SNP site, CAPS molecular marker closely linked to the maximum relative root length trait after salt stress of wheat and application

Abstract

The application discloses a SNP site, a CAPS molecular marker and application which are closely linked to a wheat salt stress post-maximum relative root length character, and belongs to the technical field of wheat genetic molecular breeding.The SNP site is at least one of the 274th, 689th, 725th, 767th, 811th and 1038th SNP sites of a TaHSFA2-2A gene sequence.The six SNPs exist in two haplotypes, namely haplotype Hap-2A-1 and haplotype Hap-2A-2, and the two haplotypes of the six SNPs are linked.Through correlation analysis and natural population verification, the maximum relative root length of wheat germination stage salt stress of Hap-2A-2 is significantly greater than that of Hap-2A-1.Therefore, the application has important application value in predicting the maximum relative root length of wheat salt stress or in preparing a reagent for predicting the maximum relative root length of wheat salt stress.

Description

TECHNICAL FIELD

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

[0002] Wheat is one of the three major food crops in the world. With the development of society and the progress of science and technology, the wheat production capacity in China has gradually developed, and the wheat planting area has increased. However, the world population is growing, and people's demand for grain yield is increasing, so the breeding goal of wheat has also changed to high yield and multi-resistance. Root is one of the most important organs of plants, which not only absorbs water and nutrient elements to transport to the aboveground part of plants to promote the growth and development of plants, but also plays an important role in the fixation of plants in soil and the interaction with soil microorganisms. Therefore, the study of the relative root length of wheat is beneficial to the screening and breeding of high-yield and high-quality resistant wheat varieties, and has important 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, screening a molecular marker related to the maximum relative root length after salt stress of wheat and using the marker to screen wheat varieties with excellent maximum relative root length after salt stress can lay a theoretical foundation for breeding high-yield and stable wheat varieties and provide a molecular assisted selection method. SUMMARY

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

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

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

[0007] The 274th position of the TaHSFA2-2A gene is C and T polymorphism, the 689th position is C and T polymorphism, the 725th position is C and T polymorphism, the 767th position is G and T polymorphism, the 811th position is T and C polymorphism, and the 1038th position is T and C polymorphism;

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

[0009] The SNP locus closely 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 a SNP locus at position 811 of the TaHSFA2-2A gene sequence; in wheat materials with a larger maximum relative root length after salt stress, the base at position 811 of the TaHSFA2-2A gene is C; and in wheat materials with a smaller maximum relative root length after salt stress, the base at position 811 of the TaHSFA2-2A gene is T.

[0011] On the basis of the above scheme, the primer pair for amplifying the molecular marker is shown in SEQ ID NO. 3 and SEQ ID NO. 4.

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

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

[0014] On the basis of the above scheme, the primer pair for detecting the SNP locus at position 811 of the TaHSFA2-2A gene sequence is shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0015] On the basis of the above scheme, a restriction enzyme SmaI is further included.

[0016] A method for predicting the maximum relative root length of wheat after salt stress, wherein the above SNP locus is detected; when the base at position 274 and / or the base at position 689 and / or the base at position 725 and / or the base at position 767 and / or the base at position 811 and / or the base at position 1038 of the TaHSFA2-2A gene sequence is C, the maximum relative root length of the wheat after salt stress is smaller; and when the base at position 274 and / or the base at position 689 and / or the base at position 725 and / or the base at position 767 and / or the base at position 811 and / or the base at position 1038 of the TaHSFA2-2A gene sequence is T, the maximum relative root length of the wheat after salt stress is larger.

[0017] On the basis of the above scheme, when detecting the SNP site at position 811 of the TaHSFA2-2A gene sequence, PCR amplification is performed using the genomic DNA of the wheat to be tested as a template and the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4. 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, through genetic variation analysis of TaHSFA2-2A, discovered molecular markers associated with the maximum relative root length trait of wheat after salt stress. These markers are located at six SNP sites at positions 274, 689, 725, 767, 811, and 1038 of SEQ ID NO.1. These six SNPs exist in two haplotypes: haplotype Hap-2A-1 (the six SNPs are C, C, C, G, T, T) and haplotype Hap-2A-2 (the six SNPs are T, T, T, T, C, C). Both haplotypes of the six SNPs are linked. Association analysis and natural population validation demonstrated that among the homozygous types of these two haplotypes, Hap-2A-2 has a significantly greater maximum relative root length during wheat germination than Hap-2A-1. The 811th nucleotide in the genomic DNA corresponding to SEQ ID NO.1 in the sequence listing is also associated with the maximum relative root length trait of wheat after salt stress. The maximum relative root length of wheat with a C at the 811th nucleotide during the germination period is significantly greater than that of wheat with a T at the same site. 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 genomic DNA in the sequence listing, wheat varieties or lines with relatively excellent maximum relative root length traits after salt stress can be quickly screened for breeding. The application of the molecular markers provided by the present invention in practice is conducive to the screening and cultivation of high-yield, high-quality, stress-resistant wheat varieties, and is of great significance for research on them. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 Schematic diagram of electrophoresis detection results of restriction endonuclease SmaI digestion products;

[0022] Figure 3 Analysis of the correlation between haplotype of wheat and the maximum relative root length trait. DETAILED DESCRIPTION

[0023] The terms used in the present application have the meanings generally understood by those of ordinary skill in the art, unless otherwise defined. The present application is described in further detail below in connection with specific examples and with reference to the data. The following examples are merely illustrative of the present application and do not in any way limit the scope of the present application.

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

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

[0026] Specific primers (TaHSFA2-2A-F and TaHSFA2-2A-R) are designed upstream and downstream of the TaHSFA2-2A gene, and PCR amplification is carried out with the genomic DNA of the wheat material as the template. The PCR amplification product is sequenced and sequence aligned.

[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 of PCR amplification is 25 μL, including 20-100 ng / μL of genomic DNA template 2.5 μL, 2.5 U Taq enzyme 12.5 μL, 10 μM forward primer 1.0 μL, 10 μM reverse primer 1.0 μL, and sterile water 8.0 μL.

[0031] The present application does not have special limitations on the specific sources of the above reagents, and the conventional commercially available products in the art can be used.

[0032] The reaction program of the PCR amplification is 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles; 72℃ final extension for 5 min, and 4℃ storage.

[0033] Sequence alignment analysis revealed that the PCR product contained two TaHSFA2-2A genomic DNAs represented by SEQ ID NO.1 and SEQ ID NO.2, and that there were six SNP sites that differed between the two. Figure 1 ), which are position 274 (with C and T polymorphisms), position 689 (with C and T polymorphisms), position 725 (with C and T polymorphisms), position 767 (with G and T polymorphisms), position 811 (with T and C polymorphisms) and position 1038 (with 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 mean maximum root length measured in the treatment group to the mean maximum root length measured in the control group; the calculation formula is: maximum relative root length = mean maximum root length of the treatment group / mean maximum root length of the control group.

[0035] Example 1

[0036] SNP sites tightly 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, T (sequence shown in SEQ ID NO. 1), the corresponding wheat is haplotype Hap-2A-1, which has a shorter maximum relative root length; when the six SNP sites are T, T, T, T, C, C (sequence shown in SEQ ID NO. 2), the corresponding wheat is haplotype Hap-2A-2, which has a longer maximum relative root length.

[0037] The nucleotide sequences 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 TaHSFA2-2A gene haplotype

[0039]

[0040] The nucleotides of the six SNP sites of the above two haplotypes all have a linkage phenomenon. As long as the nucleotide of any one site 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 in 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 for detecting the CAPS molecular marker for 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 from the wheat to be tested;

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

[0053] PCR amplification reaction system (25 μL): 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.

[0054] PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; 34 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1 min; 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 completing the 25μL PCR reaction, pre-mix 30μL of 10× NEB Buffer with 5μL of SmaI enzyme to create a 35μL system. Transfer 3μL of the PCR reaction mixture to a new PCR tube, then add 6μL of ddH2O and 1μL of the pre-mixed 35μL system mixture to create a 10μL enzyme digestion reaction. Digest at 30°C for 2 hours, followed by inactivation at 65°C for 20 minutes.

[0057] The restriction endonuclease SmaI recognizes the restriction site sequence CCC / GGG, and the PCR product is 636bp long. If the 811th base of the TaHSFA2-2A gene is C, a "CCCGGG" sequence 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, the "CCCGGG" sequence cannot be formed, and therefore it cannot be recognized by the restriction endonuclease SmaI. After the enzyme cutting reaction, the fragment remains 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 digestion product contains two DNA fragments of 199 bp and 437 bp respectively and does not contain a DNA fragment of 636 bp, or if the enzyme digestion product contains three DNA fragments of 199 bp, 437 bp, and 636 bp, then the wheat haplotype to be tested is Hap-2A-2, and the maximum relative root length of the wheat material is larger;

[0061] The electrophoresis detection results of the restriction endonuclease SmaI digestion product are shown in the figure below: Figure 2 shown.

[0062] Example 4

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

[0064] The natural population (Table 2) was genotyped using the TaHSFA2-2A-CAPS marker, and 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. Wheat genomic DNA was amplified using the TaHSFA2-2A-F and TaHSFA2-2A-R primer pairs described in Example 1. The results showed that the sequences of the PCR amplification products of the wheat tested were all SEQ ID NO. 1, except for six SNP sites. All six SNP sites satisfied the linkage relationships between the two haplotypes shown 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 and disinfected with 1% NaClO solution for 10 minutes. They were 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 description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Tightly linked to the maximum relative root length trait of wheat after salt stress TaHSFA2 -2A gene SNP application, characterized in that, Used for predicting the maximum relative root length of wheat after salt stress or for preparing a reagent for predicting the maximum relative root length of wheat after salt stress; The SNP is TaHSFA2 -SNP site at least one of positions 274, 689, 725, 767, 811, and 1038 of the 2A gene sequence; 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; described TaHSFA2 -2A gene sequence is shown in SEQ ID NO.1 or SEQ ID NO.2; when TaHSFA2 -2A gene sequence at position 274 is C and / or position 689 is C and / or position 725 is C and / or position 767 is G and / or position 811 is T and / or position 1038 is T, the maximum relative root length of the wheat after salt stress is smaller; when TaHSFA2 -When position 274 is T and / or position 689 is T and / or position 725 is T and / or position 767 is T and / or position 811 is C and / or position 1038 is C in the 2A gene sequence, the maximum relative root length of the wheat is greater after salt stress.

2. The use according to claim 1, characterized in that Amplification TaHSFA2 The primer pair for the SNP site at position 811 of the -2A gene sequence is shown in SEQ ID NO. 3 and SEQ ID NO.

4.

3. The use according to claim 1, characterized in that The reagent for predicting the maximum relative root length of wheat after salt stress comprises a primer pair for detecting the SNP site according to claim 1.

4. The use according to claim 3, characterized in that The reagent for predicting the maximum relative root length of wheat after salt stress comprises a detection TaHSFA2 -Primer pair for the SNP site at position 811 in the 2A gene sequence.

5. The use according to claim 4, characterized in that The detection TaHSFA2 The primer pair for the SNP site at position 811 of the -2A gene sequence is shown in SEQ ID NO. 3 and SEQ ID NO.

4.

6. The use according to claim 5, characterized in that The reagent for predicting the maximum relative root length of wheat after salt stress also contains restriction endonuclease SmaI.

7. A method for predicting the maximum relative root length of wheat after salt stress, characterized in that: In testing TaHSFA2 -2A gene sequence, PCR amplification was performed using the genomic DNA of the wheat to be tested as a template and the primer pair shown in SEQ ID NO.3 and SEQ ID NO.

4. The amplified product was digested with the restriction endonuclease SmaI. When the digestion product was a DNA fragment of 636 bp and did not contain two DNA fragments of 199 bp and 437 bp in size, the maximum relative root length of the wheat to be tested after salt stress was shorter; when the digestion product was two DNA fragments of 199 bp and 437 bp and did not contain a DNA fragment of 636 bp, or when the digestion product was 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 was longer.

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