CN108570517B - Specific primer related to Ning-Mai No. 9 low protein of weak gluten wheat and application of specific primer - Google Patents

Specific primer related to Ning-Mai No. 9 low protein of weak gluten wheat and application of specific primer Download PDF

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CN108570517B
CN108570517B CN201810603513.XA CN201810603513A CN108570517B CN 108570517 B CN108570517 B CN 108570517B CN 201810603513 A CN201810603513 A CN 201810603513A CN 108570517 B CN108570517 B CN 108570517B
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primer
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CN108570517A (en
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姜朋
马鸿翔
张旭
吴磊
何漪
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Jiangsu Academy of Agricultural Sciences
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/13Plant traits
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Abstract

The invention provides primers SEQ ID NO.1 and SEQ ID NO.2 related to the low protein of Ningmai No. 9 of weak gluten wheat and application thereof in identification or auxiliary identification of the protein content of weak gluten wheat grains in winter wheat areas in the middle and lower reaches of Yangtze river, wherein the primers SEQ ID NO.1 and SEQ ID NO.2 are respectively used as upstream and downstream primers, a wheat genome is used as a template for carrying out PCR amplification reaction, an amplification product is subjected to enzyme digestion by a restriction endonuclease BtsI and then subjected to agarose electrophoresis detection, and the protein content of wheat sample grains with two bands in the electrophoresis product is obviously lower than that of wheat sample grains with one band in the electrophoresis product; the detection mode is simpler, more convenient and more accurate, and the detection efficiency is higher.

Description

Specific primer related to Ning-Mai No. 9 low protein of weak gluten wheat and application of specific primer
Technical Field
The application relates to the field of wheat breeding, in particular to a specific primer related to a Ning-Mai No. 9 low-protein SNP locus of a weak gluten wheat variety and application thereof.
Background
The protein is an important component of wheat grains, not only influences the nutritional quality of wheat, but also is the basis of the processing quality of wheat. Protein content is a typical quantitative trait, the inheritance of which is controlled by multiple genes, and is susceptible to environmental influences. The traditional breeding method for selecting the protein content has the problems of large workload (measurement after grain harvest), low selection efficiency (environmental influence) and the like, and the molecular marker assisted selective breeding can select the target traits at the DNA level, so that the result is stable, the selection can be performed at the seedling stage, the cost of phenotype evaluation is reduced, and the wheat breeding efficiency is improved.
At present, many reports about protein content molecular markers and QTL positioning are distributed on multiple chromosomes. Wu Yunpeng et al detect QTL for controlling grain protein content on 3A and 3B chromosomes by using a PH 82-2/Neixiang 188 RIL population; shenwu and the like locate QTL for controlling the protein content of grains on 5 chromosomes such as 1B, 2B and the like, and the genetic contribution rate is up to 30 percent; bordes et al correlated 14 molecular markers associated with grain protein content on 15 chromosomes using 372 parts of wheat material from all over the world and 803 molecular markers; jochen et al uses 207 parts of European winter wheat as a material, and associates 115 molecular markers with 4 molecular markers related to grain protein content on 4 chromosomes. Different researchers may adopt different mapping populations and methods, and the obtained results have certain limitations, particularly the characters which are highly influenced by the environment, so that the dominant allelic variation is discovered from the variety material in a certain region, and the molecular marker is further developed, thereby having practical significance for local variety selection.
The Single Nucleotide Polymorphism (SNP) marker has the characteristics of stable heredity, large quantity, wide distribution, easy detection and the like, is suitable for detection and analysis of large quantity, has various SNP chips suitable for different animals and plants in the market at present, and plays an increasingly important role in genetic research. After the marked sites of important characters are found through genetic analysis, the marked sites need to be converted into easy-to-use molecular markers, CAPs (clean Amplified polymorphic sequences) markers based on PCR and enzyme digestion technology are one of solutions, and SNP markers can be distinguished from different allelic variations.
Protein content (< 11.5%) is an important evaluation index for weak gluten wheat. The middle and lower Yangtze river wheat areas are the dominant industrial zones of weak gluten wheat production in China, and a batch of high-quality weak gluten varieties and strains represented by Ningmai No. 9 are accumulated in breeding practice for many years, wherein a large amount of dominant allelic variation is contained, but related molecular markers and specific detection primers thereof are lacked.
The association analysis is an analysis method for identifying the relationship between a target trait and a genetic marker or a candidate gene in a certain population based on linkage disequilibrium. In recent years, this technique has been widely adopted in genetic studies such as wheat yield, quality, disease resistance, and the like. At present, no report is found on the primer research related to the Ningmai No. 9 low-protein SNP locus of the weak gluten wheat variety.
Disclosure of Invention
Aiming at the problems, 101 parts of wheat varieties in winter wheat areas in the middle and lower reaches of Yangtze river, including Ningmai No. 9, are used as materials, a high-throughput gene chip is used for obtaining genotype data, 1 site which is positioned at the end part of a 2D chromosome of the wheat and is related to protein content is detected through correlation analysis and is converted into CAPs marks, a pair of specific primers is obtained, and the primers can preliminarily distinguish the high-low protein content in weak gluten wheat (the protein content of weak gluten wheat grains is not higher than 11.5 percent according to national standards), so that help is provided for quality breeding of the wheat in the middle and lower reaches of the Yangtze river.
In order to realize the purpose, the Shenqi firstly provides a specific primer related to the weak gluten wheat Ningmai No. 9 low protein, the primer consists of an upstream primer GPC-F and a downstream primer GPC-R, the nucleotide sequence of the upstream primer GPC-F is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer GPC-R is shown as SEQ ID NO. 2.
Secondly, the invention also provides application of the specific primers SEQ ID NO.1 and SEQ ID NO.2 in identification or auxiliary identification of the protein content of the wheat grain with weak gluten in the downstream winter wheat area in Yangtze river, namely, the primers SEQ ID NO.1 and SEQ ID NO.2 are respectively used as upstream and downstream primers, a sample wheat genome is used as a template for PCR amplification reaction, an amplification product is subjected to restriction enzyme digestion by a restriction endonuclease BtsI and then subjected to agarose electrophoresis detection, and the protein content of the wheat sample grain with two bands in the electrophoresis product is obviously lower than that of the wheat sample grain with one band in the electrophoresis product.
The specific detection steps are as follows:
1) carrying out PCR amplification reaction by taking SEQ ID NO.1 and SEQ ID NO.2 as primers and taking a wheat genome as a template:
the PCR reaction system is 10 μ L: containing 10 XBuffer 1. mu.L, 15 mmol.L–1MgCl20.6μL、 2mmol· L–1dNTP 0.8μL、20ng· μL–1The upstream primer of (1) is SEQ ID NO. 11. mu.L, 20 ng. mu.L–1The downstream primer of (1) is SEQ ID NO. 21. mu.L, 0.1. mu.L of Taq enzyme, 50 ng. mu.L–13 mu L of template DNA and 3.5 mu L of deionized water;
the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 90s, and 36 cycles; extending for 10min at 72 ℃;
2) carrying out enzyme digestion reaction on the PCR product obtained in the step 1), and carrying out agarose electrophoresis detection on the enzyme digestion product:
the enzyme digestion reaction system is 20 mu L: comprises 1 XNEBuffer 2 muL, restriction enzyme BtsI 0.5 muL, 50 ng/muL PCR product 2 muL, and deionized water 15.5 muL;
and (3) enzyme digestion process: the enzyme was cleaved at 55 ℃ for 15 min.
And if the electrophoresis product has one band or two bands, the wheat sample grain protein content with two bands is obviously lower than that with one band in the electrophoresis product.
In addition, the invention also provides a kit for identifying or assisting in identifying the content of Ningmai No. 9 related wheat grain protein, and the kit comprises specific primers SEQ ID No.1 and SEQ ID No.2 and a restriction endonuclease BtsI.
In the present invention, the technical term "winter wheat zone in the middle and lower reaches of Yangtze river" area scope relates to: the north is the Huaihe river, the west to west mountainous regions and Hunan west hilly areas, the winter to east China, the south to south mountains, most of Jiangsu, Anhui, Hubei and Hunan provinces, Shanghai, Zhejiang, Jiangxi and Henan Xinyang areas (see Chengshun Henhe et al, wheat in south China, Jiangsu scientific and technological Press).
The invention develops specific primers GPC-F and GPC-R of the SNP locus wsnp _ Ex _ c14779_22892053 related to the low protein of Ningmai No. 9 of the high-quality weak gluten wheat variety, can be used for auxiliary identification of the content of the grain protein in Ningmai No. 9 related wheat, and has the advantages of simpler and more accurate detection mode and higher detection efficiency compared with high-throughput molecular detection.
Drawings
FIG. 1 is the electrophoresis chart of the PCR-enzyme digestion product of the wheat sample in example 2;
FIG. 2 is the electrophoresis diagram of the PCR reaction of the target fragment of example 3;
FIG. 3 is the electrophoresis chart of the cleavage product of the PCR reaction product of example 3.
Detailed Description
Example 1 screening of SNP sites related to Nimbin No. 9 Low protein of Weak gluten wheat variety and design of primers
In the embodiment, 101 wheat varieties in winter wheat areas in the middle and lower reaches of the Yangtze river are taken as materials (Table 1), 2015-2016 and 2016-2017 are used for planting all the materials in test bases and Liuhe test bases in the agricultural courtyard of Jiangsu province in two continuous growing seasons, and 3 rows of the plot fields are used for planting. Seeds are respectively harvested in 2016 and 2017 and dried in the air, and the protein content of the seeds is determined according to the GB/T24899-2010 method.
TABLE 1 wheat variety
DNA was isolated from young leaves of test material plants by CTAB method, Genome scanning was performed on the test material using Illumina 9k gene chip (for specific information, see: Cavanagh et al, 2013, Genome-wide compatible coverage areas for selection in genetic animals and cultures), and genotype data was initially processed using Excel 2016.
Finally, 3754 SNP sites with deletion rate of less than 10%, minimum gene frequency of > 5% and genome reference positions are obtained.
Then, the R language program package GAPIT based on the compressed mixed linear model is adopted to perform phenotype-genotype correlation analysis, the effect of each marker is estimated, the marker is considered to be correlated with the target trait when P <0.01, and the marker sites correlated in a plurality of environments are counted.
Finally, a relatively stable protein content-related site wsnp _ Ex _ C14779_22892053 is obtained, which is located at the end of the wheat 2D chromosome and has allelic variation of T/C. In the breeding of weak gluten wheat, T carried by Ningmai No. 9 is dominant allelic variation and has negative regulation and control effect on the protein content of grains.
Further developing CAPs markers aiming at the site wsnp _ Ex _ c14779_22892053, designing an upstream primer and a downstream primer as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively:
upstream primer GPC-F (SEQ ID NO. 1): 5'-GCACTTCCACCATGAACTCCTTCTC-3', respectively;
downstream primer GPC-R (SEQ ID NO. 2): 5'-TGCGGTATCTCCACAACCTCCAA-3' are provided.
Example 2 wheat genotype detection
The detection steps are as follows:
24 varieties (shown in table 2) are randomly selected from the 101 test materials described in example 1 to be subjected to PCR amplification and enzyme digestion reaction in sequence, the enzyme digestion products are subjected to agarose electrophoresis detection, the detection result is shown in table 1, and the detection result is compared with the detection result of the gene chip.
TABLE 2 detection materials
1. PCR reaction
The PCR reaction system was 10. mu.L, containing 10 XBuffer 1. mu.L, 15 mmol. multidot.L–1MgCl20.6 μL、2mmol· L–1dNTP 0.8μL(Takara)、20ng· μ L–11. mu.L of each of the upstream and downstream primers, 0.1. mu.L of Taq enzyme (Takara), and 50 ng. mu.L of each of the upstream and downstream primers–13 mu L of template DNA (template extracted by CTAB method) and 3.5 mu L of deionized water;
the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 90s, and 36 cycles; extending for 10min at 72 ℃;
2. enzyme digestion reaction
The PCR product is cut by restriction enzyme BtsI, the cutting reaction system is 20 mu L, and the cutting reaction system comprises 1 XNEBuffer 2 mu L, restriction enzyme BtsI 0.5 mu L, 50 ng/mu L PCR product 2 mu L and deionized water 15.5 mu L;
the system is placed at 55 ℃ for enzyme digestion reaction for 15 min.
And (3) carrying out 1% agarose electrophoresis detection on the enzyme digestion product, wherein the detection result is shown in figure 1, and the material names of lanes 1-24 in figure 1 correspond to those in table 2 one by one. Lanes 3, 13, 21 and 22 in FIG. 1 are not cut, and their allelic variation is C, and the rest is T, which is completely consistent with the chip detection result, further verifying the above primer application.
Example 3 detection of protein content correlation in Ningmai No. 9 advanced Breeding Material
From the advanced generation breeding population (generation F7), the parents all have ningli wheat No. 9, and the combination information, phenotype and genotype are shown in table 3:
TABLE 3 names of materials of high-generation breeding populations
The detection method comprises the following steps:
1. PCR reaction
The PCR reaction system was 10. mu.L, containing 10 XBuffer 1. mu.L, 15 mmol. multidot.L–1MgCl20.6 μL、2mmol· L–1dNTP 0.8μL(Takara)、20ng· μL–11. mu.L of each of the upstream and downstream primers, 0.1. mu.L of Taq enzyme (Takara), and 50 ng. mu.L of each of the upstream and downstream primers–13 mu L of template DNA (CTAB method) and 3.5 mu L of deionized water;
the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 90s, and 36 cycles; extending for 10min at 72 ℃;
the PCR amplification product is detected by 1% agarose electrophoresis, and the size of the obtained target fragment is 253bp (FIG. 2), and in FIG. 2, the names of materials in lanes 1-12 are shown in Table 2 in sequence.
2. Enzyme digestion reaction
The PCR product is cut by restriction enzyme BtsI, the cutting reaction system is 20 mu L, and the cutting reaction system comprises 1 XNEBuffer 2 mu L, restriction enzyme BtsI 0.5 mu L, 50 ng/mu L PCR product 2 mu L and deionized water 15.5 mu L;
the system is placed at 55 ℃ for enzyme digestion reaction for 15 min.
The enzyme digestion product was detected by 1% agarose electrophoresis, and the detection results are shown in FIG. 3, and in FIG. 3, the names of materials in lanes 1-12 are shown in Table 3.
Example wheat material base sequences were 7 parts of T and 5 parts of C, and the results of the T test showed that the grain protein content of the 7 parts of material with allelic variation T was significantly lower than that of the 5 parts of material with C and reached a very significant level (table 4).
TABLE 4
The recognition sequence of the enzyme BtsI isGCAG<T>GTherefore, when the base sequence of the SNP is T, the PCR product is cleaved, and when it is C, it is not cleaved. Theoretically, if the enzyme is completely cleaved, the 253bp PCR product will be cleaved into two bands of 119bp +134bp, but the 253bp band will not exist. In practical practice, the applicant selects agarose electrophoresis which is simple in operation but low in resolution, cannot separate two bands of 119bp and 134bp, and the enzyme digestion is incomplete, but a material with a base sequence of SNP T is still incompletely digested, two bands are shown (lanes 1-7 in FIG. 3), and a material with a base sequence of C is only one band (lanes 8-12 in FIG. 3), so that two allelic variations are distinguished.
The above examples illustrate that primers SEQ ID No.1 and SEQ ID No.2 designed according to SNP site wsnp _ Ex _ c14779_22892053 at 2D chromosome end of wheat genome can be used for weak gluten wheat breeding, when agarose electrophoresis results show two bands, the SNP site at 2D chromosome end of wheat material is indicated to carry T genotype, which has negative regulation and control effect on grain protein content; when the agarose electrophoresis result shows a band, the method has positive regulation and control effect on the protein content of grains.
Sequence listing
<110> agricultural science and academy of Jiangsu province
<120> specific primer related to Ningmai No. 9 low protein of weak gluten wheat and application thereof
<141> 2018-06-12
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gcacttccac catgaactcc ttctc 25
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgcggtatct ccacaacctc caa 23

Claims (1)

1. An application of a specific primer related to the low protein of Ningmai No. 9 of weak gluten wheat in identifying or assisting in identifying the protein content of weak gluten wheat grains in winter wheat areas at the middle and lower reaches of Yangtze river;
the primer consists of an upstream primer GPC-F and a downstream primer GPC-R, wherein the nucleotide sequence of the upstream primer GPC-F is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer GPC-R is shown as SEQ ID NO. 2;
the application comprises the following specific steps:
respectively taking SEQ ID NO.1 and SEQ ID NO.2 as upstream and downstream primers, taking a wheat genome as a template to perform PCR amplification reaction, carrying out restriction enzyme BtsI enzyme digestion on an amplification product, and then carrying out agarose electrophoresis detection, wherein the content of wheat sample seed protein with two bands of 119bp and 134bp in the electrophoresis product is obviously lower than that of wheat sample seed protein with one band of 253bp in the electrophoresis product;
the winter wheat area in the middle and lower reaches of the Yangtze river refers to: the areas of the northern Yanghe, the west to the west of the Hubei mountain areas, the Hunan hilly areas, the winter to the east China and the south to the south of the Yangtze river contain most of Jiangsu, Anhui, Hubei and Hunan provinces, and the Shanghai, Zhejiang, Jiangxi and Henan Xinyang areas.
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