阅读说明：本技术 小麦单株穗数及耐热性性状相关snp位点及其应用 (Single plant spike number and heat resistance character related SNP site of wheat and application thereof ) 是由 刘西岗 张琦 郭琳 李永鹏 景瑞莲 于 2021-09-02 设计创作，主要内容包括：本发明公开了一种小麦单株穗数及耐热性性状相关SNP位点及其应用,包括用于鉴定或辅助鉴定小麦单株穗数及耐热性性状的试剂盒、引物、相关的分子标记,以及上述元素在鉴定或辅助鉴定小麦单株穗数及耐热性性状中的用途。本发明为小麦的分子标记辅助选择育种提供了一个新方法,在培育高产小麦品种和研究中具有重要意义。(The invention discloses a wheat single plant spike number and heat resistance character related SNP site and application thereof, which comprises a kit, a primer and related molecular markers for identifying or assisting in identifying the wheat single plant spike number and the heat resistance character, and application of the elements in identifying or assisting in identifying the wheat single plant spike number and the heat resistance character. The invention provides a new method for the molecular marker-assisted selective breeding of wheat, and has important significance in cultivating high-yield wheat varieties and researching.)

1. A kit for detecting single nucleotide polymorphism of SNP site in wheat genome, wherein the SNP site corresponds to 1875 th base from 5' end of sequence shown in SEQ ID NO. 1.

2. The kit of claim 1, wherein: the nucleotide at the SNP site is G or C; when the nucleotide at the SNP site is G/G pure, the corresponding genotype is A; when the nucleotide at the SNP site is C/C pure, the corresponding genotype is B.

3. The kit of claim 1, wherein: the kit comprises a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3 in a sequence table and/or a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO.5 in the sequence table.

4. A primer, which is used for detecting the single nucleotide polymorphism of SNP loci in a wheat genome, wherein the SNP loci correspond to 1875 th base from the 5' end of a sequence shown in SEQ ID NO. 1; the nucleotide at the SNP site is G or C; when the nucleotide at the SNP site is G/G pure, the corresponding genotype is A; when the nucleotide at the SNP site is C/C pure, the corresponding genotype is B.

5. The primer of claim 4, wherein: the primers are a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3 in the sequence table and/or a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO.5 in the sequence table.

6. A molecular marker whose nucleotide sequence is the sequence of 5 'end 1772-1899 bit in SEQ ID NO.1 and/or whose nucleotide sequence is the sequence of 5' end 1647-2001 bit in SEQ ID NO. 1.

7. Use of the kit of claim 1, 2 or 3, the primer of claim 4 or 5, and the molecular marker of claim 6 for identification or assisted identification of wheat single ear number and heat tolerance trait.

8. Use of the kit of claim 1 or 2 or 3, the primer of claim 4 or 5, the molecular marker of claim 6 in wheat breeding.

9. A method for identifying or assisting in identifying the spike number and the heat resistance property of a single wheat plant is characterized by comprising the following steps: the method comprises the following steps:

A. carrying out PCR amplification on any section of DNA fragment containing the following SNP sites in the genome DNA of the wheat to be detected, and carrying out enzyme digestion identification on the PCR amplification product; the SNP locus corresponds to the 1875 th base from the 5' end of the sequence shown in SEQ ID NO. 1;

B. determining the genotype of the wheat to be detected, wherein when the nucleotide at the SNP site is G/G pure, the corresponding genotype is A; when the nucleotide at the SNP site is C/C pure, the corresponding genotype is B;

C. determining the single-plant ear number and the heat resistance character of the wheat to be detected according to the genotype of the wheat to be detected and the following standards: c-1, the number of spikes of each plant is as follows: the wheat homozygous for genotype A is smaller/candidate than the wheat homozygous for genotype B; c-2, the heat resistance is as follows: wheat homozygous for genotype a is stronger/candidate stronger than wheat homozygous for genotype b.

10. The method for identifying or assisting in identifying the number of ears per wheat and the heat resistance traits as claimed in claim 9, wherein: in the step A:

the DNA fragment amplified by the PCR is 1772-1899bp at the 5' end in SEQ ID NO. 1; the specific primer pair for PCR amplification is a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3, and a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO. 5;

the enzyme digestion comprises the following steps: taking wheat genome DNA as a template, and taking the primers 1F and 1R as a primer pair to amplify to obtain a PCR product; the PCR product is obtainedDiluting the product by 100 times, and taking the product as a template and taking the primers 2F and 2R as a primer pair to amplify to obtain a PCR product; using restriction endonucleasesSalI, enzyme digestion of a PCR product;

in the step B:

if the PCR product can not be cut, the nucleotide polymorphism site is G/G, and the genotype is A; if the PCR product can be cut, the nucleotide polymorphism site is C/C, and the genotype is B.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a single spike number and heat resistance related SNP (single nucleotide polymorphism) site of wheat and application thereof.

Background

Wheat (Triticum aestivum L) is one of three major food crops in China. Under the condition of limited cultivated land area, the improvement of the wheat yield per unit is an important way for meeting the continuous improvement of the future food demand and is also a strategic target for ensuring the food safety. The number of ears per unit area is one of three factors affecting the yield of wheat (Triticum aestivum), and is directly influenced by the number of ears per plant (SNPP), which is a complex quantitative trait susceptible to the environment. Therefore, the method has important application value in high-yield breeding of wheat by digging, regulating and controlling excellent allelic variation of the spike number of a single plant and developing a functional marker.

With the abundance of sequencing technologies and molecular markers (Cui et al., 2017; Liu et al.,2018), genome-wide association analysis (Chen et al., 2017; Liu et al.,2017) and linkage analysis (Xu et al.,2017) are widely applied to the excavation of Quantitative Trait Loci (QTL). For example, using the parental population constructed from high tillering and dwarf mutants, 1 quantitative trait gene locus controlling the number of ears per plant was detected on 2D chromosome (Xu et al, 2017). On chromosome 3A, 1 QTL was detected that simultaneously controlled plant height, panicle number and individual panicle number (Shah et al, 1999). In addition, single ear number QTL was also detected on the 3B, 4A and 6D chromosomes using 2 parental populations (Kumar et al, 2007). Single spike number formation in rice (Oryza sativa) several genes controlling tillering formation were cloned, such as MOC1 (monocultm 1), DLT (dwarfandlow-tillering), TE (tillerenhancer), THIS1(high tillering, reduced height, and preferability spikes 1), DWT1 (dwarftiller 1), OsTB1 (tempo branched 1), and MOC3 (monocultm 3) (Li et al, 2003; Tong et al, 2009; Lin et al, 2012; Liu et al, 2013; Wang et al, 2014; Lu et al, 2015 et al; Wang et al, 2020), etc. The gene TaD27(Dwarf27) involved in the strigolactone synthesis pathway affected how much wheat tillers (Zhao et al, 2020). The tin (tillerinhibition) gene (Spielmeryer et al, 2004), located on the short arm of the 1A chromosome, and the tin3 gene (Kurapparathy et al, 2007; 2008), derived from diploid wheat (T. monococcum), located on the 3Am chromosome, both caused the tillering bud to stop growing at the transition from vegetative to reproductive growth, resulting in the deterioration of the tillering.

The temperature is a key environmental factor for regulating and controlling the number of single wheat plant spikes. Generally, the temperature of 13-18 ℃ is suitable for tillering wheat, tillering is stopped when the temperature is too high or too low, the higher the temperature is, the faster the wheat young ear differentiation rate is, the shorter each period of the young ear differentiation time is, and the contrary results are obtained.

Although reports related to the regulation of the number of single plant spikes exist, the regulation mechanism of the number of single plant spikes of wheat is very complex and is very easily influenced by the environment, so that the currently discovered and reported gene is difficult to be applied to the genetic breeding improvement of wheat.

Disclosure of Invention

The invention aims to solve the technical problem of providing a wheat single-plant spike number and heat resistance character related SNP site and application thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows.

A kit for detecting single nucleotide polymorphism of SNP site in wheat genome, wherein the SNP site corresponds to 1875 th base from 5' end of sequence shown in SEQ ID NO. 1.

As a preferred embodiment of the present invention, the nucleotide at the SNP site is G or C; when the nucleotide at the SNP site is G/G pure, the corresponding genotype is A; when the nucleotide at the SNP site is C/C pure, the corresponding genotype is B.

As a preferred technical scheme, the kit comprises a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3 in a sequence table and/or a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO.5 in the sequence table.

A primer, which is used for detecting the single nucleotide polymorphism of SNP loci in a wheat genome, wherein the SNP loci correspond to 1875 th base from the 5' end of a sequence shown in SEQ ID NO. 1; the nucleotide at the SNP site is G or C; when the nucleotide at the SNP site is G/G pure, the corresponding genotype is A; when the nucleotide at the SNP site is C/C pure, the corresponding genotype is B.

As a preferred technical scheme, the primers are a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3 in a sequence table and/or a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO.5 in the sequence table.

A molecular marker whose nucleotide sequence is the sequence of 5 'end 1772-1899 bit in SEQ ID NO.1 and/or whose nucleotide sequence is the sequence of 5' end 1647-2001 bit in SEQ ID NO. 1.

The invention also comprises the application of the kit, the primers and the molecular marker in identification or auxiliary identification of the spike number of the wheat single plant and the heat resistance property.

The invention also comprises the application of the kit, the primer and the molecular marker in wheat breeding.

A method for identifying or assisting in identifying the spike number and the heat resistance property of a single wheat plant comprises the following steps:

A. carrying out PCR amplification on any section of DNA fragment containing the following SNP sites in the genome DNA of the wheat to be detected, and carrying out enzyme digestion identification on the PCR amplification product; the SNP locus corresponds to the 1875 th base from the 5' end of the sequence shown in SEQ ID NO. 1;

B. determining the genotype of the wheat to be detected, wherein when the nucleotide at the SNP site is G/G pure, the corresponding genotype is A; when the nucleotide at the SNP site is C/C pure, the corresponding genotype is B;

C. determining the single-plant ear number and the heat resistance character of the wheat to be detected according to the genotype of the wheat to be detected and the following standards: c-1, the number of spikes of each plant is as follows: the wheat homozygous for genotype A is smaller/candidate than the wheat homozygous for genotype B; c-2, the heat resistance is as follows: wheat homozygous for genotype a is stronger/candidate stronger than wheat homozygous for genotype b.

As a preferred technical solution of the present invention, in step a: the DNA fragment amplified by the PCR is 1772-1899bp at the 5' end in SEQ ID NO. 1; the specific primer pair for PCR amplification is a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3, and a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO. 5; the enzyme digestion comprises the following steps: taking wheat genome DNA as a template, and taking the primers 1F and 1R as a primer pair to amplify to obtain a PCR product; diluting the PCR product by 100 times, and taking the diluted PCR product as a template and taking the primers 2F and 2R as a primer pair to amplify to obtain a PCR product; the PCR product is cut by restriction enzyme SalI; in the step B: if the PCR product can not be cut, the nucleotide polymorphism site is G/G, and the genotype is A; if the PCR product can be cut, the nucleotide polymorphism site is C/C, and the genotype is B.

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in: according to the invention, 9 SNPs are discovered through genetic variation analysis of TabHLH92-5B gene promoter, intron, exon and 3' UTR in a wheat natural variation group, CAPS and dCAPS primers are designed according to the SNP (G/A) of two haplotypes of TabHLH92-5B at a 2666bp site, and the SNP has two genotypes: genotype A (G) and genotype B (A). Correlation analysis proves that the homozygous types of the two haplotypes have the following high and low single plant spike number and heat resistance: wheat homozygous for genotype a > wheat homozygous for genotype b. Experiments prove that the wheat with relatively high ear number per plant and heat resistance can be found by detecting the SNP. The invention provides a new method for the molecular marker-assisted selective breeding of wheat, and has important significance in cultivating high-yield wheat varieties and researching.

Drawings

FIG. 1 shows the result of electrophoresis detection of a dCAPS labeled enzyme-digested product developed by SNP of the present invention; wherein lane M is a molecular weight standard; lane C is a band cleaved by SacI, and Lane G is a band not cleaved by SacI. .

Detailed Description

The following examples illustrate the invention in detail. The raw materials and various devices used in the invention are conventional commercially available products, and can be directly obtained by market purchase.

It will be understood that the terms "comprises" and "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, and groups thereof.

It should also be understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

As used in this specification and the appended claims, the term "if" may be interpreted contextually as "when", "upon" or "in response to" determining "or" in response to detecting ". Similarly, the phrase "if it is determined" or "if a [ described condition or event ] is detected" may be interpreted contextually to mean "upon determining" or "in response to determining" or "upon detecting [ described condition or event ]" or "in response to detecting [ described condition or event ]".

Furthermore, in the description of the present application and the appended claims, the terms "first," "second," "third," and the like are used for distinguishing between descriptions and not necessarily for describing or implying relative importance.

Reference throughout this specification to "one embodiment" or "some embodiments," or the like, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the present application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," or the like, in various places throughout this specification are not necessarily all referring to the same embodiment, but rather "one or more but not all embodiments" unless specifically stated otherwise. The terms "comprising," "including," "having," and variations thereof mean "including, but not limited to," unless expressly specified otherwise.

The wheat material used in the following examples was from the national crop germplasm library (http:// icscaas. com. cn/jiguoku/zhongzhiku. htm), and the material information is presented in the chinese crop germplasm information web, website: http:// icgr.

Example 1 SNP associated with ear number of wheat Individual plant and PCR-restriction enzyme polymorphism detection thereof

1. Specific primer for amplifying genome segment containing wheat SNP and sequence analysis

1 SNP is found in the HSF coding region of the gene on the wheat genome, corresponding to the sequence shown in SEQ ID NO: 1 at position 1875 from the 5' end; the locus has two genotypes in the wheat natural variation population:

genotype A: g

Genotype B: c

According to the sequence difference of different wheat genomes, designing specific primers for PCR amplification of DNA fragments respectively containing the SNP sites:

F1：CCAGACAGATGGGAGTTCG(SEQ ID NO：2)；

R1：GGTTCTGTATTGCTCTTGCCAGG(SEQ ID NO：3)；

F2：ATCACAGCAGCAGGCTCTTGG(SEQ ID NO：4)；

R2：GCTGCTCCTGCCTCAGTTTCACGA(SEQ ID NO：5)。

the target sequence of PCR amplification by taking F1 and R1 as primer pairs is shown as 1647-2001 bit sequence in sequence 1 of the sequence table; the target sequences of PCR amplification by taking F2 and R2 as primer pairs are shown as sequence 1772-1899 in sequence 1 of the sequence table. Enzyme digestion analysis showed that the polymorphisms were recognized by SacI, respectively.

2. Establishment of PCR-enzyme digestion polymorphism detection and genotyping method

1) Extracting genome DNA of wheat to be detected;

2) using the genomic DNA obtained in the step 1) as a template, and performing PCR amplification by using primers F1 and R1, wherein a PCR amplification system (10 mu L) comprises: ddH₂mu.L of O, 1. mu.L of 10 XPCR Buffer, 0.3. mu. L, dNTP (2.5. mu. mol/L) of each of 0.3. mu. L, dNTP (2.5. mu. mol/L) of primer F1 (5. mu. mol/L) and primer R1 (5. mu. mol/L), 0.1. mu.L of Taq enzyme, and 0.5. mu.L of template (20 ng/. mu.L).

PCR amplification conditions were 94 ℃ for 4 min; 30s at 94 ℃, 30s at 56 ℃, 30s at 72 ℃ and 32 times of circulation; storing at 72 deg.C for 10min and 16 deg.C.

3) Diluting the PCR product obtained in the step 2) by 100 times, and performing PCR amplification by using primers F1 and R1 by taking the diluted PCR product as a template, wherein a PCR amplification system (10 mu L) is as follows: ddH₂mu.L of O, 1. mu.L of 10 XPCR Buffer, 0.3. mu. L, dNTP (2.5. mu. mol/L) of each of 0.3. mu. L, dNTP (2.5. mu. mol/L) of primer F2 (5. mu. mol/L) and primer R2 (5. mu. mol/L), 0.1. mu.L of Taq enzyme, and 0.5. mu.L of template (20 ng/. mu.L).

PCR amplification conditions were 94 ℃ for 4 min; 30s at 94 ℃, 30s at 56 ℃, 10s at 72 ℃ and 32 times of circulation; storing at 72 deg.C for 10min and 16 deg.C.

4) Carrying out enzyme digestion on the PCR product obtained in the step 3) by using SacI to obtain a digestion product, carrying out 4% agarose gel electrophoresis detection, recording whether the PCR product is cut into two fragments, and judging and recording the condition of the wheat to be detected at the site according to the following method:

if the enzyme digestion product is a fragment, the wheat to be detected is homozygous for G (shown as G/G) at the site (a lane G in figure 1);

if the enzyme digestion product is two fragments, the wheat to be detected is C homozygous (shown as C/C) wheat at the site (a lane C in figure 1).

5) According to the results of step 4), wheat was classified into I, II types in the case of the site as follows:

i: G/G (i.e., genotype A homozygous);

II: C/C (i.e., genotype B homozygous);

the "/" is preceded by a case on one homologous chromosome and the "/" is followed by a case on the other homologous chromosome.

3. Typing natural population by using dCAPs marker and performing correlation analysis on traits of spike number of single plant

And (3) taking 323 parts of hexaploid wheat in a natural population as the wheat to be tested, parting according to the method in the step 2, and randomly performing sequencing verification on the amplification products of part of wheat, wherein the results are shown in table 1.

TABLE 1 situation of the polymorphic sites in the wheat Natural population

Example 2

In 2015, the natural population wheat was planted in dry land of the experimental farm of the institute of crop science of agricultural sciences (beijing cisterm), in 2016, in dry land and water land of the experimental farm of the institute of crop science of agricultural sciences (beijing cisterm and changping), the number of single ears of each wheat variety was investigated, the number of single ears and the condition of the polymorphic sites were analyzed by association with tassel2.1 software, and a mixed linear model + population structure (MLM + (Q + K)) method was selected for analysis, with P <0.05 as a significance level, and the results are shown in table 2.

TABLE 2 correlation analysis results of WFZP-A gene polymorphic site conditions and individual plant spike number in natural population

The correlation analysis results in table 2 show that the difference in the number of ears per plant of the two types formed by the natural population composed of 323 hexaploid wheat shown in table 1 reaches a significant level (P < 0.05). Wherein the number of wheat single plant spikes of the type II is higher than that of the type I. In several environments, the number of single ears of wheat material of type II is 0.93, 0.74, 0.71, 1.32 higher than that of wheat of type I, respectively, and type II is an excellent genotype for increasing the number of single ears of wheat.

Example 3

The results of the association analysis in example 2 revealed that 4 thermal environments were found out among 10 environments examined, and the difference in the number of ears per plant among the 3 thermal environments was extremely significant (P <0.05), so that it was considered that the HSF-a gene and heat are highly associated with each other, and therefore, the 4 environments were classified into hot and normal according to the results of the association analysis, and the numbers of ears per plant of both genotypes were counted, and the results are shown in table 3.

TABLE 3 results of the number difference between two haplotypes per plant spike for HSF-A gene in four related environments

The results in Table 3 show that type I is reduced by 19.06% from type II under normal conditions, and 10.15% from type I to type II under hot conditions, in summary type I is a low panicle number, high heat tolerant genotype and type II is a high panicle number, low heat tolerant genotype.

In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and reference may be made to the related descriptions of other embodiments for parts that are not described or illustrated in a certain embodiment.

In summary, the present invention finds that there is a SNP corresponding to 1875 from 5' end in sequence table sequence 1 by analyzing genetic variation of HSF gene coding region in wheat natural variation population, and there are two genotypes of this SNP: genotype A (G) and genotype B (C). Correlation analysis proves that the size of the spike number of each plant in the homozygous types of the two haplotypes is as follows: the wheat with genotype A homozygous is less than wheat with genotype B homozygous, and the wheat with genotype A homozygous has stronger heat resistance than wheat with genotype B homozygous. Experiments prove that the wheat with relatively high single-plant spike number and strong heat resistance can be found by detecting the SNP. The invention provides a new method for the molecular marker-assisted selective breeding of wheat, and has important significance in cultivating high-yield wheat varieties and researching.

The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not substantially depart from the spirit and scope of the embodiments of the present invention, and are intended to be included within the scope of the present invention.

Sequence listing

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