CN113584216A

CN113584216A - KASP marker development of wheat grain weight gene TaCYP78A16 and application thereof

Info

Publication number: CN113584216A
Application number: CN202111030709.2A
Authority: CN
Inventors: 吕亮杰; 李辉; 陈希勇; 赵爱菊; 关中泼; 刘玉平
Original assignee: Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Current assignee: Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Priority date: 2021-05-17
Filing date: 2021-09-03
Publication date: 2021-11-02
Anticipated expiration: 2041-09-03
Also published as: CN113122652A; CN113584216B

Abstract

The invention discloses a method for developing KASP marker of wheat grain weight gene TaCYP78A16 and application, identification or auxiliary identification of wheat grain weight. The method comprises the following steps: detecting the genotype of the wheat to be detected, and identifying or assisting in identifying the grain weight of the wheat according to the genotype of the wheat to be detected; the genotype is the genotype of KASP _78A16 locus in the wheat genome; the KASP _78A16 site is a SNP site in wheat genome, is located at the 51 st nucleotide of SEQ ID No.4 on the 5A chromosome of wheat, and the nucleotide type is A or T. Experiments prove that the grain weight of the wheat variety with the KASP _78A16 site AA genotype is obviously higher than or is candidate to be higher than that of the wheat variety to be detected with the KASP _78A16 site TT genotype, which indicates that the KASP _78A16 site is an SNP molecular marker related to the grain weight of the wheat.

Description

KASP marker development of wheat grain weight gene TaCYP78A16 and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to the development of KASP markers of wheat grain weight genes TaCYP78A16 and application thereof.

Background

Wheat (Triticum aestivum) is widely planted worldwide and is one of three main grain crops in China, and can be used for making bread, steamed bread, biscuits, noodles and other foods after being processed and can be made into beer, alcohol, white spirit or biofuel and the like after being fermented (Zhangying, Wangxingmin, Zhouyu, and the like, the international hotspot of current wheat research [ J ] science and technology guide, 2014, 32 (13): 64-69.). Increasing wheat yield is an important condition for meeting population growth and improving people's living standard, so increasing yield is the first breeding target of breeders, and the number of panicles per unit area, the number of grains per panicle and the thousand-grain weight are important genetic factors of wheat yield (Wang Jing. research on agronomic traits of wheat related to yield [ J ]. rural economy and technology, 2018, 29 (018): 23.). In the last 60 th century, the application of dwarf genes has opened a green revolution of grains, and the yield of grains is greatly improved by enhancing the lodging resistance of wheat. In recent years, many wheat yield-related genes and their functions have been analyzed with the development of molecular biology techniques such as high-density molecular labeling and gene sequencing (Gupta P K, Mir R, Mohan A, et al. wheat Genomics: Present Status and Future genes [ J ]. International Journal of Plant Genomics, 2008 (1687-5370): 896451.). thousand-Grain Weight genes are mainly located in 1A, 1B, second homology group, 3A, 3D, 4A, 5D, sixth homology group and seventh homology group, such as genes TaSus1-7A, 6-SFT-A2, TaGASR-A1, TaTGW-7A, TaMOC1-7A, TaSAP1-A1 (Wheat sucrose synthase Gene TaSus1, association analysis of TaSus2 with thousand-Grain Weight [ D ].2012 ], Yue A, Li A, Mao X, et al. identification and maintenance of a functional of a marker 20156-SFT-A2 Associated with Weight Grain in Weight of protein [ J ]. structural, Brussing, 35(2) J-63. origin, III. g, III. F, III. sub.7, III. sub.g. K.S. protein, protein K-S. K, protein K-S.S. K.S. K. protein, 2016, 7(e0145970).

The molecular Marker Assisted Selection (MAS) is a novel breeding mode combining modern molecular biology and traditional genetic breeding, and can select target plants from DNA level at any time of plant development by using molecular markers (Tanksley et al, RFLP mapping in plant breeding: New tools for an old science.1989, Biotechnology, 7: 257 materials 263), thereby making up for many defects in traditional breeding and becoming an effective way for solving the problem of difficult variety breeding. The molecular marker-assisted selection by using a high-throughput molecular detection platform is an effective means for increasing yield breeding accuracy and improving breeding efficiency.

Single-nucleotide polymorphism (SNP) refers to DNA sequence polymorphism at the genome level caused by variation at the Single nucleotide level in a genome nucleotide sequence, and mainly includes deletion, insertion, conversion, transversion and the like of a Single base (thaliana group and the like, research on SNP molecular markers and application progress.2012, chinese agricultural bulletin, 28 (12): 154-. The marker designed based on the SNP locus is a third-generation molecular marker developed on the basis, and the marker type has low mutation frequency and high genetic stability; the loci are rich and the distribution is wide; rapid detection, large-scale screening and the like. Compared with the second generation molecular marker, the KASP marker designed based on the SNP locus does not need to be typed according to the size of a DNA fragment, and can get rid of the detection method of the traditional gel electrophoresis, which has the relatively complicated steps, low throughput and higher price. Therefore, the method is more suitable for the high-flux molecular detection platform which is rapidly developed at the present stage.

Although many of the above-mentioned linkage markers of wheat yield-related genes can be used for molecular marker-assisted selection, most of them are SSR markers, InDel markers or enzyme digestion markers, and their detection efficiency is low, and at the same time, they may produce aerosol to pollute the environment, and they are not suitable for high-throughput molecular detection platform (Huming. wheat grain weight-related genes TaTGW6, TaTGW-7A clone and their functional marker development [ D ]. 2016.; Miji mountain, Musper. origin, Mulberry and others wheat grain weight genes TaCwi-A1 functional markers CWI22, CWI21 verification and application [ J ]. Chinese agricultural science, 2014, 47 (13): 2671-2679.). Therefore, the development of the wheat yield KASP molecular marker suitable for the high-throughput molecular detection platform has important significance for popularizing the application of the molecular marker technology and improving the breeding efficiency and the breeding level of wheat in China.

Disclosure of Invention

The invention provides a method for identifying or assisting in identifying wheat grain weight, which comprises the steps of detecting the genotype of wheat to be detected, and identifying or assisting in identifying the wheat grain weight according to the genotype of the wheat to be detected; the genotype is the genotype of KASP _78A16 locus in the wheat genome; the KASP _78A16 locus is an SNP locus in a wheat genome, is positioned AT the 51 st nucleotide of SEQ ID No.4 on the 5A chromosome of wheat, has the nucleotide type of A or T, and the grain weight of the wheat to be detected with the KASP _78A16 locus as TT genotype is lower than or is candidate to be lower than that of the wheat to be detected with the KASP _78A16 locus as AT genotype or AA genotype; the wheat to be detected with the genotype of the KASP _78A16 site as an AT genotype and the wheat to be detected with the genotype of the KASP _78A16 site as an AA genotype have no significant difference in grain weight; wherein, the TT genotype represents that the nucleotide type of the KASP _78A16 site in the wheat genome is homozygote of T; the AA genotype represents that the nucleotide type of the KASP _78A16 site in the wheat genome is homozygous for A; the AT genotype indicates that the nucleotide type of the KASP _78A16 site in the wheat genome is a heterozygous type of A and T.

The application of the method in wheat breeding also belongs to the protection scope of the invention.

The invention also provides application of a substance for detecting the polymorphism or genotype of the KASP _78A16 site. The application is the application in identifying or assisting in identifying the wheat grain weight, the application in preparing products for identifying or assisting in identifying the wheat grain weight, or the application in assisting in breeding wheat or preparing products for assisting in breeding wheat.

The invention also provides a wheat breeding method, which comprises selecting wheat with AA or AT as the genotype of the KASP _78A16 site as a parent to breed, wherein the AA genotype represents the homozygote of A in the nucleotide type of the KASP _78A16 site in the wheat genome; the AT genotype indicates that the nucleotide type of the KASP _78A16 site in the wheat genome is a heterozygous type of A and T.

In the above, the wheat breeding may be the cultivation of high grain weight wheat.

Products containing a substance that detects the polymorphism or genotype of the KASP _78A16 site are also within the scope of the invention.

The product is any one of C1) -C3):

C1) products for detecting single nucleotide polymorphisms or genotypes associated with wheat grain weight;

C2) identifying or assisting in identifying a product of wheat grain weight;

C3) the product is used for wheat auxiliary breeding.

Above, the substance for detecting the polymorphism or genotype of the KASP _78a16 site may be D1), D2) or D3) as follows:

D1) the substance for detecting the polymorphism or genotype of the KASP _78A16 site contains PCR primers for amplifying the wheat genomic DNA fragment including the KASP _78A16 site;

D2) the substance for detecting the polymorphism or genotype of the KASP _78A16 locus is a PCR reagent containing the PCR primer;

D3) a kit containing the PCR primer described in D1) or the PCR reagent described in D2).

As described above, the PCR primer may be a primer set consisting of a single-stranded DNA whose nucleotide sequence is the 22 nd to 42 th positions of SEQ ID No.1 of the sequence Listing, a single-stranded DNA whose nucleotide sequence is the 22 nd to 42 th positions of SEQ ID No.2 of the sequence Listing, and a single-stranded DNA whose nucleotide sequence is SEQ ID No.3 of the sequence Listing.

Optionally, the PCR primer is a primer group consisting of a single-stranded DNA shown by SEQ ID No.1 in the sequence table, a single-stranded DNA shown by SEQ ID No.2 in the sequence table and a single-stranded DNA shown by SEQ ID No.3 in the sequence table.

In the above applications, methods and products, the PCR primers may or may not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and that can be attached to a nucleic acid. Labels include, but are not limited to, dyes; radiolabels, e.g.³²P; binding moieties such as biotin (biotin); haptens such as Digoxin (DIG); a luminescent, phosphorescent, or fluorescent moiety; and a fluorescent dye alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). Labels can provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Labels can be charged moieties (positive or negative) or alternatively, can be charge neutral. The label may comprise or be combined with a nucleic acid or protein sequence, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is detected directly (e.g., direct sequence read) without a label.

The invention provides development and application of an SNP molecular marker tightly linked with a wheat grain weight related gene TaCYP78A16, wherein the molecular marker is an SNP molecular marker KASP _78A16 tightly linked with a wheat grain weight related gene TaCYP78A 16. The molecular marker can detect 527492344 th base of 5A chromosome of wheat genome in high flux. The method provided by the invention is used for carrying out genotype identification on the SNP molecular marker closely linked with the wheat grain weight related gene TaCYP78A16, for example, a KASP technology is applied, the method has the advantages of simple and convenient operation, low cost, short detection period, stable marker, environmental protection and the like, can accurately detect the wheat grain weight related gene TaCYP78A16, and has important significance for promoting wheat yield breeding.

In one embodiment of the invention, the KASP technique was used to identify the genotype at the KASP _78a16 locus in 26 wheat varieties. Experiments prove that the maximum grain weight of the homozygous wheat variety with the KASP _78A16 site A is 50.43, the minimum value is 41.25, the average value is 46.26, the median is 46.24, the maximum grain weight of the homozygous wheat variety with the KASP _78A16 site T is 46.67, the minimum value is 35.61, the average value is 42.89, the median is 43.25, the grain weight of the wheat variety to be detected with the AA genotype is obviously higher than or is candidate to be higher than that of the wheat variety to be detected with the TT genotype, the KASP _78A16 site is an SNP molecular marker related to the grain weight of the wheat, and the method can be used for identifying or assisting in identifying the grain weight of the wheat, can be used for screening high-grain-weight wheat varieties and can be used for assisting in breeding by the wheat molecular marker. The polymorphism of KASP _78A16 site is directly expressed in DNA form and can be detected in each tissue and each development stage of wheat, which is favorable for conveniently and rapidly predicting wheat grain weight. In practical application, in order to improve the accuracy, the substance for detecting KASP _78A16 site polymorphism and genotype can be combined with other substances (such as substances for detecting other single nucleotide polymorphism or genotype related to wheat grain weight) to prepare products for identifying wheat high grain weight varieties.

Drawings

FIG. 1 is a typing chart of example 1, in which TT-FAM indicates the genotype TT and AA-VIC indicates the genotype AA, using a molecular marker KASP _78A16 to detect 26 test wheat variety materials.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The wheat varieties used in the examples described below are commercially available.

The inventor of the invention selects a SNP locus related to wheat grain weight, which is closely linked with a TaCYP78A16 gene (genbank accession number of nucleotide sequence MH572527, 1 month and 20 days in 2019) and anchored on the 527492344 th position (http: html, genome version Triticum aestivum IWGSC (Genomic sequence)), the SNP site was named KASP _78A16, and its flanking sequence is shown in SEQ ID No.4, w is a or t. KASP _78A16 is located at position 51 of SEQ ID No.4 where the nucleotide species is A or T.

The TT genotype indicates that the nucleotide species of KASP _78A16 is homozygous for T; the AA genotype indicates that the nucleotide type of KASP _78A16 is homozygous for A; the AT genotype indicates that the nucleotide type of KASP _78A16 is a hybrid of A and T.

Example 1 development of KASP marker closely linked to wheat grain weight-related Gene TaCYP78A16

1. Designing a primer: the 100bp flanking sequences of the right and left sides are extracted from the KASP _78A16 site, and preferably 3 sets of KASP primers are designed by using Primer5.0 software. After detection using the ArrayTape platform from Douglas Scientific, 1 set of KASP primers with good polymorphism was selected for subsequent validation.

The KASP primers used to detect KASP _78a16 are specifically as follows:

primer X: 5'-gaaggtcggagtcaacggattGCCATACATACATGCACGGAA-3' (SEQ ID No.1, lower case letter portion is specific fluorescent tag sequence VIC);

primer Y: 5'-gaaggtgaccaagttcatgctGCCATACATACATGCACGGAT-3' (SEQ ID No.2, lower case letter portion is specific fluorescent tag sequence FAM);

Primer R：5’-CGACGTACCGTACATGCTTGAG-3’(SEQ ID No.3)。

DNA extraction: extracting genome DNA from wheat leaves by a conventional CTAB method.

KASP reaction test

SNP marker amplification and reaction system:

(1) and (3) detecting by using a fluorescent quantitative PCR instrument AB-Q6 Flex:

the detection reaction system of the 5-mu-L PCR fluorescence quantitative instrument comprises: 50ng of genomic DNA, and 0.07. mu.L of a Primer mixture (preferably, the ratio of the Primer mixture: forward primers Primer X and Primer Y is 100 pmol. multidot.L)^-112 μ L each, reverse primerPrimer R 100pmol·L^-1 30μL，ddH₂O46. mu.L, which can achieve the same detection purpose by using other reasonable primer mixture ratios), 2.5. mu.L of LGC company 2 XKASP Mix (Low Rox). Editing a sample table, executing a running program and storing data according to an AB-Q6 instrument operation manual of the fluorescent quantitative PCR instrument.

The above reaction system is the preferred reaction system of AB-Q6 Flex, and other reasonable reaction systems can also achieve the same detection purpose.

(2) The selection of the ArrayTape platform from Douglas Scientific

1.6 u L PCR ArrayTape platform detection reaction system includes: genomic DNA 50 ng/. mu.L 0.8. mu.L, Primer mix 0.03. mu.L (preferred Primer mix ratio: forward primers Primer X, Primer Y100 pmol. L)^-112. mu.L each, reverse Primer R100 pmol. multidot.L^-1 30μL，ddH₂O46. mu.L, which can achieve the same detection object by using other reasonable primer mixture ratios), LGC 2 XKASP Mix (Std Rox) 0.8. mu.L. According to an ArrayTape platform instrument operation manual, a sample table is compiled, a program is operated, and data are read.

The above reaction system is the preferable reaction system of the ArrayTape platform of Douglas Scientific company, and other reasonable reaction systems can achieve the same detection purpose.

Note: the above is a recommended detection method, and other detection methods capable of achieving the same detection purpose can also be applied to the molecular marker-assisted breeding process of the marker.

Wherein, the 2 XKASP Mix consists of a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B, high fidelity Taq enzyme, dNTP and Mg²⁺And the like. The nucleotide sequence of the fluorescent probe A is as follows: 5'-GAAGGTCGGAGTCAACGGATT-3', connecting a VIC fluorescent group at the 5 ' end; the nucleotide sequence of the fluorescent probe B is as follows: 5'-GAAGGTGACCAAGTTCATGCT-3', wherein the 5 ' end is connected with a FAM fluorescent group; the nucleotide sequence of the quenching probe A is as follows: 5'-AATCCGTTGACTCCGACCTTC-3', wherein the 3 ' end is connected with a quenching group BHQ; the nucleotide sequence of the quenching probe B is as follows: 5'-AGCATGAACTTGGTCACCTTC-3', wherein the 3 ' end is connected with a quenching groupClique BHQ.

And (3) amplification procedure: pre-denaturation at 95 ℃ for 10min for 1 cycle; denaturation at 95 ℃ for 20s, annealing at 55-62 ℃ (preferably 55 ℃) for 60s, setting 40 cycles.

The experiment was carried out while setting a blank control (NTC) without adding template DNA to the reaction system, and 1 or more blank controls were set for each plate.

Analyzing the scanning data, determining the genotype of the KASP _78A16 locus in the wheat genome to be detected (namely, detecting whether the 527492344 th base of the chromosome 5A of the wheat genome is A or T), and if the fluorescence signal data of the amplification product of the wheat to be detected is analyzed to be close to the X axis (VIC signal) by Douglas genotyping software, determining that the genotype of the KASP _78A16 locus in the wheat genome to be detected is AA homozygous (namely, the 527492344 th base of the chromosome 5A of the wheat genome is AA homozygous); if the fluorescence signal data of the amplification product of the wheat to be detected is analyzed to be close to a Y axis (FAM signal) by Douglas genotyping software, the genotype of the KASP-78A 16 locus in the wheat genome to be detected is TT homozygosity (namely, the 527492344 th base of the No. 5A chromosome of the wheat genome is TT homozygosity); if the fluorescence signal data of the amplification product of the wheat to be detected is positioned between the X axis and the Y axis (VIC and FAM signals) through Douglas genotyping software analysis, the genotype of the KASP _78A16 site in the wheat genome to be detected is an AT heterozygous type (namely, the 527492344 th base of the No. 5A chromosome of the wheat genome is the AT heterozygous type). The samples shown black in the lower left corner are blank controls.

5. Label typing data analysis

In order to verify the reliability of the KASP _78A16 marker, the thousand kernel weight was measured on 26 wheat variety materials, and the method for measuring the thousand kernel weight is disclosed in the literature (Kong faith, Cheng Rui, e.g., Zhang Liwei, etc.. construction of wheat kernel weight major QTL near isogenic lines and evaluation of effects [ J ] wheat crop science, 2017 (03): 30-36.). The genotype of the material was detected using the ArrayTape platform from Douglas Scientific, and the detection method, reaction system and amplification procedure were carried out according to the preferred protocol described above.

The amplification results show that KASP _78A16 marker can obtain stable PCR products in 26 materials, and two allelic sites of AA-VIC and TT-FAM can be detected (FIG. 1). Counting the grain weight data of the tested varieties (table 1), wherein the maximum value of the grain weight of the AA genotype variety is 50.43, the minimum value is 41.25, the average value is 46.26, and the median is 46.24; the maximum grain weight of the TT genotype variety is 46.67, the minimum grain weight is 35.61, the average value is 42.89, and the median is 43.25. "analysis of variance" by software Microsoft EXCEL 2010: the one-way anova method analyzes the data in table 1, and as a result, F (value 7.7) > F crit (value 4.26) and P value 0.05, the grain weights of the two genotype materials are significantly different. From this, it can be seen that the grain weight of the variety carrying the AA genotype is larger.

Therefore, the KASP _78A16 marker can be used for molecular marker assisted breeding of wheat grain weight related gene TaCYP78A 16.

TABLE 1.26 grain weight and genotype information for test wheat varieties

Example 2 application of KASP marker closely linked with wheat grain weight related gene TaCYP78A16 in molecular marker-assisted selection of wheat high grain weight plants

To test the utility of the KASP _78A16 marker of the present invention, wheat variety Jimai 44 (as female parent) described in Table 1 was hybridized with Gao you 8901 (as male parent) to prepare F2 isolate, KASP marker test and thousand kernel weight weighing were performed on the F2 isolate (see Table 2 for test results), and the marker test and the thousand kernel weight measurement were performed according to the method described in example 1.

Counting the grain weight data of the tested variety, wherein in 48 isolated population single plants, the thousand grain weight of the AA or AT genotype variety has the maximum value of 52.11, the minimum value of 35.61, the average value of 45.69 and the median of 45.52; the thousand kernel weight of the TT genotype variety has a maximum value of 47.43, a minimum value of 35.91, an average value of 41.79 and a median of 42.13. "analysis of variance" by software Microsoft EXCEL 2010: the results of analyzing the data in table 2 by the method of one-way anova indicate that F (value 10.27) > F crit (value 4.05) and P value 0.01, and that the grain weight is very significantly different between the AA or AT genotype varieties and the TT genotype varieties. According to the results, the grain weight of the variety carrying the genotype A is larger, which shows that the marker KASP _78A16 has higher practicability in screening wheat high grain weight plants.

TABLE 2 phenotypic and genotypic information for wheat segregating populations

Note: ' indicates no detection signal.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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Claims

1. The method for identifying or assisting in identifying the grain weight of wheat is characterized by comprising the following steps: detecting the genotype of wheat to be detected, and identifying or assisting in identifying the grain weight of the wheat according to the genotype of the wheat to be detected; the genotype is the genotype of KASP _78A16 locus in the wheat genome; the KASP _78A16 locus is a SNP locus in wheat genome, is located at the 51 st nucleotide of SEQ ID No.4 on the 5A chromosome of wheat, and the nucleotide type is A or T,

the grain weight of the wheat to be detected with the genotype of the KASP _78A16 site as TT genotype is lower than or is lower than the grain weight of the wheat to be detected with the genotype of the KASP _78A16 site as AT genotype or AA genotype; the wheat to be detected with the genotype of the KASP _78A16 site as an AT genotype and the wheat to be detected with the genotype of the KASP _78A16 site as an AA genotype have no significant difference in grain weight; wherein, the TT genotype represents that the nucleotide type of the KASP _78A16 site in the wheat genome is homozygote of T; the AA genotype represents that the nucleotide type of the KASP _78A16 site in the wheat genome is homozygous for A; the AT genotype indicates that the nucleotide type of the KASP _78A16 site in the wheat genome is a heterozygous type of A and T.

2. Use of the method of claim 1 in wheat breeding.

3. Use of a substance that detects a polymorphism or genotype at KASP _78a16 locus as defined in claim 1 for identifying or aiding in the identification of wheat grain weight.

4. Use of a substance that detects a polymorphism or genotype at the KASP _78a16 locus as defined in claim 1 in the preparation of a product for identifying or aiding in the identification of wheat grain weight.

5. Use of a substance for detecting the polymorphism or genotype of KASP _78a16 locus as defined in claim 1 in wheat assisted breeding or in the production of wheat assisted breeding products.

6. A method of breeding wheat, comprising selecting as a parent wheat having as an AA the genotype of KASP _78A16 locus as defined in claim 1, said AA genotype representing the homozygous type A for the nucleotide species AT KASP _78A16 locus in the wheat genome, or an AT; the AT genotype indicates that the nucleotide type of the KASP _78A16 site in the wheat genome is a heterozygous type of A and T.

7. A product containing a substance for detecting the polymorphism or genotype of KASP _78A16 locus as defined in claim 1, which is any one of C1) -C3):

C2) identifying or assisting in identifying a product of wheat grain weight;

C3) the product is used for wheat auxiliary breeding.

8. Use according to any one of claims 3 to 5 or a product according to claim 7, wherein: the substance for detecting the polymorphism or genotype of KASP _78A16 site according to claim 1 is D1), D2) or D3) as follows:

D1) the substance for detecting the polymorphism or genotype of KASP _78A16 site according to claim 1 comprising PCR primers for amplifying a wheat genomic DNA fragment including said KASP _78A16 site;

D2) the substance for detecting the polymorphism or genotype of KASP _78A16 site according to claim 1 is a PCR reagent containing the PCR primer;

9. Use or product according to claim 8, characterized in that: the PCR primer is a primer group consisting of single-stranded DNA with a nucleotide sequence of 22 th to 42 th positions of SEQ ID No.1 in a sequence table, single-stranded DNA with a nucleotide sequence of 22 nd to 42 th positions of SEQ ID No.2 in the sequence table and single-stranded DNA with a nucleotide sequence of SEQ ID No.3 in the sequence table.

10. Use or product according to claim 8 or 9, characterized in that: the PCR primer is a primer group consisting of a single-stranded DNA shown by SEQ ID No.1 in a sequence table, a single-stranded DNA shown by SEQ ID No.2 in the sequence table and a single-stranded DNA shown by SEQ ID No.3 in the sequence table.