CN112593006B

CN112593006B - Wheat green-extending character main effect QTL locus, KASP primer closely linked with same and application of KASP primer

Info

Publication number: CN112593006B
Application number: CN202011627459.6A
Authority: CN
Inventors: 任天恒; 范涛; 李治; 谭飞泉; 任正隆
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-09-14
Anticipated expiration: 2040-12-31
Also published as: CN112593006A

Abstract

The invention discloses a wheat greening-extending character main effect QTL locus, a KASP primer tightly linked with the same and application of the same, wherein the main effect QTL locus QSg.sau-2B.1 and QSg.sau-6A.2 are respectively positioned on 2B chromosomes and 6A chromosomes of wheat; wherein, the physical position of QSg.sau-2B.1 on the reference genome of Chinese spring is between 58.83-65.47 Mb; physical position of QSg.sau-6A.2 on the reference genome of Chinese spring is 77.78-82.96 Mb. The invention discloses two main effect QTLs for controlling the green-extending character on the chromosomes of wheat 2B and 6A for the first time, the green-extending character controlled by the two QTLs has high value for high-yield breeding of wheat, lays a foundation for further cloning the green-extending gene and high-yield breeding of wheat, and provides a method for molecular marker-assisted breeding by utilizing the developed KASP molecular marker.

Description

Wheat green-extending character main effect QTL locus, KASP primer closely linked with same and application of KASP primer

Technical Field

The invention relates to a wheat greening-extending character major QTL locus, in particular to a wheat greening-extending character major QTL locus, a KASP primer closely linked with the same and application of the KASP primer.

Background

In the world of today, due to the influence of many factors, such as the reduction of cultivated land area, the increase of population and climate change, the food yield still cannot meet the demand of people, and the improvement of the crop yield is still the hot direction of research of many breeders. According to predictions, yields in 24% -39% of major crop regions worldwide will be stagnant or reduced, especially in regions where abiotic stress is a major constraint. In addition, due to the energy crisis caused by the shortage of petroleum resources, the production of alcohol and other energy sources by using grains has become an important way to solve the energy shortage, which further increases the pressure of grain supply. Therefore, how to realize further yield increase of crops is a practical problem in front of numerous breeders, and needs to be solved urgently.

The common wheat is not only one of three main grain crops in the world, but also the second main grain crop in China, and the yield of the common wheat is closely related to the national grain safety. Since the beginning of the fifties of the last century, a plurality of dwarf genes are successively discovered in rice and wheat and are utilized to carry out genetic breeding improvement, so that the yield of crops is remarkably improved. The dwarf crops not only have the characteristic of lodging resistance, but also distribute more organic matters into grains, and the purpose of improving the yield is achieved by improving the harvest index of the crops. In the past decades, the harvest index of the main cereal crops in China has been improved significantly, but the space for the rise is very limited, and the increase of the economic yield of crops in the future depends on the increase of the biological yield. The green plant leaves perform photosynthesis by capturing light energy to assimilate carbon dioxide and water into organic matters, so that the photosynthesis and the biological yield of the plants are closely related. Research shows that the improvement of the photosynthetic efficiency can effectively increase the biomass of crops.

The wheat variety Chuannong 18 is approved by the variety approval committee of Sichuan province in 2003, has the characteristics of strong tillering capability, high spike number, high thousand kernel weight and high yield, and also has the characteristics of greenness extending characters (Tanyuan, Zhangianqiong, ren Zheng Long. "harmonized type" new wheat variety yield potential and the research on the composition thereof [ J ] academic newspaper of Sichuan agriculture university, 2003(03): 189-. The Green-extending character means that the leaf senescence is effectively delayed after the crops bloom, the leaves keep Green, the grain filling speed is enhanced, the photosynthesis time is prolonged, and the character is translated into Stay Green internationally. At present, the international research on the green-extending character is mainly divided into two main categories: namely functional stay green and decorative stay green. The former is that leaves can be photosynthetic while keeping green to produce organic matter, the latter is the opposite, only leaves keep green and do not photosynthetic. In wheat studies it has been found that leaves which delay senescence can increase carbon fixation by 11% for only two days. Therefore, the method has important significance for high-yield breeding of crops only by functional green-extending. The test shows that the YangLu variety Chuan nong 18 cultivated for many years in the subject group belongs to a functional YangLu variety, and has high yield potential and large yield increase amplitude (Chen JB, Liang Y, Hu XY, Wang XX, Zhang HQ, Ren ZL, Luo PG. physical characteristics of "stand green" in vitro culture of strain and field growth regulation. acta physiology of plant, 2010,32: 875-.

The method for assisting breeding by utilizing the molecular marker can well overcome the defects of long breeding period, high breeding cost and low efficiency of the traditional breeding method, allows target characters to be selected in early generations, shortens the breeding process, and is convenient, rapid, accurate and flexible. With the continuous development of sequencing technology, Single Nucleotide Polymorphism (SNP) molecular markers widely existing in plants are also increasingly discovered and widely applied to genetic research of various traits of crops, including current common SNP gene chips such as: such as 9K, 90K, 15K, 660K, 55K, 820K, 35K and 50K SNP gene chips (Sun C, Dong Z, ZHao L, et al. the Wheat 660K SNP array definitions for marker-assisted selection in polyploid Wheat [ J ]. Plant Biotechnology Journal,2020,18 (6)). Competitive Allele-Specific PCR (KASP) technology, one of the mainstream SNP genotyping methods at home and abroad, is used for performing accurate biallelic determination on SNP sites according to the Specific matching of terminal bases of primers. Compared with a Taqman method, the technology has the advantages of low cost, flexibility, rapidness, high accuracy and obvious advantages in large-scale marking detection work.

Therefore, based on the KASP technology, the KASP molecular marker closely linked with the Chuannong 18 lingering green character gene is developed, which not only has important significance for the super-high yield breeding of wheat, but also lays theoretical foundation for researching the anti-aging physiological characteristics of crops.

Disclosure of Invention

The invention aims to provide a wheat green-extending character main effect QTL locus, a KASP primer closely linked with the same and application of the main effect QTL locus QSg.sau-2B.1 and QSg.sau-6A.2, wherein the green-extending character controlled by the main effect QTL locus QSg.sau-2B.1 and QSg.sau-6A.2 has high value for high-yield breeding of wheat.

In order to achieve the aim, the invention provides a wheat green-extending character main effect QTL locus QSg.sau-2B.1 and QSg.sau-6A.2, wherein the main effect QTL locus QSg.sau-2B.1 and QSg.sau-6A.2 are respectively positioned on 2B chromosomes and 6A chromosomes of wheat; wherein, the physical position of the main effect QTL locus QSg.sau-2B.1 on the Chinese spring reference genome IWGSC RefSeqv1.0 is between 58.83 and 65.47Mb, and SNP molecules on two sides of the main effect QTL locus are marked as AX-110965907 and AX-111729522; the physical position of a main effect QTL site QSg.sau-6A.2 on a Chinese spring reference genome IWGSC Refseq v1.0 is 77.78-82.96Mb, and SNP molecules on two sides of the main effect QTL site are marked as AX-110040743 and AX-109444699.

Another objective of the invention is to provide KASP primers for SNP molecular markers AX-111729522 and AX-110040743, KASP primer for SNP molecular marker AX-111729522, whose nucleotide sequence of forward primer 1 is shown in SEQ ID NO.3, nucleotide sequence of forward primer 2 is shown in SEQ ID NO.4, and nucleotide sequence of reverse primer is shown in SEQ ID NO. 5; for the KASP primer of the SNP molecular marker AX-110040743, the nucleotide sequence of the forward primer 1 is shown as SEQ ID NO.6, the nucleotide sequence of the forward primer 2 is shown as SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 8; wherein, in KASP primers aiming at SNP molecular markers AX-111729522 and AX-110040743, the 5' ends of the forward primer 1 and the forward primer 2 are connected with different fluorescent tag sequences.

Preferably, the 5' end of the forward primer 1 is connected with an F probe, and the nucleotide sequence of the F probe is shown as SEQ ID NO. 9; the 5' end of the forward primer 2 is connected with an H probe, and the nucleotide sequence of the H probe is shown in SEQ ID NO. 10.

The invention also aims to provide a kit for identifying the main effect QTL sites QSg.sau-2B.1 and QSg.sau-6A.2 of the wheat stay green character, wherein the primer in the kit is the KASP primer.

Another object of the present invention is to provide a method for identifying wheat stay green trait major QTL sites QSg.sau-2B.1 and QSg.sau-6A.2, which comprises: the KASP primer is used for carrying out fluorescence quantitative PCR amplification, and the wheat material to be detected is genotyped according to the PCR amplification result, which comprises the following steps: if the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-111729522 but does not contain the fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-111729522, the wheat material to be detected contains the main effect QTL site QSg.sau-2 B.1; if the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-111729522 but does not have a fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-111729522, the wheat material to be detected does not contain the main effect QTL site QSg.sau-2 B.1; if the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-110040743 but does not contain the fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-110040743, the wheat material to be detected contains the main effect QTL site QSg.sau-6 A.2; if the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-110040743 but does not have a fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-110040743, the wheat material to be detected does not contain the main effect QTL site QSg.sau-6 A.2.

Preferably, the genotype of the wheat material to be detected containing the wheat lingering green character main effect QTL locus QSg.sau-2B.1 and QSg.sau-6A.2 is recorded as a D genotype, the genotype of the wheat material to be detected containing no wheat lingering green character main effect QTL locus QSg.sau-2B.1 and QSg.sau-6A.2 is recorded as an A genotype, and the lingering green character of the D genotype wheat strain is obviously higher than that of the A genotype wheat strain.

Preferably, the reaction system for the fluorescent quantitative PCR amplification comprises: KASP Mastermix, the KASP primer, the genome DNA of the wheat material to be detected and RNase-free deionized water; wherein, in the KASP primer, the volume ratio of the forward primer 1 to the forward primer 2 to the reverse primer is 2:2: 5.

Preferably, the reaction procedure of the fluorescent quantitative PCR amplification comprises: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20 s; annealing and extending at 61 ℃ for 40s, circulating for 10 times, and reducing the annealing and extending temperature by 0.6 ℃ in each circulation; denaturation at 95 ℃ for 20 s; annealing and extending for 40s at 55 ℃; circulating for 30 times; keeping the temperature at 25 ℃ and collecting fluorescence signals.

Another object of the present invention is to provide the use of KASP primers designed for SNP molecular markers AX-111729522 and AX-110040743, which comprises any one of the following: (1) the application of the gene locus is used for detecting main effect QTL sites QSg.sau-2B.1 and QSg.sau-6A.2 of wheat green extending characters and positioning green extending genes; (2) the application in breeding and creating wheat resources with different green-extending characters; (3) the application of the method in promoting the convergent breeding between the wheat green-extending character major QTL locus QSg.sau-2B.1 and QSg.sau-6A.2 and other excellent character loci of wheat;

(4) the application of the polypeptide in researching the genetic mechanism and the physiological mechanism of the anti-aging physiological phenomenon of wheat after flowering.

The wheat green-extending character main effect QTL locus, the KASP primer closely linked with the same and the application thereof have the following advantages:

the invention discloses two main effect QTL QSg.sau-2B.1 and QSg.sau-6A.2 for controlling the green-extending character on

wheat

2B and 6A chromosomes for the first time, and the green-extending character controlled by the two QTL has high value for high-yield breeding of wheat. The invention lays a foundation for further cloning the lingering green gene and breeding wheat with high yield, provides a method for carrying out molecular marker assisted breeding by utilizing the developed KASP molecular marker, and has important academic significance for researching the mechanism of delaying senescence of plants.

In addition, the invention also discloses two KASP molecular markers KASP-AX-111729522 and KASP-AX-110040743 which are respectively closely linked with the two QTLs and present the characteristic of coseparation markers, the two pairs of KASP primers can promote the application of the green extending character in high yield breeding of wheat, and phenotype identification work can be greatly reduced in breeding by means of the two pairs of molecular markers, the breeding cost is saved, and the breeding efficiency is improved.

Drawings

FIG. 1 shows the location on the chromosome of two major QTL QSg.sau-2B.1 and QSg.sau-6A.2 controlling the stay green trait.

FIG. 2 shows the results of fluorescent quantitative PCR genotyping of Chuannon 18 and T1208 of parents with KASP molecular markers KASP-AX-111729522 (a in FIG. 2) and KASP-AX-110040743 (b in FIG. 2).

FIG. 3 shows the results of fluorescence quantitative PCR genotyping of 18 XT 1208 high generation inbred progeny of Chuannon by KASP molecular markers KASP-AX-111729522 (a in FIG. 3) and KASP-AX-110040743 (b in FIG. 3).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Experimental example 1 acquisition of stay green trait major QTL QSg.sau-2B.1 and QSg.sau-6A.2 and KASP molecular markers linked thereto respectively

(1) Hybridizing a wheat variety Chuannong 18 with the green-extending character as a female parent and a wheat material T1208 without the green-extending character as a male parent to obtain F₁Generation, F₁Selfing to obtain F₂A generation, obtaining a high-generation selfing population containing 371 lines by a single-seed-transmission method;

(2) 371 strains (F) constructed by Chuannong 18 XT 1208 using 55K SNP gene chip₁₁-F₁₃) The genome of the recombinant inbred population is scanned, and a linkage genetic map is constructed, wherein the genetic linkage map covers 21 chromosomes of wheat and has the total length of 4192.62 cM;

(3) the data of the stay green phenotype of each strain was investigated when segregation of the stay green trait was present in the population at late wheat flowering in 2014 2015, 2015 2016, 2016 2017 and 2018 2019. Collection and analysis of relevant phenotypic data: grading the green-extending character according to 1 to 9 grades by grading the green-extending grade of the field population, wherein 1 represents that the flag leaves are completely withered and yellow; 2, the leaves are completely withered and yellow, and the base parts of the leaves are occasionally green; 3 represents basically withered yellow and little green; 4 represents base green; 5 represents base 1/3 green; 6 represents 1/2 green; 7 represents 2/3 green; 8 represents a basic green color, and leaves are slightly withered and yellow; and 9 indicates that the leaves are all green. By combining the lingering phenotype data under four environments, QTL positioning is carried out by using software IciMapping 4.1, and finally main QTL QSg.sau-2B.1 and QSg.sau-6A.2 which are both derived from Chuannon 18 and control the lingering trait are positioned on the 2B chromosome and the 6A chromosome of wheat, the average LOD values are respectively 16.33 and 20.53, and the average explained phenotype variation rate is respectively 11.43 percent and 15.27 percent. SNP molecular markers at both sides of QSg.sau-2B.1 are AX-110965907(SEQ ID NO.14, the base at the 36 th position has C or G polymorphism, indicated by n in the sequence table) and AX-111729522(SEQ ID NO.1, the base at the 36 th position has A or T polymorphism, indicated by n in the sequence table), the physical position on the Chinese spring reference genome is 58.83-65.47Mb, while the SNP molecular markers at both sides of QSg.sau-6A.2 are AX-110040743(SEQ ID NO.2, the base at the 36 th position has A or G polymorphism, indicated by n in the sequence table) and AX-109444699(SEQ ID NO.15, the base at the 36 th position has C or T polymorphism, indicated by n in the sequence table), the physical position on the Chinese spring reference genome is 77.78-82.96 Mb;

(4) SNP molecular markers in QTL QSg.sau-2B.1 and QSg.sau-6A.2 intervals are searched in the genetic linkage map respectively, two SNP molecular markers, namely AX-110965907 and AX-111729522, are found in the QSg.sau-2B.1 interval, and one SNP molecular marker, namely AX-110040743, is found in the QSg.sau-6A.2 interval. Then, the sequences of the SNP molecular markers are designed into KASP primers on a Polymarker website, and the sequences of the 3 SNP markers are converted into KASP primers, wherein the related primer sequences are shown in a table 1: each pair of primers consists of 3 sequences, namely: forward primer 1: f probe + amplification primer sequence; forward primer 2: h probe + amplification primer sequence; reverse primer: amplifying the primer sequence; wherein, the nucleotide sequences of the F probe and the H probe are as follows:

f probe (SEQ ID NO. 9): 5'-GAAGGTGACCAAGTTCATGCT-3' (bindable FAM fluorophore)

H probe (SEQ ID NO. 10): 5'-GAAGGTCGGAGTCAACGGATT-3' (to which HEX fluorophores can be attached).

Table 1 shows three pairs of KASP primer sequences

Experimental example 2 screening of KASP molecular marker polymorphic primers between parents

(1) Extracting DNA of parent Chuannong 18 and T1208 trefoil stages by adopting a CTAB method;

(2) and performing fluorescent quantitative PCR amplification by using the synthesized primers, and performing polymorphism analysis on the parent wheat according to the PCR amplification result. Selected reaction system for fluorescent quantitative PCR amplification: KASP Mastermix 4.5. mu.L, KASPAssay Mix 2. mu.L, 50ng template DNA, RNase-free deionized water were added in a total amount of 10. mu.L, wherein KASPAssay Mix contained nucleotide sequences for each pair of primers as shown in Table 1 and the volume ratio of the three primers was 2:2:5, respectively;

the fluorescent quantitative PCR reaction procedure is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20 s; annealing and extending at 61 ℃ for 40s, circulating for 10 times, and reducing the annealing and extending temperature by 0.6 ℃ in each circulation; denaturation at 95 ℃ for 20 s; annealing and extending for 40s at 55 ℃; circulating for 30 times; preserving the temperature at 25 ℃, and collecting a fluorescence signal;

(3) screening of polymorphic molecular markers among parents, namely, for the molecular marker of QTL QSg.sau-2B.1, a pair of molecular markers with polymorphism between parents Chuannon 18 and T1208 are named as KASP-AX-111729522, the genotyping result of the parents is shown as a in figure 2, the fluorescence type of the genotype of the parent Chuannon 18 is FAM fluorophore type, and the fluorescence type of the genotype of T1208 is HEX fluorophore type. For the molecular marker of QTL QSg.sau-6A.2, a pair of molecular markers with polymorphism between the parents Chuannon 18 and T1208 are named as KASP-AX-110040743, the genotyping result of the parents is shown as b in figure 2, the fluorescence type of the genotype of the parents Chuannon 18 is HEX fluorophore type, and the fluorescence type of the genotype of T1208 is FAM fluorophore type.

Experimental example 3 application of KASP-AX-111729522 and KASP-AX-110040743 molecular markers in Chuannong 18 XT 1208 high-generation inbred population

The same genotyping method as above is adopted to randomly select 131 strains (including two parents) in the Chuannong 18 XT 1208 high-generation inbred population for genotyping by using KASP-AX-111729522 and KASP-AX-110040743 molecular markers. The results of the genotyping part of the KASP-AX-111729522 primer in the population are shown as a in FIG. 3, the results of the genotyping part of the KASP-AX-110040743 primer in the population are shown as b in FIG. 3, the genotyping results are in four categories, and the genotype A indicates that neither QSg.sau-2B.1 nor QSg.sau-6A.2 contains; genotype B indicates that QSg.sau-2B.1 contains only QSg.sau-6 A.2; genotype C indicates that QSg.sau-6A.2 alone does not contain QSg.sau-2 B.1; d genotype means that both QSg.sau-2B.1 and QSg.sau-6A.2 are contained. Through the analysis of the independent sample t test result, the improvement of the D genotype (38 lines), namely the line simultaneously containing two QTLs for controlling the stay green character on the stay green character is higher than that of the A genotype (31 lines), namely the line not containing the two QTLs for controlling the stay green character. Compared with the genotype A, the phenotype of the green-extending character is improved by 3.42, 3.99, 2.9 and 1.77 in 2014, 2015, 2016 and 2018 respectively by the genotype D.

TABLE 2 application results of KASP-AX-111729522 and KASP-AX-110040743 molecular markers in Chuannon 18 XT 1208 high generation inbred population

Note: genotype A means that neither QSg.sau-2B.1 nor QSg.sau-6A.2 contain it; genotype B indicates that QSg.sau-2B.1 contains only QSg.sau-6 A.2; genotype C indicates that QSg.sau-6A.2 alone does not contain QSg.sau-2 B.1; d genotype means that both QSg.sau-2B.1 and QSg.sau-6A.2 are contained; the values in the table are table-type values, which are graded on a scale of 1-9, based on the above-described grading of the degree of greenness of the blades.

Table 3 shows the results of t test

Note: the numbers in brackets indicate the number of lines carrying the corresponding genotypic material; genotype A means that neither QSg.sau-2B.1 nor QSg.sau-6A.2 contain it; d genotype means that both QSg.sau-2B.1 and QSg.sau-6A.2 are contained.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

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Claims

1. KASP primers aiming at SNP molecular markers AX-111729522 and AX-110040743, which is characterized in that, for the KASP primer aiming at SNP molecular marker AX-111729522, the nucleotide sequence of AX-111729522 is shown as SEQ ID NO.1, the nucleotide sequence of a forward primer 1 is shown as SEQ ID NO.3, the nucleotide sequence of a forward primer 2 is shown as SEQ ID NO.4, and the nucleotide sequence of a reverse primer is shown as SEQ ID NO. 5;

for the KASP primer of the SNP molecular marker AX-110040743, the nucleotide sequence of AX-110040743 is shown as SEQ ID NO.2, the nucleotide sequence of the forward primer 1 is shown as SEQ ID NO.6, the nucleotide sequence of the forward primer 2 is shown as SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 8;

wherein, in KASP primers aiming at SNP molecular markers AX-111729522 and AX-110040743, the 5' ends of the forward primer 1 and the forward primer 2 are connected with different fluorescent tag sequences.

2. The KASP primer of claim 1, wherein the forward primer 1 is linked at its 5' end to an F probe, the nucleotide sequence of which is shown in SEQ ID No. 9; the 5' end of the forward primer 2 is connected with an H probe, and the nucleotide sequence of the H probe is shown in SEQ ID NO. 10.

3. A method for identifying a wheat greenness-extending trait, the method comprising:

the KASP primer of claim 1 or 2 for use in fluorescent quantitative PCR amplification, and genotyping wheat material to be detected based on the PCR amplification result, wherein the primer comprises:

if the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-111729522, but does not contain a fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-111729522, contains a fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-110040743, but does not contain a fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-110040743, the genotype of the wheat material to be detected is marked as the B genotype;

if the wheat material to be detected has a fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-110040743, does not contain the fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-110040743, contains the fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-111729522 and does not contain the fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-111729522, the genotype of the wheat material to be detected is marked as the C genotype;

if the wheat material to be detected simultaneously contains the fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-111729522, but does not contain the fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-111729522, and contains the fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-110040743, but does not contain the fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-110040743, the genotype of the wheat material to be detected is marked as the D genotype;

if the wheat material to be detected contains the fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-111729522, but does not contain the fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-111729522, and contains the fluorescent signal of the fluorescent probe of the forward primer 1 of the KASP primer aiming at the SNP molecular marker AX-110040743 but does not contain the fluorescent signal of the fluorescent probe of the forward primer 2 of the KASP primer aiming at the SNP molecular marker AX-110040743, the genotype of the wheat material to be detected is marked as the genotype A;

the green extending character of the D genotype wheat strain is higher than that of the A genotype wheat strain.

4. The method of claim 3, wherein the reaction system for the fluorescent quantitative PCR amplification comprises: KASP Mastermix, KASP primer as claimed in claim 1 or 2, genomic DNA of wheat material to be tested, RNase-free deionized water; wherein, in the KASP primer, the volume ratio of the forward primer 1 to the forward primer 2 to the reverse primer is 2:2: 5.

5. The method of claim 3, wherein the reaction sequence of the fluorescent quantitative PCR amplification comprises: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 20 s; annealing and extending at 61 ℃ for 40s, circulating for 10 times, and reducing the annealing and extending temperature by 0.6 ℃ in each circulation; denaturation at 95 ℃ for 20 s; annealing and extending for 40s at 55 ℃; circulating for 30 times; keeping the temperature at 25 ℃ and collecting fluorescence signals.

6. The use of KASP primers designed for the SNP molecular markers AX-111729522 and AX-110040743 according to claim 1, comprising any one of:

(1) the application of the wheat greening character detection reagent in detecting the wheat greening character;

(2) the application in breeding and creating wheat resources with different green-extending characters;

(3) the application of the gene in the research of the genetic mechanism and the physiological mechanism of the anti-aging physiological phenomenon of wheat after flowering;

wherein the nucleotide sequence of AX-111729522 is shown in SEQ ID NO. 1; the nucleotide sequence of AX-110040743 is shown in SEQ ID NO. 2.