CN112813184B

CN112813184B - Molecular marker on wheat TaGS1.1-6A promoter and application thereof

Info

Publication number: CN112813184B
Application number: CN202110175788.XA
Authority: CN
Inventors: 童依平; 滕婉; 王亚州; 欧阳翔; 何雪; 赵学强
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2021-02-09
Filing date: 2021-02-09
Publication date: 2022-09-06
Anticipated expiration: 2041-02-09
Also published as: CN112813184A

Abstract

The invention discloses a molecular marker on a wheat TaGS1.1-6A promoter and application thereof. The molecular marker can identify the promoter type of the TaGS1.1-6A gene of wheat, and the promoter type of the TaGS1.1-6A gene is TaGS1.1-6A containing an MITE insertion sequence ^HapI Or TaGS1.1-6A without MITE insertion sequence ^HapII . Experiments prove that the promoter type of the TaGS1.1-6A gene is TaGS1.1-6A ^HapII The promoter type of the TaGS1.1-6A gene of the wheat is TaGS1.1-6A ^HapI Compared with wheat, the TaGS1.1-6A has higher expression level, higher grain yield, more grains per ear, higher yield and higher nitrogen utilization efficiency. The molecular marker can assist wheat breeding and promote the breeding of varieties with high nitrogen utilization efficiency and high yield.

Description

Molecular marker on wheat TaGS1.1-6A promoter and application thereof

Technical Field

The invention relates to a molecular marker for identifying a wheat TaGS1.1-6A promoter in the field of biotechnology and application thereof.

Background

Wheat is a main grain crop in China and all over the world, and the improvement of the wheat yield plays an extremely important role in guaranteeing the grain safety. The wheat yield is determined by the number of grains per unit area, the number of grains per spike and the thousand grain weight, and increasing the number of grains per spike is an important way for improving the wheat yield. The grain number per ear of wheat is genetically controlled and also influenced by environmental factors. The cultivation measures such as variety breeding, water and fertilizer operation and the like can obviously increase the grain number per spike so as to improve the wheat yield. In the aspect of variety breeding, increasing the grain number per ear is always one of the main targets of wheat breeders.

The nitrogen fertilizer can increase the number of grains per ear and the number of grains per ear of wheat, thereby increasingAnd (4) adding the yield. Therefore, the nitrogen is efficiently absorbed and utilized, so that the spike number and the grain number of the wheat are increased, and the yield of the wheat is increased. The nitrogen absorbed and utilized by crops is mainly ammonium nitrogen and nitrate nitrogen. After entering the plant body, the ammonium nitrogen can be directly used for amino acid synthesis. The utilization of nitrate nitrogen firstly adopts two steps of reduction reaction: NO is first converted in the cytoplasm by Nitrate Reductase (NR) ₃ ^- Reduction to NO ₂ ^- Then transferred to chloroplasts and reduced to NH by nitrite reductase (NiR) ₄ ⁺ And then used for synthesizing amino acid. In the synthesis of amino acids, NH ₄ ⁺ Glutamine (Gln) and glutamic acid (Glu) are first synthesized catalyzed by Glutamine Synthetase (GS) and glutamic acid synthase (GOGAT), which are nitrogen donors for all major amino acids, nucleic acids, and nitrogen-containing compounds such as chlorophyll (Lea, 1993). It can be seen that GS catalyzes the first step reaction of ammonium ions into amino acids, and is a key position in the synthetic pathway of amino acids. GS is divided into two subclasses in plants, GS1 and GS2, located in the cytoplasm and chloroplast, respectively. The A, B and D genomes of common wheat each contain 3 GS1 genes and 1 GS2 gene. In the GS1 gene of wheat, only the expression of TaGS1.1 is up-regulated by low nitrogen stress. The method has important significance for improving the nitrogen utilization efficiency and the yield traits of the wheat by excavating excellent allelic variation of the GS gene in the wheat germplasm resources.

Disclosure of Invention

The invention aims to solve the technical problem of how to identify the wheat yield and the nitrogen utilization efficiency.

In order to solve the technical problems, the invention firstly provides a molecular marker on a wheat TaGS1.1-6A promoter.

The molecular marker on the wheat TaGS1.1-6A promoter provided by the invention is a DNA molecule obtained by taking the genome DNA of wheat to be detected as a template and adopting a primer pair A1 for amplification; the A1 consists of single-stranded DNA (deoxyribonucleic acid) named as P1 and P2, the P1 is the single-stranded DNA which is specifically combined with the upstream position 2094 of the double-stranded DNA shown in the sequence 1, and the P2 is the single-stranded DNA which is specifically combined with the downstream position 2310 of the double-stranded DNA shown in the sequence 1; the wheat to be detected is a homozygous inbred line.

The polymorphism of the molecular marker can be that the position corresponding to the 2094 th to 2310 th positions of the sequence 1 in the wheat genome is the 2094 th to 2310 th positions of the sequence 1 or the 2094 th to 2310 th positions of the deletion sequence 1.

The 2089 th to 2309 th sites of sequence No. 1 in the sequence table are miniature inverted-repeat transposable elements (MITEs) on the TaGS1.1-6A promoter.

In the above molecular marker, the P1 may be a single-stranded DNA represented by 2049 th to 2069 th positions of sequence 1 in the sequence table or 2038 th to 2058 th positions of sequence 2 in the sequence table, and the P2 may be a single-stranded DNA reverse-complementary to a sequence represented by 2838 th to 2858 th positions of sequence 1 in the sequence table or a sequence represented by 2614 th to 2634 th positions of sequence 2 in the sequence table.

In order to solve the technical problems, the invention also provides a method for identifying the wheat TaGS1.1-6A gene promoter type.

The method for identifying the TaGS1.1-6A gene promoter type of the wheat provided by the invention is characterized in that the wheat to be detected is a homozygous inbred line, and the TaGS1.1-6A gene promoter type is TaGS1.1-6A ^HapI Or TaGS1.1-6A ^HapII The method is as follows I or II:

i, including the following K1) and K2):

K1) taking the genome DNA of the wheat to be detected as a template, and carrying out PCR amplification on the primer pair A1 to obtain a PCR product;

K2) detecting the PCR product obtained in the step K1), and determining the promoter type of the TaGS1.1-6A gene of the wheat to be detected according to the PCR product:

the wheat TaGS1.1-6A gene promoter type to be detected, of which the PCR product contains a DNA fragment 1, is TaGS1.1-6A ^HapI (ii) a The PCR product does not contain the promoter type TaGS1.1-6A of the TaGS1.1-6A gene of the wheat to be detected of the DNA fragment 1 ^HapII (ii) a The DNA fragment 1 is a DNA fragment from 2094 th site to 2310 th site of a sequence 1 in a sequence table;

II, including the following L1) and L2):

l1) performing PCR amplification by using the genome DNA of the wheat to be detected as a template and adopting a primer pair consisting of P1 and P2 to obtain a PCR product; the P1 is a single-stranded DNA shown in 2049 th to 2069 th positions of a sequence 1 in the sequence table or 2038 th to 2058 th positions of a sequence 2 in the sequence table, and the P2 is a single-stranded DNA which is reversely complementary with a sequence shown in 2838 th to 2858 th positions of the sequence 1 in the sequence table or a sequence shown in 2614 th to 2634 th positions of the sequence 2;

l2) the following L21) or L22):

l21) detecting the size of the PCR product obtained in the step L1), and determining the TaGS1.1-6A gene promoter type of the wheat to be detected according to the size of the PCR product:

the PCR product only contains 810bp DNA fragments, and the promoter type of the TaGS1.1-6A gene of the wheat to be detected is TaGS1.1-6A ^HapI (ii) a The promoter type of the TaGS1.1-6A gene of the wheat to be detected, of which the PCR product only contains 597bp DNA segments, is TaGS1.1-6A ^HapII ；

L22) detecting the sequence of the PCR product obtained in the step L1), and determining the promoter type of the TaGS1.1-6A gene of the wheat to be detected according to the PCR product:

the PCR product only contains the DNA fragment shown in the 2049 th to 2858 th sites of the sequence 1 in the sequence table, and the promoter type of the TaGS1.1-6A gene of the wheat to be detected, which does not contain the DNA fragment shown in the 2038 th and 2634 th sites of the sequence 2 in the sequence table, is TaGS1.1-6A ^HapI (ii) a The PCR product only contains the DNA fragment shown by the 2038-2634 th site of the sequence 2 of the sequence table and the DNA fragment shown by the 2049-2858 th site of the sequence 1 of the sequence table, and the promoter type of the TaGS1.1-6A gene of the wheat to be detected is TaGS1.1-6A ^HapII 。

In the method for identifying the promoter type of TaGS1.1-6A gene, the PCR amplification system for performing the PCR amplification may contain dNTPs for dATP, dTTP, dCTP and dGTP, DNA polymerase and/or PCR reaction buffer.

In the above method for identifying the promoter type of the TaGS1.1-6A gene, the reaction conditions for performing the PCR amplification may be: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 1.5min, and circulation for 36 times; extending for 6min at 72 ℃, and keeping the temperature at 4 ℃.

In the above method for identifying the promoter type of TaGS1.1-6A gene, the promoter type of TaGS1.1-6A gene is TaGS1.1-6A ^HapI Wheat (containing indels highly coincident with MITE), TaGS1.1-6A gene promoter type is TaGS1.1-6A ^HapII Compared with wheat (without indels highly coincident with MITE), TaGS1.1-6A has lower expression level, lower seed yield, less spike grain number and lower nitrogen utilization efficiency. The difference is obvious under the conditions of high nitrogen and low nitrogen, and the nitrogen content in the nutrient solution under the condition of nutrient solution culture under the high nitrogen condition is 2.0 mmol/L; under the field test condition, the nitrogen fertilizer application amount of the wheat in the whole growth period is 180kg N/ha; the nitrogen content in the nutrient solution under the low-nitrogen condition is 0.2mmol/L under the nutrient solution culture condition; under the field test condition, the nitrogen fertilizer application amount in the whole growth period of the wheat is 60kg N/ha.

In order to solve the technical problems, the invention also provides a method for identifying or assisting in identifying the expression quantity and/or grain yield and/or grain number per ear and/or yield and/or nitrogen utilization efficiency of the wheat TaGS1.1-6A.

The method for identifying or assisting in identifying the expression quantity and/or grain yield and/or grain number per ear and/or yield and/or nitrogen utilization efficiency of homozygous wheat TaGS1.1-6A provided by the invention is 1) or 2) as follows:

1) identifying the TaGS1.1-6A gene promoter type of the wheat to be detected according to the method;

2) determining the expression quantity and/or grain yield and/or grain number per ear and/or yield and/or nitrogen utilization efficiency of the TaGS1.1-6A of the wheat to be detected according to the TaGS1.1-6A gene promoter type: the promoter type of the TaGS1.1-6A gene is TaGS1.1-6A ^HapI The wheat of (2) and TaGS1.1-6A gene promoter type are TaGS1.1-6A ^HapII Compared with wheat, the TaGS1.1-6A has the advantages of lower expression level, lower grain yield, fewer grains per ear, lower yield and lower nitrogen utilization efficiency.

The system for carrying out the PCR amplification in the method for identifying or assisting in identifying the expression quantity and/or grain yield and/or grain number per ear and/or yield and/or nitrogen utilization efficiency of TaGS1.1-6A of homozygous wheat can be the system for carrying out the PCR amplification in the method for identifying the TaGS1.1-6A gene promoter type, and the reaction condition for carrying out the PCR amplification can be the reaction condition for carrying out the PCR amplification in the method for identifying the TaGS1.1-6A gene promoter 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 TaGS1.1-6A expression level and/or higher grain yield and/or more grain number per ear and/or higher yield and/or higher nitrogen utilization efficiency.

The method for identifying or assisting in identifying the stable genetic wheat with higher TaGS1.1-6A expression level and/or higher grain yield and/or more grain per ear and/or higher yield and/or higher nitrogen utilization efficiency provided by the invention is I or II as follows:

I. including M1) and M2) as follows:

m1) carrying out PCR amplification by using the A1 to obtain a PCR product by using the genome DNA of the wheat to be detected as a template;

m2) detecting the PCR product obtained in the step M1), if the PCR product corresponds to a DNA fragment which does not contain the sequence 1 from the 2094 th position to the 2310 th position, the wheat to be detected is or is candidate to be stable genetic wheat with higher expression level of TaGS1.1-6A and/or higher grain yield and/or more spike grain number and/or higher yield and/or higher nitrogen utilization efficiency;

II. Including the following N1) and N2):

n1) using the wheat genome DNA to be detected as a template, and carrying out PCR amplification by adopting the A1 to obtain a PCR product;

n2) as follows N21) or N22):

n21) detecting the size of the PCR product obtained in the step N1), wherein if the PCR product only contains 597bp DNA fragments and does not contain 810bp DNA fragments, the wheat to be detected is or is selected as stable genetic wheat with higher TaGS1.1-6A expression quantity and/or higher grain yield and/or more spike grain number and/or higher yield and/or higher nitrogen utilization efficiency;

n22) detecting the sequence of the PCR product obtained in the step N1), if the PCR product contains the DNA fragment shown in the sequence 2 of the sequence table and does not contain the DNA fragment shown in the sequence 1 of the sequence table, the wheat to be detected is or is selected as stable genetic wheat with higher expression level of TaGS1.1-6A, higher grain yield, more spike grain number, higher yield and/or higher nitrogen utilization efficiency.

The system for performing the PCR amplification in the method for identifying or assisting in identifying the stable genetic wheat with the TaGS1.1-6A, which has the advantages of higher expression level and/or higher grain yield and/or more grain number per ear and/or higher yield and/or higher nitrogen utilization efficiency, can be the system for performing the PCR amplification in the method for identifying the TaGS1.1-6A gene promoter type, and the reaction condition for performing the PCR amplification can be the reaction condition for performing the PCR amplification in the method for identifying the TaGS1.1-6A gene promoter type.

In order to solve the technical problems, the invention also provides a primer pair for identifying or assisting in identifying the expression quantity and/or the grain yield and/or the grain number per ear and/or the yield of the wheat TaGS1.1-6A and/or the nitrogen utilization efficiency.

The primer pair for identifying or assisting in identifying the expression quantity and/or grain yield and/or spike grain number and/or yield of homozygous wheat TaGS1.1-6A and/or higher nitrogen utilization efficiency is the primer pair A1.

In order to solve the technical problems, the invention also provides a system for identifying or assisting in identifying the expression quantity and/or grain yield and/or grain number per ear and/or yield and/or nitrogen utilization efficiency of homozygous wheat TaGS1.1-6A.

The system for identifying or assisting in identifying the expression quantity and/or grain yield and/or spike grain number and/or yield and/or nitrogen utilization efficiency of the wheat TaGS1.1-6A consists of X1 and X2; the X1 is the primer pair A1, and the X2 is a reagent and/or an 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 above system, the A1 and the reagents required for PCR amplification can be packaged separately. The two single-stranded DNAs in a1 can be packaged independently. Each reagent required for PCR amplification can be packaged independently.

The system for identifying or assisting in identifying the expression quantity and/or grain yield and/or spike grain number and/or yield and/or nitrogen utilization efficiency of the wheat TaGS1.1-6A can also be a system only containing the A1 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 wheat TaGS1.1-6A gene promoter type;

h2, and application of the molecular marker in identification or auxiliary identification of expression quantity and/or grain yield and/or spike grain number and/or yield and/or nitrogen utilization efficiency of wheat TaGS1.1-6A;

h3, and application of the molecular marker in identification or assisted identification of stable genetic wheat with higher TaGS1.1-6A expression level and/or higher grain yield and/or more grain number per ear and/or higher yield and/or higher nitrogen utilization efficiency;

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

h5, and the application of the method for identifying the wheat TaGS1.1-6A gene promoter type in identifying or assisting in identifying the expression quantity and/or grain yield and/or grain number per ear and/or yield and/or nitrogen utilization efficiency of wheat TaGS1.1-6A;

h6, and the application of the method for identifying the TaGS1.1-6A gene promoter type of wheat in identifying or assisting in identifying stable genetic wheat with higher TaGS1.1-6A expression quantity and/or higher grain yield and/or more grain number per ear and/or higher yield and/or higher nitrogen utilization efficiency;

h7, the application of the method for identifying the TaGS1.1-6A gene promoter type of wheat in wheat breeding;

h8, and the application of the method for identifying or assisting in identifying the expression quantity and/or grain yield and/or grain number per ear and/or yield and/or nitrogen utilization efficiency of the wheat TaGS1.1-6A in wheat breeding;

h9, and the application of the method for identifying or assisting in identifying stable genetic wheat with higher TaGS1.1-6A expression level and/or higher grain yield and/or more grain number per ear and/or higher yield and/or higher nitrogen utilization efficiency in wheat breeding;

h10, and an application of the primer pair or the system in preparing a reagent or a kit for identifying or assisting in identifying the expression quantity and/or grain yield and/or grain number per ear and/or yield and/or nitrogen utilization efficiency of wheat TaGS1.1-6A;

h11, the application of the primer pair or the system in identifying or assisting in identifying the wheat TaGS1.1-6A gene promoter type;

h12, and application of the primer pair or the system in identification or auxiliary identification of the expression quantity and/or grain yield and/or ear grain number and/or yield and/or nitrogen utilization efficiency of wheat TaGS1.1-6A;

h13, and the application of the primer pair or the system in identification or assisted identification of stable genetic wheat with higher TaGS1.1-6A expression level and/or higher grain yield and/or more grain number per ear and/or higher yield and/or higher nitrogen utilization efficiency;

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

In the above application, the TaGS1.1-6A gene promoter type is TaGS1.1-6A ^HapI The wheat (containing MITE) and TaGS1.1-6A gene promoter type is TaGS1.1-6A ^HapII Compared with wheat (without MITE), TaGS1.1-6A has lower expression level, lower grain yield, fewer grains per ear, lower yield and lower nitrogen utilization efficiency. The difference is obvious under the conditions of high nitrogen and low nitrogen, and the nitrogen content in the nutrient solution under the nutrient solution culture condition under the high nitrogen condition in the embodiment of the invention is 2.0 mmol/L; under the field test condition, the nitrogen fertilizer application amount of the wheat in the whole growth period is 180kg N/ha; the nitrogen content in the nutrient solution under the low-nitrogen condition is 0.2mmol/L under the nutrient solution culture condition; under the field test condition, the nitrogen fertilizer application amount of the wheat in the whole growth period is 60kg N/ha.

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

The wheat breeding method provided by the invention identifies the TaGS1.1-6A gene promoter type of wheat according to the method for identifying the TaGS1.1-6A gene promoter type, and selects the TaGS1.1-6A gene promoter type as TaGS1.1-6A ^HapII The wheat of (2) is used as a parent for breeding.

In the present invention, the number of moles of the two single-stranded DNAs of A1 may be 1: 1.

In the present invention, when the size of the PCR product is detected, the detection can be performed by electrophoresis, and the PCR product contains a DNA fragment of 810bp and can show a band between 750bp and 1000 bp. The PCR product contains 597bp DNA fragment which can show a band between 500bp and 750 bp.

The invention also provides a DNA molecule of 2089 th to 2309 th positions of the sequence 1.

Experiments prove that the molecular marker can be used for identifying the expression quantity and/or the grain yield and/or the ear grain number and/or the yield and/or the nitrogen utilization efficiency of the wheat TaGS1.1-6A, the two PCR products obtained by carrying out PCR amplification by using the primer pair of the invention and taking the wheat genome with different expression quantities and/or grain yields and/or ear grain numbers and/or yields and/or nitrogen utilization efficiencies of the TaGS1.1-6A as a template correspond to the expression quantity and/or the grain yields and/or the ear grain numbers and/or the yields and/or the nitrogen utilization efficiencies of the wheat TaGS1.1-6A, and when the PCR product of the wheat contains a DNA fragment (810bp) shown in a sequence 1 and does not contain a DNA fragment (597bp) shown in a sequence 2, the wheat shows that the expression quantity and/or the grain yields of the wheat TaGS1.1.1-6A are lower and/or the ear grain yields are lower and/or the ears grain numbers are less and/or the yields are lower The amount is lower and/or the nitrogen utilization efficiency is lower, when the PCR product of the wheat contains a DNA fragment (597bp) shown in a sequence 2 and does not contain a DNA fragment (810bp) shown in a sequence 1, the wheat shows that the TaGS1.1-6A expression amount is higher, the grain yield is higher, the grain number per ear is larger, the yield is higher and/or the nitrogen utilization efficiency is higher.

Drawings

FIG. 1 is an electrophoretogram of the middle of example 1 of the present invention for detecting the presence or absence of insertion of the promoter region MITE of TaGS1.1-6A in different wheat varieties by PCR amplification-gel electrophoresis, wherein LY502 is a glutenin 502, LX is a liangxing 99, J411 is Beijing 411, and XY54 is Xiaoyan 54.

FIG. 2 is a graph showing the correlation between the insertion of MITE in the TaGS1.1-6A promoter region and the expression of TaGS1.1-6A in the root and aerial parts of wheat at the seedling stage in the experiment of the nutrient solution culture method of example 3 of the present invention, in which HapI represents 6 BC carrying MITE insertion ₃ F ₄ Means. + -. SD, HapII of the lines represent 6 BC not carrying a MITE insertion ₃ F ₄ Means. + -. SD of the strain. High nitrogen and low nitrogen represent treatments with nitrogen content of 2.0mmol/L and 0.2mmol/L, respectively, in the nutrient solution. And respectively show that the difference between HapI and HapII reaches P significantly<0.05(*)and P<0.01(×) level.

FIG. 3 is a graph showing the correlation analysis result of the expression level of TaGS1.1-6A in TaGS1.1-6A promoter region MITE and TaGS1.1-6A in Taglans leaf at heading stage in the field experiment of example 3 of the present invention, in which a in FIG. 3 is the result of high nitrogen treatment, b in FIG. 3 is the result of low nitrogen treatment, and HapI indicates 8 BC carrying MITE insertion ₃ F ₄ Means. + -. SD, HapII of the lines represent 7 BC not carrying a MITE insertion ₃ F ₄ Means. + -. SD of the strain. And indicate that the difference between HapI and HapII, respectively, significantly reached P<0.05(*)and P<0.01(×) level.

FIG. 4 is a graph showing the correlation between the MITE insertion in TaGS1.1-6A promoter region and wheat yield traits in the field experiment of example 3, wherein HapI indicates 101 BC carrying MITE insertion ₃ F ₄ Means. + -. SD, HapII of the lines indicate 88 BC not carrying a MITE insertion ₃ F ₄ Means. + -. SD of the strain. And respectively show that the difference between HapI and HapII reaches P significantly<0.05(*)and P<0.01(×) level.

FIG. 5 is a graph showing the correlation between the MITE insertion in TaGS1.1-6A promoter region and the nitrogen utilization in wheat in the field experiment of example 3, in which HapI indicates 12 BC carrying MITE insertion ₃ F ₄ Means. + -. SD, HapII of the lines represent 12 BC not carrying a MITE insertion ₃ F ₄ Means. + -. SD of the strain. And indicate that the difference between HapI and HapII, respectively, significantly reached P<0.05(*)and P<0.01(×) level.

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.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

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 Xiao-Yi 54 in the following examples was selected by the institute of genetics and developmental biology of the academy of sciences of China, and approved by the Committee for the approval of crop varieties in Shaanxi province in 2000. The method is described in non-patent documents of engineering peak construction, horse as a civilian, Chengzun, Humeijun, Shenwei, Lizhengping, Tongping, Libubingu, Xiaoyan 54, Jing 411 and filial generations thereof for stabilizing and optimizing dynamic change of photosynthetic characteristics of strains [ J ] crop report, 2009,35(06):1051 plus 1058 ], and public can be obtained from institute of genetics and developmental biology of Chinese academy of sciences to repeat experiments of the application and cannot be used for other purposes.

The Liangxing 99 is a wheat variety cultivated by Liangxing seed research institute of Dezhou, Shandong, and the variety is examined and approved by the national Committee for crop variety in 2006, and the variety number is national examined wheat 2006016. The method is described in non-patent literature "Liujun Sheng, Wang Fu Jun, Liupu. super high-yield high-quality new wheat variety-Liangxing 99[ J ] agricultural science and technology communication, 2006(07): 60", and the public can be obtained from research institute of genetics and developmental biology of Chinese academy of sciences, so that the application experiment can be repeated, and the method cannot be used for other purposes.

The wheat variety Jing 411 in the following examples was selected and bred by the seed management station of Beijing, approved by the Committee for examining and deciding crop varieties in Beijing in 1991, approved by the Committee for examining and deciding crop varieties in Shanxi province in 1992, and approved by the Committee for examining and deciding crop varieties in the nation in 1993. Variety registration number: GS 02002-1992. The method is described in non-patent literature, "project construction peak, horse is civilian, ChengGenyun, Humeijun, Shenallowed steel, Li Zhengsheng, Tongyiping, Lishun, Li hongwei, Xiaoyan 54, Jing 411 and filial generations thereof stabilize dynamic changes of photosynthetic characteristics of preferred strains [ J ]. crop reports, 2009,35(06):1051 one 1058", which can be obtained from institute of genetics and developmental biology of Chinese academy of sciences to repeat the application experiments and cannot be used for other purposes.

Wheat variety Luyuan 502 in the following examples was selected and bred by the Ministry of agricultural application and research institute of atomic energy of agricultural sciences of Shandong province, the institute of crop science of Chinese agricultural sciences, approved by the 2011 national committee for approval of crop varieties, and the number of the national approval was national approval wheat 2011016. The method is described in non-patent documents of "Lixinhua, Lipeng, high national strength, Zhang Fengyun, Sun bright column", high-yield widely-adapted new wheat variety Ruyuan 502 and a breeding strategy thereof [ J ]. Shandong agricultural science, 2013,45(04):32-34 ", and the public can obtain from the institute of genetics and developmental biology of Chinese academy of sciences to repeat the experiment of the application and cannot be used for other purposes.

In the following examples, the vectors pEASY-Blunt (CB101) and Trans-T1 competent cells (CD501) were all products of Takara Shuzo.

The preparation method of the liquid LB culture medium in the following examples is (taking 1L as an example): 10g of trypsin (Tryptone, Oxoid, L0042B), 5g of Yeast Extract (Yeast Extract, Oxoid, LP0021B), 10g of sodium chloride, dissolved in ddH ₂ The volume in O is constant to 1L.

The preparation method of the solid LB medium in the following examples is (taking 1L as an example): 10g of trypsin (Tryptone, Oxoid, L0042B), 5g of Yeast Extract (Yeast Extract, Oxoid, LP0021B), 10g of sodium chloride, 10g of Agar (Agar, Japan), dissolved in ddH ₂ The volume in O is up to 1L.

Example 1

The invention firstly clones TaGS1.1-6A gene promoter region (TaGS1.1-6Apro) on common wheat 6A chromosome, finds that sequences of TaGS1.1-6Apro of different wheat varieties have sequence variation, the length of the indel is 217bp, and the indel is highly overlapped with a miniature inverted-repeat transposable element (MITE) of about 221 bp. TaGS1.1-6Apro containing MITE insertion in promoter region was named TaGS1.1-6A ^HapI The nucleotide sequence is shown as 1 st-3247 th site of sequence table sequence 1 (clone is from Xiaomai variety Xiaoyan 54, 3158 nd-3160 th site of sequence 1 is ATG), wherein the indel sequence is from 2094 th-2310 th site of sequence 1, the sequence of MITE is from 2089 th-2309 th site of sequence 1(ii) a TaGS1.1-6Apro containing no MITE insertion was named TaGS1.1-6A ^HapII The nucleotide sequence is shown as 1 st to 3022 nd sites in the sequence 2 of the sequence table (cloned from Jing 411 of Xiaomai variety, and 2933 th to 2935 th sites in the sequence 2 are ATG). The specific process is as follows:

cloning of TaGS1.1-6A promoter sequence in common wheat

2 common wheat varieties of Elytrigia tritici 54 and Jing 411 are taken as experimental materials to clone the TaGS1.1-6A promoter sequence. The specific process is as follows:

1. wheat genome DNA extraction

Extracting the genome DNA of each variety of wheat, and storing at-20 ℃ for later use.

PCR amplification

1) PCR reaction System (Toyobo, KFX-101, containing 2 XPCR buffer for KOD FX, 2mM dNTPs and KOD FX (1.0U/. mu.L))

Genomic DNA (100 ng/. mu.L): 2 mu L of the solution;

Primer F(10μM)：1.5μL；

Primer R(10μM)：1.5μL；

2×PCR buffer for KOD FX：25μL；

2mM dNTPs：10μL；

KOD FX(1.0U/μL)：1μL；

ddH ₂ O：9μL。

the primers for cloning the promoter sequence of TaGS1.1-6A in wheat are as follows:

Primer F：5′-TGTGGCTAGAACCAAACACCCACCTAAC-3′；

Primer R：5′-AGGAAGGAGGACGTACCATATGTACTCG-3′。

2) PCR procedure

At 98 ℃ for 5 min; 98 ℃, 30s, 62 ℃, 30s, 68 ℃, 4min, 36 cycles; keeping the temperature at 68 ℃ for 10min and 10 ℃.

PCR product gel recovery and sequencing

The obtained PCR product is detected by 1% agarose gel electrophoresis and is connected into a pEASY-Blunt vector for sequencing, and the sequence shows that the sequence is 1 st to 3247 th sites of a sequence 1 or 1 st to 3022 th sites of a sequence 2 in a sequence table.

Method for detecting MITE insertion of TaGS1.1-6A promoter region in different wheat varieties

The extraction method of wheat genome DNA is shown in the step one. And judging whether the MITE of the TaGS1.1-6A promoter region in different wheat varieties is inserted or not by using a PCR amplification method according to polymorphism of the length of the amplified fragment.

The PCR amplification primers are as follows:

an upstream primer: 5'-ATTCCCCCGGTGCTTCAAACG-3' (as shown in 2049 th to 2069 th bits of sequence 1 in the sequence table or 2038 th to 2058 th bits of sequence 2 in the sequence table);

a downstream primer: 5'-AGTCTGTTCGACCGGGGGTAG-3' (reverse complement of the sequence from 2838 th to 2858 th of the sequence in the sequence table or from 2614 th to 2634 th of the sequence 2).

The PCR reaction system is as follows:

genomic DNA (100 ng/. mu.L): 1.5 mu L;

Primer F(10μM)：0.5μL；

Primer R(10μM)：0.5μL；

2×Taq PCR StarMix with Loading Dye(Genstar,A012-100)：10μL；

ddH ₂ O：7.5μL

PCR reaction procedure:

94 ℃ for 5 min; 94 ℃, 30s, 60 ℃, 30s, 72 ℃, 1.5min, 36 cycles; keeping the temperature at 72 ℃, 6min and 4 ℃.

The polymorphism of the length of the amplified fragment was detected by electrophoresis on 1% agarose gel.

The electrophoresis result is shown in figure 1, and the electrophoresis and sequencing results show that the size of the amplified fragments in the Xiaoyan 54 and Liangxing 99 wheat varieties is 810bp (the specific sequence is shown as 2049 th-2858 th bits of a sequence 1 in a sequence table, comprises 2089 th-2309 th bits of the sequence 1 in the sequence table and contains 2094 th-2310 th bits of indels in the sequence 1 in the sequence table), and TaGS1.1-6Apro is TaGS1.1-6A ^HapI (the promoter region contains MITE insertion and contains indels from 2094 th to 2310 th of the sequence 1); the amplified fragment size in the wheat varieties Jing 411 and Luyuan 502 is 597bp (the specific sequence is the 2038 th and 2634 th site of the sequence 2 in the sequence table), and the TaGS1.1-6Apro is TaGS1.1-6A ^HapII (the promoter region does not contain a MITE insertion, does not contain sequence 12094-indel at 2310).

Example 2

The TaGS1.1-6Apro of the Elytrigia tritici 54 is TaGS1.1-6A ^HapI (promoter region contains MITE insertion); TaGS1.1-6Apro of Jing 411 is TaGS1.1-6A ^HapII (the promoter region does not contain a MITE insertion). Homozygotes in the "Elytrigs tritici 54/Jing 411" RIL population (Ren et al, 2012) were identified in multi-point field trials for years for phenotypes of yield traits and nitrogen utilization-related traits for high-nitrogen and low-nitrogen treatments to analyze the association of different TaGS1.1-6Apro types with wheat variety phenotypes.

The TaGS1.1-6Apro type of the RIL population of Xiao-El 54/Jing 411 is detected according to the method of step two in example 1.

The yield and the phenotype identification test of the nitrogen utilization related traits of the RIL population of the Elytrigs tritici 54/Jing 411 are positioned at an agricultural ecosystem test station (Hebei province) of the agriculture resource research center of the institute of genetics and developmental biology of Chinese academy of sciences and a Pingchun province test station of the genetic and developmental biology of Chinese academy of sciences.

The fertilizer application sets two treatments, high nitrogen and low nitrogen. Applying urea to a nitrogen fertilizer, applying base fertilizer to 8kg of nitrogen per mu in high-nitrogen treatment, and topdressing to 4kg of nitrogen per mu in a jointing stage; the low-nitrogen treatment only takes 4kg N/mu as base fertilizer, and no additional nitrogen fertilizer is needed. The phosphate fertilizer adopts heavy superphosphate with the dosage of 6kg P ₂ O ₅ Per mu, the fertilizer is applied as a base fertilizer. Each test material in the field test is randomly arranged in sequence, each test material in each repetition is sown for 2 rows, the row spacing of sowing is 5cm, the row spacing is 23cm, and the sowing depth is 3-5 cm.

The wheat harvest stage is used for investigating the biological yield, the grain yield, the spike number, the main spike grain number, the main spike weight and the thousand kernel weight. And measuring the total nitrogen concentration of the grains and the straws by a Kjeldahl method, and calculating the cumulative amount of nitrogen and the utilization efficiency of the nitrogen according to the dry matter weight and the nitrogen content.

Straw biomass (biological yield (g/plant) -kernel yield (g/plant)

The total nitrogen accumulation (g/plant) is the straw biomass x the straw total nitrogen content + the kernel yield x the kernel total nitrogen content nitrogen utilization efficiency (g kernel/g N) is the kernel yield/total nitrogen accumulation.

The presence or absence of the insertion of MITE in the TaGS1.1-6A promoter region was used for correlation analysis with phenotypic traits. The results are shown in Table 1.

TABLE 1 correlation of TaGS1.1-6A with yield and nitrogen utilization related traits in "Elytrigs tritici 54/Jing 411" RIL population

Note: the data are means + -SD with TaGS1.1-6A ^HapI The RIL of (1) contains 89 of TaGS1.1-6A ^HapII Has 87 RILs.

The analysis result shows that TaGS1.1-6A ^HapII Significant correlation was made with higher grain yield, primary ear number, primary ear weight, and nitrogen utilization efficiency.

Example 3

In order to further clarify the association between TaGS1.1-6Apro variation and traits and nitrogen utilization-related traits, TaGS1.1-6A-carrying ^HapII (promoter region does not contain MITE insertion) wheat variety Rouygen 502 as donor parent, carrying TaGS1.1-6A ^HapI (promoter region containing MITE insertion) wheat variety Liangxing 99 as recurrent parent, creating BC ₃ F ₄ Group of plants from the BC ₃ F ₄ The population was used as experimental material.

Nutrient solution culture method

Selecting consistent plump wheat seeds with 10% H ₂ O ₂ Sterilizing for 20min, washing with clear water for several times, placing the seeds uniformly in a culture dish paved with filter paper, adding appropriate amount of saturated CaSO ₄ Carrying out dark germination in an incubator at 23 ℃ for 1 day, transferring the seedlings into a culture tray with a gauze, carrying out tap water culture for 6 days, picking the seedlings with consistent growth, removing endosperm, transferring the seedlings into an 8L water culture box, and continuing culture. The nutrient solution culture experiment is divided into two nitrogen treatment levels of high nitrogen and low nitrogen (the formula of the nutrient solution is shown in a table 2). Each treatment was set to 3 replicatesThe culture medium was changed every 2 days. The culture temperature is as follows: 20 ℃; light period: 15h of illumination/9 h; the illumination intensity is as follows: 405 μmol m ^-2 s ^-1 . Collecting root and overground part samples after 3 weeks of culture to analyze the gene expression quantity of the wheat in the seedling stage.

TABLE 2 wheat hydroponics test nutrient solution formula

Method for detecting TaGS1.1-6A expression quantity in wheat

Detecting the expression quantity of TaGS1.1-6A in different wheat tissues by a fluorescent real-time quantitative PCR (qRT-PCR) method.

1. Extraction of total RNA from wheat

Extracting total RNA of a sample to be detected, detecting the concentration and quality of the RNA, and detecting the purity of the RNA according to the absorbance values at 260nm and 280nm, wherein the ratio of OD260/OD280 of pure RNA is close to 2.0 (the ratio is preferably between 1.9 and 2.1). The quality and size of the RNA were checked by electrophoresis on a 1% agarose gel. mu.L of RNA was aspirated, 3. mu.L of RNase-free water was added, and 1. mu.L of loading buffer was added and denaturation was carried out at 65 ℃ for 5 min. Undegraded RNA bands were intact and non-diffuse.

Preparation of cDNA template

According to eastern China (ReverTra)

qPCR RT Master Mix with gDNA Remover, FSQ-301) reverse transcription kit to carry out reverse transcription to obtain cDNA, and storing or diluting the cDNA by 20 times at-20 ℃ for common PCR or real-time quantitative PCR (qRT-PCR) amplification reaction.

qRT-PCR reaction

qRT-PCR reaction System:

three technical replicates were set for each sample. 2 x SYBR Green I Master (Roche,

480 SYBR Green I Master，4707516001)。

the primers for identifying the relative expression quantity of the TaGS1.1-6A gene are as follows:

Primer F：5′-TGCCGTGGAGAAGCTCAAGCTC-3′；

Primer R：5′-TGCGACGCCCCAGCTGAAGGT-3′。

the internal standard gene (TaActin) primers are as follows:

Primer F：5′-ACCTTCAGTTGCCCAGCAAT-3′；

Primer R：5′-CAGAGTCGAGCACAATACCAGTTG-3′。

a) qRT-PCR reaction program:

95℃，2min；95℃，15s，60℃，15s，72℃，20s，45cycles；95℃，5s，65℃，1min，40℃，20s。

the instrument used for Real-Time PCR was Roche, LightCycler480 II.

After the reaction is finished, the amplification curve and the dissolution curve of qRT-PCR are confirmed, and the specificity of the PCR reaction can be confirmed through analysis of the dissolution curve. Ct mean was calculated. And determining the relative expression quantity of the target gene according to the expression quantity of the corresponding reference gene. The relative expression of the genes is calculated by the formula:

relative expression amount of 2 ^-ΔCt ，△Ct＝Ct _{Target gene} -Ct _{Internal standard gene} 。

The results show that TaGS1.1-6A is carried in the seedling stage of wheat, in the ground parts treated by high nitrogen and low nitrogen and in the root systems treated by low nitrogen ^HapII BC of ₃ F ₄ The expression quantity of the strain TaGS1.1-6A is obviously higher than that of the strain carrying TaGS1.1-6A ^HapI BC of ₃ F ₄ The expression level of the strain, TaGS1.1-6A ^HapII Is obviously related to the higher TaGS1.1-6A expression level of root systems and overground parts, and is particularly shown in figure 2.

2. Field test

"Rugen 502// Liangxing 99" BC ₃ F ₄ The phenotypic identification test of yield and nitrogen utilization related traits of segregating populations was located in northern agricultural science and technology park (Hebei Zhao county) of the institute of agriculture and forestry science and research, Shijiazhuang, Hebei province.

The fertilizer application sets two treatments, high nitrogen and low nitrogen. Applying urea to a nitrogen fertilizer, applying base fertilizer to 8kg of the nitrogen fertilizer per mu in a high-nitrogen treatment process, and topdressing to 4kg of the nitrogen fertilizer per mu in a jointing stage; the low-nitrogen treatment only takes 4kg N/mu as base fertilizer, and no additional application of nitrogen fertilizer is needed. The phosphate fertilizer adopts heavy superphosphate with the dosage of 6kg P ₂ O ₅ Applied as base fertilizer per mu. In the field test, each test material is arranged randomly in sequence, each test material is sown in 2 rows in each repetition, the sowing plant spacing is 5cm, the row spacing is 23cm, and the sowing depth is 3-5 cm.

And collecting flag leaves at heading stages of high nitrogen treatment and low nitrogen treatment to detect the expression quantity of TaGS1.1.

The wheat harvest stage was investigated for biological yield, grain yield, ear number, main ear number and thousand kernel weight. And (3) measuring the total nitrogen concentration of the grains and the straws by using a Kjeldahl nitrogen determination method, and calculating the cumulative amount of nitrogen and the utilization efficiency of the nitrogen according to the weight of dry matters and the nitrogen content.

Straw biomass (biological yield (g/plant) -kernel yield (g/plant)

In field experiments, the "Rugen 502// Liangxing 99" BC was used ₃ F ₄ The segregating population is subjected to phenotype identification on yield and nitrogen utilization related characters under the conditions of high-nitrogen treatment and low-nitrogen treatment, and is subjected to correlation analysis with the genotype of TaGS1.1-6A.

The results show that: under the condition of high nitrogen, carrying TaGS1.1-6A ^HapII BC of ₃ F ₄ The expression quantity of the strain TaGS1.1-6A is obviously higher than that of the strain carrying TaGS1.1-6A ^HapI BC of (a) ₃ F ₄ The expression level of the strain (FIG. 3, panel a); under the condition of low nitrogen, carrying TaGS1.1-6A ^HapII BC of ₃ F ₄ The expression quantity of the strain TaGS1.1-6A is higher than that of the strain TaGS1.1-6AHaving TaGS1.1-6A ^HapI BC of ₃ F ₄ The strains expressed, but the differences did not reach significant levels (FIG. 3, panel b). TaGS1.1-6A ^HapII Significantly correlated with higher kernel yield and kernel number per ear (fig. 4), and significantly correlated with higher nitrogen utilization efficiency (fig. 5).

These results show that the TaGS1.1-6A promoter does not contain MITE insertion, which is beneficial to increasing the expression quantity of TaGS1.1-6A of root systems and overground parts, increasing the grain number per ear and the grain yield under different nitrogen application levels, and increasing the nitrogen utilization efficiency. The TaGS1.1-6A promoter sequence variation can be used for breeding new wheat varieties for improving the nitrogen utilization efficiency and the grain yield of wheat.

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 made possible within the scope of the claims attached below.

Reference to the literature

Lea PJ(1993).Nitrogen Metabolism.In Plant biochemistry and Molecular Biology,P.Lea and R.Leegood,eds(New York:John Wiley&sons),pp.155-180.

Ren YZ,He X,Liu DC,Li JJ,Zhao XQ,Li B,Tong YP,Zhang AM,Li ZS.(2012).Major quantitative trait loci for seminal root morphology of wheat seedlings.Molecular Breeding 30,139-148.

Chengfeng, Ma is Min, Chen Genyun, Humeijun, Shenyou, Li Zhengsheng, Tongyiping, Lishun, Li hongwei Xiaoyan 54 and Jing 411 and their filial generation stabilize dynamic change of photosynthetic property of preferred strain [ J ]. crop science and report, 2009,35(06):1051 one-wall 1058

Liujun liter, Wang Fu Jun, Liupu, a new high-yield high-quality wheat variety-Liang xing 99[ J ] agricultural science and technology communication, 2006(07):60.

Li Xinhua, Li Peng, high national strength, Zhang Fengyun, Sun bright column, Luyuan 502, a new high-yield Guangdong wheat variety, and a breeding strategy thereof [ J ]. Shandong agricultural science, 2013,45(04):32-34.

Sequence listing

<110> institute of genetics and developmental biology of the Chinese academy of sciences

Molecular marker on <120> wheat TaGS1.1-6A promoter and application thereof

<130> GNCSY210028

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3247

<212> DNA

<213> wheat (Triticum aestivum)

<400> 1

tgtggctaga accaaacacc cacctaactt gatcaaactt gactaaggtg aggtgtggca 60

aagtgtgtca atatgtgcct ggaaaccaaa cagcccctat gtgacactat tttattttta 120

ttcggcttgt taaactatgt tgtttatttg gtttgaaact atatgtttgc ttgtgaaaat 180

ttgtgcatat ttgttgaaaa agagcggcca aacgtccacg ccgacaaatg taactcggcg 240

ccttaggcgc accgccgact caaatttcaa atcgagtgga cgccgaacgg gcggtcgacc 300

caaacggaca aaagcaccgt ccgtttaggt cgacgcgttg gagttgctct aagagcaatc 360

ctttttgcga agtgttcaga gacaaagctg aagtaaacgt gcactagaaa tgttcccaat 420

gaaaatagag atacattttc atgtctgata ctccttccgt cccataatgt aatgtgtatc 480

caacagagac tcataaactc tcaccaagat tattgcaaaa gccatccata tttataatat 540

caaatatata ccatgtgaat tgatatattt catgctgaat ccaatgatgt tgattctggt 600

attttggatg ctaattagtt tctctataaa ctcggtcaaa gattacaatg ttttgggacg 660

gagagggtat tattttggat gcattattcc ctccgctcta actcacttgg gagtggatta 720

caaggtggaa gtgttgactc caccgaaata tgagaactcg acagagcggt taggatcaca 780

cgatggggcc accgtctaga tatgagtgtt aacggtggcg gggtcaagag gaacaagcaa 840

aatattaaaa tgaaaagttg agtaatatag atatatgtat tttttggttt tcctcctcct 900

aaaatcagag tcatggcaaa tccttcttct tccttctcga ccttcttctt tttctttctt 960

cttctttctc tttcttcaag atatataatt ttcatatatg tttgtatcgg tttgaaaatt 1020

atttcattgg ttatattggc cctcacattt tcatattact atgatgtttt ttacttataa 1080

cggaggcgta gttgaaggta ctagtggaca aaaaggcgga agtataggca ccaccgaggt 1140

agaagacctc catttagcga ttaggatcat gacgttgagc caccgactga tcgatgacga 1200

caatggaact gagagaaacg aataaaatat aaaacgcgac caagatttca tggtcaatcc 1260

ttctccctcc ttctcgacat cttcttccta gtgtcatcac cgcattctta ggctcgccgt 1320

ataggggcca atccttcttc cttctcagcc atcttcttca tttcatcatc accgcctcat 1380

cctcagtcca accttgcatc atagctaggc actgccttct caggcttgtt atatatgggc 1440

ctttggctcc gatggacgct ccaaccatag ccgctctaaa ccctgacccc taacttcgac 1500

agggtctcct ctttgggact aaagcactcg cacgtggttt cctctcatag catcatcggc 1560

aaatttacgc tcccgcaacc ctaaccgaca ccgatggaat ccaatttttt ttcaaccaca 1620

acaaagaaaa aaaggcaatc agctgaataa taataccccc tgcaaaaaaa agctgaatag 1680

taatatcaaa tgcaaacttt catatatatc gcttgggata aaatctacca ggagtaaccg 1740

attcgggcag atacccgcac cggatcagca gccggcatgc caaccaagat tcacatcaat 1800

caaacgtgga aagataagat cccgcacgga atgcgcaaat gggtcacgtg tgtggtggcg 1860

ctgccgtcgc accagttcat accccagctt tttttttcct gaaccggcga tcgattcacc 1920

gcagctttcc ttttcgctca cctcacgtgc gctccacctc gtcatttccc tcctcgtgac 1980

tcctgcggtt acaaaccccg attttgtggt aattttgaac ttatcaaaaa gcacaccgct 2040

ggagaaagat tcccccggtg cttcaaacgt ggctctccgg agtaataagt actccctccg 2100

tccgggtgta taagtcattc gcgtagttct aggtcatcga tttgagaaat taaatatgtg 2160

ttatatgtca tgaaaagtat accactagat tttcacacgg atgtagtttc taaatatata 2220

ttttttgtca catataatac atatttagat agttaaatta ttgacctaga actacgcgaa 2280

tgacttatac accgggacgg aggtagtact aaaaaaaaga cacggagtcg cttcattttt 2340

gcgcctaata aatctgcaag aatttatgtg tgcacgcaga aatccgcgag attcccctgc 2400

gggtgaagat tctctgaatc tgcacccccc ccccactccc actggcgcat gaggtgggcg 2460

gcattaggtg gaagcatgat taaattccag caaatactca atcaatctat tccagctttg 2520

gactcctctc ccactccaaa gcaaaacgtt gaaaaataaa aaaaagaaaa aaaaactact 2580

actttacttt actttacccc tatgctcgga actgataatc aaatctccca gcagcaccac 2640

accatcgaca aagctgccgc ccgcaacgca ctaaaaaaaa cattttactt atttttttca 2700

gcaacaaaac gcgcgttttc ccagactccg tattccgtac cgtacggctg acagccgggc 2760

ccatgcgccg cgtcggtccc gcaggtcatc gtcgtgggag cgccggcagc gggtgcggga 2820

taggtaacat cgcgcaccta cccccggtcg aacagactgc cggaaataaa atattttatt 2880

tttatcccgc gaaggagacg tcgccacggc ggagtccgca gagagataaa gatatctttg 2940

tgagagagag aaagcgccgc acgtatagcg gcggtacacg gatacgcacc gcgcgcaacg 3000

actttccctg tagcagctgc tcctgccggg tgcgtgctgg gtcgtcgcca ttatatagcc 3060

gccctcccga tctccccttg gcctcgcacc acccccaccc gccttccttc ctgccttcct 3120

gccctcagtc agccggagcc ggattgctcc tcccgccatg gcgctcctca ccgatctcct 3180

caacctcgac ctcaccgact ccacggagaa gatcatcgcc gagtacatat ggtacgtcct 3240

ccttcct 3247

<210> 2

<211> 3022

<212> DNA

<213> wheat (Triticum aestivum)

<400> 2

tgtggctaga accaaacacc cacctaactt gatcaaactt gactaaggtg aggtgtggca 60

gagtgtgtca atatgtggct ggaaaccaaa cagcccctat gtgacactat tttattttta 120

ttcggcttgt taaactatgt tgtttatttg gtttgaaact atatgtttgc ttgtgaaaat 180

ttgtgcatat ttgttgaaaa gagcggccaa acgtccacgc cgacaaatat aactcggcgc 240

cttaggcgca ccgccgactc aaatctcaaa tctagtggac gccgaacggg cggtcgatcc 300

aaacggacaa aagcaccgtc cgtttaggtc gacgcgttgg agttgctcta agagcaaccc 360

tttttgcgaa gtgttcagag acaaagctga agtaaacgtg cactagaaat gtacccaatg 420

aaaatagaga tacattttca tgtctgatac tccttccgtc ccataatgta atgtgtatcc 480

aacggagact cataaactct caccaagatt attgcaaaag ccatccatat ttataatatc 540

aaatatatac catgtgaatt gatatatttc atgctgaatc caatgatgtt gattctggta 600

ttttggatgc taattagttt ctctataaac tcggtcaaag attacaatgt tttgggatgg 660

agagggtatt attttggatg cattattccc tccgctctaa ctcacttggg agtggattac 720

aaggtggaag tgttgactcc accgaaatat gagaactcga cagagcggtt aggatcacac 780

gatggggcca ccgtctagat atgagtgtta acggtggcgg ggtcaagagg aacaagcaaa 840

atattaaaat gaaaagttga gtaatataga tatatgtatt ttttggtttt cctcctccta 900

aaatcagagt catggcaaat ccttcttctt ccttctcgac cttcttcttt ttctttcttc 960

ttcttcctct ttcttcaaga tatataattt tcatatatgt ttgtatcggt ttgaaaatta 1020

tttcattggt tatattggcc ctcacatttt catattacta tgatgttttt tacttataac 1080

ggaggcgtag ttgaaggtac tagtggacaa aaaggcggaa gtataggcac caccgaggta 1140

gaagacctcc atttagcgat taggatcatg acgttgagcc accgactgat cgatgacgac 1200

aatggagctg agagaaacga ataaaatata aaacgcgacc aagatttcat ggtcaatcct 1260

tctccctcct tctcgacatc ttcttcctag cgtcatcacc gcattcttag gctcgccgta 1320

taggggccaa tccttcttcc ttctcagcca tcttcttcat ttcatcatca ccgcctcatc 1380

ctcagtccaa ccttgcatca tagctaggca ctgccttctc aggcttgtca tatatgggcc 1440

tttggctctg atggacgctc caaccatagc cgccctaaac cctgacccct aacttcgaca 1500

gggtctcctc tttgggacta aagcactcgc acgtggtttc ctctcatagc atcatcggca 1560

aatttacgct cccgcaaccc taatcgacac cgatggaatc caattttttt tcaaccacaa 1620

caaagaaaaa aaaggcaatc agctgaataa taataccccc tgcaaaaaaa agctgaatag 1680

taatatcaaa tgcaaacttt catatatatc gcttgggata aaatctacca ggagtaaccg 1740

attcgggcag atacccgcac cggatcagca gccggcatgc caaccaagat tcacatcaat 1800

caaacgtgga aagataagat cccgcacgga atgcgcaaat gggtcacgtg tgtggtggcg 1860

ctgccgtcgc accagttcat accccagctt tttttttcct gaaccggcga cacctttcct 1920

tttcgctcac ctcacgtgcg ctccacctcg tcatttccct cctcgtgact cctgcggtta 1980

caaaccccga ttttgtggta attttgaact tatcaaaaag cacaccgctg gagaaacatt 2040

cccccggtgc ttcaaacgtg gctctccgga gtaataagta ctaaaaaaaa agacacggag 2100

tcgcttaatt tttgcgccta ataaatctgc aagaatttat gtgtgcacgc agaaatccgc 2160

gagattcccc tgcgggtgaa gattctctga atctgcaccc cccccccccc ccactcccac 2220

tggcgcatga ggtgggcggc attaggtgga agcatgatta aattccagca aatactcaat 2280

caatctattc cagctttgga ctcctctccc actccaaagc aaaacgttga aaaataaaag 2340

aaagaaaaaa aactactact ttactttact ttacccctat gctcggaact gataatcaaa 2400

tctcccagca gcaccacacc atcgacaaag ctgccgcccg caacgcacta aaaaaaacat 2460

tttacttatt tttttcagca acaaacgcgc gttttcccag actccgtatt ccgtaccgta 2520

cggctgacag ccgggcccat gcgccgcgtc ggtcccgcag gtcatcgtcg tgggagcgcc 2580

ggcagcgggt gcgggatagg taacatcgcg cacctacccc cggtcgaaca gactaccgga 2640

aataaatatt ttatttttat cccgcgaagg agacgtcgcc acggcggagt ccgcagagag 2700

ataaagatat ctttgtgaga gagagaaagc gccgcgcgta tagcggcggt acacggatac 2760

gcaccgcgcg caacgagttt ccctgtagca gctgctcctg ccgggtgcgt gctgggtcgt 2820

cgccattata tagccgccct cccgatctcc ccttggcctc gcaccacccc cacccgcctt 2880

ccttcctgcc ttcctgcccc cagtcagccg gagccggatt gctcctcccg ccatggcgct 2940

cctcaccgat ctcctcaacc tcgacctcac cgactccacg gagaagatca tcgccgagta 3000

catatggtac gtcctccttc ct 3022

Claims

1. Wheat (Triticum aestivum L.) as one of raw materialsTaGS1.1-6AThe molecular marker is characterized in that the molecular marker is a DNA molecule obtained by taking the genome DNA of wheat to be detected as a template and amplifying A1 by adopting a primer pair; the primer pair A1 consists of single-stranded DNA named P1 and P2; the P1 is shown as 2049 th to 2069 th bits of a sequence 1 in the sequence table or 2038 th to 2058 th bits of a sequence 2 in the sequence tableThe P2 is a single-stranded DNA which is reversely complementary with the sequence shown in 2838 th to 2858 th positions of the sequence 1 or the sequence shown in 2614 th to 2634 th positions of the sequence 2; the wheat to be detected is a homozygous inbred line.

2. The method for identifying the wheat TaGS1.1-6A gene promoter type is characterized in that: the wheat to be detected is a homozygous inbred line, andTaGS1.1-6Athe gene promoter type isTaGS1.1-6A ^HapI OrTaGS1.1-6A ^HapII The method comprises the following K1) and K2):

K1) carrying out PCR amplification on the primer pair A1 in claim 1 by using the genomic DNA of wheat to be detected as a template to obtain a PCR product;

K2) detecting the PCR product obtained in the step K1), and determining the wheat to be detected according to the PCR productTaGS1.1-6AGene promoter types:

the PCR product contains DNA fragment 1 of the wheat to be detectedTaGS1.1-6AThe gene promoter type isTaGS1.1- 6A ^HapI (ii) a The PCR product does not contain the wheat to be detected with the DNA fragment 1TaGS1.1-6AThe gene promoter type isTaGS1.1-6A ^HapII (ii) a The DNA fragment 1 is a DNA fragment from 2094 th to 2310 th site of a sequence 1 in a sequence table.

3. Identification or assisted identification of homozygous wheatTaGS1.1-6AThe expression amount and/or grain yield and/or grain number per ear and/or nitrogen utilization efficiency of (a), characterized in that: the following 1) or 2):

1) method for identifying wheat to be tested according to claim 2TaGS1.1-6AA gene promoter type;

2) according toTaGS1.1-6ADetermination of the type of Gene promoter in wheat to be testedTaGS1.1-6AExpression level and/or grain yield and/or grain number per ear and/or nitrogen utilization efficiency of (a): the describedTaGS1.1-6AThe gene promoter type isTaGS1.1-6A ^HapI Of wheat, withTaGS1.1-6AThe gene promoter type isTaGS1.1-6A ^HapII Compared with the wheat, the wheat flour has the advantages that,TaGS1.1-6Athe expression amount of the nitrogen-containing compound is low, the yield of grains is low, the grain number of grains per spike is small, and the nitrogen utilization efficiency is low.

4. Identification or assisted identificationTaGS1.1-6AThe method for stably inheriting wheat with higher expression amount and/or higher grain yield and/or more grain number per ear and/or higher nitrogen utilization efficiency is characterized in that: including M1) and M2) as follows:

m1) carrying out PCR amplification on the A1 by using the genome DNA of the wheat to be detected as a template and adopting the primer pair A1 as claimed in claim 1 to obtain a PCR product;

m2) detecting the PCR product obtained in the step M1), if the PCR product corresponds to a DNA fragment which does not contain the sequence 1 from the 2094 th to 2310 th sites, the wheat to be detected is or is selected asTaGS1.1-6AThe expression level of the wheat is higher, the grain yield is higher, the grain number per ear is more, and/or the nitrogen utilization efficiency is higher.

5. Identification or assisted identification of homozygous wheatTaGS1.1-6AThe expression amount and/or grain yield and/or grain number per ear and/or nitrogen utilization efficiency of the primer pair, characterized in that: is the primer pair A1 as set forth in claim 1.

6. Identification or assisted identification of homozygous wheatTaGS1.1-6AThe expression amount and/or grain yield and/or grain number per ear and/or nitrogen utilization efficiency of (a), characterized in that: consists of X1 and X2; the X1 is the primer pair A1 as claimed in claim 1, and the X2 is a reagent and/or an instrument required for PCR amplification.

7. Any one of the following applications H1-H14:

h1, the molecular marker of claim 1 in identification or auxiliary identification of wheatTaGS1.1-6AUse in gene promoter types;

h2, the molecular marker of claim 1 in identification or auxiliary identification of wheatTaGS1.1-6AThe expression amount and/or the grain yield and/or the grain number per ear and/or the nitrogen utilization efficiency;

h3, the molecular marker of claim 1 in identification or assisted identificationTaGS1.1-6AThe expression level of the wheat is higher, or the grain yield is higher, or the ear grain number is more, or the nitrogen utilization efficiency is higher;

h4, the use of the molecular marker of claim 1 in wheat breeding;

h5, wheat identified according to claim 2TaGS1.1-6AMethod for identifying or assisting in identifying wheat by using gene promoter typeTaGS1.1-6AThe expression amount and/or the grain yield and/or the grain number per ear and/or the nitrogen utilization efficiency;

h6, wheat identified according to claim 2TaGS1.1-6AMethod for identifying or assisting in identifying gene promoter typesTaGS1.1-6AThe expression level of the wheat is higher, or the grain yield is higher, or the ear grain number is more, or the nitrogen utilization efficiency is higher;

h7, wheat identified according to claim 2TaGS1.1-6AThe application of the gene promoter type method in wheat breeding;

h8, wheat identified or assisted by claim 3TaGS1.1-6AThe expression amount and/or grain yield and/or grain number per ear and/or nitrogen utilization efficiency of the wheat;

h9, the identification or the auxiliary identification of claim 4TaGS1.1-6AThe method for stably inheriting wheat with higher expression quantity and/or higher grain yield and/or more grain number per ear and/or higher nitrogen utilization efficiency is applied to wheat breeding;

h10, the primer pair of claim 5 or the system of claim 6 in preparation of identification or auxiliary identification of wheatTaGS1.1-6AThe expression amount and/or the grain yield and/or the grain number per ear and/or the nitrogen utilization efficiency of the nitrogen-containing compound;

h11, the primer pair of claim 5 or the system of claim 6 in the identification or auxiliary identification of wheatTaGS1.1-6AUse in gene promoter types;

h12, the primer pair of claim 5 or the system of claim 6 in the identification or auxiliary identification of wheatTaGS1.1-6AThe expression amount and/or the grain yield and/or the grain number per ear and/or the nitrogen utilization efficiency;

h13, the primer pair of claim 5 or the system of claim 6 in identification or assisted identificationTaGS1.1-6AThe expression level of the wheat is higher, the grain yield is higher, the grain number per ear is more, and/or the nitrogen utilization efficiency is higher;

h14, the primer pair of claim 5 or the system of claim 6, for use in wheat breeding.

8. A method of breeding wheat characterized by the fact that it is identified according to claim 2TaGS1.1-6AMethod for identifying wheat by gene promoter typeTaGS1.1-6AGene promoter type, selectionTaGS1.1-6AThe gene promoter type isTaGS1.1- 6A ^HapII The wheat of (2) is used as a parent for breeding.