Wheat thousand seed weight related A1440G SNP site and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site related to thousand seed weight of wheat and application of the SNP site. The SNP site is the 1440th site from the 5'tail end of SEQ ID NO: 1 on a wheat genome, and is named as A1440G SNP site; the site has A / G polymorphism. The invention provides a method for screening or auxiliary screening of wheat with thousand grain weight based on the SNP site, develops a CAPS molecular marker, and provides a breeding method of wheat with high thousand grain weight based on the CAPS molecular marker. The invention has important significance on wheat molecular marker assisted breeding.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of molecular marker breeding, and particularly relates to an A1440G SNP site related to wheat thousand-grain weight and application thereof. BACKGROUND

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in China. Increasing wheat yield per unit is an important way to meet the increasing demand for food, and is also a strategic goal to ensure food security. Ear number, grain number per ear and thousand-grain weight are three elements of wheat yield, and coordinating the relationship among the three elements and improving the level of the three elements are the key to wheat high-yield breeding. Compared with ear number and grain number per ear, thousand-grain weight has the greatest impact on wheat yield, and is less affected by the environment. Therefore, improving thousand-grain weight is an important way to increase wheat yield.

[0003] At present, researchers have located a large number of QTLs for regulating wheat thousand-grain weight. For example, Su et al. used a “Shixin828 x Kenong2007” RIL population to detect 21 QTLs for regulating wheat thousand-grain weight on chromosomes 1A, 1B, 2A, 2B, 2D, 3A, 3B, 4B, 5A, 5B, 6A, 6D and 7A; MeCartney et al. used a “Opata85 x W7984” RIL population to detect QTL sites on chromosomes 1A, 2A, 2B, 3B, 6D and 7A; Su et al. detected a major QTL for regulating wheat thousand-grain weight on chromosome 7A and named it TaTKW-7AL, which explained 19.7% of the phenotypic variation of wheat thousand-grain weight; Huang et al. used a “AC Karma x 87E03” DH population to detect QTL sites for regulating wheat thousand-grain weight on chromosomes 2B, 2D, 3B, 4B, 4D and 6A, and one of the sites had a contribution rate as high as 26.3%; and Li et al. used a recombinant inbred line population to detect QTL sites for regulating wheat thousand-grain weight on chromosomes 1D, 3B, 5D, 6A and 7D. However, since the phenotypic contribution rates of most QTLs are small and the QTLs have poor reproducibility in different years and environments, the QTLs are difficult to apply to genetic improvement of wheat thousand-grain weight.

[0004] CAPS marker is also called PCR-RFLP (restriction fragment length polymorphism polymerase chain reaction), which is a kind of co-dominant molecular marker based on PCR, and reveals the restriction length variation information of specific PCR fragments. The basic principle is to use PCR to amplify the target DNA, and then use specific restriction endonuclease to cut the amplified product into different size fragments, which can be distinguished directly on the gel electrophoresis. Different alleles have different distribution of restriction enzyme cutting sites, and different length of DNA fragment bands are generated. The advantage is to avoid the cumbersome transfer and hybridization steps in RFLP analysis, and to maintain the accuracy of RFLP analysis. However, the SNP is less likely to be located at the restriction enzyme cutting site, therefore, the dCAPS marker is proposed, that is, by introducing mismatched bases in the amplification primer based on the CAPS marker, combining the SNP site to introduce a new restriction endonuclease action site, and generating a similar polymorphism as the CAPS marker.

[0005] Therefore, by mining and applying high-quality thousand-grain-weight SNP sites, technical support can be provided for cultivating high-yield and stable-yield wheat varieties, helping to improve the yield level of wheat, and having important practical significance for guaranteeing food security and promoting sustainable agricultural development. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a wheat thousand-grain-weight related A1440G SNP site and its application.

[0007] To solve the above technical problems, the technical solutions adopted by the present application are as follows.

[0008] A method for screening or assisting in screening the thousand-grain-weight of wheat, which is based on the specific genotype of a single nucleotide polymorphism site in the wheat genome to screen or assist in screening the thousand-grain-weight trait of wheat.

[0009] As a preferred technical solution of the present application, the single nucleotide polymorphism site corresponds to the 1440th position from the 5' end of SEQ ID NO: 1, which is named A1440G SNP site; the genotype of the A1440G SNP site includes AA or GG; when the nucleotide at the site is A / A homozygous, it corresponds to genotype I wheat; when the nucleotide at the site is G / G homozygous, it corresponds to genotype II wheat; the thousand-grain-weight of genotype I homozygous wheat is greater than or candidate greater than the thousand-grain-weight of genotype II homozygous wheat.

[0010] As a preferred technical solution of the present application, the method for detecting the genotype of A1440G SNP site in the wheat genome as AA or GG is as follows A) or B):

[0011] A) sequencing;

[0012] B) using a primer pair to perform PCR amplification on the wheat genomic DNA to be tested, and then using a restriction endonuclease to cut the amplification product and detecting the cut product.

[0013] As a preferred technical solution of the present application, the primer pair is a primer pair F1 and R1 consisting of SEQ ID NO: 2 and SEQ ID NO: 3, and a primer pair F2 and R2 consisting of SEQ ID NO: 4 and SEQ ID NO: 5; and the restriction endonuclease is specifically FokI enzyme.

[0014] As a preferred technical solution of the present application, the primer pair F1 and R1 is used to perform PCR amplification on the wheat genomic DNA to be tested to obtain a PCR amplification product P1, and the PCR amplification product P1 is diluted 10 times as a template to perform PCR amplification using a primer pair B consisting of primers F2 and R2 to obtain a PCR amplification product P2; and then a restriction endonuclease is used to cut the amplification product and detect the cut product.

[0015] If the cut product is a DNA fragment of 190 bp in length, the genotype of the A1440G SNP site in the wheat genomic DNA to be tested is AA, i.e., the genotype of the wheat to be tested based on the TaAN1-2B gene is genotype I.

[0016] If the cut product is a DNA fragment of 170 bp in length, the genotype of the A1440G SNP site in the wheat genomic DNA to be tested is GG, i.e., the genotype of the wheat to be tested based on the TaAN1-2B gene is genotype II.

[0017] On the other hand, the present application also comprises the use of the above method in wheat breeding.

[0018] On the other hand, the present application also comprises a method for wheat breeding, which is method A or method B:

[0019] The method A comprises the following steps: selecting a wheat material with a genotype AA of the A1440G SNP site as a parent for breeding;

[0020] The method B comprises the following steps: screening a high-thousand-grain-weight wheat as a parent for breeding according to the above method.

[0021] On the other hand, the present application also comprises a primer pair for detecting the above SNP site, which comprises a primer pair F1 and R1 consisting of SEQ ID NO: 2 and SEQ ID NO: 3, and a primer pair F2 and R2 consisting of SEQ ID NO: 4 and SEQ ID NO: 5.

[0022] In another aspect, the present application also comprises a reagent or a kit for screening or assisting in screening the thousand kernel weight trait of wheat, which is used for detecting the SNP site, and at least comprises the primer pair and the restriction endonuclease.

[0023] In the last aspect, the present application also comprises the application of the primer pair or the reagent or the kit in any one of the following (1)-(4):

[0024] (1) screening or assisting in screening wheat with different thousand kernel weight;

[0025] (2) identifying or assisting in identifying the thousand kernel weight of wheat;

[0026] (3) identifying or assisting in identifying the genotype of the TaAN1-2B gene of wheat;

[0027] (4) wheat breeding.

[0028] The beneficial effects produced by the above technical solutions are that the present application firstly provides a SNP site related to the thousand kernel weight trait of wheat, i.e. the A1440G SNP site, which is the 1440th site from the 5' end of SEQ ID NO: 1 on the 2B chromosome of the wheat genome, and the genotype of the site is AA or GG. The present application further develops a CAPS molecular marker based on the SNP site, which is used for detecting the single nucleotide polymorphism of the SNP site in the wheat genome, and provides a method for screening or assisting in screening the related traits of the thousand kernel weight of the wheat to be tested. Finally, the present application finds through a large number of experimental verification of wheat materials that the thousand kernel weight of the wheat to be tested with the genotype AA is significantly higher than that of the wheat to be tested with the genotype GG. The present application has important application value in the process of molecular marker assisted breeding of wheat. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a schematic diagram for comparing the grain phenotype and agronomic traits of wild type Kenong 9204 and mutant Taan1 in the embodiments of the present application.

[0030] Figure 2 It is a schematic diagram of TaAN1-2B haplotype and SNP site.

[0031] Figure 3 It is a schematic diagram of the genotype detection results of the TaAN1-2B gene of part of wheat varieties in a natural population; wherein M is a molecular weight standard; lane A is a band that cannot be cut by FokI; and lane G is a band that is cut by FokI.

[0032] Figure 4 It is a schematic diagram for distinguishing the excellent haplotype of TaAN1-2B high thousand kernel weight varieties; wherein A is a schematic diagram for distinguishing the thousand kernel weight haplotype in 2015; and B is a schematic diagram for distinguishing the thousand kernel weight haplotype in 2016. Detailed Implementation

[0033] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are conventional commercially available products and can be directly obtained through market purchase. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. It should be understood that, as used in this specification and appended claims, the term "comprising" indicates the presence of the described feature, integral, step, operation, element, and / or component, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or collections thereof. It should also be understood that the term "and / or" as used in this specification and appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0034] As used in this specification and the appended claims, the term "if" may be interpreted, depending on the context, as "when," "once," "in response to determination," or "in response to detection." Similarly, the phrases "if determined" or "if [the described condition or event] is detected" may be interpreted, depending on the context, as meaning "once determined," "in response to determination," "once [the described condition or event]," or "in response to detection." Furthermore, in the description of this specification and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance. References to "one embodiment" or "some embodiments" described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in yet other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms “including,” “comprising,” “having,” and variations thereof all mean “including but not limited to,” unless otherwise specifically emphasized.

[0035] Example 1: Discovery of the A1440G SNP site and development of CAPS molecular markers

[0036] I. Discovery of the A1440G SNP site

[0037] The application locates a gene TaAN1-2B associated with thousand-grain weight on a 2B chromosome by the method of whole genome association analysis of an EMS population of Triticum L. cultivar 9204, and creates a Taan1 frameshift mutation homozygous strain with stable inheritance by using Crispr-cas9 technology. Figure 1 As shown in the drawings, the Taan1 mutant has the phenotypes of smaller grain length, grain width and thousand-grain weight.

[0038] The application uses HapgeneR software to identify haplotypes of the exon capture sequencing results of the natural population, and the results show that there are 10 SNP polymorphic variation sites in the gene region of TaAN-1-2B, forming three main haplotypes, as shown in the drawings. Figure 2 Through analysis of a large number of wheat varieties, a SNP site related to the thousand-grain weight trait of wheat is found. The SNP site is located on the 2B chromosome of the wheat genome, at the 1440th position from the 5' end of SEQ ID NO: 1, and is marked as A1440G SNP. The genotype of the SNP site is AA or GG.

[0039] II. Development of CAPS molecular markers

[0040] Based on the A1440G SNP site in step one, a CAPS molecular marker is developed, and the primer sequences are as follows:

[0041] Primer F1: 5'-CGGTTAGCATGCATGTGAAGGAT-3' (SEQ ID NO: 2)

[0042] Primer R1: 5'-ATTACATCCTCAGTAGCGATTAGTAGCT-3' (SEQ ID NO: 3)

[0043] Primer F2: 5'-TTACTCCCTCCGCCTCAAAATGCAAGACG-3' (SEQ ID NO: 4)

[0044] Primer R2: 5'-GCGACATGCATGTTGCATCTTTGGTGGTTATAC-3' (SEQ ID NO: 5)

[0045] The target sequence amplified by primer pair F1 and R1 is shown in SEQ ID NO: 1 from the 1095th to the 2067th position from the 5' end.

[0046] The amplified product is digested with restriction endonuclease FokI:

[0047] If the digestion product is a 190bp long DNA fragment, the genotype of the A1440G SNP site is AA;

[0048] If the enzyme digestion product is a 170bp long DNA fragment, the genotype of the A1440G SNP site is GG.

[0049] Example 2, Application of CAPS molecular marker

[0050] Test materials: 193 wheat varieties (all hexaploid).

[0051] I. Genotype detection

[0052] 1. Extraction of wheat genomic DNA

[0053] Genomic DNA of the test wheat varieties was extracted.

[0054] 2. PCR amplification and enzyme digestion

[0055] The genomic DNA of the test wheat in step 1 was used as a template, and primer pair A and primer pair B were used for PCR amplification to obtain PCR amplification product P1.

[0056] The reaction system was 10 μL, which was composed of 3.6 μL ddH2O, 5 μL 2×Taq enzyme Mix, 0.2 μL primer F1 aqueous solution (concentration of 10 μmol / L), 0.2 μL primer R1 aqueous solution (concentration of 10 μmol / L), and 1 μL genomic DNA of the test wheat (concentration of 20 ng / μL). 2×Taq enzyme Mix was a product of Nanjing Novozyme Co., Ltd., with product catalog number P131. The reaction conditions were: 95℃ for 3 min; 95℃ for 15 s, 66℃ for 15 s, 72℃ for 20 s, 32 cycles; 72℃ for 10 min; 16℃ storage.

[0057] 10-fold diluted PCR amplification product P1 was used as a template, and primer pair C and primer pair D were used for PCR amplification to obtain PCR amplification product P2.

[0058] The reaction system was 10 μL, which was composed of 3.6 μL ddH2O, 5 μL 2×Taq enzyme Mix, 0.2 μL primer F2 aqueous solution (concentration of 10 μmol / L), 0.2 μL primer R2 aqueous solution (concentration of 10 μmol / L), and 1 μL diluted PCR amplification product P1. The reaction conditions were: 95℃ for 3 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 5 s, 35 cycles; 72℃ for 10 min; 16℃ storage.

[0059] The obtained PCR amplification product P2 was subjected to enzyme digestion with restriction enzyme FokI to obtain the enzyme digestion product.

[0060] 3. Detection of enzyme digestion product

[0061] The enzyme digestion product was detected by 4% agarose gel electrophoresis.

[0062] If the enzyme digestion product is a DNA fragment of 190 bp in length, the genotype of the A1440G SNP site of the wheat material is AA, i.e., the genotype of the wheat to be tested based on the TaAN1-2B gene is genotype I; if the enzyme digestion product is a DNA fragment of 170 bp in length, the genotype of the A1440G SNP site of the wheat material is GG, i.e., the genotype of the wheat to be tested based on the TaAN1-2B gene is genotype II.

[0063] The detection results show that the genotype of 27 wheat varieties based on the TaAN1-2B gene is genotype II, and the genotype of 170 wheat varieties based on the TaAN1-2B gene is genotype I. The names of the wheat varieties and the thousand-grain weight data are shown in Table 1. Some detection results are shown in FIG. 1. Figure 3 From left to right, P2 uncut product, Bainong 160, Shi 4185, Hanxuan No. 3, Heng 7228, Huaimai 18, Luohan No. 11; among them, genotype I is A-type wheat variety, and genotype II is G-type wheat variety.

[0064] Table 1 Names of wheat varieties and thousand-grain weight data

[0065]

[0066]

[0067]

[0068]

[0069] II. Correlation analysis of the genotype of the A1440G SNP site and the yield-related agronomic traits of wheat

[0070] The wheat varieties were planted in two years, respectively, and the average thousand-grain weight of the two genotypes of wheat was counted after harvesting. The statistical results are shown in Table 2.

[0071] Table 2 Average thousand-grain weight of two genotypes of wheat

[0072]

[0073] Note: P value is the significance level of the correlation analysis, “*” indicates P < 0.05, and “**” indicates P < 0.01.

[0074] The genotype of the wheat TaAN1-2B gene in the natural population was associated with the thousand-grain weight by selecting a t-test method by using Graphpad Prism 9.0 software, and the results are shown in Table 2, Figure 4 .

[0075] The results show that the thousand-grain weight of "wheat with genotype I" is higher than or candidate higher than that of "wheat with genotype II" in the natural population of 193 wheat varieties; the study on the natural population shows that genotype I is an excellent genotype for improving the thousand-grain weight of wheat.

[0076] The above-described embodiments are only used to illustrate the technical solutions of the present application, rather than limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. A method of screening or assisting in the screening of wheat thousand kernel weight, characterised by: Screening or assisted screening of wheat based on specific genotypes of single nucleotide polymorphism sites in wheat genome for the thousand kernel weight trait.

2. The method of claim 1, wherein: The single nucleotide polymorphism site corresponds to the 1440th position from the 5' end of SEQ ID NO: 1, named A1440G SNP site; the genotype of the A1440G SNP site includes AA or GG; when the nucleotide at the site is A / A homozygous, it corresponds to genotype I wheat; when the nucleotide at the site is G / G homozygous, it corresponds to genotype II wheat; the thousand kernel weight of genotype I homozygous wheat is greater than or candidate greater than that of genotype II homozygous wheat.

3. The method of claim 2, wherein: The method for detecting whether the genotype of the A1440G SNP site in the wheat genome is AA or GG is as follows A) or B): A) sequencing; B) PCR amplification of the wheat genome DNA to be tested with a primer pair, and then enzyme digestion of the amplification product to detect the enzyme digestion product.

4. The method of claim 3, wherein: The primer pair is primer pair F1 and R1 consisting of SEQ ID NO: 2 and SEQ ID NO: 3, and primer pair F2 and R2 consisting of SEQ ID NO: 4 and SEQ ID NO: 5; and the restriction enzyme is specifically FokI enzyme.

5. The method of claim 4, wherein: The wheat genome DNA to be tested is subjected to PCR amplification with primer pair F1 and R1 in claim 4 to obtain PCR amplification product P1, 10-fold dilution of the PCR amplification product P1 is used as a template for PCR amplification with primer pair B consisting of primers F2 and R2 to obtain PCR amplification product P2; and the amplification product is subjected to enzyme digestion with a restriction enzyme to detect the enzyme digestion product: If the enzyme digestion product is a 190bp long DNA fragment, the genotype of the A1440G SNP site in the wheat genome to be tested is AA, i.e. the genotype of the wheat to be tested based on TaAN1-2B gene is genotype I; If the enzyme digestion product is a 170bp long DNA fragment, the genotype of the A1440G SNP site in the wheat genome to be tested is GG, i.e. the genotype of the wheat to be tested based on TaAN1-2B gene is genotype II.

6. Use of the method of any one of claims 1-5 in wheat breeding.

7. A method for wheat breeding, which is method A or method B: The method A comprises the following steps: selecting wheat material with genotype AA of A1440G SNP site as a parent for breeding; The method B comprises the following steps: screening high thousand kernel weight wheat as a parent for breeding according to the method of any one of claims 1-6.

8. A pair of primers, characterized in that: The primer pair for detecting the SNP site in claim 2 comprises primer pair F1 and R1 consisting of SEQ ID NO: 2 and SEQ ID NO: 3, and primer pair F2 and R2 consisting of SEQ ID NO: 4 and SEQ ID NO:

5.

9. An agent or kit for screening or aiding in the screening of a wheat thousand kernel weight trait, characterized in that: The reagent or kit for detecting the SNP site in claim 2 comprises at least the primer pair and the restriction enzyme in claim 4.

10. Use of the primer pair or the reagent or kit in any one of the following (1)-(4): (1) screening or assisting in screening different thousand-grain-weight wheat; (2) identifying or assisting in identifying wheat thousand-grain-weight; (3) identifying or assisting in identifying the genotype of wheat TaAN1-2B gene; (4) wheat breeding.

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