CN102382895A - PCR system for identifying or assisting in identifying genes of wheat for Sinkiang stretched noodle quality - Google Patents
PCR system for identifying or assisting in identifying genes of wheat for Sinkiang stretched noodle quality Download PDFInfo
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Abstract
The invention discloses a PCR (Polymerase Chain Reaction) system for identifying and assisting in identifying genes of wheat for Sinkiang stretched noodle quality and application thereof. Primer pair groups of the PCR system of the invention are 1) or 2): 1) a primer pair group A and a primer pair group B; 2) the primer pair group A; three primer pairs of the primer pair group A sequentially consist of two DNA single strands shown by a sequence 1 and a sequence 2, two DNA single strands shown by a sequence 3 and a sequence 4, and two DNA single strands shown by a sequence 5 and a sequence 6; three primer pairs of the primer pair group B sequentially consist of two DNA single strands shown by a sequence 7 and a sequence 8, two DNA single stands shown by a sequence 9 and a sequence 10, and two DNA single strands shown by a sequence 11 and a sequence 12. The primer pairs of the groups of the primer pair groups are not mutually restrained and mispaired, so that a group of genes can be identified by the PCR at a time at the same temperature, in addition, the results are reliable, the repeatability is good, the specificity is strong, the cost is low, the time consumption is short, and evidences are provided for seed selection of excellent wheat and improvement of processing quality.
Description
Technical field
The present invention relates to a kind ofly be used to identify or the PCR system and the application thereof of assistant identification wheat hand-pulled noodles quality trait gene, particularly a kind of be used to identify or the multiple PCR primer of the hand-pulled noodles color and luster trait related gene of assistant identification Winter Wheat in Xinjiang to be measured and gluten quality trait genes involved to group, test kit and application thereof.
Background technology
Noodles are traditional foods of northern China resident, are one of daily staple foods of people.In 2000 years of development processes, noodles have formed its characteristic form in different regions.In Xinjiang region, hand-pulled noodles is one of local daily staple food, is loved by the people.As the wet white salt noodles of aquatic foods, the Xinjiang hand-pulled noodles is with its making, instant, and the mouthfeel bullet is tough, smooth, and plurality of advantages such as botargo is abundant, delicious food are walked out Xinjiang region gradually, is accepted and likes by the people of all parts of the country.According to statistics, 80% of the YO of Xinjiang region whole meal flour is used to make hand-pulled noodles.But, to compare with the status that the Xinjiang hand-pulled noodles becomes more and more important in people's diet, the method that the evaluation of Xinjiang hand-pulled noodles quality still combines with traditional instrument analysis and finished product sensory evaluation is main, has directly limited its development.Therefore, create trace, high-quality hand-pulled noodles competition method fast, significant for processing, the evaluation of cultivation, seed selection and the high-quality hand-pulled noodles of hand-pulled noodles specific breed.
In recent years, along with growth in the living standard, people require increasingly highly to Xinjiang hand-pulled noodles quality, and correlation of attributes research has obtained paying attention to.Research at present mainly concentrates on the screening of quality article indexs such as the abrasive dust relevant with the high-quality hand-pulled noodles, protein, starch, dough rheological characteristics, the seed selection aspect of the improvement of evaluation method and hand-pulled noodles specific breed.Because Xinjiang hand-pulled noodles quality constituent element is complicated, relative index is more, so pass through the research of Xinjiang hand-pulled noodles method for evaluating quality, the index that filters out effectively is applied to its quality evaluation, becomes the key link of Xinjiang hand-pulled noodles quality-improving.
Xinjiang hand-pulled noodles evaluation index mainly comprises noodle color and sensory evaluation two portions.Noodle color is except that the influence that receives milling characters indexs such as wheat flour extraction rate, protein contnt and flour particle index; Yellow pigment (YP) content and polyphenoloxidase (PPO) are active also to have remarkably influenced (Hu Fengling to it; He Zhonghu; The Markers for Detection of wheat breed Yellow pigment content such as Ge Jiangui and polyphenol oxidase activity gene. wheat crops journal [J] .2011,31 (1): 47-53.).According to judgement criteria (Mu Peiyuan; Sang Wei, Wang Liang, etc. processing quality characteristic and tailored flour quality evaluation Study of indexes thereof that the commercially available whole meal flour in Xinjiang is made the Xinjiang hand-pulled noodles. wheat crops journal [J] .2007; 27 (6): 1034-1041.); Hand-pulled noodles best color and luster in high-quality Xinjiang is a brilliant white, and the active low kind of low YP content and PPO has whiteness and color proterties preferably.Seed or flour YP content mainly receive phytoene synthase (PSY) effect gene; Whiteness high negative correlation (Kruger J E, Matsuo R R, Presten K.A comparison of methods for the prediction of Cantonese noodle colour [J] .Canadian Journal of Plant Science with flour and dough; 1992; 72:1021-1029.), degree high-positive correlation yellow with it, low YP content gene type is Psy-A1b and Psy-B1b; The PPO activity mainly receives the Ppo effect gene; Can explain 50%～70% of noodles (group) browning variation; Be major cause (the Kruger J E that causes noodles (group) color browning; Hatcher D W, De Pauw R.A whole seed assay for polyphenol oxidase in Canadian prairie spring wheats and its usefulness as a measure of noodle darkening [J] .Cereal Chemistry, 1994; 71:324-326.), seed or flour PPO low activity genotype are Ppo-A1b and Ppo-D1a.Therefore, to the evaluation of low YP content and PPO low activity target gene type, can be used as the method for Xinjiang hand-pulled noodles color and luster fast prediction.Psy-A1a is high Yellow pigment content genotype, and Psy-A1b is low Yellow pigment content genotype, and Psy-A1c genotype kind is rarer, and wherein Psy-A1b helps improving the whiteness to the Xinjiang hand-pulled noodles, is hand-pulled noodles high-quality gene.The Ppo-D1a in PpoD1 site is the active genotype of medium low PPO, can slow down the noodles browning.
The hand-pulled noodles sensory evaluation mainly comprises projects such as hand-pulled noodles feel, noodles quality, mouthfeel.Research shows; Quality traits such as protein properties, starch property and grain hardness; Can determine sensory evaluation result (Yun S H, Quail K, Moss R.Physicochemical properties of Australian wheat flours for white salted noodles [J] the .Journal of Cereal Sciences of hand-pulled noodles through the influence of dough rheological properties and starch pasting characteristic to a certain extent; 1996,23:181-189.).
Proteinic quantity and quality have material impact to noodle quality; Its quality mainly is meant the formation and the ratio of prolamine and glutenin in the flour; Different protein constitutes has given wheat grain protein different quality; Wherein, the composition of high-molecular-weight glutelin subunit (HMW-GS) and low-molecular-weight glutenin subunit (LMW-GS) and the visco-elasticity and the extensibility of dough are closely related, have determined the quality of noodles to a certain extent.The flour that is fit to Xinjiang hand-pulled noodles making is generally middle muscle, and has extensibility preferably.Lu waits the people quietly and discovers (Lu Jing; Huang Tianrong; Nie Li wheat high-molecular-weight glutelin subunit and 1B/1R translocation line are to the influence [J] of Xinjiang hand-pulled noodles quality trait. Xinjiang agricultural sciences, 2010,47 (10): 1909-1917.); HMW-GS 7+8,5+10,2+10 etc. have the forward effect to the quality of Xinjiang hand-pulled noodles, but the high-quality HMW-GS and the high-quality LMW-GS in Glu-A1 site are not explained.The HMW-GS of high-quality hand-pulled noodles kind and the Markers for Detection of LMW-GS are found, except that the high-quality hand-pulled noodles subunit type of having found, 2
*With subunit types such as Glu-B3a the quality of Xinjiang hand-pulled noodles also there is certain forward effect.The Ax2 in Glu-A1 site
*2 of genetic expression
*Subunit is through improving gluten strength (Payne P I; Lawrence G J.Catalogue of alleles for the complex gene loci, Glu-A1, Glu-B1; And Glu-D1 which code for high molecular weight subunits of glutenin in hexaploid wheat [J] .Cereal Research Communications; 1983,11:29-35.), the hand-pulled noodles quality there is certain contribution.In addition, the 1B/1R transposition also has remarkably influenced to the protein characteristic of wheat.The 1B the short arm of a chromosome of wheat is replaced and formation wheat-rye 1B/1R translocation line by the 1R the short arm of a chromosome of rye.Transposition makes LMW-GS on the common wheat 1B the short arm of a chromosome (Glu-B3 site) and prolamine (Gli-B1 site) disappearance; Or replaced by the secaline of inferior quality (secalin genes encoding); Cause the increase of dough viscosity, intensity to reduce; Dough rheological properties and noodle quality all have extremely significant negative impact (Wieser H, Kieffer R, Lelley T.The influence of 1B/1R chromosome translocation on gluten protein composition and technological properties of bread wheat [J] .J Sci Food Agric; 2000,80:1640-1647.).Lu Jing (Nie Li wheat high-molecular-weight glutelin subunit and 1B/1R translocation line are to the influence [J] of Xinjiang hand-pulled noodles quality trait for Lu Jing, Huang Tianrong. the Xinjiang agricultural sciences; 2010; 47 (10): 1909-1917.) grade is discovered, compares with non-1B/1R translocation line (not containing ω-secalin gene), and each item processing quality of 1B/1R translocation line (containing ω-secalin gene) all significantly reduces; Can not satisfy the requirement that the high-quality hand-pulled noodles is made mostly, in the hand-pulled noodles quality breeding of Xinjiang, should eliminate.
The major gene puroindoline of grain hardness is positioned at the 5D the short arm of a chromosome, is divided into two types of Pina and Pinb, and its Different Variation has significant effects to wheat milling characters and food-processing quality.(Chen Feng, Shang Xiaoli, Dong Zhongdong such as Chen; Deng. the variation of the historical kind puroindoline of Chinese wheat allele detects [J]. the wheat crops journal; 2011,31 (3): 389-394) research shows that Pinb-D1b is higher than Pina-D1b type flour extraction rate, ash is low; Have better milling characters, the former steamed bun, noodles and bread processing quality also all are superior to Pina-D1b and wild-type simultaneously.
The traditional evaluation method of Xinjiang hand-pulled noodles needs multiple analytical instrument, amount of samples is big, the mensuration cycle is long, and detection when being difficult to realize lot of materials, a plurality of index, has restricted the quality-improving and the evaluation of Xinjiang hand-pulled noodles.Along with the clone of wheat processing correlation of attributes gene and the exploitation of molecule marker thereof, utilize Auele Specific Primer that the article plasmagene is carried out the method that fast PCR detects and be used widely.But the quality-improving of Xinjiang hand-pulled noodles relates to a plurality of genes, and traditional P CR method generally once can only detect one or a few gene, is difficult to satisfy seed selection and follow-up screening thereof, the processing of hand-pulled noodles specific breed, the demand of evaluation.
Summary of the invention
The purpose of this invention is to provide be used to identify or the multiple PCR primer of assistant identification wheat hand-pulled noodles to be measured quality trait genes involved to group, test kit, and utilize said multiple PCR primer group to be identified or the method for assistant identification wheat hand-pulled noodles to be measured quality trait.
Provided by the present inventionly be used to identify or the multiple PCR primer of assistant identification wheat hand-pulled noodles to be measured quality trait genes involved is following a1 to group) or primer a2) to group:
A1) primer of group A and primer being made up of group B primer is to organizing;
A2) primer is to group A;
To all being specific to hand-pulled noodles color and luster genes involved, specifically right by two primers that are specific to a Psy-A1 gene and primer being specific to the Ppo-D1 gene is to forming to the primer of group among the A for said primer; Said primer to the primer of group among the B to all being specific to hand-pulled noodles gluten strength, gluten quality and milling characters genes involved, specifically by being specific to Ax2
*A primer of gene is right, is specific to the right and primer being specific to ω-secalin gene of a primer of Pinb-D1b gene to forming;
Said primer is among the group A, and to 1, and the primer that sequence 3 and two single stranded DNAs shown in the sequence 4 are formed is to 2 to the primer that is respectively sequence in the sequence table 1 and two single stranded DNAs shown in the sequence 2 and forms for two primers of the said Psy-A1 of being specific to gene; A said primer that is specific to the Ppo-D1 gene to the primer formed for sequence in the sequence table 5 and two single stranded DNAs shown in the sequence 6 to 3;
Said primer is to organizing among the B the said Ax2 that is specific to
*A primer of gene to the primer formed for sequence in the sequence table 7 and two single stranded DNAs shown in the sequence 8 to 4; A said primer that is specific to the PinbD1b gene to the primer formed for sequence in the sequence table 9 and two single stranded DNAs shown in the sequence 10 to 5; A said primer that is specific to ω-secalin gene to the primer formed for sequence in the sequence table 11 and two single stranded DNAs shown in the sequence 12 to 6.
Wherein, sequence 1 is made up of 20 Nucleotide; Sequence 2 is made up of 22 Nucleotide; Sequence 3 is made up of 20 Nucleotide; Sequence 4 is made up of 20 Nucleotide; Sequence 5 is made up of 20 Nucleotide; Sequence 6 is made up of 20 Nucleotide; Sequence 7 is made up of 21 Nucleotide; Sequence 8 is made up of 20 Nucleotide; Sequence 9 is made up of 21 Nucleotide; Sequence 10 is made up of 18 Nucleotide; Sequence 11 is made up of 20 Nucleotide; Sequence 12 is made up of 20 Nucleotide.
Among the present invention, said Psy-A1 gene has three allelotrope, is respectively Psy-A1a, Psy-A1b and Psy-A1c; Said Ppo-D1 gene has two allelotrope, is respectively Ppo-D1a and Ppo-D1b.
Said primer uses at PCR reaction system moderate right two primers of each primer among the group A, and said primer is to 1, said primer to 2 with said primer be 1: 4: 1 to 3 mol ratios in the PCR reaction system; Said special primer uses at PCR reaction system moderate right two primers of each primer among the group B, and said primer is to 4, said primer to 5 with said primer be 1: 1: 1 to 6 mol ratios in the PCR reaction system.
Contain and saidly be used to identify or the multiple PCR primer of assistant identification wheat hand-pulled noodles to be measured quality trait genes involved also belongs to protection scope of the present invention to the test kit of group.
Saidly be used to identify or the multiple PCR primer of assistant identification wheat hand-pulled noodles to be measured quality trait genes involved also belongs to protection scope of the present invention to the preparation method of group.This preparation method specifically can comprise with said be used for identifying or the multiple PCR primer of assistant identification wheat hand-pulled noodles to be measured quality trait genes involved to right said two single stranded DNAs of each primer of the group step of packing separately respectively.
The preparation method of said test kit also belongs to protection scope of the present invention.This preparation method specifically can comprise the steps: with said be used for identifying or the multiple PCR primer of assistant identification wheat hand-pulled noodles to be measured quality trait genes involved to right said two single stranded DNAs of each primer of group respectively separately after the packing, and at least a being packaged in the same test kit in the following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
Identify or assistant identification wheat to be measured contain any in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c, contain in Ppo-D1a and these two kinds of genes of Ppo-D1b anyly, and whether contain Ax2
*At least a method in these three kinds of genes of gene, Pinb-D1b gene and ω-secalin gene also belongs to protection scope of the present invention.
This method comprises the step of following (1) and (2):
Said (1) is following A) and B):
A) genomic dna with wheat to be measured is a template, with said primer group A is carried out the PCR reaction, and the annealing temperature of said PCR reaction is 60 ℃;
B) genomic dna with wheat to be measured is a template; With said primer group B is carried out the PCR reaction; The annealing temperature of said PCR reaction is from 62 ℃; Whenever carry out 1 PCR reaction cycle and descend 0.1 ℃, in an embodiment of the present invention, group B is carried out 35 circulations of PCR reaction having carried out altogether with said primer.
(2) detect the size of the PCR product that step (1) obtains; According to following method according to the PCR product confirm said wheat to be measured contain any in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c, contain in Ppo-D1a and these two kinds of genes of Ppo-D1b anyly, and whether contain Ax2
*At least a in these three kinds of genes of gene, Pinb-D1b gene and ω-secalin gene:
With said primer group A is carried out in the product of PCR reaction, if contain two dna fragmentations of size for 1686bp and 194bp simultaneously, said wheat to be measured contains the Psy-A1a gene or the candidate is contained the Psy-A1a gene; If contain two dna fragmentations of size for 1686bp and 231bp simultaneously, said wheat to be measured contains the Psy-A1b gene or the candidate is contained the Psy-A1b gene; If contain two dna fragmentations of size for 1001bp and 194bp simultaneously, said wheat to be measured contains the Psy-A1c gene or the candidate is contained the Psy-A1c gene; Be the dna fragmentation of 713bp if contain size, said wheat to be measured contains the Ppo-D1a gene or the candidate is contained the Ppo-D1a gene; Be not the dna fragmentation of 713bp if do not contain size, said wheat to be measured contains the Ppo-D1b gene or the candidate is contained the Ppo-D1b gene;
In the product that carry out PCR reaction of said primer to group B, be the dna fragmentation of 1319bp if contain size, said wheat to be measured contains Ax2
*Gene or candidate are contained Ax2
*Gene; Be not the dna fragmentation of 1319bp if do not contain size, said wheat to be measured does not contain Ax2
*Gene or candidate are not contained Ax2
*Gene; Be the dna fragmentation of 250bp if contain size, said wheat to be measured contains the Pinb-D1b gene or the candidate is contained the Pinb-D1b gene; Be not the dna fragmentation of 250bp if do not contain size, said wheat to be measured does not contain the Pinb-D1b gene or the candidate is not contained the Pinb-D1b gene; Be that the dna fragmentation of 1067bp, said wheat to be measured contain ω-secalin gene or the candidate is contained ω-secalin gene if contain size; Be not that the dna fragmentation of 1067bp, said wheat to be measured do not contain ω-secalin gene or the candidate is not contained ω-secalin gene if do not contain size.
Identify or assistant identification wheat to be measured contains in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c anyly, and contain that any method also belongs to protection scope of the present invention in Ppo-D1a and these two kinds of genes of Ppo-D1b.
This method comprises the step of following (1) and (2):
(1) genomic dna with wheat to be measured is a template, with said primer group A is carried out the PCR reaction, and the annealing temperature of said PCR reaction is 60 ℃;
(2) detect the size of the PCR product that step (1) obtains; It is any to confirm that according to the PCR product said wheat to be measured contains in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c according to following method, and contains in Ppo-D1a and these two kinds of genes of Ppo-D1b any:
With said primer group A is carried out in the product of PCR reaction, if contain two dna fragmentations of size for 1686bp and 194bp simultaneously, said wheat to be measured contains the Psy-A1a gene or the candidate is contained the Psy-A1a gene; If contain two dna fragmentations of size for 1686bp and 231bp simultaneously, said wheat to be measured contains the Psy-A1b gene or the candidate is contained the Psy-A1b gene; If contain two dna fragmentations of size for 1001bp and 194bp simultaneously, said wheat to be measured contains the Psy-A1c gene or the candidate is contained the Psy-A1c gene; Be the dna fragmentation of 713bp if contain size, said wheat to be measured contains the Ppo-D1a gene or the candidate is contained the Ppo-D1a gene; Be not the dna fragmentation of 713bp if do not contain size, said wheat to be measured contains the Ppo-D1b gene or the candidate is contained the Ppo-D1b gene.
Another object of the present invention provides a kind of method of cultivating the hand-pulled noodles wheat breed.
This method comprise adopt following a)-e) at least a wheat step of carrying out breeding as the parent:
A) contain or the candidate is contained the Psy-A1b gene;
B) contain or the candidate is contained the Ppo-D1a gene;
C) contain or the candidate is contained Ax2
*Gene;
D) contain or the candidate is contained the Pinb-D1b gene;
E) do not contain or the candidate is not contained ω-secalin gene.
From numerous wheat breeds, select to satisfy a)-e) at least one method of wheat; Be above-mentioned evaluation or assistant identification wheat to be measured contain any in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c, contain in Ppo-D1a and these two kinds of genes of Ppo-D1b anyly, and whether contain Ax2
*At least a method in these three kinds of genes of gene, Pinb-D1b gene and ω-secalin gene.It is many more that selected wheat satisfies above-mentioned a)-e) item number in five, representes the suitable more making hand-pulled noodles of selected wheat.
In an embodiment of the present invention; Said wheat to be measured is specially Winter Wheat in Xinjiang, like new winter of wheat breed (being) No. 16, new winter No. 20, Ji wheat 26, lay down 54 for a short time, crust winter No. 2, new Ukraine 83, new Ukraine 84, north are 11, big by 1885, new winter of swallow No. 24, new winter No. 27, new winter No. 28, Kui winter No. 4, Ji wheat 24, Ji wheat 31, stone winter No. 8,89-2012, new winter No. 22, new winter No. 23, ligusticumic are excellent 8901, No. 18, HD04-23,79-18, new winter No. 14, new winter, 85 (1), 89 (35), 80182, flower 91-26 and 89-44 etc.Utilize the present invention that above-mentioned wheat breed (being) is identified or assistant identification after; Find that wheat breed Ji wheat 31 has hand-pulled noodles color and luster high-quality gene Psy-A1b and Ppo-D1a simultaneously, the wheat breed stone winter has hand-pulled noodles gluten strength, gluten quality and milling characters high-quality Gene A x2 No. 8
*In the time of with Pinb-D1b, do not contain the ω-secalin gene of influential hand-pulled noodles quality again.
Primer provided by the present invention to group A or primer to each primer of group B between do not have mutual restraining effect and mispairing, detect reliable results, the good reproducibility of kind (being), cost is low.Primer provided by the present invention is all selected same annealing temperature to group A or primer for use to group B, has realized that PCR of uniform temp accomplishes the discriminating of one group of gene, and high specificity, testing process weak point consuming time.The present invention is contained primer group A or primer are used for the parent's evaluation of Wheat Breeding for Quality and the polymerization of filial generation high-quality gene to the multiplex PCR system of organizing B; Will improve the efficient of high-quality hand-pulled noodles wheat special variety evaluation and seed selection, accelerate the processing quality improvement of hand-pulled noodles wheat special kind.
Description of drawings
Fig. 1 is the multiplex PCR agarose gel electrophoresis figure of Psy-A1a, Psy-A1b, Psy-A1c and Ppo-D1a genetic marker.
Fig. 2 is Ax2
*, Pinb-D1b and ω-secalin genetic marker multiplex PCR agarose gel electrophoresis figure.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The multiplex PCR of embodiment 1, hand-pulled noodles color and luster genes involved detects
The multiple PCR primer of a present embodiment and primer being specific to Ppo-D1 gene right by two primers that are specific to the Psy-A1 gene to group is to forming; Wherein, to 1, and the primer that sequence 3 and two single stranded DNAs shown in the sequence 4 are formed is to 2 to the primer that is respectively sequence in the sequence table 1 and two single stranded DNAs shown in the sequence 2 and forms for two primers of the said Psy-A1 of being specific to gene; A said primer that is specific to the Ppo-D1 gene to the primer formed for sequence in the sequence table 5 and two single stranded DNAs shown in the sequence 6 to 3.
The multiple PCR primer of present embodiment can detect 5 kinds of genotype Psy-A1a, Psy-A1b, Psy-A1c, Ppo-D1a and Ppo-D1b of Psy-A1 and two gene locuss of Ppo-D1 simultaneously to group.The material that contains the Psy-A1a gene, PCR amplify two bands of 1686bp and 194bp simultaneously; The material that contains the Psy-A1b gene, PCR amplify two bands of 1686bp and 231bp simultaneously; The material that contains the Psy-A1c gene, PCR amplify two bands of 1001bp and 194bp simultaneously; The material that contains the Ppo-D1a gene, pcr amplification go out the band of 713bp; The material that contains the Ppo-D1b gene, pcr amplification do not have the band of 713bp.
One, the multiplex PCR of known type wheat breed hand-pulled noodles color and luster genes involved amplification
1, the preparation of wheat cdna group
Employing CTAB method is carried out the extraction of genomic dna to the wheat breed (being) of 4 parts of known types, obtains the genomic dna corresponding to the wheat breed (being) of 4 parts of known types, as the template of hand-pulled noodles color and luster genes involved multiplex PCR amplification.Wherein, the wheat breed of 4 parts of known types (being) is new winter No. 16, new winter No. 20, Ji wheat 26 and lays down 54 for a short time, (Wang Liang such as Wang Liang; Mu Peiyuan, Xu Hongjun, etc. the Molecular Detection [J] of Xinjiang wheat breed Yellow pigment content gene (Psy-A1) allelic variation. the wheat crops journal; 2009,29 (4): 782-786. Wang Liang, Mu Peiyuan; Xu Hongjun, etc. the distribution [J] of polyphenoloxidase in the wheat breed of Xinjiang (PPO) active gene allelic variation. wheat crops journal, 2008; 28 (5): 766-771.) Psy-A1 and the Ppo-D1 gene genotype of these four wheat breeds (being) have been carried out Markers for Detection, seen table 1 for details.
The genotype of table 1 known type wheat breed (being) hand-pulled noodles color and luster genes involved
2, multiplex PCR amplification wheat breed hand-pulled noodles color and luster genes involved
Genomic dna with the wheat breed (being) of 4 parts of known types of above-mentioned steps 1 preparation is respectively template, utilizes the primer in the table 2 that compsn is carried out the multiplex PCR amplification.
It is 20 μ L that PCR reacts total system; Wherein, Dna profiling 100-200ng; MasterMix (containing archaeal dna polymerase, PCR reaction buffer, 4 kinds of dNTP) (Beijing health is the century bio tech ltd) 10 μ L (making that the concentration of every kind of dNTP is 200 μ M in the PCR reaction system of 20 μ L), each primer at the final concentration that PCR reacts in total system is: the sequence 1 of primer in to 1 is 0.05 μ M with two dna single chains shown in the sequence 2 at the final concentration that PCR reacts in total system; Primer is 0.2 μ M with two dna single chains shown in the sequence 4 at the final concentration that PCR reacts in total system to the sequence 3 in 2; Primer is 0.05 μ M with two dna single chains shown in the sequence 6 at the final concentration that PCR reacts in total system to the sequence 5 in 3.
The PCR response procedures is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 35s, 60 ℃ of annealing 35s, 72 ℃ are extended 1min30s, and amplified reaction carries out 37 circulations; 72 ℃ are extended 8min; 4 ℃, insulation finishes.
Pcr amplification carries out on PTC-200 type PCR appearance, carries out electrophoresis with 1 * TAE at 2% sepharose, and the voltage during electrophoresis is 150V, and electrophoresis time is 40min, and electrophoresis finishes through under gel imaging system, observing photograph behind the ethidium bromide staining.
Table 2 hand-pulled noodles color and luster genes involved amplimer sequence and expection amplified fragments size thereof
Annotate: " Psy-A1a or Psy-A1c/Psy-A1b " and " 194/231 " expression: be the dna fragmentation of 194bp if contain size in the PCR product, then the Psy-A1 gene genotype is Psy-A1a or Psy-A1c; Be the dna fragmentation of 231bp if contain size in the PCR product, then the Psy-A1 gene genotype is Psy-A1b; " Psy-A1a or Psy-A1b/Psy-A1c " and " 1686/1001 " expression: be the dna fragmentation of 1686bp if contain size in the PCR product, then the Psy-A1 gene genotype is Psy-A1a or Psy-A1b; Be the dna fragmentation of 1001bp if contain size in the PCR product, then the Psy-A1 gene genotype is Psy-A1c; " Ppo-D1a/Ppo-D1b " and " 713/-" expression: be the dna fragmentation of 713bp if contain size in the PCR product, then the Ppo-D1 gene genotype is Ppo-D1a; Be not the dna fragmentation of 713bp if do not contain size in the PCR product, then the Ppo-D1 gene genotype is Ppo-D1b.
3, the multiplex PCR amplification of known type wheat breed hand-pulled noodles color and luster genes involved
Electrophoresis result is shown in the swimming lane 1-swimming lane 4 of Fig. 1: new winter of wheat breed (being) No. 20 is after above-mentioned multiplex PCR amplification; Obtain three bands that size is about 1686bp, 194bp and 713bp, this new winter of explanation wheat breed (being) is contained Psy-A1a and Ppo-D1a gene No. 20; New winter of wheat breed (being) No. 16 obtains two bands that size is about 1686bp and 194bp after above-mentioned multiplex PCR amplification, newly the winter is contained Psy-A1a and Ppo-D1b gene No. 16 to this explanation wheat breed (being); Wheat breed (being) Ji wheat 26 obtains three bands that size is about 1686bp, 231bp and 713bp after above-mentioned multiplex PCR amplification, this explanation wheat breed (being) Ji wheat 26 contains Psy-A1b and Ppo-D1a gene; Wheat breed (being) lays down 54 for a short time after the amplification of above-mentioned multiplex PCR, obtains two bands that size is about 1686bp and 231bp, and this explains that wheat breed (being) is laid down for a short time and 54 contains Psy-A1b and Ppo-D1b gene.The above results and table 1 are in full accord, the multiple PCR primer of this explanation present embodiment to group can be simultaneously, 5 kinds of genotype Psy-A1a, Psy-A1b, Psy-A1c, Ppo-D1a and Ppo-D1b in accurate detection wheat breed hand-pulled noodles color and luster genes involved Psy-A1 and two sites of Ppo-D1.This will help selecting and contain Psy-A1b and genotypic wheat breed of Ppo-D1a or flour simultaneously, carry out hand-pulled noodles color and luster genetic improvement or evaluation.
Two, the multiplex PCR of testing gene type wheat breed hand-pulled noodles color and luster genes involved amplification
1, the preparation of wheat cdna group
Employing CTAB method is carried out the extraction of genomic dna to the wheat breed (being) of 13 parts of testing gene types, obtains the genomic dna corresponding to the wheat breed (being) of 13 parts of testing gene types, as the template of hand-pulled noodles color and luster genes involved multiplex PCR amplification.Wherein, the wheat breed of 13 parts of testing gene types (being) is for crust winter No. 2, new Ukraine 83, new Ukraine 84, north are 11, big by 1885, new winter of swallow No. 24, new winter No. 27, new winter No. 28, Kui winter No. 4, Ji wheat 24, Ji wheat 31, stone winter No. 8 and 89-2012.
2, multiplex PCR amplification wheat breed hand-pulled noodles color and luster genes involved
Genomic dna with the wheat breed (being) of 13 parts of testing gene types of above-mentioned steps 1 preparation is respectively template, utilizes the primer in the table 2 that compsn is carried out the multiplex PCR amplification.
PCR reaction system and response procedures are with in the step 12.
3, the multiplex PCR amplification of testing gene type wheat breed hand-pulled noodles color and luster genes involved
Electrophoresis result is shown in the swimming lane 5-swimming lane 17 of Fig. 1: wheat breed (being) crust winter No. 2 obtains two bands that size is about 1001bp and 194bp after above-mentioned multiplex PCR amplification, and this explanation wheat breed (being) clings to winter and contains Psy-A1c and Ppo-D1b gene No. 2; The new Ukraine 83 of wheat breed (being), new Ukraine 84, north are 11, big by 1885, new winter of swallow No. 24, new winter No. 27, new winter No. 28, Ji wheat 24 and 89-2012 be after above-mentioned multiplex PCR amplification; All obtain three bands that size is about 1686bp, 194bp and 713bp, the new Ukraine 83 of this explanation wheat breed (being), new Ukraine 84, north are 11, swallow is big by 1885, newly winter No. 24, new winter No. 27, new winter No. 28, Ji wheat 24 and 89-2012 all contain Psy-A1a and Ppo-D1a gene; Wheat breed (being) Kui winter No. 4 and stone winter No. 8 all obtain two bands that size is about 1686bp and 194bp after above-mentioned multiplex PCR amplification, this explanation wheat breed (being) Kui winter No. 4 and stone winter are all contained Psy-A1a and Ppo-D1b gene No. 8; Wheat breed (being) Ji wheat 31 obtains three bands that size is about 1686bp, 231bp and 713bp after above-mentioned multiplex PCR amplification, this explanation wheat breed (being) Ji wheat 31 contains Psy-A1b and Ppo-D1a gene.The above results and traditional substance PCR result are consistent.And the multiplex PCR amplified production carries out sequence alignment through reclaiming, checking order with target gene, is homologous sequence.Through gene sequencing, further confirmed above-mentioned multiplex PCR result's accuracy.
The multiplex PCR of embodiment 2, hand-pulled noodles gluten strength, gluten quality and milling characters genes involved detects
The multiple PCR primer of present embodiment is to organizing by being specific to Ax2
*A primer of gene is right, is specific to the right and primer being specific to ω-secalin gene of a primer of Pinb-D1b gene to forming; Wherein, the said Ax2 that is specific to
*A primer of gene to the primer formed for sequence in the sequence table 7 and two single stranded DNAs shown in the sequence 8 to 4; A said primer that is specific to the PinbD1b gene to the primer formed for sequence in the sequence table 9 and two single stranded DNAs shown in the sequence 10 to 5; A said primer that is specific to ω-secalin gene to the primer formed for sequence in the sequence table 11 and two single stranded DNAs shown in the sequence 12 to 6.
The multiple PCR primer of present embodiment can be used for detecting simultaneously Ax2 to group
*, Pinb-D1b and ω-secalin gene.Contain Ax2 in the Glu-A1 site
*In the material of gene, pcr amplification goes out size and is the band of 1319bp; In the material that contains the Pinb-D1b gene, pcr amplification goes out size and is the band of 250bp; In the material that contains ω-secalin gene, pcr amplification goes out size and is the band of 1067bp.
One, the multiplex PCR of known type wheat breed hand-pulled noodles gluten strength, gluten quality and milling characters genes involved amplification
1, the preparation of wheat cdna group
Employing CTAB method is carried out the extraction of genomic dna to the wheat breed (being) of 5 parts of known types; Obtain genomic dna, as the template of hand-pulled noodles gluten strength, gluten quality and the amplification of milling characters genes involved multiplex PCR corresponding to the wheat breed (being) of 5 parts of known types.Wherein, the wheat breed of 5 parts of known types (being) is followed successively by excellent 8901, HD04-23 of new winter No. 22, new winter No. 23, ligusticumic and 79-18.(Wang Liang, Mu Peiyuan, Xu Hongjun such as Wang Liang; Deng. Xinjiang wheat breed hmw glutenin subunit compositional analysis [J]. wheat crops journal, 2008,28 (3): 430-435. Wang Liang; Mu Peiyuan, Sang Wei, etc. the Molecular Detection [J] of Xinjiang wheat breed grain hardness and the variation of puroindoline allele. the wheat crops journal; 2010,30 (1): 17-22. Wang Liang, Mu Peiyuan; Xu Hongjun, etc. the multiplex PCR of Xinjiang wheat 1BL/1RS transposition and Dx5 gene detects and gluten attributional analysis [J]. wheat crops journal, 2011; 31 (3):, and whether contain ω-secalin gene and carried out Markers for Detection, seen table 3 for details 469-474.) to the Glu-A1 and the Pinb-D1 gene genotype of these five wheat breeds (being).
The genotype of table 3 known type wheat breed (being) hand-pulled noodles gluten strength, gluten quality and milling characters genes involved
Annotate: "+" expression contains ω-secalin gene; "-" expression does not contain Ax2
*Gene or do not contain ω-secalin gene.
2, multiplex PCR amplification wheat breed hand-pulled noodles gluten strength, gluten quality and milling characters genes involved
Genomic dna with the wheat breed (being) of 5 parts of known types of above-mentioned steps 1 preparation is respectively template, utilizes the primer in the table 4 that compsn is carried out the multiplex PCR amplification.
It is 20 μ L that PCR reacts total system; Wherein, Dna profiling 100-200ng; MasterMix (containing archaeal dna polymerase, PCR reaction buffer, 4 kinds of dNTP) (Beijing health is the century bio tech ltd) 10 μ L (making that the concentration of every kind of dNTP is 200 μ M in the PCR reaction system of 20 μ L) are 0.1 μ M as every single stranded DNA of primer at the final concentration that PCR reacts in total system.
The PCR response procedures is: 95 ℃ of preparatory sex change 5min; 94 ℃ of sex change 45s, annealing temperature is from 62 ℃, and along with the carrying out of PCR reaction, 1 said annealing temperature of circulation of every increase descends 0.1 ℃, and each round-robin annealing time is 60s, and 72 ℃ are extended 1min, and amplified reaction carries out 35 circulations; 72 ℃ are extended 8min; 4 ℃, insulation finishes.
Pcr amplification carries out on PTC-200 type PCR appearance, carries out electrophoresis with 1 * TAE at 2% sepharose, and the voltage during electrophoresis is 160V, and electrophoresis time is 30min, and electrophoresis finishes through under gel imaging system, observing photograph behind the ethidium bromide staining.
Table 4 hand-pulled noodles gluten strength, gluten quality and milling characters genes involved amplimer sequence and expection amplified fragments size
3, the multiplex PCR amplification of known type wheat breed hand-pulled noodles gluten strength, gluten quality and milling characters genes involved
Electrophoresis result is shown in the swimming lane 1-swimming lane 5 of Fig. 2: new winter of wheat breed (being) No. 22 and HD04-23 are after above-mentioned multiplex PCR amplification; All obtain two bands that size is about 1319bp and 250bp, new winter of this explanation wheat breed (being) No. 22 and HD04-23 all contain Ax2
*Gene and Pinb-D1b gene; Wheat breed (being) ligusticumic excellent 8901 is after above-mentioned multiplex PCR amplification, and no amplified band occurs, and this explanation wheat breed (being) ligusticumic excellent 8901 does not contain Ax2
*, Pinb-D1b and ω-secalin gene; New winter of wheat breed (being) No. 23 obtains the band that size is about 250bp after above-mentioned multiplex PCR amplification, the new winter of this explanation wheat breed (being) is contained the PinbD1b gene No. 23; Wheat breed (being) 79-18 obtains two bands that size is about 250bp and 1067bp after above-mentioned multiplex PCR amplification, this explanation wheat breed (being) 79-18 contains PinbD1b gene and ω-secalin gene.The above results and table 3 are in full accord, the multiple PCR primer of this explanation present embodiment to group can the while, accurate detection wheat breed hand-pulled noodles gluten strength, gluten quality and milling characters genes involved Ax2
*, Pinb-D1b and ω-secalin, whether assess sample has the needed gluten strength of the high-quality hand-pulled noodles of making, gluten quality and milling characters.
Two, the multiplex PCR of testing gene type wheat breed hand-pulled noodles gluten strength, gluten quality and milling characters genes involved amplification
1, the preparation of wheat cdna group
Employing CTAB method is carried out the extraction of genomic dna to the wheat breed (being) of 12 parts of testing gene types, obtains the genomic dna corresponding to the wheat breed (being) of 12 parts of testing gene types, as the template of hand-pulled noodles color and luster genes involved multiplex PCR amplification.Wherein, the wheat breed of 12 parts of testing gene types (being) is new winter No. 14, new winter No. 24, new winter No. 28, new winter No. 18, Ji wheat 26, Kui winter No. 4, stone winter No. 8,85 (1), 89 (35), 80182, flower 91-26 and 89-44.
2, multiplex PCR amplification wheat breed hand-pulled noodles gluten strength, gluten quality and milling characters genes involved
Genomic dna with the wheat breed (being) of 12 parts of testing gene types of above-mentioned steps 1 preparation is respectively template, utilizes the primer in the table 4 that compsn is carried out the multiplex PCR amplification.
PCR reaction system and response procedures are with in the step 12.
3, the multiplex PCR amplification of testing gene type wheat breed hand-pulled noodles gluten strength, gluten quality and milling characters genes involved
Electrophoresis result is shown in the swimming lane 6-swimming lane 17 of Fig. 2: new winter of wheat breed (being) No. 14 obtains the band that size is about 1319bp after above-mentioned multiplex PCR amplification, and the new winter of this explanation wheat breed (being) is contained Ax2 No. 14
*Gene; New winter of wheat breed (being) No. 24 obtains the band that size is about 1067bp after above-mentioned multiplex PCR amplification, the new winter of this explanation wheat breed (being) is contained ω-secalin gene No. 24; New winter of wheat breed (being) No. 28 all obtains the band that size is about 250bp with 89 (35) after above-mentioned multiplex PCR amplification, and newly winter No. 28 and 89 (35) is all contained the Pinb-D1b gene to this explanation wheat breed (being); New winter of wheat breed (being) No. 18, Ji wheat 26,80182 and 89-44 all do not amplify any band after above-mentioned multiplex PCR amplification, new winter of this explanation wheat breed (being) No. 18, Ji wheat 26,80182 and 89-44 all do not contain Ax2
*, Pinb-D1b and ω-secalin gene; Wheat breed (being) Kui winter No. 4 and flower 91-26 all obtain three bands that size is about 1319bp, 250bp and 1067bp after above-mentioned multiplex PCR amplification, this explains wheat breed (being) Kui winter No. 4 and spends 91-26 all to contain Ax2
*, Pinb-D1b and ω-secalin gene; Wheat breed (being) stone winter No. 8 obtains two bands that size is about 1319bp and 250bp after above-mentioned multiplex PCR amplification, this explains that wheat breed (being) stone winter contains Ax2 No. 8
*Gene and Pinb-D1b gene; Wheat breed (being) 85 (1) obtains two bands that size is about 250bp and 1067bp after above-mentioned multiplex PCR amplification, this explanation wheat breed (being) 85 (1) contains Pinb-D1b and ω-secalin gene.The above results and traditional substance PCR result are consistent.And the multiplex PCR amplified production carries out sequence alignment through reclaiming, checking order with target gene, is homologous sequence.Through gene sequencing, further confirmed above-mentioned multiplex PCR result's accuracy.
Claims (10)
1. be used to identify or the multiple PCR primer of assistant identification wheat hand-pulled noodles to be measured quality trait genes involved to group, said hand-pulled noodles quality trait genes involved is Psy-A1 gene, Ppo-D1 gene, Ax2
*Gene, Pinb-D1b gene and ω-secalin gene is characterized in that: said multiple PCR primer is made up of group B group A and primer primer group; A said primer and primer being specific to said Ppo-D1 gene right by two primers that are specific to said Psy-A1 gene to group A is to forming; Said primer is to organizing B by being specific to said Ax2
*A primer of gene is right, is specific to the right and primer being specific to said ω-secalin gene of a primer of said Pinb-D1b gene to forming;
Said primer is among the group A, and to 1, and the primer that sequence 3 and two single stranded DNAs shown in the sequence 4 are formed is to 2 to the primer that is respectively sequence in the sequence table 1 and two single stranded DNAs shown in the sequence 2 and forms for two primers of the said Psy-A1 of being specific to gene; A said primer that is specific to the Ppo-D1 gene to the primer formed for sequence in the sequence table 5 and two single stranded DNAs shown in the sequence 6 to 3;
Said primer is to organizing among the B the said Ax2 that is specific to
*A primer of gene to the primer formed for sequence in the sequence table 7 and two single stranded DNAs shown in the sequence 8 to 4; A said primer that is specific to the Pinb-D1b gene to the primer formed for sequence in the sequence table 9 and two single stranded DNAs shown in the sequence 10 to 5; A said primer that is specific to ω-secalin gene to the primer formed for sequence in the sequence table 11 and two single stranded DNAs shown in the sequence 12 to 6.
2. be used to identify or the multiple PCR primer of assistant identification wheat hand-pulled noodles to be measured quality trait genes involved to group; For the said primer in the claim 1 to the group A; An and primer being specific to Ppo-D1 gene right by two primers that are specific to the Psy-A1 gene is to forming;
To 1, and the primer that sequence 3 and two single stranded DNAs shown in the sequence 4 are formed is to 2 to the primer that is respectively sequence in the sequence table 1 and two single stranded DNAs shown in the sequence 2 and forms for two primers of the said Psy-A1 of being specific to gene; A said primer that is specific to the Ppo-D1 gene to the primer formed for sequence in the sequence table 5 and two single stranded DNAs shown in the sequence 6 to 3.
3. primer according to claim 1 and 2 is to group; It is characterized in that: said primer uses at PCR reaction system moderate two right primers of each primer among the group A; And said primer is to 1, said primer to 2 with said primer be 1: 4: 1 to 3 mol ratios in the PCR reaction system; Said special primer uses at PCR reaction system moderate right two primers of each primer among the group B, and said primer is to 4, said primer to 5 with said primer be 1: 1: 1 to 6 mol ratios in the PCR reaction system.
4. contain the test kit of arbitrary said primer to organizing among the claim 1-3.
5. arbitrary said primer is characterized in that the preparation method of group among the claim 1-3: said preparation method comprises the step that arbitrary said primer among the claim 1-3 is packed separately respectively right said two single stranded DNAs of each primer in the group.
6. the preparation method of the said PCR test kit of claim 4; Comprise the steps: with arbitrary described primer among the claim 1-3 to right said two single stranded DNAs of each primer in the group respectively separately after the packing and at least a being packaged in the same test kit in the following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
7. identify or assistant identification wheat to be measured contain any in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c, contain in Ppo-D1a and these two kinds of genes of Ppo-D1b anyly, and whether contain Ax2
*At least a method in these three kinds of genes of gene, Pinb-D1b gene and ω-secalin gene, it is characterized in that: said method comprises the step of following (1) and (2):
Said (1) is following A) and B):
A) genomic dna with wheat to be measured is a template, with claim 1 or 3 said primers the said primer in the group is carried out the PCR reaction to group A, and the annealing temperature of said PCR reaction is 60 ℃;
B) genomic dna with wheat to be measured is a template, with claim 1 or 3 said primers primer described in the group is carried out the PCR reaction to group B, and the annealing temperature of said PCR reaction is from 62 ℃, whenever carries out 1 PCR reaction cycle and descends 0.1 ℃;
(2) detect the size of the PCR product that step (1) obtains; According to following method according to the PCR product confirm said wheat to be measured contain any in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c, contain in Ppo-D1a and these two kinds of genes of Ppo-D1b anyly, and whether contain Ax2
*At least a in these three kinds of genes of gene, Pinb-D1b gene and ω-secalin gene:
With said primer group A is carried out in the product of PCR reaction, if contain two dna fragmentations of size for 1686bp and 194bp simultaneously, said wheat to be measured contains the Psy-A1a gene or the candidate is contained the Psy-A1a gene; If contain two dna fragmentations of size for 1686bp and 231bp simultaneously, said wheat to be measured contains the Psy-A1b gene or the candidate is contained the Psy-A1b gene; If contain two dna fragmentations of size for 1001bp and 194bp simultaneously, said wheat to be measured contains the Psy-A1c gene or the candidate is contained the Psy-A1c gene; Be the dna fragmentation of 713bp if contain size, said wheat to be measured contains the Ppo-D1a gene or the candidate is contained the Ppo-D1a gene; Be not the dna fragmentation of 713bp if do not contain size, said wheat to be measured contains the Ppo-D1b gene or the candidate is contained the Ppo-D1b gene;
In the product that carry out PCR reaction of said primer to group B, be the dna fragmentation of 1319bp if contain size, said wheat to be measured contains Ax2
*Gene or candidate are contained Ax2
*Gene; Be not the dna fragmentation of 1319bp if do not contain size, said wheat to be measured does not contain Ax2
*Gene or candidate are not contained Ax2
*Gene; Be the dna fragmentation of 250bp if contain size, said wheat to be measured contains the Pinb-D1b gene or the candidate is contained the Pinb-D1b gene; Be not the dna fragmentation of 250bp if do not contain size, said wheat to be measured does not contain the Pinb-D1b gene or the candidate is not contained the Pinb-D1b gene; Be that the dna fragmentation of 1067bp, said wheat to be measured contain ω-secalin gene or the candidate is contained ω-secalin gene if contain size; Be not that the dna fragmentation of 1067bp, said wheat to be measured do not contain ω-secalin gene or the candidate is not contained ω-secalin gene if do not contain size.
8. evaluation or assistant identification wheat to be measured contain in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c any; And contain any method in Ppo-D1a and these two kinds of genes of Ppo-D1b, it is characterized in that: said method comprises the step of following (1) and (2):
(1) genomic dna with wheat to be measured is a template, with claim 2 or 3 said primers the said primer in the group is carried out the PCR reaction to group A, and the annealing temperature of said PCR reaction is 60 ℃;
(2) detect the size of the PCR product that step (1) obtains; It is any to confirm that according to the PCR product said wheat to be measured contains in these three kinds of genes of Psy-A1a, Psy-A1b and Psy-A1c according to following method, and contains in Ppo-D1a and these two kinds of genes of Ppo-D1b any:
With said primer group A is carried out in the product of PCR reaction, if contain two dna fragmentations of size for 1686bp and 194bp simultaneously, said wheat to be measured contains the Psy-A1a gene or the candidate is contained the Psy-A1a gene; If contain two dna fragmentations of size for 1686bp and 231bp simultaneously, said wheat to be measured contains the Psy-A1b gene or the candidate is contained the Psy-A1b gene; If contain two dna fragmentations of size for 1001bp and 194bp simultaneously, said wheat to be measured contains the Psy-A1c gene or the candidate is contained the Psy-A1c gene; Be the dna fragmentation of 713bp if contain size, said wheat to be measured contains the Ppo-D1a gene or the candidate is contained the Ppo-D1a gene; Be not the dna fragmentation of 713bp if do not contain size, said wheat to be measured contains the Ppo-D1b gene or the candidate is contained the Ppo-D1b gene.
9. method of cultivating the hand-pulled noodles wheat breed, comprise employing through the method for claim 7 identify obtain following a)-e) at least a wheat step of carrying out breeding as the parent:
A) contain or the candidate is contained the Psy-A1b gene;
B) contain or the candidate is contained the Ppo-D1a gene;
C) contain or the candidate is contained Ax2
*Gene;
D) contain or the candidate is contained the Pinb-D1b gene;
E) do not contain or the candidate is not contained ω-secalin gene.
10. according to arbitrary described method among the claim 7-9, it is characterized in that: said wheat is a Winter Wheat in Xinjiang.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103571951A (en) * | 2013-10-24 | 2014-02-12 | 安康学院 | Polymerase chain reaction (PCR) system for identifying related genes of wheat flour color |
| CN103571951B (en) * | 2013-10-24 | 2015-07-01 | 安康学院 | Polymerase chain reaction (PCR) system for identifying related genes of wheat flour color |
| CN104894285A (en) * | 2015-06-29 | 2015-09-09 | 天津市农业生物技术研究中心 | Method for detecting genes Cf-9 and I-2 according to dual-PCR (polymerase chain reaction) technology |
| CN105112546A (en) * | 2015-09-23 | 2015-12-02 | 中国农业科学院作物科学研究所 | Primer set for detecting functional genes of wheat on basis of KASP [competitive allele specific PCR (polymerase chain reaction)] technology and application of set primer |
| CN105112546B (en) * | 2015-09-23 | 2018-01-09 | 中国农业科学院作物科学研究所 | Primer set and its application based on KASP technology for detection wheat functional genes |
| CN114854902A (en) * | 2022-06-27 | 2022-08-05 | 河南省作物分子育种研究院 | Wheat molecular marker 5668 and application thereof in grain hardness improvement |
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