a SNP site tightly linked to a maximum relative root length trait of wheat after salt stress, a CAPS molecular marker and an application thereof. Background Art

[0002] Wheat is one of the three major food crops in the world. With the development of society and the advancement of science and technology, China's wheat production capacity has gradually developed and the wheat planting area has increased. However, the current world population is growing, people's demand for the yield of food crops is increasing, and the goal of wheat breeding has also shifted to high-yield and multi-resistant types. Roots are one of the most important organs of plants. They can not only absorb water and nutrients and transmit them to the aboveground parts of plants to promote plant growth and development, but also play an important role in the fixation of plants in the soil and the interaction with soil microorganisms. Therefore, studying the relative root length of wheat is conducive to the screening and cultivation of high-yield, high-quality and stress-resistant wheat varieties, and its research is of great significance.

[0003] With the rapid development of high-throughput sequencing technology and the increasing quality of wheat genome reference sequences, nucleotide polymorphism markers are widely used in the identification of wheat population genotypes. Therefore, molecular markers related to the maximum relative root length of wheat after salt stress were screened out, and the markers were used to screen wheat varieties with excellent maximum relative root length traits after salt stress, laying a theoretical foundation for breeding high-yield and stable-yield wheat varieties and providing molecular-assisted selection methods. Summary of the invention

[0004] In view of the problems existing in the prior art, the object of the present invention is to provide a SNP site tightly linked to the maximum relative root length trait of wheat after salt stress, a CAPS molecular marker and an application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A SNP site that is tightly linked to the maximum relative root length trait of wheat after salt stress, wherein the SNP site is at least one of the 274th, 689th, 725th, 767th, 811th, and 1038th positions of the TaHSFA2-2A gene sequence;

[0007] The TaHSFA2-2A gene has C and T polymorphisms at position 274, C and T polymorphisms at position 689, C and T polymorphisms at position 725, G and T polymorphisms at position 767, T and C polymorphisms at position 811, and T and C polymorphisms at position 1038;

[0008] The TaHSFA2-2A gene sequence is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0009] The application of the above SNP loci tightly linked to the maximum relative root length trait of wheat after salt stress is used to predict the maximum relative root length of wheat after salt stress or to prepare a reagent for predicting the maximum relative root length of wheat after salt stress.

[0010] A CAPS molecular marker for predicting the maximum relative root length of wheat after salt stress is the 811th SNP site of the TaHSFA2-2A gene sequence; in wheat materials with larger maximum relative root length of wheat after salt stress, the 811th base of the TaHSFA2-2A gene is C; in wheat materials with smaller maximum relative root length of wheat after salt stress, the 811th base of the TaHSFA2-2A gene is T.

[0011] Based on the above scheme, the primer pair for amplifying the molecular marker is shown as SEQ ID NO.3 and SEQ ID NO.4.

[0012] A reagent for predicting the maximum relative root length of wheat after salt stress, comprising a primer pair for detecting the above SNP site.

[0013] On the basis of the above scheme, a primer pair for detecting the SNP site at position 811 of the TaHSFA2-2A gene sequence is included.

[0014] Based on the above scheme, the primer pair for detecting the SNP site at position 811 of the TaHSFA2-2A gene sequence is shown as SEQ ID NO.3 and SEQ ID NO.4.

[0015] Based on the above scheme, restriction endonuclease SmaI is also included.

[0016] A method for predicting the maximum relative root length of wheat after salt stress, detecting the above-mentioned SNP sites; when the 274th position of the TaHSFA2-2A gene sequence is C and / or the 689th position is C and / or the 725th position is C and / or the 767th position is G and / or the 811th position is T and / or the 1038th position is T, the maximum relative root length of the wheat after salt stress is shorter; when the 274th position of the TaHSFA2-2A gene sequence is T and / or the 689th position is T and / or the 725th position is T and / or the 767th position is T and / or the 811th position is C and / or the 1038th position is C, the maximum relative root length of the wheat after salt stress is longer.

[0017] On the basis of the above scheme, when detecting the SNP site at position 811 of the TaHSFA2-2A gene sequence, the genomic DNA of the wheat to be tested is used as a template, and the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4 is used for PCR amplification. The amplified product is digested with the restriction endonuclease SmaI. When the digestion product is a DNA fragment of 636 bp and does not contain two DNA fragments of 199 bp and 437 bp, respectively, the maximum relative root length of the wheat to be tested after salt stress is shorter; when the digestion product is two DNA fragments of 199 bp and 437 bp and does not contain a DNA fragment of 636 bp, or when the digestion product is three DNA fragments of 199 bp, 437 bp and 636 bp, the maximum relative root length of the wheat to be tested after salt stress is longer.

[0018] Advantages of the technical solution of the present invention:

[0019] The present invention analyzes the genetic variation of TaHSFA2-2A, and finds molecular markers related to the maximum relative root length trait of wheat after salt stress, which are respectively located at 6 SNP sites at the 274th, 689th, 725th, 767th, 811th and 1038th positions of SEQ ID NO.1, and these 6 SNPs have two haplotypes; haplotype Hap-2A-1 (the 6 SNPs are C, C, C, G, T, T), haplotype Hap-2A-2 (the 6 SNPs are T, T, T, T, C, C), and the two haplotypes of the 6 SNPs are linked. It is proved by association analysis and natural population verification that among the homozygous types of the two haplotypes, the maximum relative root length of wheat in the germination period of Hap-2A-2 is significantly greater than that of Hap-2A-1. The 811th nucleotide in the genomic DNA corresponding to SEQ ID NO.1 in the sequence list is also related to the maximum relative root length trait of wheat after salt stress, and the maximum relative root length of wheat in the germination period with the 811th nucleotide being C is significantly greater than that of wheat with the 811th nucleotide being T. Experiments have shown that by detecting the wheat haplotype of the present invention and the 811th nucleotide in the genome corresponding to SEQ ID NO.1 in the sequence list, wheat varieties or strains with relatively excellent maximum relative root length trait after salt stress can be quickly screened out for breeding. The molecular markers provided by the present invention are applied in practice, which is conducive to the screening and cultivation of high-yield, high-quality, stress-resistant wheat varieties, and the research thereof is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of SNP sites in the TaHSFA2-2A genome sequence;

[0021] Figure 2 It is a schematic diagram of the electrophoresis detection results of the restriction endonuclease SmaI digestion products;

[0022] Figure 3 This is the association analysis between wheat haplotype and maximum relative root length trait. DETAILED DESCRIPTION

[0023] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0024] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0025] The SNP sites described in the following examples were obtained by the following method:

[0026] Specific primers (TaHSFA2-2A-F and TaHSFA2-2A-R) were designed upstream and downstream of the TaHSFA2-2A gene, and PCR amplification was performed using the genomic DNA of the wheat material as a template. The obtained PCR amplification products were sequenced and compared.

[0027] The specific primer sequences are as follows:

[0028] TaHSFA2-2A-F (forward primer): 5′-CCAAGCTTGCATTATAAGTCTCCT-3′ (SEQ ID NO. 5);

[0029] TaHSFA2-2A-R (reverse primer): 5'-CACTACTAGTTCTATCCAGGGT-3' (SEQ ID NO. 6).

[0030] The reaction system for PCR amplification is 25 μL, including 2.5 μL of 20-100 ng / μL genomic DNA template, 12.5 μL of 2.5UTaq enzyme, 1.0 μL of 10 μM forward primer, 1.0 μL of 10 μM reverse primer, and 8.0 μL of sterile water.

[0031] The present invention has no particular limitation on the specific sources of the above reagents, and any conventional commercially available product in the art may be used.

[0032] The reaction procedure of the PCR amplification was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, 34 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0033] Sequence comparison revealed that the PCR product contained two TaHSFA2-2A genomic DNAs shown in SEQ ID NO.1 and SEQ ID NO.2, and there were six SNP sites differences between the two ( Figure 1 ), which are the 274th (C and T polymorphisms), 689th (C and T polymorphisms), 725th (C and T polymorphisms), 767th (G and T polymorphisms), 811th (T and C polymorphisms) and 1038th (T and C polymorphisms) of the TaHSFA2-2A gene, respectively.

[0034] In the following examples, the maximum relative root length refers to the ratio of the maximum root length mean value measured in the treatment group to the maximum root length mean value measured in the control group; the calculation formula is: maximum relative root length = maximum root length mean value of the treatment group / maximum root length mean value of the control group.

[0035] Example 1

[0036] The SNP sites closely linked to the maximum relative root length trait of wheat after salt stress are located at positions 274, 689, 725, 767, 811, and 1038 of the TaHSFA2-2A gene sequence shown in SEQ ID NO. 1. When the six SNP sites are C, C, C, G, T, and T in sequence (the sequence shown in SEQ ID NO. 1), the corresponding wheat is haplotype Hap-2A-1, and its maximum relative root length is smaller; when the six SNP sites are T, T, T, T, C, and C in sequence (the sequence shown in SEQ ID NO. 2), the corresponding wheat is haplotype Hap-2A-2, and its maximum relative root length is larger.

[0037] The nucleotides of the TaHSFA2-2A gene haplotype at each SNP site are shown in Table 1.

[0038] Table 1 Nucleotide of each SNP site in the haplotype of TaHSFA2-2A gene

[0039]

[0040] The nucleotides of the six SNP sites of the above two haplotypes are all linked. As long as the nucleotide of any one of the sites is determined, the nucleotides of other sites can be obtained.

[0041] SEQ ID NO.1(5'→3'):

[0042]

[0043] SEQ ID NO.2(5’→3’):

[0044]

[0045] Example 2

[0046] CAPS molecular markers for detecting maximum relative root length of wheat after salt stress

[0047] Based on one of the SNP sites described in Example 1, site 811, a CAPS molecular marker for detecting the maximum relative root length of wheat was developed.

[0048] The primer sequences of the CAPS molecular marker for detecting the maximum relative root length of wheat after salt stress are as follows: TaHSFA2-2A-CAPS-F (forward primer): 5'-TGGAACTCCTTGCTGACGAG-3' (SEQ ID NO.3); TaHSFA2-2A-CAPS-R (reverse primer): 5'-ACAGGAGTACAACTTTAGTCCA-3' (SEQ ID NO.4).

[0049] Example 3

[0050] The method for predicting the maximum relative root length of wheat after salt stress is as follows:

[0051] 1) Extracting genomic DNA of wheat to be tested;

[0052] 2) Using the extracted genomic DNA as a template, PCR amplification was performed using primers TaHSFA2-2A-CAPS-F / R to obtain PCR amplification products;

[0053] The reaction system for PCR amplification (25 μL): 2.5 μL of genomic DNA template of 20-100 ng / μL, 12.5 μL of 2.5UTaq enzyme, 1.0 μL of 10 μM forward primer, 1.0 μL of 10 μM reverse primer, and 8.0 μL of sterile water.

[0054] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, 34 cycles; final extension at 72°C for 5 min, and storage at 4°C.

[0055] 3) digesting the PCR product obtained in step 2) with restriction endonuclease SmaI to obtain a digestion product;

[0056] After the 25μl system PCR reaction is completed, 30μL 10×NEB Buffer and 5μL SmaI enzyme are mixed into a 35μL system for use. Then transfer 3μL PCR reaction solution to a new PCR tube, add 6μL ddH2O and 1μL of the 35μL system mixture prepared in advance, construct a 10μL enzyme digestion reaction system, digest at 30℃ for 2h, and inactivate at 65℃ for 20min.

[0057] The restriction site sequence recognized by the restriction endonuclease SmaI is CCC / GGG, and the length of the PCR product is 636bp. If the 811th base of the TaHSFA2-2A gene is C, a sequence of "CCCGGG" is formed. This site is recognized by the restriction endonuclease SmaI, and the PCR amplification product is cut into two DNA fragments of 199bp and 437bp. If the 811th base of the TaHSFA2-2A gene is T, a sequence of "CCCGGG" cannot be formed, and thus it cannot be recognized by the restriction endonuclease SmaI. After the enzyme cutting reaction, the fragment is still 636bp.

[0058] 4) The enzyme digestion product of step 3) is subjected to electrophoresis detection,

[0059] If the enzyme digestion product contains a DNA fragment of 636 bp in size and does not contain two DNA fragments of 199 bp and 437 bp in size, the haplotype of the wheat to be tested is Hap-2A-1, and the maximum relative root length of the wheat material after salt stress is small;

[0060] If the enzyme cleavage product contains two DNA fragments of 199 bp and 437 bp in size and does not contain a DNA fragment of 636 bp in size, or the enzyme cleavage product contains three DNA fragments of 199 bp, 437 bp and 636 bp, then the haplotype of the wheat to be tested is Hap-2A-2, and the maximum relative root length of the wheat material is larger;

[0061] The schematic diagram of the electrophoresis detection results of the restriction endonuclease SmaI digestion product is shown in Figure 2 shown.

[0062] Example 4

[0063] Association analysis between wheat haplotypes and maximum relative root length traits after salt stress

[0064] The TaHSFA2-2A-CAPS marker was used to genotype the natural population (Table 2), and the association analysis between haplotypes and root length traits was performed. The specific methods are as follows:

[0065] Detection of wheat haplotypes

[0066] A natural population containing 181 wheat varieties was used as material, and wheat haplotypes were identified according to the method of Example 3 to determine the haplotype of the wheat to be tested.

[0067] The haplotype detection results are shown in Table 2. The TaHSFA2-2A-F and TaHSFA2-2A-R primer pairs in Example 1 were used to amplify wheat genomic DNA. The results showed that the sequences of the PCR amplification products of the wheat to be tested were all SEQ ID NO.1 except for the 6 SNP sites, and the 6 SNP sites all satisfied the linkage relationship of the two haplotypes in Table 1, indicating that the TaHSFA2-2A-CAPS marker of the present invention can be used to detect haplotypes Hap-2A-1 and Hap-2A-2.

[0068] Table 2 Haplotype statistics of 181 wheat varieties in natural populations.

[0069]

[0070]

[0071] Association analysis between haplotypes and maximum relative root length traits after salt stress

[0072] Seeds of the above wheat materials with full grains and uniform size were selected, disinfected with 1% NaClO solution for 10 minutes, and then treated with distilled water and 160mM NaCl solution respectively. The maximum root length was measured on the third day, and the maximum relative root length before and after salt stress was calculated. The correlation between different haplotypes and the relative root length trait of wheat was analyzed, such as Figure 3 As shown, the P value of the TaHSFA2-2A-CAPS molecular marker and the maximum relative root length of wheat before and after salt stress during germination was 0.043, which was less than 0.05, indicating that TaHSFA2-2A-CAPS was significantly positively correlated with the maximum relative root length of wheat before and after salt stress during germination. The maximum relative root length of wheat with C at the 811th position of the TaHSFA2-2A gene before and after salt stress during germination was significantly greater than that of wheat with T at the same position, indicating that this marker can be used to breed wheat varieties with the maximum relative root length before and after salt stress during germination.

[0073] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A SNP locus closely linked to the maximum relative root length trait of wheat after salt stress, characterized in that: The SNP site is at least one of the 274th, 689th, 725th, 767th, 811th, and 1038th positions of the TaHSFA2-2A gene sequence; The TaHSFA2-2A gene has C and T polymorphisms at position 274, C and T polymorphisms at position 689, C and T polymorphisms at position 725, G and T polymorphisms at position 767, T and C polymorphisms at position 811, and T and C polymorphisms at position 1038; The TaHSFA2-2A gene sequence is shown as SEQ ID NO.1 or SEQ ID NO.

2.

2. The use of the SNP locus tightly linked to the maximum relative root length trait of wheat after salt stress according to claim 1, characterized in that: Used for predicting the maximum relative root length of wheat after salt stress or for preparing reagents for predicting the maximum relative root length of wheat after salt stress.

3. A CAPS molecular marker for predicting the maximum relative root length of wheat after salt stress, characterized in that: It is the 811th SNP site in the TaHSFA2-2A gene sequence; in the wheat materials with larger maximum relative root length after salt stress, the 811th base of the TaHSFA2-2A gene is C; in the wheat materials with smaller maximum relative root length after salt stress, the 811th base of the TaHSFA2-2A gene is T.

4. The CAPS molecular marker for predicting the maximum relative root length of wheat after salt stress according to claim 3, characterized in that: The primer pair for amplifying the molecular marker is shown in SEQ ID NO.3 and SEQ ID NO.

4.

5. A reagent for predicting the maximum relative root length of wheat after salt stress, characterized in that: Comprising a primer pair for detecting the SNP site of claim 1.

6. The reagent for predicting the maximum relative root length of wheat after salt stress according to claim 5, characterized in that: Contains a primer pair for detecting the SNP site at position 811 in the TaHSFA2-2A gene sequence.

7. The reagent for predicting the maximum relative root length of wheat after salt stress according to claim 6, characterized in that: The primer pair for detecting the SNP site at position 811 of the TaHSFA2-2A gene sequence is shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. The reagent for predicting the maximum relative root length of wheat after salt stress according to claim 7, characterized in that: Also included is the restriction endonuclease SmaI.

9. A method for predicting the maximum relative root length of wheat after salt stress, characterized in that: Detect the SNP site described in claim 1; when the 274th position of the TaHSFA2-2A gene sequence is C and / or the 689th position is C and / or the 725th position is C and / or the 767th position is G and / or the 811th position is T and / or the 1038th position is T, the maximum relative root length of the wheat after salt stress is smaller; when the 274th position of the TaHSFA2-2A gene sequence is T and / or the 689th position is T and / or the 725th position is T and / or the 767th position is T and / or the 811th position is C and / or the 1038th position is C, the maximum relative root length of the wheat after salt stress is larger.

10. The method for predicting the maximum relative root length of wheat after salt stress according to claim 9, characterized in that: When detecting the 811th SNP site of the TaHSFA2-2A gene sequence, the genomic DNA of the wheat to be tested is used as a template, and the primer pair shown in SEQID NO.3 and SEQ ID NO.4 is used for PCR amplification. The amplified product is digested with the restriction endonuclease SmaI. When the digestion product is a DNA fragment of 636 bp and does not contain two DNA fragments of 199 bp and 437 bp, respectively, the maximum relative root length of the wheat to be tested after salt stress is shorter; when the digestion product is two DNA fragments of 199 bp and 437 bp and does not contain a DNA fragment of 636 bp, or when the digestion product is three DNA fragments of 199 bp, 437 bp and 636 bp, the maximum relative root length of the wheat to be tested after salt stress is longer.

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