CN114507751A

CN114507751A - Molecular marker related to activity of superoxide dismutase of wheat grains and application of molecular marker

Info

Publication number: CN114507751A
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: 2022-05-17
Anticipated expiration: 2042-03-16
Also published as: CN114507751B

Abstract

The invention discloses a molecular marker related to the activity of superoxide dismutase of wheat grains and application thereof. The invention designs a specific primer for amplifying a wheat genome DNA fragment containing the molecular marker based on two allelic variation forms of TaSOD-A1 gene, namely TaSOD-A1a gene and TaSOD-A1b gene, and provides a method for identifying the activity of the wheat grain superoxide dismutase by using the molecular marker and the primer. The molecular marker of the invention can quickly identify the allelic variation conditions of TaSOD-A1a and TaSOD-A1b and the SOD activity thereof, quickly screen out the wheat varieties with higher SOD activity, can not only accelerate the breeding pace of new high-quality wheat varieties, but also has important theoretical significance and economic value for the auxiliary selection of the wheat varieties with high SOD activity by the molecular marker.

Description

Molecular marker related to activity of superoxide dismutase of wheat grains and application of molecular marker

Technical Field

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

Background

Superoxide dismutase (SOD) is widely distributed in microorganisms, plants and animals, can scavenge Superoxide anion free radicals in organisms, and has important influence on the nutritional quality and processing quality of the facial product due to the activity of the SOD. Superoxide dismutase has become the most focused component of researchers besides protein, and thus is becoming a research hotspot in the aspects of wheat and human health nutrition. The improvement of the nutrition and processing quality of wheat in China through a genetic approach is feasible, and the breakthrough achievement in breeding mainly depends on the discovery and utilization of key genes. Researches find that the SOD can oxidize the conjugated double bonds of carotenoid and other pigment molecules in the flour, thereby playing a role in bleaching the color of the flour product; the SOD can also degrade dietary fiber components in the flour and convert the dietary fiber components into components with prebiotics effect to improve the nutritional quality of the flour product; SOD also stabilizes protein conformation by affecting intramolecular or intermolecular sulfhydryls and disulfide bonds in the dough, resulting in more disulfide bonds, thereby improving dough rheology. SOD activity is complex quantitative character controlled by multiple genes, is greatly influenced by environment, has larger difficulty in directly selecting the SOD activity level, and has small effect of a single gene, so that the polymerization of related genes of the SOD activity by utilizing molecular markers is an important means for improving the SOD activity. The functional marker is a novel molecular marker developed according to the internal sequence polymorphism sequence of the functional gene, and theoretically, the markers from functional motifs in the gene can determine the type of target allele under different genetic backgrounds without further verification, but no functional marker related to SOD exists at present. The gene related to the SOD activity of the wheat is excavated, and the molecular marker closely linked with the gene is developed, so that an important theoretical basis can be provided for the molecular mechanism research related to the gene pyramiding breeding and quality characters, and the method has important theoretical and practical significance for cultivating new wheat varieties with high SOD activity.

The SOD activity of wheat grains is influenced by the genotype and the environment at the same time, but is mainly determined by genetic factors. At present, the excavation of the major effective site related to the wheat SOD activity is less. Wu, Baek and the like take the four-lacking body and the two-terminal body in China as materials, the Mn-SOD active gene of wheat grains is positioned on the 2 nd homologous group of wheat, the Cu/Zn-SOD active gene of the wheat grains is positioned on the long arm of the chromosome of the 7 th homologous group, and a QTL (quantitative trait locus) influencing the SOD activity in the wheat seeds is arranged on the long arm of the 2D chromosome. According to the principle of co-linearity of gramineous crop genes, the wheat SOD genes are presumed to be positioned in the 7 th homologous chromosome group by combining the positioning result of the SOD genes in rice; through correlation and linkage analysis, the important locus for controlling the SOD activity of the wheat grains is located on the 5A chromosome. Kumar et al cloned the Mn-SOD gene on wheat 6D chromosome; li Juan et al obtained cDNA sequence of Fe-SOD gene from wheat Nicotiana 19; wangcong et al used PCR technology with wheat BS366 as material and succeeded in obtaining its Cu/Zn-SOD sequence. In order to more accurately identify the genotypes of the SOD activities of different wheat varieties, it is necessary to clone the major active sites of wheat, develop a pair of dominant complementary functional markers according to the sequences of different alleles, further improve the efficiency of molecular marker assisted selective breeding, provide a feasible molecular marker for improving the SOD activities of wheat, and finally lay a certain theoretical basis for molecular marker assisted breeding of new varieties and map-based cloning.

Disclosure of Invention

The invention aims to solve the technical problem of how to identify or assist in identifying whether the wheat is high-SOD activity wheat or low-SOD activity wheat and/or detect or assist in detecting whether the wheat carries TaSOD-A1a gene or TaSOD-A1b gene. The technical problem to be solved is not limited to the technical subject as described, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.

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

A1) application in identifying the activity of the superoxide dismutase of the wheat grains;

A2) the application in identifying or assisting in identifying whether the wheat to be detected is high-SOD active wheat or low-SOD active wheat;

A3) the application in identifying or assisting in identifying the wheat varieties with low SOD activity;

A4) the application in identification or auxiliary identification of wheat varieties with high SOD activity;

A5) the application in identifying or assisting in identifying the SOD activity level in wheat grains;

A6) the application in detecting or assisting in detecting whether wheat carries TaSOD-A1a gene or TaSOD-A1b gene;

A7) the application in identifying or assisting in identifying wheat varieties carrying TaSOD-A1a genes;

A8) the application in identifying or assisting in identifying wheat varieties carrying TaSOD-A1b genes;

A9) the application in the analysis and identification of wheat germplasm resources;

A10) the application in wheat molecular assisted genetic breeding;

the molecular marker can be any one of the following:

B1) a molecular marker 1, wherein the molecular marker 1 is a DNA molecule (with the length of 1419bp) with the nucleotide sequence shown in the 140 th and 1558 th positions of SEQ ID No. 5;

B2) a molecular marker 2, wherein the nucleotide sequence of the molecular marker 2 is a DNA molecule (the length is 2567bp) shown by 127-2693 of SEQ ID No. 6;

B3) a molecular marker composition consisting of said molecular marker 1 and said molecular marker 2.

Further, the identification of the activity of the wheat grain superoxide dismutase can be used for identifying the activity of the wheat grain superoxide dismutase, namely the activity of the wheat grain superoxide dismutase is low when the activity of the wheat grain superoxide dismutase is less than or equal to 1728.05U/g; when the SOD activity is greater than or equal to 1814.19U/g, the SOD activity is high.

The nucleotide sequence of the TaSOD-A1a gene is shown in SEQ ID No.5, the 64-177, 348-499, 1179-1243, 1452-1511, 1604-1741, 1819-1943, 2003-2287, 2425-2558, 2675-2707, 2805-2967 of 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 in SEQ ID No.6, the 64-177, 348-499, 1179-1243, 1452-1511, 1604-1741, 1819-1943, 2003-2287, 2425-2558, 2675-2707, 2805-2967 of 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 variation forms of the TaSOD-A1 gene in wheat.

The 1419bp DNA molecule amplified by the upstream primer 1F having the nucleotide sequence of SEQ ID No.1 and the downstream primer 1R having the nucleotide sequence of SEQ ID No.2 (i.e., position 140-1558 of SEQ ID No. 5) was designated as molecular marker 1.

The 2567bp DNA molecule (i.e., position 127 and 2693 of SEQ ID No. 6) amplified by the upstream primer 2F having the nucleotide sequence of SEQ ID No.3 and the downstream primer 2R having the nucleotide sequence of SEQ ID No.4 was designated as molecular marker 2.

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

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

C1) the primer pair 1 comprises a forward primer F1 and a reverse primer R1, wherein the forward primer F1 is a single-stranded DNA with a nucleotide sequence of SEQ ID No.1, and the reverse primer R1 is a single-stranded DNA with a nucleotide sequence of SEQ ID No. 2;

C2) the primer pair 2 consists of a forward primer F2 and a reverse primer R2, wherein the forward primer F2 is a single-stranded DNA with the nucleotide sequence of SEQ ID No.2, and the reverse primer R2 is a single-stranded DNA with the nucleotide sequence of SEQ ID No. 3;

C3) a primer pair composition consisting of the primer pair 1 in C1) and the primer pair 2 in C2).

The molecular marker, and/or the PCR primer are also in the protection scope of the invention.

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

Further, the reagent can be a PCR reagent for identifying the activity of the wheat grain superoxide dismutase.

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

(1) a kit 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 reagent set 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 amplification²⁺One or more of (a).

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 active wheat or low SOD active wheat;

D2) a method for identifying or assisting in identifying the SOD activity level in wheat grains;

D3) a method for detecting or detecting in an auxiliary way whether wheat carries TaSOD-A1a gene or TaSOD-A1b gene;

characterized in that the method comprises the following steps:

E1) performing PCR amplification by using the primer pair 1, the primer pair 2 or the primer pair composition by using the wheat genome DNA to be identified as a template to obtain a PCR product;

E2) and identifying whether the wheat is high-SOD active wheat or low-SOD active wheat or identifying the SOD activity in wheat grains or detecting whether the wheat carries TaSOD-A1a gene or TaSOD-A1b gene according to the PCR product.

In the 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 the PCR amplification is as follows: about 80ng of template DNA, 0.25. mu.l of Taq DNA Polymerase (TIANGEN, code: ET101), 2.5. mu.l of 10 XTaq Buffer I (TIANGEN, code: ET101), 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 supplemented with sterile ultrapure water to 25. mu.l.

Further, the reaction procedure for performing PCR amplification using the primer pair 1 is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 63.7 ℃ for 30s, and extension at 72 ℃ for 1.5min for 35 cycles; finally, extending for 8min at 72 ℃; keeping the temperature at 12 ℃. Sequencing the obtained PCR amplification products respectively.

Further, the reaction procedure for performing PCR amplification using the primer pair 2 is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 61 ℃ for 30s, and extension at 72 ℃ for 2.5min for 35 cycles; finally, extending for 8min at 72 ℃; keeping the temperature at 12 ℃. 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 active wheat or low SOD active wheat 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 is or is candidate to be the wheat with high SOD activity; if the PCR product does not contain the molecular marker 1, the wheat to be identified is or is candidate to be the wheat with low SOD activity;

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

when the primer pair composition (primer pair 1 and primer pair 2) is used 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 the wheat with low SOD activity; 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 the wheat with high SOD activity;

F2) the identification of the SOD activity in the wheat grains according to the PCR product is as follows:

when the primer pair 1 is used for PCR amplification, the SOD activity of the PCR product in the wheat grain to be identified containing the molecular marker 1 is higher than that of the PCR product in the wheat grain to be identified not containing the molecular marker 1;

when the primer pair 2 is used for carrying out PCR amplification, the SOD activity of the PCR product in the wheat grains to be identified containing the molecular marker 2 is lower than the SOD activity of the PCR product in the wheat grains to be identified not containing the molecular marker 2;

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

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 the 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 the 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 used 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 the 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-SOD active wheat or low-SOD active wheat according to the PCR product is:

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

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

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 1419bp fragment and the PCR amplification product of the primer pair 2 does not contain a 2567bp fragment, the wheat to be detected is or is selected as 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 selected as the wheat with low SOD activity;

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

when the primer pair 1 is used for PCR amplification, the SOD activity of the PCR amplification product in the wheat grain to be identified containing the 1419bp segment is higher than the SOD activity of the PCR product in the wheat grain to be identified containing no 1419bp segment;

when the primer pair 2 is used for carrying out PCR amplification, the SOD activity of the PCR amplification product in the wheat grains to be identified, which contain the fragments with the size of 2567bp, is lower than the SOD activity of the PCR amplification product in the wheat grains to be identified, which do not contain the fragments with the size of 2567 bp;

when the primer pair composition (the primer pair 1 and the primer pair 2) is used for carrying out PCR amplification, the PCR amplification product of the primer pair 2 does not contain a fragment with the size of 2567bp, the SOD activity of the to-be-identified wheat grain of which the PCR amplification product of the primer pair 1 contains a fragment with the size of 1419bp is higher than that of the to-be-identified wheat grain of which the PCR amplification product of the primer pair 1 does not contain a fragment with the size of 1419bp, and the PCR amplification product of the primer pair 2 contains a fragment with the 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 1419bp fragment, the wheat to be identified carries the TaSOD-A1a gene; if the PCR amplification product does not contain a 1419bp fragment, 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 the TaSOD-A1b gene; if the PCR amplification product does not contain a fragment with the size of 2567bp, the wheat to be identified carries 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 1419bp fragment and the PCR amplification product of the primer pair 2 does not contain a 2567bp fragment, the wheat to be identified carries the TaSOD-A1a gene; if the PCR amplification product of the primer pair 2 contains a 2567bp fragment and the PCR amplification product of the primer pair 1 does not contain a 1419bp fragment, 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 methods:

(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 the PCR amplification product of the primer pair 1 contains a 1419bp fragment, and the PCR amplification product of the primer pair 2 does not contain a 2567bp fragment;

(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 the PCR amplification product contains a 1419bp fragment;

(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 the 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 the 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 the 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 the wheat D6 contains the molecular marker 1;

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

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

(1) the method comprises the following steps: breeding by taking wheat E1 as a parent; the wheat E1 meets 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 the PCR amplification product of the primer pair 1 does not contain a 1419bp fragment, and the PCR amplification product of the primer pair 2 contains a 2567bp fragment;

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

(3) the method comprises the following steps: breeding by taking wheat E3 as a parent; the wheat E3 meets the following conditions: performing PCR amplification on the genomic DNA of the wheat E3 by using the primer pair 2, wherein the 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 meets the following conditions: the genomic DNA of the 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 meets the following conditions: the genome DNA of the 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 meets the following conditions: the genomic DNA of the wheat E6 does not contain the molecular marker 1;

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

Further, in the method, the method for detecting whether the genomic DNA of wheat to be identified contains the molecular marker 1 can specifically be as follows: performing PCR amplification on the primer pair 1 by using the genomic DNA of the wheat to be identified as a template to obtain a PCR amplification product; if the PCR amplification product contains a 1419bp fragment, the genomic DNA of the wheat to be identified contains the molecular marker 1; if the PCR amplification product does not contain a 1419bp fragment, 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 wheat to be identified contains the molecular marker 2 can be specifically as follows: performing PCR amplification by using the primer pair 2 by using the genomic DNA of the wheat to be identified as a template to obtain a PCR amplification product; if the PCR amplification product contains a 2567bp fragment, the genomic DNA of the wheat to be identified contains the molecular marker 2; if the PCR amplification product does not contain a fragment with the size of 2567bp, the genome DNA of the wheat to be identified does not contain the molecular marker 2.

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

The invention also provides the application of the molecular marker, and/or the PCR primer, and/or the reagent or the kit in cultivating a high SOD active wheat variety and/or cultivating a low SOD active wheat variety;

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

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

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

Wheat to be identified as described herein includes wheat germplasm resources, genetic populations, or breeding material.

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

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

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

The SOD activity as described herein means superoxide dismutase activity.

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

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

Experiments prove that: the molecular marker of the invention can quickly identify the allelic variation conditions of TaSOD-A1a and TaSOD-A1b and the SOD activity thereof, and can quickly screen out wheat varieties (materials) with higher SOD activity, thereby not only accelerating the breeding pace of new high-quality wheat varieties, but also having important theoretical significance and economic value for the auxiliary selection of the wheat varieties with high SOD activity by utilizing the molecular marker.

Drawings

FIG. 1 is an electrophoretogram of a portion of a sample. Wherein, the samples in the lanes 1-10 are Gao Cheng 8901, Shaan 715, Wenzhou 8425B, Linmai No.2, Zhong892, China spring, Zhongmai 871, Yumai No. 7, Zhouma 11 and Lankou No.2 of the Xiaomai variety in Table 2. M is Trans 5K DNA Marker.

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.

And performing basic data statistical analysis and independent sample T test on the SOD activity of the wheat grains by adopting SPSS 25.0 software. And judging whether the variances of the two groups of data are equal according to Leven test of a variance equation, and then, according to whether the variances are equal, looking at the corresponding T value and the significance level. If P is greater than 0.05, the mean values of the two groups of data have no significant difference and have no statistical significance; if P is less than 0.05, the mean difference between the two groups of data is shown, and the statistical significance is achieved.

Example 1 method for identifying Superoxide dismutase (SOD) activity of wheat grains

Molecular marker for identifying activity of superoxide dismutase of wheat grains

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

The other is shown as SEQ ID No.6 and is named allele TaSOD-A1 b; SEQ ID No.6 from the 5' end 64-177, 348-.

2. Designing a specific primer pair 1 and a specific primer pair 2 based on two allelic variation forms of the TaSOD-A1 gene as follows:

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

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

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

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

Theoretically, the primer pair 1 can amplify the target fragment with the size of 1419bp (i.e. the 140-th 1558 th position of SEQ ID No. 5) by using the allele TaSOD-A1a shown in the 140-th 1558 th position of SEQ ID No.5 as a template, and cannot amplify the target fragment with the size of 1419bp by using the allele TaSOD-A1b shown in the 140-th 1558 th position of SEQ ID No.6 as a template.

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

Method for identifying activity of superoxide dismutase of wheat grains

1. And extracting the genome DNA of the wheat grains to be detected.

2. And (3) performing PCR amplification by respectively adopting the obtained genome DNA as a template and the primer pair 1 and/or the primer pair 2 obtained in the step one to obtain a PCR amplification product.

The reaction system of PCR amplification is as follows: about 80ng of template DNA, 0.25. mu.L of Taq DNA Polymerase (TIANGEN, code: ET101), 2.5. mu.L of 10 XTaq Buffer I (TIANGEN, code: ET101), 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 supplemented with sterile ultrapure water to 25. mu.L.

The reaction procedure for PCR amplification of primer pair 1 was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 63.7 ℃ for 30s, and extension at 72 ℃ for 1.5min for 35 cycles; finally, extending for 8min at 72 ℃; keeping the temperature at 12 ℃. Sequencing the obtained PCR amplification products respectively.

The reaction procedure for PCR amplification of primer pair 2 was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 61 ℃ for 30s, and extension at 72 ℃ for 2.5min for 35 cycles; finally, extending for 8min at 72 ℃; keeping the temperature at 12 ℃. Sequencing the obtained PCR amplification products respectively.

The amplification product was electrophoresed through a 1.0% agarose gel, 1 XTAE electrophoresis buffer system and then observed under a gel imager. Recovering the amplified target band, connecting by using a pEasy-T5 Zero Cloning Kit, transforming Trans-T1 escherichia coli competent cells, screening positive monoclones for detection, ensuring the accuracy of the sequence by 3 times of each sample, and confirming the size and the sequence of a PCR amplification product.

3. Defining wheat grain superoxide dismutase (SOD) activity:

when the SOD activity is less than or equal to 1728.05U/g, the wheat grains are wheat with low SOD activity;

when the SOD activity is greater than or equal to 1814.19U/g, the wheat grains are wheat with high SOD activity.

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

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

(2) If the PCR amplification product of the primer pair 1 contains a 1419bp fragment and/or the PCR amplification product of the primer pair 2 does not contain a 2567bp fragment, the wheat to be detected carries the allele TaSOD-A1 a;

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 the allele TaSOD-A1 b.

Wherein the nucleotide sequence of the 1419bp fragment is 140 th and 1558 th positions of SEQ ID No. 5; the nucleotide sequence of the 2567bp fragment is 127-2693 of SEQ ID No. 6.

Example 2 validation of molecular markers

Experimental materials: 128 parts of the Chinese winter wheat major cultivar (Table 2) can be obtained from the national germplasm resources library of the institute of crop science of Chinese academy of agricultural sciences.

The method for identifying the superoxide dismutase activity of the wheat grains described in example 1 is adopted to verify 128 parts of Chinese winter wheat main-pushing varieties. Meanwhile, planting each wheat variety in a Sinkiang agricultural academy of sciences (Marna laboratory station), detecting the SOD activity of the wheat grains after harvesting, repeatedly detecting the SOD activity of each wheat material twice, if the error of the detection results of the two times exceeds 10%, repeatedly detecting for the third time, and averaging the results.

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

(1) reagent

0.05mol/L Phosphate Buffered Saline (PBS) pH 7.8, 130mmol/L methionine (Met) solution, 750. mu. mol/L LNBT solution, 20. mu. mol/L riboflavin solution, 100. mu. mol/L EDTA-Na₂Solution, SOD extraction medium.

(2) Extraction of crude SOD enzyme

Weighing 0.5g of whole wheat flour (obtained by grinding and crushing whole grain wheat) into a10 m L centrifuge tube, adding 5m L SOD extraction medium (0.05 mol/L phosphoric acid buffer solution containing 1% polyvinylpyrrolidone and having pH of 7.8) pre-cooled at 4 deg.C, placing on a vortex mixer, and oscillating to make the buffer solution fully contact with the whole wheat flour; placing the centrifugal tube with the sample on a shaking table in an ice bath, and shaking for 2 hours; then centrifuging for 15min at 4 ℃ and 10000 rpm; and (4) sucking the supernatant, immediately putting the supernatant into a refrigerator at 4 ℃ to obtain SOD crude enzyme liquid, and analyzing for later use.

(3) SOD Activity measurement

5 cleaned and dried finger-shaped tubes (required to have good transparency) are taken for each treatment, the serial numbers are (i) - (v), each reagent and enzyme solution (SOD crude enzyme solution) are added according to the table 1, and the total volume of the reaction system is 3 ml. After mixing, the tube is placed in the dark, and the other tubes are reacted for 20min at 25 ℃ under 4500lx sunlight (the conditions of all the tubes are required to be consistent, and the reaction time is properly adjusted according to the reaction color and the enzyme activity of the control tube under light). After the reaction, the reaction was stopped by covering the reaction with a black cloth.

TABLE 1 amounts of reagents

And (4) blank zeroing the number (v) tube, measuring the absorbance of the reaction solution of the number (v) tube to (iv) tube at 560nm, and recording the measurement data.

(4) Calculation of results

The SOD activity unit is known to be expressed as 50% inhibition of photochemical reduction of NBT as one enzyme activity unit, and was calculated as follows.

In the formula: total SOD activity is expressed in enzyme units per gram 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: the amount of crude enzyme solution (mL) was measured

W: fresh weight of sample (g)

The SOD activity detection results of the wheat seeds of each variety are shown in Table 2: PCR amplification products obtained by amplifying 128 Chinese winter wheat varieties (lines) are divided into two types according to the size: a 1419bp (corresponding to the primer pair 1) fragment, wherein the nucleotide sequence of the 1419bp fragment is the 140 nd-1558 th position of SEQ ID No. 5; the other is a 2567bp (corresponding to primer pair 2) fragment, and the nucleotide sequence of the 2567bp fragment is 127-2693 of SEQ ID No. 6. The electrophoretogram of a part of the sample is shown in FIG. 1 (wherein, the samples in lanes 1-10 are respectively Gao city 8901, Shaan 715, Wen 8425B, Craimai No.2, Zhong892, China spring, Zhongmai 871, Yumai No. 7, Zhongmai No. 11, and Lanke No. 2). 128 wheat varieties (lines): 86 varieties carry allele TaSOD-A1a, and the average value of SOD activity is 1814.19U/g; 42 varieties carry allele TaSOD-A1b, and the average value of SOD activity is 1728.05U/g; the independent sample T test is carried out by SPSS 25.0 software, and the result shows that the SOD activity of the wheat variety carrying the allele TaSOD-A1a is higher than that of the wheat variety carrying the allele TaSOD-A1b, and the two have obvious difference (P < 0.05).

The results show that: 47 of 86 wheat varieties carrying the allele TaSOD-A1a are high SOD active varieties, namely the accuracy rate of identifying the high SOD active wheat is 55% by the aid of whether the wheat varieties carry the allele TaSOD-A1 a; 27 wheat varieties with low SOD activity in 42 wheat varieties carrying the allele TaSOD-A1b are identified by whether the wheat varieties carry the allele TaSOD-A1b, namely, the accuracy rate of the wheat with low SOD activity is 65 percent.

TABLE 2 PCR test results and SOD Activity test results for various wheat varieties

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.

SEQUENCE LISTING

<110> Sinkiang university of agriculture

<120> molecular marker related to activity of superoxide dismutase of wheat grains 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. Any one of the following uses of a molecular marker and/or a substance for detecting the molecular marker:

A10) the application in wheat molecular assisted genetic breeding;

the molecular marker is any one of the following:

B1) a molecular marker 1, wherein the molecular marker 1 is a DNA molecule with a nucleotide sequence shown in the 140 th and 1558 th positions of SEQ ID No. 5;

B2) a molecular marker 2, wherein the molecular marker 2 is a DNA molecule with a nucleotide sequence shown in the 127 nd-2693 site of SEQ ID No. 6;

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

3. The use according to claim 2, wherein the PCR primer is any one of the following:

4. Any of the molecular markers set forth in claim 1, and/or any of the PCR primers set forth in claim 3.

5. A reagent or kit comprising the PCR primer of claim 3.

6. Any one of the following methods:

characterized in that the method comprises the following steps:

E1) performing PCR amplification by using the wheat genome DNA to be identified as a template and using the primer pair 1, the primer pair 2 or the primer pair composition in the claim 3 to obtain a PCR product;

7. The method of claim 6, wherein the E2) is F1), F2), or F3):

when the primer pair 1 as claimed in claim 3 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 the wheat with high SOD activity; if the PCR product does not contain the molecular marker 1, the wheat to be identified is or is candidate to be the wheat with low SOD activity;

when the primer pair 2 as claimed in claim 3 is used for PCR amplification, if the PCR product contains the molecular marker 2, the wheat to be identified is or is candidate to be the wheat with low SOD activity; if the PCR product does not contain the molecular marker 2, the wheat to be identified is or is candidate to be the wheat with high SOD activity;

when the composition is subjected to PCR amplification by using the primers as set forth in claim 3, 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 selected as wheat with low SOD activity; 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 the wheat with high SOD activity;

when the primer pair 1 as claimed in claim 3 is used for PCR amplification, the SOD activity of the PCR product in the wheat grain to be identified containing the molecular marker 1 is higher than that of the PCR product in the wheat grain to be identified not containing the molecular marker 1;

when the primer pair 2 as claimed in claim 3 is used for PCR amplification, the SOD activity of the PCR product in the wheat grain to be identified containing the molecular marker 2 is lower than that of the PCR product in the wheat grain to be identified not containing the molecular marker 2;

when the primer pair composition is used for PCR amplification, the PCR product contains the molecular marker 1, and the SOD activity in the wheat grains to be identified without the molecular marker 2 is higher than the SOD activity in the wheat grains to be identified without the molecular marker 1 and containing the molecular marker 2;

when PCR amplification is carried out using the primer pair 1 as described in claim 3, if the PCR product contains the molecular marker 1, the wheat to be identified carries the 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 PCR amplification is carried out using the primer pair 2 as set forth in claim 3, if the PCR product contains the molecular marker 2, the wheat to be identified carries the 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 composition is subjected to PCR amplification by using the primers as set forth in claim 3, 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.

8. The use of the molecular marker of claim 1, and/or the PCR primer of claim 2 or 3, and/or the reagent or the kit of claim 5 for breeding wheat varieties with high SOD activity and/or wheat varieties with low SOD activity.

9. The use of the reagent or kit of claim 5 in the identification of wheat grain superoxide dismutase activity, and/or in the analysis and identification of wheat germplasm resources, and/or in wheat molecular assisted genetic breeding.

10. PCR primers for amplifying the molecular marker as described in claim 1.