CN114507751B

CN114507751B - Molecular marker related to superoxide dismutase activity of wheat seeds and application thereof

Info

Publication number: CN114507751B
Application number: CN202210257614.2A
Authority: CN
Inventors: 耿洪伟; 任毅; 程宇坤; 孙玲
Original assignee: Xinjiang Agricultural University
Current assignee: Xinjiang Agricultural University
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2023-07-21
Anticipated expiration: 2042-03-16
Also published as: CN114507751A

Abstract

The invention discloses a molecular marker related to the activity of superoxide dismutase of wheat grains and application thereof. The invention designs specific primers for amplifying wheat genome DNA fragments containing the molecular markers based on two allelic variation forms of TaSOD-A1a and TaSOD-A1b genes of the TaSOD-A1 gene and provides a method for identifying the activity of superoxide dismutase of wheat seeds by utilizing the molecular markers and the primers. The molecular marker can rapidly identify the allelic variation of TaSOD-A1a and TaSOD-A1b and the SOD activity thereof, rapidly screen out wheat varieties with higher SOD activity, accelerate the growth pace of new varieties of high-quality wheat, and have important theoretical significance and economic value for auxiliary selection of high-SOD activity wheat varieties by the molecular marker.

Description

Molecular marker related to superoxide dismutase activity of wheat seeds and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a molecular marker related to the activity of superoxide dismutase of wheat seeds and application thereof.

Background

Superoxide dismutase (Superoxide dismutase, SOD) is widely distributed in microorganisms, plants and animals, and can remove superoxide anion free radicals in organisms, and the activity of superoxide dismutase has important influence on the nutritional quality and processing quality of the flour products. Superoxide dismutase has now become the component of greatest interest to researchers in addition to proteins, and thus has also gradually become a research hotspot in wheat and human nutrition. The improvement of the nutrition and processing quality of wheat in China through genetic approaches is feasible, and the breakthrough achievement in breeding mainly depends on the development and utilization of key genes. The research shows that SOD can oxidize conjugated double bonds of carotenoid and other pigment molecules in flour, thereby bleaching the color of flour products; SOD can degrade dietary fiber components in flour and is converted into components with prebiotic effect to improve the nutritional quality of flour products; SOD also improves the rheological properties of the dough by affecting intramolecular or intermolecular sulfhydryl and disulfide bonds in the dough, allowing it to produce more disulfide bonds, stabilizing the conformation of the protein. The SOD activity is a complex quantitative character controlled by multiple genes, is greatly influenced by environment, has great difficulty in directly selecting the SOD activity, has small effect of a single gene, and is an important means for improving the SOD activity by polymerizing the gene related to the SOD activity through molecular markers. Functional markers are novel molecular markers developed according to functional gene internal sequence polymorphism sequences, and theoretically, the types of target alleles can be determined under different genetic backgrounds without further verification of the functional motifs in genes, but functional markers related to SOD are not available at present. The method has the advantages that the related gene of the SOD activity of the wheat is excavated, the molecular marker closely linked with the gene is developed, an important theoretical basis is provided for the research of molecular mechanism related to the polymerization breeding and quality character of the gene, and important theoretical and practical significance is provided for the cultivation of new varieties of the wheat with high SOD activity.

The SOD activity of wheat kernel is affected by both genotype and environment, but is mainly determined by genetic factors. At present, the excavation of major sites related to the SOD activity of wheat is less. Wu, baek and the like use the fountains and double end bodies of China as materials, a wheat grain Mn-SOD active gene is positioned on a wheat 2 nd homologous group, a wheat grain Cu/Zn-SOD active gene is positioned on a chromosome long arm of a7 th homologous group, and a QTL site influencing SOD activity in wheat seeds exists on a 2D chromosome long arm. According to the gene colinear principle of gramineous crops, the positioning result of SOD genes in rice is combined to infer that the wheat SOD genes are positioned in the 7 th homologous chromosome group; through association and linkage analysis, an important site for controlling the SOD activity of the wheat grain is located on a 5A chromosome. Kumar et al cloned the Mn-SOD gene on the wheat 6D chromosome; li Juan and the like obtain the cDNA sequence of the Fe-SOD gene by using wheat tobacco grower 19 as a material; wang Peng et al used PCR technology with wheat BS366 as the material and successfully obtained its Cu/Zn-SOD sequence. In order to more accurately identify genotypes of SOD activities of different varieties of wheat, it is necessary to perform gene cloning on major sites of the wheat, develop a pair of dominant complementary functional markers according to sequences of different alleles, further improve efficiency of molecular marker assisted selection breeding, provide feasible molecular markers for improving the SOD activities of the wheat, and finally lay a certain theoretical foundation for molecular marker assisted breeding and cultivation of new varieties and map cloning.

Disclosure of Invention

The invention aims to solve the technical problems of identifying or assisting in identifying whether wheat is high SOD activity wheat or low SOD activity wheat and/or detecting or assisting in detecting whether wheat carries TaSOD-A1a gene or TaSOD-A1b gene. The technical problems to be solved are not limited to the technical subject matter as described, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

In order to solve the technical problems, the invention firstly provides a molecular marker related to the activity of wheat grain superoxide dismutase (Superoxide dismutase, SOD) and/or any one of the following applications of detecting the molecular marker substances:

a1 Use in the identification of wheat grain superoxide dismutase activity;

a2 The application of the method in identifying or assisting in identifying whether the wheat to be detected is high SOD activity wheat or low SOD activity wheat;

a3 The application of the method in the identification or auxiliary identification of low SOD activity wheat varieties;

a4 The application of the method in identifying or assisting in identifying the wheat variety with high SOD activity;

a5 The application of the method in the identification or auxiliary identification of the SOD activity in the wheat grains;

a6 The application of the kit in detecting or assisting in detecting whether the wheat carries the TaSOD-A1a gene or the TaSOD-A1b gene;

a7 The application of the gene in identifying or assisting in identifying the wheat variety carrying the TaSOD-A1a gene;

a8 The application of the gene in identifying or assisting in identifying the wheat variety carrying the TaSOD-A1b gene;

a9 Use in analysis and identification of wheat germplasm resources;

a10 Application in wheat molecular assisted genetic breeding;

the molecular marker may be any of the following:

b1 Molecular marker 1, wherein the molecular marker 1 is a DNA molecule (with the length of 1419 bp) with the nucleotide sequence shown in 140 th-1558 th positions of SEQ ID No. 5;

b2 Molecular marker 2, wherein the molecular marker 2 is a DNA molecule (with the length of 2567 bp) with the nucleotide sequence shown in 127 th-2693 th positions of SEQ ID No. 6;

b3 A molecular marker composition consisting of the molecular marker 1 and the molecular marker 2.

Further, the identification of the superoxide dismutase activity of the wheat seeds can be the identification of the level of the superoxide dismutase activity of the wheat seeds, namely, the SOD activity is low when the SOD activity is less than or equal to 1728.05U/g; when the SOD activity is equal to or higher than 1814.19U/g, the SOD activity is high.

The nucleotide sequence of the TaSOD-A1a gene is shown as SEQ ID No.5, and the 64-177, 348-499, 1179-1243, 1452-1511, 1604-1741, 1819-1943, 2003-2287, 2425-2558, 2675-2707 and 2805-2967 of the SEQ ID No.5 are exon sequences, and 178-347, 500-1178, 1244-1451, 1512-1603, 1742-1818, 1944-2002, 2288-2424, 2559-2674 and 2708-2804 are intron sequences.

The nucleotide sequence of the TaSOD-A1b gene is shown as SEQ ID No.6, and the 64-177, 348-499, 1179-1243, 1452-1511, 1604-1741, 1819-1943, 2003-2287, 2425-2558, 2675-2707 and 2805-2967 of the SEQ ID No.6 are exon sequences, and 178-347, 500-1178, 1244-1451, 1512-1603, 1742-1818, 1944-2002, 2288-2424, 2559-2674 and 2708-2804 are intron sequences.

The TaSOD-A1a gene and the TaSOD-A1b gene are two allelic variant forms of the TaSOD-A1 gene in wheat.

A1419 bp DNA molecule amplified from an upstream primer 1F having the nucleotide sequence of SEQ ID No.1 and a downstream primer 1R having the nucleotide sequence of SEQ ID No.2 (i.e., positions 140-1558 of SEQ ID No. 5) was designated as molecular marker 1.

A2567 bp DNA molecule amplified from an upstream primer 2F having the nucleotide sequence of SEQ ID No.3 and a downstream primer 2R having the nucleotide sequence of SEQ ID No.4 (i.e., positions 127-2693 of SEQ ID No. 6) was designated as molecular marker 2.

In the above application, the substance contains PCR primers for amplifying the wheat genomic DNA fragment containing the molecular marker.

In the above application, the PCR primer may be any of the following:

c1 A primer pair 1, wherein the primer pair 1 consists of a forward primer F1 and a reverse primer R1, the forward primer F1 is single-stranded DNA with a nucleotide sequence of SEQ ID No.1, and the reverse primer R1 is single-stranded DNA with a nucleotide sequence of SEQ ID No. 2;

c2 A primer pair 2, wherein the primer pair 2 consists of a forward primer F2 and a reverse primer R2, the forward primer F2 is single-stranded DNA with a nucleotide sequence of SEQ ID No.3, and the reverse primer R2 is single-stranded DNA with a nucleotide sequence of SEQ ID No. 4;

c3 A primer pair composition consisting of the primer pair 1 of C1) and the primer pair 2 of C2).

The molecular markers, and/or the PCR primers are also within the scope of the present invention.

The invention also provides a reagent or a kit containing the PCR primer.

Further, the reagent may be a PCR reagent for identifying the superoxide dismutase activity of wheat kernel.

The PCR reagent for identifying the activity of the superoxide dismutase of the wheat seeds can be specifically any one of the following:

(1) A kit of reagents consisting of PCR reagent 1 and PCR reagent 2;

(2) PCR reagent 1 or PCR reagent 2;

the PCR reagent 1 comprises the primer pair 1; the PCR reagent 2 comprises the primer pair 2.

In the PCR reagent, each primer pair in the kit can be independently packaged, and each primer can be independently packaged.

Further, the kit also comprises Taq DNA polymerase, dNTP, PCR buffer solution and Mg required by PCR amplification ²⁺ One or more of the following.

The various reagent components of the kit may be present in separate containers or may be pre-combined in whole or in part into a reagent mixture.

The invention also provides any one of the following methods:

d1 A method for identifying or assisting in identifying whether wheat is high SOD activity wheat or low SOD activity wheat;

d2 A method for identifying or assisting in identifying the SOD activity in wheat grains;

d3 A method for detecting or assisting in detecting whether wheat carries a TaSOD-A1a gene or a TaSOD-A1b gene;

characterized in that the method comprises the steps of:

e1 Using the wheat genome DNA to be identified as a template, and carrying out PCR amplification by using the primer pair 1, the primer pair 2 or the primer pair composition to obtain a PCR product;

e2 Whether the wheat is high SOD activity wheat or low SOD activity wheat or whether the wheat has SOD activity in wheat grains or whether the wheat carries TaSOD-A1a gene or TaSOD-A1b gene is identified according to the PCR product.

In the above method, the annealing temperature of the PCR amplification is 63.7℃for the primer pair 1 and 61℃for the primer pair 2.

Further, the reaction system of PCR amplification is as follows: about 80ng,Taq DNA Polymerase (TIANGEN, code: ET 101) of the template DNA was 0.25. Mu.l, 2.5. Mu.l (TIANGEN, code: ET 101) of 10 XTaq Buffer I, 1. Mu.l of dNTP mix (TIANGEN, code: CD111,2.5mM each), 1. Mu.l of each of the upstream primer and the downstream primer, and the reaction system was replenished with sterile ultrapure water to 25. Mu.l.

Further, the reaction procedure for PCR amplification using the primer set 1 is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 63.7℃for 30s, extension at 72℃for 1.5min for 35 cycles; finally, the mixture is extended for 8min at 72 ℃; preserving heat at 12 ℃. And sequencing the obtained PCR amplification products respectively.

Further, the reaction procedure for PCR amplification using the primer set 2 is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 61℃for 30s, extension at 72℃for 2.5min for 35 cycles; finally, the mixture is extended for 8min at 72 ℃; preserving heat at 12 ℃. And sequencing the obtained PCR amplification products respectively.

In the above method, the E2) may be F1), F2) or F3):

f1 The identification of whether the wheat is high SOD activity wheat or low SOD activity wheat according to the PCR product is:

when the primer pair 1 is used for PCR amplification, if the PCR product contains the molecular marker 1, the wheat to be identified is or is candidate to be high SOD activity wheat; if the PCR product does not contain the molecular marker 1, the wheat to be identified is or is candidate to be low SOD activity wheat;

when the primer pair 2 is utilized for PCR amplification, if the PCR product contains the molecular marker 2, the wheat to be identified is or is candidate to be low SOD activity wheat; if the PCR product does not contain the molecular marker 2, the wheat to be identified is or is candidate to be high SOD activity wheat;

when the primer pair composition (primer pair 1 and primer pair 2) is utilized for PCR amplification, if the PCR product contains the molecular marker 2 and does not contain the molecular marker 1, the wheat to be identified is or is candidate to be low SOD activity wheat; if the PCR product contains the molecular marker 1 and does not contain the molecular marker 2, the wheat to be identified is or is candidate to be high SOD activity wheat;

f2 The SOD activity in the wheat grains is identified according to the PCR products:

when the primer pair 1 is used for PCR amplification, the SOD activity in the wheat grain to be identified containing the molecular marker 1 in the PCR product is higher than the SOD activity in the wheat grain to be identified containing the molecular marker 1 in the PCR product;

when the primer pair 2 is used for PCR amplification, the SOD activity in the wheat grain to be identified, which contains the molecular marker 2, of the PCR product is lower than the SOD activity in the wheat grain to be identified, which does not contain the molecular marker 2 of the PCR product;

when the primer pair composition (primer pair 1 and primer pair 2) is used for PCR amplification, the SOD activity in the wheat grain to be identified, which contains the molecular marker 1 and does not contain the molecular marker 2, is higher than the SOD activity in the wheat grain to be identified, which contains the molecular marker 2 and does not contain the molecular marker 1, by the PCR product;

f3 The detection of whether the wheat carries the TaSOD-A1a gene or the TaSOD-A1b gene according to the PCR product is as follows:

when the primer pair 1 is used for PCR amplification, if the PCR product contains the molecular marker 1, the wheat to be identified carries a TaSOD-A1a gene; if the PCR product does not contain the molecular marker 1, the wheat to be identified carries a TaSOD-A1b gene;

when the primer pair 2 is used for PCR amplification, if the PCR product contains the molecular marker 2, the wheat to be identified carries a TaSOD-A1b gene; if the PCR product does not contain the molecular marker 2, the wheat to be identified carries a TaSOD-A1a gene;

when the primer pair composition (primer pair 1 and primer pair 2) is utilized for PCR amplification, if the PCR product contains the molecular marker 2 and does not contain the molecular marker 1, the wheat to be identified carries a TaSOD-A1b gene; if the PCR product contains the molecular marker 1 and does not contain the molecular marker 2, the wheat to be identified carries the TaSOD-A1a gene.

Further, the identification of whether the wheat is high or low SOD activity based on the PCR product is:

when the primer pair 1 is used for PCR amplification, if the PCR amplification product contains a fragment with the size of 1419bp, the wheat to be detected is or is candidate to be high SOD activity wheat; if the PCR amplification product does not contain a fragment with the size of 1419bp, the wheat to be detected is or is candidate to be low SOD activity wheat;

when the primer pair 2 is used for PCR amplification, if the PCR amplification product contains a fragment with the size of 2567bp, the wheat to be detected is or is candidate to be low SOD activity wheat; if the PCR amplification product does not contain a fragment with the size of 2567bp, the wheat to be detected is or is candidate to be high SOD activity wheat;

when the primer pair composition (the primer pair 1 and the primer pair 2) is used for PCR amplification, if the PCR amplified product of the primer pair 1 contains a fragment with the size of 1419bp and the PCR amplified product of the primer pair 2 does not contain a fragment with the size of 2567bp, the wheat to be detected is or is candidate to be high SOD activity wheat; if the PCR amplification product of the primer pair 2 contains a fragment with the size of 2567bp and the PCR amplification product of the primer pair 1 does not contain a fragment with the size of 1419bp, the wheat to be detected is or is candidate to be low SOD activity wheat;

further, the identification of the SOD activity in the wheat grain according to the PCR product is as follows:

when the primer pair 1 is used for PCR amplification, the SOD activity in the wheat grain to be identified, of which the PCR amplification product contains a fragment with the size of 1419bp, is higher than the SOD activity in the wheat grain to be identified, of which the PCR product does not contain a fragment with the size of 1419 bp;

when the primer pair 2 is used for PCR amplification, the SOD activity in the wheat grain to be identified, of which the PCR amplification product contains a fragment with the size of 2567bp, is lower than that in the wheat grain to be identified, of which the PCR product does not contain a fragment with the size of 2567 bp;

when the primer pair composition (primer pair 1 and primer pair 2) is used for PCR amplification, the PCR amplification product of the primer pair 2 does not contain a fragment with a size of 2567bp, and the SOD activity in wheat grains to be identified, of which the PCR amplification product of the primer pair 1 contains a fragment with a size of 1419bp, is higher than the SOD activity in wheat grains to be identified, of which the PCR amplification product of the primer pair 1 does not contain a fragment with a size of 1419bp, and the PCR amplification product of the primer pair 2 contains a fragment with a size of 2567 bp;

further, the detection of whether the wheat carries the TaSOD-A1a gene or the TaSOD-A1b gene according to the PCR product is as follows:

when the primer pair 1 is used for PCR amplification, if the PCR amplification product contains a fragment with the size of 1419bp, the wheat to be identified carries a TaSOD-A1a gene; if the PCR amplification product does not contain a fragment with the size of 1419bp, the wheat to be identified carries a TaSOD-A1b gene;

when the primer pair 2 is used for PCR amplification, if the PCR amplification product contains a fragment with the size of 2567bp, the wheat to be identified carries a TaSOD-A1b gene; if the PCR amplification product does not contain a fragment with the size of 2567bp, the wheat to be identified carries a TaSOD-A1a gene;

when the primer pair composition (primer pair 1 and primer pair 2) is used for PCR amplification, if the PCR amplification product of the primer pair 1 contains a fragment with the size of 1419bp and the PCR amplification product of the primer pair 2 does not contain a fragment with the size of 2567bp, the wheat to be identified carries the TaSOD-A1a gene; if the PCR amplification product of the primer pair 2 contains a fragment with the size of 2567bp and the PCR amplification product of the primer pair 1 does not contain a fragment with the size of 1419bp, the wheat to be identified carries the TaSOD-A1b gene.

The invention also provides a method for cultivating the wheat variety with high SOD activity, which can be any one of the following steps:

(1) The method comprises the following steps: breeding by taking wheat D1 as a parent; the wheat D1 satisfies the following conditions: performing PCR amplification on the genomic DNA of the wheat D1 by using the primer pair 1 and the primer pair 2 respectively, wherein a PCR amplification product of the primer pair 1 contains a fragment with the size of 1419bp, and a PCR amplification product of the primer pair 2 does not contain a fragment with the size of 2567 bp;

(2) The method comprises the following steps: breeding by taking wheat D2 as a parent; the wheat D2 satisfies the following conditions: performing PCR amplification on the genomic DNA of the wheat D2 by using the primer pair 1, wherein a PCR amplification product contains a fragment with the size of 1419 bp;

(3) The method comprises the following steps: breeding by taking wheat D3 as a parent; the wheat D3 meets the following conditions: performing PCR amplification on the genomic DNA of the wheat D3 by using the primer pair 2, wherein a PCR amplification product does not contain a fragment with the size of 2567 bp;

(4) The method comprises the following steps: breeding by taking wheat D4 as a parent; the wheat D4 satisfies the following conditions: the genomic DNA of wheat D4 contains the molecular marker 1 and does not contain the molecular marker 2;

(5) The method comprises the following steps: breeding by taking wheat D5 as a parent; the wheat D5 satisfies the following conditions: the genomic DNA of wheat D5 does not contain the molecular marker 2;

(6) The method comprises the following steps: breeding by taking wheat D6 as a parent; the wheat D6 satisfies the following conditions: the genomic DNA of wheat D6 contains the molecular marker 1;

the SOD activity in the wheat variety with high SOD activity is larger than or equal to 1814.19U/g.

The invention also provides a method for cultivating the wheat with low SOD activity, which can be any one of the following steps:

(1) The method comprises the following steps: breeding by taking wheat E1 as a parent; the wheat E1 satisfies the following conditions: performing PCR amplification on the genomic DNA of the wheat E1 by using the primer pair 1 and the primer pair 2 respectively, wherein a PCR amplification product of the primer pair 1 does not contain a fragment with the size of 1419bp, and a PCR amplification product of the primer pair 2 contains a fragment with the size of 2567 bp;

(2) The method comprises the following steps: breeding by taking wheat E2 as a parent; the wheat E2 satisfies the following conditions: performing PCR amplification on the genomic DNA of the wheat E2 by using the primer pair 1, wherein a PCR amplification product does not contain a fragment with the size of 1419 bp;

(3) The method comprises the following steps: breeding by taking wheat E3 as a parent; the wheat E3 satisfies the following conditions: performing PCR amplification on the genomic DNA of the wheat E3 by using the primer pair 2, wherein a PCR amplification product contains a fragment with the size of 2567 bp;

(4) The method comprises the following steps: breeding by taking wheat E4 as a parent; the wheat E4 satisfies the following conditions: the genomic DNA of wheat E4 contains the molecular marker 2 and does not contain the molecular marker 1;

(5) The method comprises the following steps: breeding by taking wheat E5 as a parent; the wheat E5 satisfies the following conditions: the genomic DNA of wheat E5 contains the molecular marker 2;

(6) The method comprises the following steps: breeding by taking wheat E6 as a parent; the wheat E6 satisfies the following conditions: the genomic DNA of wheat E6 does not contain the molecular marker 1;

the SOD activity in the low SOD activity wheat is less than or equal to 1728.05U/g.

Further, in the method, the method for detecting whether the genomic DNA of the wheat to be identified contains the molecular marker 1 may specifically be as follows: taking the genomic DNA of the wheat to be identified as a template, and carrying out PCR amplification by using the primer pair 1 to obtain a PCR amplification product; if the PCR amplification product contains a fragment with the size of 1419bp, the genomic DNA of the wheat to be identified contains the molecular marker 1; if the PCR amplification product does not contain a fragment with the size of 1419bp, the genomic DNA of the wheat to be identified does not contain the molecular marker 1.

Further, in the method, the method for detecting whether the genomic DNA of the wheat to be identified contains the molecular marker 2 may specifically be as follows: taking the genomic DNA of the wheat to be identified as a template, and carrying out PCR amplification by using the primer pair 2 to obtain a PCR amplification product; if the PCR amplification product contains a fragment with the size of 2567bp, the genomic DNA of the wheat to be identified contains the molecular marker 2; if the PCR amplification product does not contain a 2567bp fragment, the genomic DNA of the wheat to be identified does not contain the molecular marker 2.

Specifically, in the invention, the fragment with the size of 1419bp is specifically 140-1558 of SEQ ID No. 5; the 2567bp fragment is specifically the 127 th-2693 th bit of SEQ ID No. 6.

The invention also provides application of the molecular marker and/or the PCR primer and/or the reagent or the kit in cultivating wheat varieties with high SOD activity and/or wheat varieties with low SOD activity;

the SOD activity in the low SOD activity wheat variety is less than or equal to 1728.05U/g;

The invention also provides application of the reagent or the kit in identifying the superoxide dismutase activity of wheat grains, and/or in analyzing and identifying wheat germplasm resources, and/or in wheat molecular assisted genetic breeding.

PCR primers for amplifying the molecular markers are also within the scope of the present invention.

The wheat to be identified herein includes wheat germplasm resources, genetic populations, or breeding materials.

The molecular assisted genetic breeding described herein can be the selection of wheat varieties with high SOD activity and/or the selection of wheat varieties with low SOD activity.

The SOD activity in the low SOD activity wheat grain is less than or equal to 1728.05U/g;

the SOD activity in the wheat grain with high SOD activity is equal to or more than 1814.19U/g.

SOD activity as described herein all refer to superoxide dismutase activity.

In the invention, the SOD activity of wheat is reflected by detecting the SOD activity of wheat grains.

In the above, the wheat to be tested may be specifically the wheat in table 2.

Experiments prove that: the molecular marker can rapidly identify the allelic variation of TaSOD-A1a and TaSOD-A1b and the SOD activity thereof, and the molecular marker can rapidly screen out wheat varieties (materials) with higher SOD activity, thereby accelerating the growth pace of high-quality wheat new varieties and having important theoretical significance and economic value for auxiliary selection of the high-SOD activity wheat varieties by using the molecular marker.

Drawings

FIG. 1 is an electrophoretogram of a portion of a sample. Lanes 1-10 are shown in Table 2 as wheat varieties of Ligusticum sinense 8901, shaanxi 715, zhou 8425B, linmai No.2, zhong892, chinese spring, zhongmai 871, yumai No. 7, zhou Mai, and Lancole No.2, respectively. M is a Trans 5K DNA Marker.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

And adopting SPSS 25.0 software to carry out basic data statistical analysis and independent sample T test on the SOD activity of the wheat grains. And judging whether the variances of the two groups of data are equal according to the level test of the variance equation, and then looking at the corresponding T value and significance level according to whether the variances are equal. If P is more than 0.05, the mean value of the two groups of data is proved to have no significant difference and no statistical significance; if P <0.05, mean differences between the two sets of data are statistically significant.

Example 1 methods for identifying the Activity of wheat seed superoxide dismutase (Superoxide dismutase, SOD)

1. Molecular marker for identifying superoxide dismutase activity of wheat grains

1. Two allelic variants of the TaSOD-A1 gene exist in wheat: one shown in SEQ ID No.5, designated as allele TaSOD-A1a; the SEQ ID No.5 shows the exon sequences from the 5' -end 64-177, 348-499, 1179-1243, 1452-1511, 1604-1741, 1819-1943, 2003-2287, 2425-2558, 2675-2707, 2805-2967, and the intron sequences 178-347, 500-1178, 1244-1451, 1512-1603, 1742-1818, 1944-2002, 2288-2424, 2559-2674, 2708-2804.

The other is shown as SEQ ID No.6 and named as allele TaSOD-A1b; the SEQ ID No.6 shows that the SEQ ID Nos. 64-177, 348-499, 1179-1243, 1452-1511, 1604-1741, 1819-1943, 2003-2287, 2425-2558, 2675-2707 and 2805-2967 are exon sequences, and 178-347, 500-1178, 1244-1451, 1512-1603, 1742-1818, 1944-2002, 2288-2424, 2559-2674 and 2708-2804 are intron sequences.

2. Specific primer pair 1 and primer pair 2 were designed based on two allelic variants of the TaSOD-A1 gene as follows:

upstream primer 1F of primer pair 1 (SEQ ID No. 1): 5'-GCAAACAGCTTATGCTTTCC-3';

downstream primer 1R of primer pair 1 (SEQ ID No. 2): 5'-GCTGTAAACAAGTGCAACGC-3';

upstream primer 2F (SEQ ID No. 3) of primer pair 2: 5'-GGATCTGAAGATGGCAAACCGT-3';

downstream primer 2R of primer pair 2 (SEQ ID No. 4): 5'-GCTGCCAACTTCAACAGTCC-3'.

Theoretically, the primer pair 1 uses the allele TaSOD-A1a shown in 140-1558 of SEQ ID No.5 as a template, can amplify to obtain a target fragment with 1419bp (namely 140-1558 of SEQ ID No. 5), and uses the allele TaSOD-A1b shown in 140-1558 of SEQ ID No.6 as a template, cannot amplify to obtain the target fragment with 1419 bp.

Similarly, the primer pair 2 uses the allele TaSOD-A1b shown in the 127 th-2693 th positions of SEQ ID No.6 as a template, can amplify to obtain a target fragment with the size of 2567bp (namely the 127 th-2693 th positions of SEQ ID No. 6), and uses the allele TaBahd-A1a shown in the 127 th-2693 th positions of SEQ ID No.5 as the template, cannot amplify to obtain the target fragment with the size of 2567 bp.

2. Method for identifying superoxide dismutase activity of wheat seeds

1. Extracting genome DNA of wheat grains to be detected.

2. And (3) taking the obtained genome DNA as a template, and respectively adopting the primer pair 1 and/or the primer pair 2 obtained in the step (I) to carry out PCR amplification to obtain PCR amplification products.

The PCR amplification reaction system is as follows: about 80ng,Taq DNA Polymerase (TIANGEN, code: ET 101) of the template DNA was 0.25. Mu.L, 2.5. Mu.L (TIANGEN, code: ET 101) of 10 XTaq Buffer I, 1. Mu.L of dNTP mix (TIANGEN, code: CD111,2.5mM each), 1. Mu.L of each of the upstream primer and the downstream primer, and the reaction system was replenished with sterile ultrapure water to 25. Mu.L.

The reaction procedure for PCR amplification of primer pair 1 was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 63.7℃for 30s, extension at 72℃for 1.5min for 35 cycles; finally, the mixture is extended for 8min at 72 ℃; preserving heat at 12 ℃. And sequencing the obtained PCR amplification products respectively.

The reaction procedure for PCR amplification of primer pair 2 was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 61℃for 30s, extension at 72℃for 2.5min for 35 cycles; finally, the mixture is extended for 8min at 72 ℃; preserving heat at 12 ℃. And sequencing the obtained PCR amplification products respectively.

The amplified products were electrophoresed through a 1.0% agarose gel, 1×TAE electrophoresis buffer system, and then observed under a gel imager. Recovering amplified target bands, connecting by using a pEasy-T5 Zero Cloning Kit, transforming Trans-T1 escherichia coli competent cells, screening positive monoclonal and delivering and measuring, and repeating 3 times of each sample to ensure the accuracy of sequences, thereby confirming the size and the sequence of PCR amplified products.

3. Definition of wheat grain superoxide dismutase (SOD) activity:

when the SOD activity is less than or equal to 1728.05U/g of the grain, the wheat grain is a low SOD activity wheat;

when the SOD activity is larger than or equal to 1814.19U/g grain, the wheat grain is a high SOD activity wheat.

(1) If the PCR amplification product of the primer pair 1 contains a fragment with the size of 1419bp and/or the PCR amplification product of the primer pair 2 does not contain a fragment with the size of 2567bp, the wheat to be detected is wheat with high SOD activity;

if the PCR amplified product of the primer pair 2 contains a fragment with the size of 2567bp and/or the PCR amplified product of the primer pair 1 does not contain a fragment with the size of 1419bp, the wheat to be detected is wheat with low SOD activity.

(2) If the PCR amplification product of the primer pair 1 contains a fragment with the size of 1419bp and/or the PCR amplification product of the primer pair 2 does not contain a fragment with the size of 2567bp, the wheat to be detected carries an allele TaSOD-A1a;

if the PCR amplification product of the primer pair 2 contains a fragment with the size of 2567bp and/or the PCR amplification product of the primer pair 1 does not contain a fragment with the size of 1419bp, the wheat to be detected carries an allele TaSOD-A1b.

Wherein the nucleotide sequence of the fragment with the size of 1419bp is 140 th-1558 th site of SEQ ID No. 5; the nucleotide sequence of the 2567bp fragment is 127 th-2693 th bit of SEQ ID No. 6.

Example 2 verification of molecular markers

Experimental materials: 128 Chinese winter wheat main variety (Table 2) can be obtained from the national germplasm resource library of the national academy of agricultural sciences.

128 Chinese winter wheat master varieties were verified by the method for identifying the superoxide dismutase activity of wheat seeds described in example 1. Meanwhile, planting each wheat variety in a Malus laboratory station of Xinjiang agricultural academy of sciences, detecting the SOD activity of wheat seeds after harvesting, repeatedly detecting the SOD activity of each wheat material twice, and if the error of the two detection results exceeds 10%, repeatedly detecting for the third time, and taking the average value of the results.

The method for measuring SOD activity of wheat grains is as follows:

(1) Reagent(s)

0.05mol/L Phosphate Buffer (PBS) with pH=7.8, 130mmol/L methionine (Met) solution, 750. Mu. Mol/LNBT solution, 20. Mu. Mol/L riboflavin solution, 100. Mu. Mol/LEDTA-Na ₂ Solution, SOD extraction medium.

(2) Extraction of SOD crude enzyme liquid

Weighing 0.5g of whole wheat flour (whole grain wheat is ground and crushed to obtain whole wheat flour), putting the whole wheat flour into a10 m L centrifuge tube, adding 5m L of pre-cooled SOD extraction medium (0.05 mol/L phosphate buffer solution containing 1% polyvinylpyrrolidone and having pH of 7.8) into the centrifuge tube, and putting the mixture on a mixer for oscillation to enable the buffer solution to fully contact with the whole wheat flour; placing the centrifuge tube with the sample on a shaking table in an ice bath manner, and shaking for 2 hours; then centrifuging at 4 ℃ and 10000rpm for 15min; and sucking the supernatant, immediately placing the supernatant into a refrigerator at the temperature of 4 ℃ to obtain SOD crude enzyme liquid, and analyzing the SOD crude enzyme liquid for later use.

(3) SOD activity determination

For each treatment, 5 washed and dried finger-shaped tubes (required to have good transparency) were used, the numbers were (1) to (5), and each reagent and enzyme solution (SOD crude enzyme solution) were added according to Table 1, and the total volume of the reaction system was 3ml. After mixing, the tube (5) was placed in the dark, and the other tubes were reacted at 25℃under 4500lx sunlight for 20min (the light receiving conditions of the tubes were required to be uniform, and the reaction time was appropriately adjusted depending on the reaction color and enzyme activity of the control tube under light). After the reaction, the reaction was stopped by masking with black cloth.

Table 1 amounts of reagents

The absorbance of the reaction solution of the (1) to (4) tubes was measured at 560nm with the (5) tube as a blank for zeroing, and the measurement data were recorded.

(4) Result calculation

SOD activity is known to be expressed as 50% of the unit of enzyme activity inhibiting the photochemical reduction of NBT, calculated as follows.

Wherein: SOD total activity is expressed in enzyme units per gram of fresh weight of sample (U/g)

Ao: absorbance of control tube under light

As: absorbance of sample measuring tube

V: total volume of sample extract (mL)

Vs: measurement of crude enzyme solution amount (mL)

W: fresh weight of sample (g)

The results of the SOD activity detection of each variety of wheat grain are shown in Table 2: PCR amplified products obtained by amplifying 128 Chinese winter wheat varieties (lines) are divided into two types according to the size: a fragment of 1419bp (corresponding primer pair 1), the nucleotide sequence of the fragment of 1419bp being positions 140-1558 of SEQ ID No. 5; the other is 2567bp (corresponding to primer pair 2), and the nucleotide sequence of the 2567bp fragment is 127-2693 of SEQ ID No. 6. The electrophoresis pattern of some samples is shown in FIG. 1 (wherein, lane 1-10 samples are shown in table wheat variety ligusticum 8901, shan 715, zhou 8425B, linmai No.2, zhong892, chinese spring, zhongmai 871, yumai No. 7, zhou Mai, lanco No.2, respectively). Of the 128 wheat varieties (lines): 86 varieties carry allele TaSOD-A1a, and the SOD activity average value is 1814.19U/g;42 varieties carry alleles TaSOD-A1b, and the SOD activity average value is 1728.05U/g; independent sample T-test with SPSS 25.0 software showed that the SOD activity of wheat varieties carrying allele TaSOD-A1a was higher than that of wheat varieties carrying allele TaSOD-A1b, with significant differences (P < 0.05).

The results show that: of 86 wheat varieties carrying the allele TaSOD-A1a, 47 are varieties with high SOD activity, namely the accuracy rate of the auxiliary identification of the wheat with high SOD activity is 55% by carrying the allele TaSOD-A1a; of the 42 wheat varieties carrying the allele TaSOD-A1b, 27 are low SOD active varieties, namely the accuracy of the auxiliary identification of the low SOD active wheat by carrying the allele TaSOD-A1b is 65%.

TABLE 2 PCR detection results and SOD Activity detection results for each wheat variety

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The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, it will be appreciated that the invention may 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.

SEQUENCE LISTING

<110> Xinjiang agricultural university

<120> a molecular marker related to superoxide dismutase activity of wheat grain and application thereof

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

gcaaacagct tatgctttcc 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

gctgtaaaca agtgcaacgc 20

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ggatctgaag atggcaaacc gt 22

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

gctgccaact tcaacagtcc 20

<210> 5

<211> 3121

<212> DNA

<213> wheat (Triticum aestivum)

<400> 5

ggtgttcatc cagtgggtct tgatcatcag gtggaattgc ttcccagtat gaaagtacta 60

gctgtcgcgc gtgccaattc tgacacatta atggccacaa gtgggatatt acctaatgat 120

tcaagtggat ctgaagatgg caaacagctt atgctttcca cttgtaaaat ctctgaggta 180

cactttcacc atggtgtgag tattttatct gcaactatat gcacttgaaa tatatagctt 240

tgaggcatat aaatcatgtt tttattatgt acctcttgtt ccatatcgtt cttttttatt 300

caatgtaacc attgttgcct ctttattttt atttttttta tttctaggct tgggaaggtg 360

ggccactttt tgattgtgat gggaaccttt ttggcatgaa cctttctttg gttgcggaaa 420

gaacttcctt cgtgccaagg agtacaatta tcaaatggtt ggagaaattt aagccaaaaa 480

gggaagttga gaggttcagg tatatcttgt gtgttttctt gtatttttgc ttatgtttgt 540

ccagcactaa ttccatttgt gctaggctat attgattacc aaacatttcg aggcatatta 600

attttgcaag aatctggatc gtgggcgtag ggtgtcattc cttttgtggt tttatgcatc 660

acacttgtta tttccttgca cttttcattg tttatttatc ccttcataac aacttgcttg 720

ttggctgcct ctaccaagaa tactcatcat tacttcaaag ctgaagttag tagctttttt 780

gttcctgctt tatatttatg tatggtgttc gtagtttctt cccacttccc ttttttatcc 840

tcaggtaaca cattaatgtt gaatgtactc cctccgttcc ataattcttg tggaactaaa 900

accacgacga gaattataga acggagggag tagtagattt ttctaatctc atcctgtttc 960

ggacttaaaa tgtggtaaat agttggattt atgatctttt cctgacccta taggttctgt 1020

atagtgcaat tgctcagttc tcctcacaac gtagtcctct ttcttctctt gcaattctgt 1080

taatgtagat gttgcgtagc gctgatttgg tctattttac ttctggttcg tactcatggg 1140

gatgtaaaat ttattccaag tttactatgt ctttccagtt cccacaagga tatgaaaata 1200

cagaggccag tgcagcccat gccatattgc acctatgcaa gaggtgagca cacagccaga 1260

tatattttgc tgttgtgcat tttctcatca tttctagtga tattgtttca taagatcctc 1320

acaagtgatg caaacttagc atttcacgta agctgtttag ccttttttat agttgtactc 1380

tcatggtgtc aatgctaatc agctaacctt attacactat cttcatgggt gttttcaact 1440

tggggacata gacagatctg gtgatctaga ctccttgggt tatccaaagc catcaaaaac 1500

cattgtcagt ggtgagttga tgcttataag tcggtactgc gttgcacttg tttacagctt 1560

tcctctacca ctgactacta ttgtgccatc taacattgaa cagagggctt ggttttggtt 1620

aataccttcg aagagacttt tggtgacata tatgattctg gtaaaggtat ctggagccaa 1680

ctcacagaaa tgggttctca tagtttacct cgacgagttg tcgcactcgc ttcattcagc 1740

ggtaatatct cagtaatgtg ccttttacta ttccacattt gaacttggct gtcaatcaaa 1800

actgcggtat atatacagga gaaaggaggt ttttcgcatg cacaggctta tttattgaat 1860

ggaatggatg cacccccatt ctgacttcag cgagtttggt tagtgatcct aaagatggaa 1920

acaagattgc tgaaaagttg aaggttggtt cttgatgtgg tccttacatt ttctgcttaa 1980

gtctcacaag tgccatttgc agattgaagt tttgcttcca aacaataaat gcaaagaagg 2040

ggaattgcga tattgtaatt tacactacaa tgttgctctt gtcgttgtca aggatttctg 2100

ctgtcttcgt ccagttagaa ttcatgaaga ttggtggaat cctctaattc tggaagattc 2160

caatgtagta gctgtggggc gctgcttcaa atctggcagg ttaatggctg cacatgggag 2220

actaactgac tggtcaggcg tgcttgacta cagagatctt aggtactcca gttgtaagat 2280

cactaaggtt agtactgctc gtggagccgt gggttaatct tatttgatgt atccgtcaca 2340

tgatgtacta gaccatatct ctttggcaaa acaaatggca acttctccat tcatattgct 2400

ttaatgacac cttttgtttg ctaggctggg attggtgggc ctctgattga ctttaaaggg 2460

agatttgttg gcatgaactt ctatgataag aaaataggaa ccccgttctt gttcagggat 2520

tgtattattc gagtgctggc acattttaag gaaaatgggt atgtacactg actttttgtt 2580

tggcatattc aactgcagga cttgaaattt tgtgttttat caaagagaaa aagaaaaact 2640

ttgcattaaa tctggcctac tgtgtttgac acaggactgt tgaagttggc agcggtaaac 2700

caatcaggtg tgcattatat ttactcatca ctttgatgtt ttgcataaac aaaatctgcc 2760

gataacgtta ctgtgtgtgg aaaatgattg gataatggaa ataggtggcc ggtgcccata 2820

ccgtgttggc gtcatccgga cgatgagctc aaagattcgc tcccacttgg ccacggggaa 2880

aaatctggga ggtatggatt cacatatgtg gatggagaca gggttgacaa ccatagatct 2940

cgtcatgccc ttgatctgct ccgttgaatt tgcgctctcc atgtgttgaa cctgtttgtt 3000

tgagctgtta gaagttatct ctcatcaagc catatctcaa tgtttggacc aatattatgg 3060

tgttccattt atgagagtga atatacttca tctagagctc tggtggtgtt tgctagtttc 3120

t 3121

<210> 6

<211> 3121

<212> DNA

<213> wheat (Triticum aestivum)

<400> 6

ggtgttcatc cagtgggtct tgatcatcag gtggaattgc ttcccagtat gaaagtacta 60

gctgtcgcgc gtgccaattc tgacacatta atggccacaa gtgggatatt acctaatgat 120

tcaagtggat ctgaagatgg caaacagttt atgctttcca cttgtaaaat ctctgaggta 180

cactttcacc atggtgtgag tattttatct gcaactatat gcacttgaaa tatatagctc 240

tgaggcatat aaatcatgtt tttattatgt acctcttgtt ccatatcgtt cttttttatt 300

caatgtaacc attgttgcct ctttattttt atttttttta tttctaggct tgggaaggtg 360

ggccactttt tgattgtgat gggaaccttt ttggcatgaa cctttctttg gttgcggaaa 420

gaacttcctt cgtgccaagg agtacaatta tcaaatggtt ggagaaattt aagccaaaaa 480

gggaagttga gaggttcagg tatatcttgt gtgttttctt gtatttttgc ttatgtttgt 540

ccagcactaa ttccatttgt gctaggctat attgattacc aaacatttcg aggcatatta 600

attttgcaag aatctggatc gtgggcgtag ggtgtcattc cttttgtggt tttatgcatc 660

acacttgtta tttccttgca cttttcattg tttatttatc ccttcataac aacttgcttg 720

ttggctgcct ctaccaagaa tactcatcat tacttcaaag ctgaagttag tagctttttt 780

gttcctgctt tatatttatg tatggtgttc gtagtttctt cccacttccc ttttttatcc 840

tcaggtaaca cattaatgtt gaatgtactc cctccgttcc ataattcttg tggaactaaa 900

accacgacga gaattataga acggagggag tagtagattt ttctaatctc atcctgtttc 960

ggacttaaaa tgtggtaaat agttggattt atgatctttt cctgacccta taggttctgt 1020

atagtgcaat tgctcagttc tcctcacaac gtagtcctct ttcttctctt gcaattctgt 1080

taatgtagat gttgcgtagc gctgatttgg tctattttac ttctggttcg tactcatggg 1140

gatgtaaaat ttattccaag tttactatgt ctttccagtt cccacaagga tatgaaaata 1200

cagaggccag tgcagcccat gccatattgc acctatgcaa gaggtgagca cacagccaga 1260

tatattttgc tgttgtgcat tttctcatca tttctagtga tattgtttca taagatcctc 1320

acaagtgatg caaacttagc atttcacgta agctgtttag ccttttttat agttgtactc 1380

tcatggtgtc aatgctaatc agctaacctt attacactat cttcatgggt gttttcaact 1440

tggggacata gacagatctg gtgatctaga ctccttgggt tatccaaagc catcaaaaac 1500

cattgtcagt ggtgagttga tgcttataag tcggtactgc gttgcacttg tttacagctt 1560

tcctctacca ctgactacta ttgtgccatc taacattgaa cagagggctt ggttttggtt 1620

aataccttcg aagagacttt tggtgacata tatgattctg gtaaaggtat ctggagccaa 1680

ctcacagaaa tgggttctca tagtttacct cgacgagttg tcgcactcgc ttcattcagc 1740

ggtaatatct cagtaatgtg ccttttacta ttccacattt gaacttggct gtcaatcaaa 1800

actgcggtat atatacagga gaaaggaggt ttttcgcatg cacaggctta tttattgaat 1860

ggaatggatg cacccccatt ctgacttcag cgagtttggt tagtgatcct aaagatggaa 1920

acaagattgc tgaaaagttg aaggttggtt cttgatgtgg cccttacatt ttctgcttaa 1980

gtctcacaag tgccatttgc agattgaagt tttgcttcca aacaataaat gcaaagaagg 2040

ggaattgcga tattgtaatt tacactacaa tgttgctctt gtcgttgtca aggatttctg 2100

ctgtcttcgt ccagttagaa ttcatgaaga ttggtggaat cctctaattc tggaagattc 2160

caatgtagta gctgtggggc gctgcttcaa atctggcagg ttaatggctg cacatgggag 2220

actaactgac tggtcaggcg tgcttgacta cagagatctt aggtactcca gttgtaagat 2280

cactaaggtt agtactgctc gtggagccgt gggttaatct tatttgatgt atccgtcaca 2340

tgatgtacta gaccatatct ctttggcaaa acaaatggca acttctccat tcatattgct 2400

ttaatgacac cttttgtttg ctaggctggg attggtgggc ctctgattga ctttaaaggg 2460

agatttgttg gcatgaactt ctatgataag aaaataggaa ccccgttctt gttcagggat 2520

tgtattattc gagtgctggc acattttaag gaaaatgggt atgtacactg actttttgtt 2580

tggcatattc aactgcagga cttgaaattt tgtgttttat caaagagaaa aagaaaaact 2640

ttgcattaaa tctggcctac tgtgtttgac acaggactgt tgaagttggc agcggtaaac 2700

caatcaggtg tgcattatat ttactcatca ctttgatgtt ttgcataaac aaaatctgcc 2760

gataacgtta ctgtgtgtgg aaaatgattg gataatggaa gtaggtggcc ggtgcccata 2820

ccgtgttggc gtcatccgga cgatgagctc aaagattcgc tcccacttgg ccacggggaa 2880

aaatctggga ggtatggatt cacatatgtg gatggagaca gggttgacaa ccatagatct 2940

cgtcatgccc ttgatctgct ccgttgaatt tgcgctctcc atgtgttgaa cctgtttgtt 3000

tgagctgtta gaagttatct ctcatcaagc catatctcaa tgtttggacc aatattatgg 3060

tgttccattt atgagagtga atatacttca tctagagctc tggtggtgtt tgctagtttc 3120

t 3121

Claims

1. The use of any of the following molecular markers:

a1 Use in the identification of wheat grain superoxide dismutase activity;

a3 In detecting or assisting in detecting that the wheat is portableTaSOD-A1aThe gene is alsoTaSOD-A1bApplication in genes;

the molecular marker is a molecular marker composition, the molecular marker composition consists of a molecular marker 1 and a molecular marker 2, and the molecular marker 1 is a DNA molecule with a nucleotide sequence shown in 140 th-1558 th positions of SEQ ID No. 5; the molecular marker 2 is a DNA molecule with a nucleotide sequence shown in 127 th-2693 th positions of SEQ ID No. 6;

when the SOD activity is less than or equal to 1728.05U/g, the SOD activity is low; when the SOD activity is higher than or equal to 1814.19U/g, the SOD activity is high; the saidTaSOD-A1aThe nucleotide sequence of the gene is shown as SEQ ID No.5, and theTaSOD-A1bThe nucleotide sequence of the gene is shown as SEQ ID No. 6.

2. Use of any one of the following for detecting a molecularly imprinted substance as defined in claim 1:

b1 Use in the identification of wheat grain superoxide dismutase activity;

b2 The application of the method in identifying or assisting in identifying whether the wheat to be detected is high SOD activity wheat or low SOD activity wheat;

b3 In detecting or assisting in detecting that the wheat is portableTaSOD-A1aThe gene is alsoTaSOD-A1bApplication in genes;

3. The use according to claim 2, wherein the substance comprises PCR primers for amplifying a wheat genomic DNA fragment comprising the molecular marker of claim 1.

4. The use according to claim 3, wherein the PCR primer is a primer pair composition consisting of primer pair 1 and primer pair 2, the primer pair 1 consisting of forward primer F1 and reverse primer R1, the forward primer F1 being single-stranded DNA having the nucleotide sequence of SEQ ID No.1, the reverse primer R1 being single-stranded DNA having the nucleotide sequence of SEQ ID No. 2; the primer pair 2 consists of a forward primer F2 and a reverse primer R2, wherein the forward primer F2 is single-stranded DNA with a nucleotide sequence of SEQ ID No.3, and the reverse primer R2 is single-stranded DNA with a nucleotide sequence of SEQ ID No. 4.

5. The molecular marker of claim 1 or the PCR primer of claim 4.

6. A reagent or kit comprising the PCR primer of claim 4.

7. A method of any one of:

d2 A test or auxiliary test of whether wheat is portableTaSOD-A1aThe gene is alsoTaSOD-A1bA method of gene;

characterized in that the method comprises the steps of:

e1 Using the wheat genome DNA to be identified as a template, and carrying out PCR amplification by using the PCR primer of claim 4 to obtain a PCR product;

e2 Identifying whether wheat is high SOD activity wheat or low SOD activity wheat or detecting whether wheat is carried according to the PCR productTaSOD-A1aThe gene is alsoTaSOD-A1bA gene;

8. The method of claim 7, wherein E2) is F1) or F2):

when performing PCR amplification using the PCR primer described in claim 4, if the PCR product contains the molecular marker 2 and does not contain the molecular marker 1, the wheat to be identified is or is candidate to be low SOD activity wheat; if the PCR product contains the molecular marker 1 and does not contain the molecular marker 2, the wheat to be identified is or is candidate to be high SOD activity wheat;

f2 The detection of wheat carrying based on the PCR productTaSOD-A1aThe gene is alsoTaSOD-A1bThe gene is as follows:

when PCR amplification is performed using the PCR primer as set forth in claim 4, if the PCR product contains the molecular marker 2 and does not contain the molecular marker 1, wheat to be identified is carried withTaSOD-A1bA gene; if the PCR product contains the molecular marker 1 and does not contain the molecular marker 2, the wheat to be identified carriesTaSOD-A1aAnd (3) a gene.

9. Use of the molecular marker of claim 1, the PCR primer of claim 3 or 4, or the reagent or kit of claim 6 for breeding high SOD activity wheat varieties and/or breeding low SOD activity wheat varieties, when SOD activity is equal to or less than 1728.05U/g; when the SOD activity is equal to or higher than 1814.19U/g, the SOD activity is high.

10. Use of the reagent or kit of claim 6 for identifying superoxide dismutase activity of wheat kernel.