SNP-a1473g associated with thousand kernel weight of wheat and application thereof

Abstract

The application discloses a SNP-A1473G related to a thousand-grain weight of wheat and an application thereof. The SNP site corresponds to the 1473th base from the 5' end of a sequence shown in SEQ ID NO:1. When the site is AA homozygous, the corresponding genotype is I; when the site is GG homozygous, the corresponding genotype is II; and the size of the thousand-grain weight is: the wheat with the genotype I homozygous is larger than or candidate larger than the wheat with the genotype II homozygous. The SNP has higher effectiveness and potential application value. The wheat with higher thousand-grain weight can be found by detecting the SNP, and the application has important value in the research or application of cultivating high-yield wheat varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to SNP-A1473G related to the thousand-grain weight of wheat and its applications. Background Technology

[0002] Wheat (Triticum aestivum L.) is one of my country's most important food crops. Increasing wheat yield per unit area is a crucial way to meet the ever-increasing demand for food and a strategic goal to ensure food security. The number of ears, the number of grains per ear, and the thousand-grain weight are the three key factors constituting wheat yield. Coordinating the relationship between these three factors and improving their levels is crucial for high-yield wheat breeding. Compared to the number of ears and the number of grains per ear, the thousand-grain weight has the greatest impact on wheat yield and is relatively less affected by environmental factors. Therefore, improving the thousand-grain weight is an important way to increase wheat yield.

[0003] Currently, researchers have identified a large number of QTLs regulating thousand-grain weight in wheat. For example, Su et al. used the “Shixin828 x Kenong2007” RIL population to detect a total of 21 thousand-grain weight QTLs on chromosomes 1A, 1B, 2A, 2B, 2D, 3A, 3B, 4B, 5A, 5B, 6A, 6D, and 7A; MeCartney et al. used the “Opata85 x W7984” RIL population to detect QTL loci located on chromosomes 1A, 2A, 2B, 3B, 6D, and 7A; Su et al. detected a major thousand-grain weight QTL on chromosome 7A and named it TaTKW-7AL, which explained 19.7% of the thousand-grain weight phenotypic variation; Huang et al. used the “AC Karma x In the DH population of “87E03”, QTL loci for thousand-grain weight were detected on chromosomes 2B, 2D, 3B, 4B, 4D, and 6A, with one locus contributing as much as 26.3%. Li et al. detected QTL loci controlling thousand-grain weight on chromosomes 1D, 3B, 5D, 6A, and 7D using a recombinant inbred line population. However, because the phenotypic contribution of most QTLs is small and their repeatability is poor across different years and environments, QTLs are difficult to apply to the genetic improvement of wheat thousand-grain weight.

[0004] CAPS markers, also known as PCR-RFLP (Restriction Fragment Length Polymorphism Polymerase Chain Reaction), are a class of codominant molecular markers based on PCR. They reveal information about the restriction length variations of specific PCR fragments. The basic principle is to amplify the target DNA using PCR, then digest the amplified product with specific restriction endonucleases to cut it into fragments of different sizes, which are directly distinguished on gel electrophoresis. Different alleles have different distributions of restriction enzyme sites, producing DNA fragment bands of different lengths. The advantage is that it avoids the cumbersome transfer and hybridization steps of RFLP analysis while maintaining the accuracy of RFLP analysis. However, it is rare for SNPs to be located precisely at restriction enzyme sites. Therefore, dCAPS markers were proposed. These markers, based on CAPS markers, introduce mismatched bases into the amplification primers, binding to SNP sites to introduce new restriction endonuclease action sites, producing polymorphisms similar to CAPS markers.

[0005] Although many QTLs associated with wheat thousand-grain weight have been identified, most QTLs have a small phenotypic contribution, require additive effects to be expressed, and have poor repeatability across different years and environments. Therefore, these QTLs are difficult to apply to the genetic improvement of wheat thousand-grain weight. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide SNP-A1473G related to the thousand-grain weight of wheat and its application.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0008] A SNP site associated with the thousand-grain weight of wheat, wherein the SNP site corresponds to the 1473rd base from the 5' end of the sequence shown in SEQ ID NO: 1. When this site is homozygous for AA, it corresponds to genotype I; when this site is homozygous for GG, it corresponds to genotype II. The thousand-grain weight is: wheat homozygous for genotype I is greater than or candidate to be greater than wheat homozygous for genotype II.

[0009] On the other hand, the present invention also includes a reagent or kit for identifying or assisting in the identification of the thousand-grain weight trait of wheat. The reagent or kit is used for the above-mentioned SNP sites. The reagent or kit contains a PCR amplification specific primer combination and enzyme digestion component corresponding to the SNP sites, as well as template DNA, buffer, dNTPs and other necessary components for gene detection.

[0010] As a preferred embodiment of the present invention, the target DNA fragment for PCR amplification of the reagent or kit is designed to be the 5' end 931-2597bp in SEQ ID NO: 1.

[0011] As a preferred embodiment of the present invention, the PCR amplification specific primer combination includes: primer pair F1 and R1 composed of SEQ ID NO: 4 and SEQ ID NO: 5, and primer pair F2 and R2 composed of SEQ ID NO: 2 and SEQ ID NO: 3.

[0012] As a preferred embodiment of the present invention, the enzyme digestion component is the restriction endonuclease Hpy188I.

[0013] On the other hand, the present invention also includes a method for identifying or assisting in the identification of wheat thousand-grain weight in the early stage of breeding. Based on the above-mentioned SNP sites, in the early stage of molecular marker-assisted selection breeding, primers are designed to amplify any DNA fragment containing the SNP sites in the genomic DNA of the wheat to be tested by PCR. The wheat genotype is identified by enzyme digestion of the PCR amplification product, and the wheat thousand-grain weight phenotype is identified or assisted in the identification based on the following correlation between genotype and phenotype: wheat homozygous for genotype I is greater than or candidate greater than wheat homozygous for genotype II.

[0014] As a preferred embodiment of the present invention, the DNA fragment amplified by PCR is the 5' end 931-2597bp of SEQ ID NO: 1; the specific primer pair for PCR amplification is primer pair F1 and R1 composed of SEQ ID NO: 4 and SEQ ID NO: 5, and primer pair F2 and R2 composed of SEQ ID NO: 2 and SEQ ID NO: 3; the restriction endonuclease Hpy188I is used for enzyme digestion.

[0015] As a preferred embodiment of the present invention, the enzyme digestion includes the following steps: using wheat genomic DNA as a template, amplifying with primers F1 and R1 to obtain PCR product P1; diluting this PCR product P1 10 times, using it as a template, amplifying with primers F2 and R2 to obtain PCR product P2; digesting PCR product P2 with the restriction endonuclease Hpy188I; if PCR product P2 can be cleaved, the nucleotide polymorphism site is AA, and the genotype is I; if the PCR product cannot be cleaved, the nucleotide polymorphism site is GG, and the genotype is II; the thousand-grain weight is: wheat homozygous for genotype I is greater than or candidate greater than wheat homozygous for genotype II.

[0016] On the other hand, the present invention also includes the use of the above-mentioned wheat SNP sites, which is to screen or assist in screening the thousand-grain weight phenotype of wheat in the early stage of molecular marker-assisted selection breeding.

[0017] Finally, the present invention also includes a primer combination comprising primer pair F1 and R1 consisting of SEQ ID NO: 4 and SEQ ID NO: 5, and primer pair F2 and R2 consisting of SEQ ID NO: 2 and SEQ ID NO: 3, which is used to detect the above-mentioned SNP sites.

[0018] The beneficial effects of adopting the above technical solution are as follows: The research and development team of this invention, through genetic variation analysis of wheat natural variant populations, discovered a SNP corresponding to position 1473 from the 5' end of sequence listing 1. This SNP has two genotypes: genotype I (A) and genotype II (G). Association analysis showed that, in the homozygous types of these two genotypes, the thousand-grain weight is: wheat homozygous for genotype I has a greater or candidate greater weight than wheat homozygous for genotype II. This invention also provides a dCAPS marker for detecting the SNP. Experiments have shown that by detecting this SNP, wheat with a higher thousand-grain weight can be identified. This invention provides a new method for molecular marker-assisted selection breeding of wheat, which is of great significance in breeding high-yielding wheat varieties or in research. The SNP site developed in this invention not only expands the genetic resource tools for wheat, but also, through our scientific research experiments and data statistics, has been verified to have good and broad application potential. Attached Figure Description

[0019] Figure 1 This is a comparison of the grain phenotype and agronomic traits of wild-type Kenong 9204 and mutant Tag6 in the embodiments of the present invention.

[0020] Figure 2 The results show the genotype detection of the TaG6-7B gene in some wheat varieties in a natural population; where M is the molecular weight standard; lane A is the band that can be cleaved by Hpy188I; and lane G is the band that cannot be cleaved by Hpy188I.

[0021] Figure 3 This is a schematic diagram showing the association between gene polymorphism sites and thousand-grain weight in a natural population.

[0022] Figure 4 Images of wheat harvested in a field in Shunyi District, Beijing.

[0023] Figure 5 To measure the thousand-grain weight of wheat using professional seed-copying instruments. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] The sequencing data of the wheat materials used in the following examples were all exon capture sequencing data. Material information can be found on the China Crop Germplasm Information Network (website: http: / / icgr.caas.net.cn). Since the wheat materials are all cultivated varieties, they are generally assumed to be highly homozygous plant materials with homozygous genotypes.

[0027] Example 1: Discovery of A1473G SNP in wheat TaG6-7B gene and establishment of wheat genotyping method based on TaG6-7B gene.

[0028] 1.1 Discovery of the wheat thousand-grain weight gene TaG6-7B

[0029] This invention utilizes genome-wide association analysis (GWAS) at the EMS population level of the wheat variety Kenong 9204 to locate the TaG6-7B gene associated with thousand-grain weight on chromosome 7B; and employs CRISPR-Cas9 technology to create homozygous lines with stable inheritance of the Tag6 frameshift mutation. For example... Figure 1 As shown, the Tag6 mutant exhibits a phenotype with smaller grain length, grain width, and thousand-grain weight.

[0030] 1.2 Discovery of the A1473G SNP in the wheat TaG6-7B gene

[0031] The inventors of this invention used HapgeneR software to perform haplotype identification on the natural population exon capture sequencing results. The results showed that there were 10 SNP polymorphic variant sites in the gene region of TaAN-1-7B.

[0032] A SNP site, named A1473G SNP, was discovered in the wheat TaG6-7B gene (nucleotide sequence shown in SEQ ID NO: 1). A1473G SNP is located at position 1473 from the 5' end of SEQ ID NO: 1, and has genotypes of AA homozygous (genotype I) and GG homozygous (genotype II). Since genomic DNA is composed of two antisense complementary single-stranded DNA molecules forming a double-stranded DNA molecule, the DNA molecule encoding the protein is generally named the sense DNA molecule; the antisense DNA molecule is named the antisense DNA molecule. The genotype of A1473G SNP is the sense DNA genotype.

[0033] 1.3 Synthesize primer pair A and primer pair B for amplifying the target sequence including the A1473G SNP.

[0034] Primer pair A and primer pair B were designed and synthesized for amplifying the target sequence including the A1473G SNP. Primer pair A consists of primer F1 and primer R1. Primer pair B consists of primer F2 and primer R2 (where SEQ ID NO: 3 introduces a mismatched base).

[0035] The nucleotide sequences of each primer are as follows:

[0036] Primer F2: 5′-GTCCCATAATATAAGTGCGTTTTTGACAATAGTGTAGTCTG-3′ (SEQ ID NO: 2)

[0037] Primer R2: 5′- CTGACAGCTCTACCATCATAAATCTGCTGTGTTGT-3′ (SEQ ID NO: 3)

[0038] Primer F1: 5′-GAAGCGTCTTCTTGTTAGATTTAAACTCGTGGTTTGC-3′ (SEQ ID NO: 4)

[0039] Primer R1: 5′- CCAAGACCCAAAAGTAGGTGATCACTGGCACA-3′ (SEQ ID NO: 5)

[0040] The target sequence for primer pair A amplification is shown in positions 931-2597 from the 5' end of SEQ ID NO: 1.

[0041] Example 2: Establishment of a genotyping method for wheat based on the TaG6-7B gene

[0042] a. Extract genomic DNA from the wheat to be tested.

[0043] b. Using the genomic DNA of the wheat to be tested from step a as a template, PCR amplification was performed using primer pair A composed of primers F1 and R1 to obtain PCR amplification product P1.

[0044] The reaction system consisted of 10 μL of ddH2O, 5 μL of 2×Taq enzyme Mix, 0.4 μL of primer F1 aqueous solution (concentration of 10 μmol / L), 0.4 μL of primer R1 aqueous solution (concentration of 10 μmol / L), and 1 μL of genomic DNA from wheat to be tested (concentration of 20 ng / μL).

[0045] 2×Taq enzyme Mix is ​​a product of Nanjing Novizan Co., Ltd., with product catalog number P131.

[0046] The reaction conditions were: 95℃ for 3 min; 95℃ for 15 s, 63℃ for 15 s, 72℃ for 20 s, 32 cycles; 72℃ for 10 min; and stored at 16℃.

[0047] c. After completing step b, use the diluted PCR amplification product P1 (1 volume of PCR amplification product P1 mixed with 9 volumes of water) as a template, and perform PCR amplification using primer pair B composed of primer F2 and primer R2 to obtain PCR amplification product P2.

[0048] The reaction system consisted of 10 μL of ddH2O, 5 μL of 2×Taq enzyme Mix, 0.4 μL of primer F2 aqueous solution (concentration of 10 μmol / L), 0.4 μL of primer R2 aqueous solution (concentration of 10 μmol / L), and 1 μL of dilution buffer for PCR amplification product P1.

[0049] The reaction conditions were: 95℃ for 3 min; 95℃ for 15 s, 62℃ for 15 s, 72℃ for 5 s, 34 cycles; 72℃ for 10 min; and stored at 16℃.

[0050] d. The PCR amplification product P2 obtained in step c is digested with the restriction endonuclease Hpy188I to obtain the digested product. The digested product is then detected by 3% agarose gel electrophoresis. The undigested product P2 is used as a marker, and the following judgments are made: If the digested product is band A (shown as 182bp), then the wheat A1473G SNP to be tested is homozygous AA, that is, the genotype of the wheat to be tested based on the TaG6-7B gene is genotype I; if the digested product is band B (shown as 222bp), then the wheat A1473G SNP to be tested is homozygous GG, that is, the genotype of the wheat to be tested based on the TaG6-7B gene is genotype II.

[0051] Example 3: Association analysis and verification of wheat TaG6-7B gene genotype and wheat thousand-grain weight

[0052] 3.1 Genotyping of TaG6-7B genes in wheat in natural populations

[0053] Genotyping of wheat varieties in the natural population was performed using the method described in step c of Example 2. The natural population consisted of 323 wheat varieties (all hexaploid), all of which were planted and harvested in an experimental field in Shunyi District, Beijing. Figure 4 Wheat variety names and thousand-grain weight data are detailed in Table 1. The thousand-grain weight of wheat was measured using a professional seed-copying instrument. Figure 5 .

[0054] Some test results can be found Figure 2 (From left to right: Shimai 19, Heng 4399, Xinong 6028, Luohan 7, P2 undigested product; among them, genotype I is type A wheat variety, and genotype II is type G wheat variety).

[0055] The genotypes based on the TaG6-7B gene are shown in Table 1: 55 wheat varieties were genotype II based on the TaG6-7B gene, 211 wheat varieties were genotype I based on the TaG6-7B gene, and the remaining 57 materials could not be genotyped.

[0056] Table 1. Wheat Variety Names and 1000-Grain Weight Data

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] 3.2 Association Analysis of Gene Polymorphism Sites and Thousand-Grain Weight in Natural Populations

[0064] Wheat varieties were planted in the field of the experimental base in Shunyi District, Beijing in October 2014 and October 2015, respectively, and harvested in mid-June 2015 and mid-June 2016, respectively. The average thousand-grain weight of the two genotypes of wheat was counted after harvest. The statistical results are shown in Table 2.

[0065] Table 2. Association analysis results between gene polymorphism sites in natural populations and thousand-grain weight.

[0066]

[0067] Note: P-value represents the significance level of the association analysis. "*" indicates P < 0.05, and "**" indicates P < 0.01.

[0068] The association between the genotype of the wheat TaG6-7B gene and thousand-grain weight in a natural population was analyzed using the t-test method with Graphpad Prism 9.0 software. The results are shown in Table 2. Figure 3 .

[0069] The results showed that in a natural population of 323 wheat varieties, the thousand-grain weight of wheat with genotype I was greater than that of wheat with genotype II; the ">" signifies a statistically significant difference. This study of the natural population indicates that genotype I is the superior genotype for increasing the thousand-grain weight of wheat.

[0070] The above results indicate that detecting the genotype of wheat based on the TaG6-7B gene can screen or assist in screening for the thousand-grain weight trait, which has important application value in the process of molecular marker-assisted breeding of wheat.

[0071] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0072] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for identifying or assisting in the identification of the thousand-grain weight of wheat in the early stages of breeding, characterized by: Based on a SNP site associated with wheat thousand-grain weight, the SNP site corresponds to the 1473rd base from the 5' end of the sequence shown in SEQ ID NO:

1. When this site is homozygous for AA, it corresponds to genotype I; when this site is homozygous for GG, it corresponds to genotype II. In the early stages of molecular marker-assisted selection breeding, primers are designed to amplify any DNA fragment containing the SNP site in the wheat genomic DNA to be tested by PCR. The wheat genotype is identified by enzyme digestion of the PCR amplification product. Based on the following correlation between genotype and phenotype, the wheat thousand-grain weight phenotype is identified or assisted in identification: wheat homozygous for genotype I is greater than or candidate greater than wheat homozygous for genotype II.

2. The method according to claim 1, characterized in that: The DNA fragment amplified by PCR is the 5' end 931-2597bp of SEQ ID NO: 1; the specific primer pairs for PCR amplification are primer pair F1 and R1 composed of SEQ ID NO: 4 and SEQ ID NO: 5, and primer pair F2 and R2 composed of SEQ ID NO: 2 and SEQ ID NO: 3; the restriction endonuclease Hpy188I is used for enzyme digestion.

3. The method according to claim 1, characterized in that: The enzyme digestion includes the following steps: using wheat genomic DNA as a template, amplifying with primers F1 and R1 to obtain PCR product P1; diluting this PCR product P1 10-fold, using it as a template, amplifying with primers F2 and R2 to obtain PCR product P2; digesting PCR product P2 with the restriction endonuclease Hpy188I; if PCR product P2 can be cleaved, the nucleotide polymorphism site is AA, and the genotype is I; if the PCR product cannot be cleaved, the nucleotide polymorphism site is GG, and the genotype is II; the thousand-grain weight is: wheat homozygous for genotype I is greater than or candidate greater than wheat homozygous for genotype II.

4. The use of a reagent for detecting SNP sites associated with wheat thousand-grain weight in wheat thousand-grain weight phenotypic screening or auxiliary screening, wherein the SNP site corresponds to the 1473rd base from the 5' end of the sequence shown in SEQ ID NO: 1, and when this site is homozygous for AA, it corresponds to genotype I; when this site is homozygous for GG, it corresponds to genotype II, characterized in that: The application is to screen or assist in screening the thousand-grain weight phenotype of wheat in the early stage of molecular marker-assisted selection breeding.

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