CN113249513A

CN113249513A - Molecular marker for identifying or assisting in identifying wheat SDS-sedimentation value and application thereof

Info

Publication number: CN113249513A
Application number: CN202110637144.8A
Authority: CN
Inventors: 李保云; 张润琪; 娄鸿耀; 郭丹丹; 徐焕文; 解超杰; 尤明山; 梁荣奇
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-06-08
Filing date: 2021-06-08
Publication date: 2021-08-13

Abstract

The invention discloses a molecular marker for identifying or assisting in identifying a wheat SDS-sedimentation value, a special primer combination thereof and application thereof in wheat breeding. Experiments prove that the molecular marker identified or assisted identified high SDS-sedimentation value haplotype has the SDS-sedimentation value which is remarkably higher than that of the low SDS-sedimentation value haplotype, so that the rapid screening of wheat strains with higher SDS-sedimentation value in early generations and different populations of wheat breeding is assisted, the breeding selection efficiency can be improved, and the quality breeding process of the wheat is accelerated.

Description

Molecular marker for identifying or assisting in identifying wheat SDS-sedimentation value and application thereof

Technical Field

The invention relates to a molecular marker for identifying or assisting in identifying a wheat SDS-sedimentation value in the field of biotechnology and application thereof.

Background

Molecular marker-assisted selection is an important technical means for progeny selection in crop breeding at present. Compared with the traditional selection technology, the method has the obvious advantages of accuracy, high efficiency, no limitation of seasons and the like. The baking quality of wheat is mainly determined by the content and quality of gluten. In wheat quality breeding, the content and quality of gluten are generally measured by SDS-sedimentation value. The main components of gluten are prolamines and gluten. Gluten determines the elasticity of dough, with high molecular weight gluten content accounting for only 5% -10% of the total protein content of wheat grain, but the most important factor in wheat processing quality (Payne et al 1981). The high molecular weight gluten is composed of N-terminal, C-terminal and central repeat regions, wherein the cysteine residues are the providers of intermolecular disulfide bonds in the gluten polymer, giving the polymer a stable linear structure and a specific spatial conformation, giving the dough good elasticity. The high molecular weight glutelins are mainly encoded by Glu-1 locus on wheat 1A, 1B and 1D genomes, wherein 1Dx2+1Dy12 subunit and 1Dx5+1Dy10 subunit encoded by the Glu-D1 locus are two more common allelic variations. Previous studies generally suggested that 1Dx5 has a key SNP near the N-terminus of the repeat compared to 1Dx2, resulting in an increase in one cysteine residue in the 1Dx5 subunit over the 1Dx2 subunit, thereby allowing more disulfide bonds to form. Thus, the 1Dx5+1Dy10 subunit is superior to the 1Dx2+1Dy12 subunit. Based on this, some molecular markers for this SNP were developed to distinguish between 1Dx2 and 1Dx5, and molecular marker assisted selection for quality breeding (D' Ovidio R and Anderson O D.1994; Ma et al.2003; Ishikawa G and Nakamura T.2007).

Reference documents:

Payne P I,Holt L M,Law C N.Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin.Theoretical and Applied Genetics,1981,63(2):129-138

D’Ovidio R and Anderson O D.PCR analysis to distinguish between alleles ofa member of a multigene family correlated with wheat bread-making quality.Theoretical and Applied Genetics,1994,88(6-7):759-763

Ma W,Zhang W,Gale K R.Multiplex-PCR typing of high molecular weight glutenin alleles in wheat.Euphytica,2003,134(1):51-60.

Ishikawa G,Nakamura T.A new co-dominant PCR-based marker to identify the high-molecular-weight glutenin subunit combination“5+10”of common wheat.Wheat Information Servise,2007,103:1-4.

disclosure of Invention

The invention aims to solve the technical problem of how to identify the SDS-sedimentation value of wheat.

In order to solve the technical problems, the invention firstly provides a molecular marker for identifying or assisting in identifying the SDS-sedimentation value of wheat.

The molecular marker for identifying or assisting in identifying the SDS-sedimentation value of wheat is named as 1DxTX, and is a DNA molecule obtained by amplifying a primer pair A and a primer pair B by taking the genomic DNA of wheat to be detected as a template;

the primer pair A consists of single-stranded DNA (deoxyribonucleic acid) named F and R1, wherein the F is the single-stranded DNA specifically combined with the 434 nd upstream position of the double-stranded DNA shown in the sequence 1, and the R1 is the single-stranded DNA specifically combined with the 434 nd nucleotide G of the double-stranded DNA shown in the sequence 1;

the primer pair B consists of single-stranded DNA (deoxyribonucleic acid) named F and R2, wherein the F is the single-stranded DNA specifically combined with the 434 nd upstream of the double-stranded DNA shown in the sequence 1, and the R2 is the single-stranded DNA specifically combined with the 434 nd nucleotide C of the double-stranded DNA shown in the sequence 1.

The wheat to be detected can be a homozygous inbred line. The wheat to be tested may also be a heterozygous line.

The polymorphism of the molecular marker can be any one of nucleotides corresponding to 434 th position of sequence 1 in wheat genome and representing C/G by letter S.

In the above molecular marker, F may be a single-stranded DNA represented by sequence 4 in the sequence table, R1 may be a single-stranded DNA represented by sequence 2 in the sequence table, and R2 may be a single-stranded DNA represented by sequence 3 in the sequence table.

In order to solve the technical problems, the invention also provides a method for identifying the haplotype of the wheat 1Dx gene.

The method for identifying the haplotype of the wheat 1Dx gene provided by the invention is characterized in that the wheat to be detected is a homozygous inbred line or a heterozygous line, the haplotype of the wheat 1Dx gene is a haplotype with a high SDS-sedimentation value or a haplotype with a low SDS-sedimentation value, and the method is as follows I or II:

i, including the following K1) and K2):

K1) respectively carrying out PCR amplification by using the primer pair A and the primer pair B by using the wheat genome DNA to be detected as a template to obtain PCR products;

K2) detecting the PCR product obtained in the step K1), and determining the haplotype type of the wheat 1Dx gene to be detected according to the PCR product:

the PCR product of the primer pair A contains a DNA fragment 1, and the haplotype type of the gene haplotype of the wheat 1Dx to be detected, which does not contain the DNA fragment 2, of the PCR product of the primer pair B is homozygous for the haplotype with high SDS-sedimentation value; the PCR product of the primer pair A does not contain a DNA fragment 1, and the haplotype of the wheat 1Dx gene to be detected containing a DNA fragment 2 of the PCR product of the primer pair B is homozygote of the haplotype with low SDS-sedimentation value; the PCR product of the primer pair A contains a DNA fragment 1, and the haplotype type of the wheat 1Dx gene to be detected, which contains a DNA fragment 2, of the PCR product of the primer pair B is a heterozygote type; the DNA fragment 1 comprises a nucleotide G corresponding to 434 th site of a sequence 1 in a sequence table; the DNA fragment 2 comprises a nucleotide C corresponding to 434 th site of a sequence 1 in a sequence table;

II, including the following L1) and L2):

l1) taking the genome DNA of the wheat to be detected as a template, and respectively carrying out PCR amplification by adopting a primer pair consisting of F and R1 and a primer pair consisting of F and R2 to obtain PCR products; the F can be a single-stranded DNA shown in a sequence 4 in a sequence table, the R1 can be a single-stranded DNA shown in a sequence 2 in the sequence table, and the R2 can be a single-stranded DNA shown in a sequence 3 in the sequence table;

l2) the following L21) or L22):

l21) detecting the PCR product obtained in the step L1), and determining the haplotype type of the wheat 1Dx gene to be detected according to the existence and the size of the PCR product:

the PCR product of the primer pair consisting of the F and the R1 contains a 453bp DNA fragment, and the haplotype type of the wheat 1Dx gene to be detected, which does not contain the 453bp DNA fragment, of the PCR product of the primer pair consisting of the F and the R2 is a high SDS-sedimentation value haplotype;

the PCR product of the primer pair consisting of the F and the R1 does not contain a 453bp DNA fragment, and the haplotype type of the wheat 1Dx gene to be detected, which contains the 453bp DNA fragment, of the PCR product of the primer pair consisting of the F and the R2 is a low SDS-sedimentation value haplotype;

the PCR product of the primer pair consisting of the F and the R1 contains a 453bp DNA fragment, and the haplotype type of the wheat 1Dx gene to be detected, which contains the 453bp DNA fragment, of the PCR product of the primer pair consisting of the F and the R2 is a heterozygote type;

l22) detecting the sequence of the PCR product obtained in the step L1), and determining the haplotype type of the wheat 1Dx gene to be detected according to the PCR product:

the haplotype of the wheat 1Dx gene to be detected, of which the 434 th bit of the sequence 1 in the sequence table corresponds to the PCR product, is homozygous for the haplotype with high SDS-sedimentation value; the haplotype of the wheat 1Dx gene to be detected, of which the 434 th bit of the sequence 1 in the sequence table corresponds to the PCR product, is homozygous for the haplotype with low SDS-sedimentation value; the haplotype of the wheat 1Dx gene to be detected, which corresponds to 434 th nucleotide of the sequence 1 in the sequence table and contains the PCR product, is a heterozygous type.

In the method for identifying the haplotype of the 1Dx gene, the PCR amplification system for PCR amplification may comprise dNTPs for dATP, dTTP, dCTP and dGTP, DNA polymerase and/or PCR reaction buffer.

In the method for identifying the haplotype of the 1Dx gene, the reaction conditions for performing the PCR amplification may be: PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.

In the method for identifying the 1Dx gene haplotype, the 1Dx gene haplotype is a wheat with a high SDS-sedimentation value haplotype, and compared with a wheat with a 1Dx gene haplotype with a low SDS-sedimentation value haplotype, the wheat with a high SDS-sedimentation value haplotype has higher SDS-sedimentation value, better gluten quality and better quality.

In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying the SDS-sedimentation value of wheat and/or the quality of wheat gluten.

The method for identifying or assisting in identifying the SDS-sedimentation value and/or the quality of gluten of the homozygous wheat provided by the invention is 1) or 2) as follows:

1) identifying the 1Dx gene haplotype type of the wheat to be detected according to the method;

2) determining the SDS-sedimentation value and/or the quality of gluten of the wheat to be detected according to the 1Dx gene haplotype type: compared with wheat with 1Dx gene haplotype type being low SDS-sedimentation value haplotype, the 1Dx gene haplotype type is wheat with high SDS-sedimentation value haplotype type, and has higher SDS-sedimentation value, better gluten quality and better quality.

The PCR amplification system in the method for identifying or assisting in identifying the SDS-sedimentation value and/or the gluten quality and/or quality of homozygous wheat may be the PCR amplification system in the method for identifying the 1Dx gene haplotype type, and the reaction conditions for performing the PCR amplification may be the PCR amplification reaction conditions in the method for identifying the 1Dx gene haplotype type.

In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality.

The method for identifying or assisting in identifying stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality provided by the invention is I or II as follows:

I. including M1) and M2) as follows:

m1) taking the genome DNA of the wheat to be detected as a template, and respectively carrying out PCR amplification by adopting the primer pair A and the primer pair B to obtain PCR products;

m2) detecting the PCR product obtained in the step M1), if the PCR product of the primer pair A contains a DNA fragment 1, the PCR product of the primer pair B does not contain a DNA fragment 2, and the wheat to be detected is or is selected as stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality; the DNA fragment 1 comprises a nucleotide G corresponding to 434 th site of a sequence 1 in a sequence table; the DNA fragment 2 comprises a nucleotide C corresponding to 434 th site of a sequence 1 in a sequence table;

II. Including the following N1) and N2):

n1) taking the genome DNA of the wheat to be detected as a template, and respectively carrying out PCR amplification by adopting a primer pair consisting of F and R1 and a primer pair consisting of F and R2 to obtain PCR products;

n2) as follows N21) or N22):

n21) detecting the size of the PCR product obtained in the step N1), if the PCR product of the primer pair consisting of the F and the R1 contains a 453bp DNA fragment, the PCR product of the primer pair consisting of the F and the R2 does not contain the 453bp DNA fragment, and the wheat to be detected is or is selected as stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality;

n22) detecting the sequence of the PCR product obtained in the step N1), if the 434 th position of the PCR product corresponding to the sequence 1 in the sequence table is G nucleotide, the wheat to be detected is or is selected as stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality.

The system for performing the PCR amplification in the method for identifying or assisting in identifying stable genetic wheat with a high SDS-sedimentation value and/or a good gluten quality and/or a good quality may be the system for performing the PCR amplification in the method for identifying a 1Dx gene haplotype type, and the reaction conditions for performing the PCR amplification may be the reaction conditions for performing the PCR amplification in the method for identifying a 1Dx gene haplotype type.

In order to solve the technical problems, the invention also provides a primer pair combination for identifying or assisting in identifying the SDS-sedimentation value and/or the quality of gluten of homozygous wheat.

The primer pair combination for identifying or assisting in identifying the SDS-sedimentation value and/or the quality of gluten of homozygous wheat provided by the invention consists of the primer pair A and the primer pair B.

In order to solve the technical problems, the invention also provides a system for identifying or assisting in identifying the SDS-sedimentation value and/or the quality of gluten of homozygous wheat.

The system for identifying or assisting in identifying the SDS-sedimentation value and/or the quality of wheat gluten consists of X1 and X2; the X1 is the primer combination, and the X2 is the reagent and/or the instrument required for PCR amplification.

In the above system, the reagents required for PCR amplification may comprise dNTPs of dATP, dTTP, dCTP and dGTP, DNA polymerase and/or PCR reaction buffer, or may be the dNTP mixture, the DNA polymerase and/or the PCR reaction buffer alone; the apparatus required for performing PCR amplification may be a PCR apparatus.

In the system, the primer pair A and the primer pair B and the reagents required for PCR amplification can be independently packaged. The primer pair A and the primer pair B are independently packaged, two single-stranded DNAs in the primer pair A can be independently packaged, and two single-stranded DNAs in the primer pair B can be independently packaged. Each reagent required for PCR amplification can be packaged independently.

The above system for identifying or assisting in identifying the SDS-sedimentation value of wheat and/or the quality and/or quality of wheat gluten may also comprise only the primer pair A and the primer pair B, and the reagent or kit required for PCR amplification.

In order to solve the technical problem, the invention also provides any one of the following applications H1-H14:

h1, and the application of the molecular marker in identifying or assisting in identifying the haplotype type of the wheat 1Dx gene;

h2, the use of said molecular marker for identifying or assisting in identifying the SDS-sedimentation value of wheat and/or the quality and/or quality of gluten;

h3, and the application of the molecular marker in identification or auxiliary identification of stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality;

h4, and the application of the molecular marker in wheat breeding;

h5, the application of the method for identifying the haplotype type of the wheat 1Dx gene in identifying or assisting in identifying the SDS-sedimentation value and/or the quality of wheat gluten;

h6, the application of the method for identifying the haplotype type of the wheat 1Dx gene in identifying or assisting in identifying stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality;

h7, the application of the method for identifying the haplotype type of the wheat 1Dx gene in wheat breeding;

h8, and the use of the method for identifying or assisting in identifying the SDS-sedimentation value and/or the quality and/or quality of wheat gluten in wheat breeding;

h9, and the application of the method for identifying or assisting in identifying stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality in wheat breeding;

h10, the primer pair combination or the system is used for preparing a reagent or a kit for identifying or assisting in identifying the SDS-sedimentation value and/or the quality of wheat gluten;

h11, the primer pair combination or the application of the system in identifying or assisting in identifying the haplotype type of the wheat 1Dx gene;

h12, the primer pair combination or the system for identifying or assisting in identifying the quality and/or quality of wheat SDS-sedimentation value and/or gluten;

h13, the primer pair combination or the system is applied to identification or auxiliary identification of stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality;

h14, the primer pair combination or the application of the system in wheat breeding.

In the above application, the 1Dx gene haplotype is a high SDS-sedimentation haplotype (corresponding to nucleotide G at position 434 in sequence 1 in the sequence table), and compared with the 1Dx gene haplotype is a low SDS-sedimentation haplotype (corresponding to nucleotide C at position 434 in sequence 1 in the sequence table), the SDS-sedimentation is higher, and the gluten has better quality and better quality.

In order to solve the technical problems, the invention also provides a wheat breeding method.

According to the wheat breeding method provided by the invention, the 1Dx gene haplotype type of the wheat is identified according to the method for identifying the 1Dx gene haplotype type, and the wheat with the 1Dx gene haplotype type being the haplotype with high SDS-sedimentation value is selected as a parent to carry out breeding.

In the present invention, the number of moles of the primer pair a and the primer pair B may be 1:1, the number of moles of the two single-stranded DNAs of the primer pair a may be 1:1, and the number of moles of the two single-stranded DNAs of the primer pair B may be 1: 1.

In the present invention, when the size of the PCR product is detected by electrophoresis, the PCR product containing the 453bp DNA fragment can show a band between 400bp and 500 bp.

The subject group study found that the key SNPs found by the predecessors did not completely distinguish between the 1Dx2 subunit and the 1Dx5 subunit. In a few varieties of 1Dx5 subunit, the same SNP as the common 1Dx2 subunit is present. However, the difference in the number of cysteine residues in the 1Dx high molecular weight glutelin subunit caused by this SNP does have a significant effect on quality. The inventor redevelops a three-primer molecular marker 1DxTX aiming at the SNP causing the difference of the number of cysteine residues of 1Dx high-molecular-weight glutenin subunits, definitely judges whether the key SNP exists or not by two times of PCR and agarose gel electrophoresis instead of distinguishing 1Dx2 from 1Dx5 so as to distinguish haplotypes, and proves that the SDS-sedimentation value of the high SDS-sedimentation-value haplotype screened by the molecular marker is remarkably higher than that of the low SDS-sedimentation-value haplotype by detecting an RIL group and a large number of existing wheat varieties. Therefore, the molecular marker can quickly screen wheat strains with higher SDS-sedimentation value in early generation and different groups of wheat breeding, improve breeding selection efficiency and accelerate the quality breeding process of wheat.

Drawings

FIG. 1 is a histogram of SDS-sedimentation frequency distribution (BLUP) of RIL population in example 1.

FIG. 2 is a graph of the results of SDS-PAGE in example 1 detecting the glutelin subunits of the parents TAA10 and XX329, wherein the ratio of CS: chinese spring; CU: 7+9, 5+10 subunit standards; t: TAA 10; x: XX 329.

FIG. 3 is a graph comparing the glutelin subunit gene 1Dx between the parents TAA10 and XX329 in example 1.

FIG. 4 is a schematic diagram of molecular markers designed according to the difference between two haplotypes of the glutelin subunit gene 1Dx in example 1.

FIG. 5 is a graph of the results of electrophoresis of the screening of RIL populations using 1DxTX in example 1. During electrophoresis, a PCR product obtained by amplifying 1DxF/1DxTR is firstly spotted in a gel hole, after the gel hole is electrified for a period of time and completely enters agarose gel, a PCR product obtained by amplifying 1DxF/1DxXR is spotted in the gel hole, wherein the former uses a marker at the left end as a standard, and the latter uses a marker at the right end as a standard.

FIG. 6 is a graph showing the results of comparison of SDS-sedimentation values of two haplotypes in each environment of the RIL population in example 1. Wherein, E1, 17 Beijing; e2, north river 17; e3, 18 beijing; e4, 18 hebei.

FIG. 7 is a graph showing the results of partial electrophoresis of the screening of natural populations using 1DxTX in example 1. During electrophoresis, a PCR product obtained by amplifying 1DxF/1DxTR is firstly spotted in a gel hole, after the gel hole is electrified for a period of time and completely enters agarose gel, a PCR product obtained by amplifying 1DxF/1DxXR is spotted in the gel hole, wherein the former uses a marker at the left end as a standard, and the latter uses a marker at the right end as a standard. In the figure, 1 is Marker; 2 is Elytrigia 534; 3 is new wheat 19; 4 is west farming 213; 5 is a short antibody 58; 6 is Kenong 2009; 7 is Nongda 3753; 8 is Nongda 152; 9 is teacher Luan 02-1; 10 is Zhongyu No. 8; and 11 is Marker.

FIG. 8 is a partial result of SDS-PAGE detecting glutelin subunits of the natural population in example 1. 1 is CS; 2 is Elytrigia 534; 3 is new wheat 19; 4 is west farming 213; 5 is a short antibody 58; 6 is Kenong 2009; 7 is Nongda 3753; 8 is Nongda 152; 9 is teacher Luan 02-1; 10 is Zhongyu No. 8.

FIG. 9 is a graph showing the results of comparing SDS-sedimentation values of two different haplotypes detected by the molecular markers of the natural population in example 1. Wherein, T: homozygous TAA10 haplotype, X: homozygous XX329 haplotypes.

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 are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The wheat variety TAA10 in the following examples was disclosed in the non-patent document "Identification of QTL for grain size and shape on the D gene of natural and synthetic allohexa-polar foods with near-polar AABB genes", wheat variety XX329 was disclosed in the non-patent document "Identification of QTL for grain size and shape on the D gene of natural and synthetic allohexa-polar foods with near-polar AABB genes", the RIL group in the following examples was disclosed in the non-patent document "public QTL mapping of wheat albumin content, Chinese university, academic thesis", the above-mentioned agricultural materials are available from China, and the materials are used only for the experiments which are repeated in connection with the present invention and are not used for other purposes.

Example 1

1. Parental selection and planting and phenotypic identification of RIL populations

TAA10 and XX329 were two wheat materials in this laboratory, differing only in the D genome, and the SDS-sedimentation value of TAA10 was very significantly higher than XX 329. TAA10 and XX329 are used as parents, and after the two parents are crossed, 7 generations of selfing are carried out to construct a RIL population containing 198 lines. The parents TAA10 and XX329, and the constructed RIL population were used as materials for planting and phenotypic identification as follows:

two-year two-point four-environment planting is carried out in the Beijing and Shijiazhuang in 2017 and 2018 respectively. Double row sowing, 2m row length, 0.2m row spacing, 3 repetitions per site. The harvested seeds are ground into whole wheat flour by a cyclone mill, 2g of the whole wheat flour is weighed, and the SDS-sedimentation value is measured by a micro-method, and the technology is repeated for three times during measurement.

Statistical analysis is carried out on SDS-sedimentation values of RIL populations and parents of the RIL populations by using R language, SPSS and EXCEL data processing software, and the results show that the SDS-sedimentation values of the populations are in continuous distribution, the absolute values of skewness and kurtosis are less than 1, the population SDS-sedimentation values accord with normal distribution, and the population SDS-sedimentation values belong to typical quantitative traits, and the method is specifically shown in figure 1 and table 1.

TABLE 1 statistical analysis of SDS-sedimentation values (BLUP) of parental and RIL populations

Note: representative significance analysis result is P <0.01

2. Identification of parent glutelin subunit, Glu-D1 site comparison and design of molecular marker

TAA10 and XX329 were two wheat materials in this laboratory, differing only in the D genome and with TAA10 having a very significantly higher SDS-sedimentation value than XX329, and these two varieties were selected as parents for subsequent testing.

The glutelin subunits of both parents were examined by SDS-PAGE (see FIG. 2), and both were found to be 7+9, 5+ 10. Retrieving the parental re-sequencing data (see FIG. 3), it was found that there is a key SNP (S at position 434 in sequence No. 1 of the sequence listing, and the letter S represents any nucleotide in C/G) in the repeat region near the N-terminus of 1Dx5 gene, which results in XX329 (the 1Dx5 gene corresponds to C at position 434 in sequence No. 1 of the sequence listing, and the corresponding haplotype is named as XX329 haplotype) having one less cysteine residue than TAA10 (the 1Dx5 gene corresponds to G at position 434 in sequence No. 1 of the sequence listing, and the corresponding haplotype is named as TAA10 haplotype), which may result in the reduction of its disulfide bond and thus the quality. Previous studies have generally used this SNP as a marker for distinguishing between the 2 and 5 subunits. The present study shows that the molecular marker is named as 1DxTX, the same as the 5 subunit, the difference of the two haplotypes of XX329 haplotype and TAA10 haplotype possibly has certain influence on the quality. Two downstream primers for the molecular marker 1DxTX were designed as shown in fig. 4, wherein the downstream primer specifically binding to the sequence corresponding to the sequence "G" at position 434 of sequence 1 was named "1 DxTR", the downstream primer specifically binding to the sequence corresponding to the sequence "C" at position 434 of sequence 1 was named "1 dxr", and a1 Dx-specific upstream primer named "1 DxF" was designed in the 1Dx upstream promoter region.

1 DxTR: 5'-GTATGAAACCTGCTGCGGAC-3' (as shown in sequence 2 of the sequence table, the binding position with the sequence 1 is position 434 and 453 of the sequence 1);

1 DxXR: 5'-GTATGAAACCTGCTGCGGAG-3' (as shown in sequence 3 of the sequence table, the binding position to the sequence 1 is position 434 and 453 of the sequence 1);

1 DxF: 5'-TAGCCAACCTTCACAATCTT-3' (as shown in sequence 4 of the sequence table, the binding position with the sequence 1 is the 1 st to 20 th position of the sequence 1).

3. Screening of RIL populations Using 1DxTX

The parental TAA10 and XX329 and the genome DNA of each wheat line in the RIL population are respectively extracted as templates, and PCR amplification is respectively carried out on two groups of primer pairs, namely 1DxF/1DxTR (a primer pair consisting of an upstream primer 1DxF and a downstream primer 1 DxTR) and 1DxF/1DxXR (a primer pair consisting of an upstream primer 1DxF and a downstream primer 1 DxXR) which are designed aiming at a molecular marker 1 DxTX.

PCR amplification System (10. mu.l System): template DNA 1-3. mu.L, 2 XPCR Mix 5. mu.L (product of Ongjingke Co.), upstream primer 1. mu.L (concentration 2-10. mu.M), downstream primer 1. mu.L (concentration 2-10. mu.M), ddH₂O make up to 10. mu.L. Other volumes of amplification system were scaled up or down.

PCR amplification procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.

And respectively carrying out 2% agarose gel electrophoresis on the two groups of PCR amplification products, dropping the PCR product obtained by amplifying 1DxF/1DxTR in a gel hole during electrophoresis, electrifying for a period of time till the PCR product completely enters agarose gel, dropping the PCR product obtained by amplifying 1DxF/1DxXR, and imaging by using a gel imager, wherein partial results are shown in figure 5: the material which can amplify a target band with the size of 453bp by using 1DxF/1DxTR is homozygous TAA10 haplotype (the 1Dx5 gene corresponds to the G at the 434 th position of the sequence 1), the material which can amplify a target band with the size of 453bp by using 1DxF/1DxXR is homozygous XX329 haplotype (the 1Dx5 gene corresponds to the C at the 434 th position of the sequence 1), and the material which can amplify a target band with the size of 453bp by using two pairs of primers is heterozygous.

The results of the molecular marker are shown in figure 6, and the SDS-sedimentation values of the homozygous TAA10 haplotype and the homozygous XX329 haplotype in the RIL group are counted, so that the SDS-sedimentation value of the homozygous TAA10 haplotype in the RIL group is very obviously higher than that of the homozygous XX329 haplotype in four environments, the homozygous TAA10 haplotype is called as a high SDS-sedimentation-value haplotype, and the homozygous XX329 haplotype is called as a low SDS-sedimentation-value haplotype.

In the RIL population of 198 lines, there were 87 families homozygous for the TAA10 haplotype and 68 families homozygous for the XX329 haplotype. Comparing the different haplotypes with the phenotype data (obtained from section 1 of this example) of the population under 4 environments (17 Hebei, 17 Beijing, 18 Hebei, 18 Beijing), it was found that the coincidence ratio of the high SDS-sedimentation haplotype to the high SDS-sedimentation phenotype was 70.5% on average, and the coincidence ratio of the low SDS-sedimentation haplotype to the SDS-low sedimentation phenotype was 72% on average. Specific data for each environment are shown in table 2.

TABLE 2 comparative analysis of genotype and phenotype (SDS-sedimentation value) for RIL populations

The above results indicate that the molecular marker 1DxTX is effective in selecting the SDS-sedimentation trait in the Recombinant Inbred Line (RIL) population constructed by TAA10 and XX 329.

4. Screening of natural populations using 1DxTX

The natural colony containing 399 cultivated varieties is planted in Beijing in 2017, sowed in a single-row area, the row length is 1m, the row spacing is 0.2m, and the sowing is repeated for 3 times.

Detecting each wheat to be detected in the natural population by using a molecular marker 1DxTX, wherein the method comprises the following steps:

extracting genome DNA of wheat to be detected as a template, and respectively carrying out PCR amplification on two groups of primer pairs, namely 1DxF/1DxTR (a primer pair consisting of an upstream primer 1DxF and a downstream primer 1 DxTR) and 1DxF/1DxXR (a primer pair consisting of an upstream primer 1DxF and a downstream primer 1 DxXR) designed aiming at a molecular marker 1 DxTX.

PCR amplification System (10. mu.l System): template DNA 1-3. mu.L, 2 XPCRmix 5. mu.L (product of Ongki Co., Ltd.), upstream primer 1. mu.L (concentration 2-10. mu.M), downstream primer 1. mu.L (concentration 2-10. mu.M), ddH₂O make up to 10. mu.L. Other volumes of amplification system were scaled up or down.

And respectively carrying out 2% agarose gel electrophoresis on the two groups of PCR amplification products, dropping the PCR product obtained by amplifying 1DxF/1DxTR in a gel hole during electrophoresis, electrifying for a period of time till the PCR product completely enters agarose gel, dropping the PCR product obtained by amplifying 1DxF/1DxXR, and imaging by using a gel imager, wherein partial results are shown in figure 7.

Meanwhile, the glutelin subunit type of each wheat to be detected is detected by SDS-PAGE, and partial results are shown in figure 8.

Comparing the 1DxTX molecular marker assay results with the SDS-sedimentation value assay results, figure 9 was obtained: the sedimentation value of the homozygous TAA10 haplotype in the natural population is also significantly higher than the homozygous XX329 haplotype, and the proportion of the homozygous TAA10 haplotype is smaller, regardless of the specific gluten subunit type. Analyzing a specific glutelin subunit population, wherein the SDS-sedimentation value of the homozygous TAA10 haplotype in the 5+10 subunit population is also remarkably higher than that of the homozygous XX329 haplotype, and the proportion of the homozygous TAA10 haplotype is higher; however, in other subunit populations, the difference in SDS-sedimentation values between the two haplotypes was not significant (probably due to the too small number of homozygous TAA10 haplotype material), and the proportion of homozygous TAA10 haplotype was low.

In this natural population, 70 of the homozygous TAA10 haplotypes and 329 of the homozygous XX329 haplotype were found, and the coincidence ratio of the high SDS-sedimentation haplotype (homozygous TAA10 haplotype) to the high SDS-sedimentation phenotype was found to be 68% and the coincidence ratio of the low SDS-sedimentation haplotype (homozygous XX329 haplotype) to the low SDS-sedimentation phenotype was found to be 55% by comparison with the SDS-sedimentation phenotype data, and the specific data for each of the different subunit populations are shown in Table 3.

TABLE 3 comparative analysis of genotype and phenotype (SDS-sedimentation value) of natural populations

The molecular marker 1DxTX can assist in identifying the SDS-sedimentation value, has strong amplification specificity and convenient detection, is a codominant marker, and can accurately identify the heterozygous genotype. The molecular marker of the invention has high amplification abundance, can be detected by agarose gel electrophoresis, and has short detection time. Therefore, the molecular marker of the invention can improve the accuracy of selection and accelerate the cultivation speed of new high-quality 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. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

<120> molecular marker for identifying or assisting in identifying wheat SDS-sedimentation value and application thereof

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<170> SIPOSequenceListing 1.0

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<213> wheat (Triticum aestivum)

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attcactgat agtccaccga gatggctaag cggttagtcc tctttgtggc ggtagtcgtc 120

gccctcgtgg ctctcaccgt cgctgaaggt gaggcctctg agcaactaca gtgtgagcgc 180

gagctccagg agctccagga gcgcgagctc aaggcatgcc agcaggtcat ggaccaacag 240

ctccgagaca ttagccccga gtgccacccc gtcgtcgtca gcccggtcgc gggacaatac 300

gagcagcaaa tcgtggtgcc gcccaagggc ggatctttct accccggcga gaccacgcca 360

ccgcagcaac tccaacaacg tatattttgg ggaatacctg cactactaaa aaggtattac 420

ccaagtgtaa cttstccgca gcaggtttca tac 453

<210> 2

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtatgaaacc tgctgcggac 20

<210> 3

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gtatgaaacc tgctgcggag 20

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Claims

1. The molecular marker for identifying or assisting in identifying the SDS-sedimentation value of wheat is characterized in that the molecular marker is a DNA molecule obtained by taking the genomic DNA of wheat to be detected as a template and amplifying a primer pair A and a primer pair B;

2. The molecular marker of claim 1, wherein: the F is a single-stranded DNA shown in a sequence 4 in a sequence table, the R1 is a single-stranded DNA shown in a sequence 2 in the sequence table, and the R2 is a single-stranded DNA shown in a sequence 3 in the sequence table.

3. The method for identifying the haplotype type of the wheat 1Dx gene is characterized by comprising the following steps: the wheat to be detected is a homozygous inbred line or a heterozygous line, the wheat 1Dx gene haplotype is a high SDS-sedimentation value haplotype or a low SDS-sedimentation value haplotype, and the method comprises the following steps of I or II:

i, including the following K1) and K2):

II, including the following L1) and L2):

l1) using the wheat genome DNA to be detected as a template, and respectively carrying out PCR amplification by using a primer pair consisting of F and R1 in claim 2 and a primer pair consisting of F and R2 in claim 2 to obtain PCR products;

l2) the following L21) or L22):

l21) detecting the size of the PCR product obtained in the step L1), and determining the haplotype type of the wheat 1Dx gene to be detected according to the size of the PCR product:

the PCR product of the primer pair consisting of the F and the R1 contains a 453bp DNA fragment, and the haplotype type of the wheat 1Dx gene to be detected, which does not contain the 453bp DNA fragment, of the PCR product of the primer pair consisting of the F and the R2 is homozygous for the haplotype with high SDS-sedimentation value;

the PCR product of the primer pair consisting of the F and the R1 does not contain a 453bp DNA fragment, and the haplotype type of the wheat 1Dx gene to be detected, which contains the 453bp DNA fragment, of the PCR product of the primer pair consisting of the F and the R2 is homozygotic with the haplotype with low SDS-sedimentation value;

4. Method for identifying or assisting in identifying the SDS-sedimentation value and/or the quality of wheat gluten, characterized in that: the following 1) or 2):

1) identifying the haplotype type of the 1Dx gene in the wheat to be tested according to the method of claim 3;

5. A method for identifying or assisting in identifying stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality is characterized in that: is I or II as follows:

I. including M1) and M2) as follows:

m1) carrying out PCR amplification by using the genome DNA of wheat to be detected as a template and adopting the primer pair A and the primer pair B of claim 1 or 2 respectively to obtain PCR products;

II. Including the following N1) and N2):

n1) using wheat genome DNA to be detected as a template, and respectively carrying out PCR amplification by using a primer pair consisting of the pair F and the R1 in the claim 1 or 2 and a primer pair consisting of the pair F and the R2 in the claim 1 or 2 to obtain PCR products;

n2) as follows N21) or N22):

6. Primer pair combination for identifying or assisting in identifying the SDS-sedimentation value and/or the quality of wheat gluten, which is characterized in that: consisting of the primer pair a according to claim 1 or 2 and the primer pair B according to claim 1 or 2.

7. System for the identification or assisted identification of the SDS-sedimentation value and/or the quality and/or quality of homozygous wheat gluten, characterized in that: consists of X1 and X2; the X1 is the primer combination of claim 6, and the X2 is the reagents and/or instruments required for PCR amplification.

8. Any one of the following H1-H14:

use of H1, the molecular marker of claim 1 or 2, for identifying or aiding in identifying a haplotype type of a wheat 1Dx gene;

use of H2, the molecular marker of claim 1 or 2, for identifying or aiding in identifying the quality and/or quality of SDS-sedimentation value and/or gluten of wheat;

use of H3, the molecular marker of claim 1 or 2, for identifying or assisting in identifying stable genetic wheat with a higher SDS-sedimentation value and/or a better quality of gluten and/or a better quality;

use of H4, the molecular marker of claim 1 or 2, in wheat breeding;

h5, use of the method for identifying haplotype types of wheat 1Dx gene of claim 3 for identifying or assisting in identifying the SDS-sedimentation value and/or the quality and/or quality of wheat gluten;

h6, the use of the method for identifying haplotype types of wheat 1Dx gene of claim 3 for identifying or assisting in identifying stable genetic wheat with higher SDS-sedimentation value and/or better gluten quality and/or better quality;

h7, the use of the method for identifying the haplotype type of the wheat 1Dx gene of claim 3 in wheat breeding;

h8, use of the method of claim 4 for identifying or aiding in identifying the SDS-sedimentation value and/or the quality and/or quality of wheat gluten in wheat breeding;

h9, use of the method of claim 5 for identifying or assisting in identifying stable genetic wheat with higher SDS-sedimentation values and/or better gluten quality and/or better quality in wheat breeding;

use of H10, the primer pair combination of claim 6 or the system of claim 7 for the preparation of a reagent or kit for identifying or assisting in identifying the quality and/or quality of SDS-sedimentation value and/or gluten of wheat;

use of H11, the primer pair combination of claim 6, or the system of claim 7 for identifying or aiding in identifying a haplotype type of a wheat 1Dx gene;

use of H12, the primer pair combination of claim 6 or the system of claim 7 for identifying or assisting in identifying the quality and/or quality of SDS-sedimentation value and/or gluten of wheat;

use of H13, the primer combination pair of claim 6 or the system of claim 7 for identifying or assisting in identifying stable genetic wheat with a higher SDS-sedimentation value and/or a better quality of gluten and/or a better quality;

h14, the primer pair combination of claim 6 or the system of claim 7, in wheat breeding.

9. A method for breeding wheat, characterized in that the method for identifying the haplotype type of the 1Dx gene according to claim 3 identifies the haplotype type of the 1Dx gene of wheat, and wheat having the haplotype type of the 1Dx gene with a high SDS-sedimentation value is selected as a parent for breeding.