CN102382895B - 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 by the people of all parts of the country and likes.According to statistics, 80% of the annual production 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 based on traditional instrument analysis and finished product sensory evaluation 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 are more and more higher to the requirement of 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 product 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 is subjected to milling characters indexs such as wheat flour extraction rate, protein content 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, Deng. 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 are subjected to phytoene synthase (PSY) effect gene, whiteness high negative correlation (Kruger J E with flour and dough, Matsuo R R, Presten K.A comparison of methods for the prediction of Cantonese noodle colour[J] .Canadian Journal of Plant Science, 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 is subjected to 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.Studies show that, quality traits such as protein properties, starch property and grain hardness, can determine sensory evaluation result (the Yun S H of hand-pulled noodles by influence to a certain extent to dough rheological characteristics and starch pasting characteristic, Quail K, Moss R.Physicochemical properties of Australian wheat flours for white salted noodles[J] .Journal of Cereal Sciences, 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. the 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 illustrated.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 by 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 dough viscosity to increase, intensity reduces, dough rheological characteristics and noodle quality all there is 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 (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. 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 every 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 such as Chen, Shang Xiaoli, Dong Zhongdong, Deng. the variation of the historical kind puroindoline of Chinese wheat allele detects [J]. the wheat crops journal, 2011,31 (3): studies show that 389-394) Pinb-D1b is lower than Pina-D1b type flour extraction rate height, ash, have better milling characters, the former steamed bun, noodles and bread processing quality also all are better than 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 product 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 PCR method generally once can only detect one or a few gene, is difficult to satisfy the demand of the seed selection of hand-pulled noodles specific breed and follow-up screening thereof, processing, 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 described 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 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 described primer; Described 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;
Described primer is among the group A, and two primers of the described Psy-A1 of being specific to gene are to being respectively two single stranded DNAs shown in the sequence 1 and sequence 2 are formed in the sequence table primer to 1, and the primer that two single stranded DNAs shown in sequence 3 and the sequence 4 are formed is to 2; A described primer that is specific to the Ppo-D1 gene to the primer formed for two single stranded DNAs shown in sequence 5 in the sequence table and the sequence 6 to 3;
Described primer is to organizing among the B the described Ax2 that is specific to
*A primer of gene to the primer formed for two single stranded DNAs shown in sequence 7 in the sequence table and the sequence 8 to 4; A described primer that is specific to the PinbD1b gene to the primer formed for two single stranded DNAs shown in sequence 9 in the sequence table and the sequence 10 to 5; A described primer that is specific to ω-secalin gene to the primer formed for two single stranded DNAs shown in sequence 11 in the sequence table and 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, described Psy-A1 gene has three allelotrope, is respectively Psy-A1a, Psy-A1b and Psy-A1c; Described Ppo-D1 gene has two allelotrope, is respectively Ppo-D1a and Ppo-D1b.
Described primer uses at PCR reaction system moderate right two primers of each primer among the group A, and described primer is to 1, described primer to 2 and described primer be 1: 4: 1 to 3 mol ratios in the PCR reaction system; Described special primer uses at PCR reaction system moderate right two primers of each primer among the group B, and described primer is to 4, described primer to 5 and described primer be 1: 1: 1 to 6 mol ratios in the PCR reaction system.
Contain and describedly 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.
Describedly 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 described 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 described two single stranded DNAs of each primer of the group step of packing separately respectively.
The preparation method of described test kit also belongs to protection scope of the present invention.This preparation method specifically can comprise the steps: with described 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 described 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 Psy-A1a, Psy-A1b and these three kinds of genes of 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):
Described (1) is following A) and B):
A) genomic dna with wheat to be measured is a template, with described primer group A is carried out the PCR reaction, and the annealing temperature of described PCR reaction is 60 ℃;
B) genomic dna with wheat to be measured is a template, with described primer group B is carried out the PCR reaction, the annealing temperature of described PCR reaction is from 62 ℃, whenever carrying out 1 PCR reaction cycle descends 0.1 ℃, in an embodiment of the present invention, with described primer group B is carried out 35 circulations of PCR reaction having carried out altogether.
(2) detect the size of the PCR product that step (1) obtains, as follows according to the PCR product determine described wheat to be measured contain any in Psy-A1a, Psy-A1b and these three kinds of genes of 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 described primer group A is carried out in the product of PCR reaction, if contain two dna fragmentations of size for 1686bp and 194bp simultaneously, described 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, described 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, described 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, described 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, described 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 described primer to group B, be the dna fragmentation of 1319bp if contain size, described 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, described 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, described 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, described 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, described 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, described 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 Psy-A1a, Psy-A1b and these three kinds of genes of 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 described primer group A is carried out the PCR reaction, and the annealing temperature of described PCR reaction is 60 ℃;
(2) detect the size of the PCR product that step (1) obtains, it is any to determine that according to the PCR product described wheat to be measured contains in Psy-A1a, Psy-A1b and these three kinds of genes of Psy-A1c as follows, and contains in Ppo-D1a and these two kinds of genes of Ppo-D1b any:
With described primer group A is carried out in the product of PCR reaction, if contain two dna fragmentations of size for 1686bp and 194bp simultaneously, described 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, described 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, described 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, described 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, described 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 Psy-A1a, Psy-A1b and these three kinds of genes of 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, represents the suitable more making hand-pulled noodles of selected wheat.
In an embodiment of the present invention, described wheat to be measured is specially Winter Wheat in Xinjiang, as 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, HD04-23,79-18, new winter No. 14, new winter No. 18,85 (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 finishes 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 if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels 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, two primers of the described Psy-A1 of being specific to gene are to being respectively two single stranded DNAs shown in the sequence 1 and sequence 2 are formed in the sequence table primer to 1, and the primer that two single stranded DNAs shown in sequence 3 and the sequence 4 are formed is to 2; A described primer that is specific to the Ppo-D1 gene to the primer formed for two single stranded DNAs shown in sequence 5 in the sequence table and 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, the new winter No. 20, Ji wheat 26 and lay down 54 for a short time, (Wang Liang such as Wang Liang, Mu Peiyuan, Xu Hongjun, Deng. 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, Deng. the distribution [J] of polyphenoloxidase in the wheat breed of Xinjiang (PPO) active gene allelic variation. the 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 composition is carried out the multiplex PCR amplification.
The PCR reaction totally is 20 μ L, wherein, dna profiling 100-200ng, MasterMix (contains 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: sequence 1 and two dna single chains sequence 2 shown in of primer in to 1 are 0.05 μ M at the final concentration that PCR reacts in total system; Primer is 0.2 μ M to sequence 3 and two the dna single chains shown in the sequence 4 in 2 at the final concentration that PCR reacts in total system; Primer is 0.05 μ M to sequence 5 and two the dna single chains shown in the sequence 6 in 3 at the final concentration that PCR reacts in total system.
The PCR response procedures is: 94 ℃ of pre-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 instrument, carries out electrophoresis with 1 * TAE at 2% sepharose, and the voltage during electrophoresis is 150V, and electrophoresis time is 40min, and electrophoresis finishes to take a picture through observing under gel imaging system 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, the new winter of this explanation wheat breed (being) is contained Psy-A1a and Ppo-D1b gene No. 16; 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 explanation wheat breed (being) is laid down for a short time and 54 contained 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 composition 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) crust winter is contained 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 is consistent with traditional substance PCR result.And the multiplex PCR amplified production carries out sequence alignment through reclaiming, checking order with target gene, is homologous sequence.By 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 described Ax2 that is specific to
*A primer of gene to the primer formed for two single stranded DNAs shown in sequence 7 in the sequence table and the sequence 8 to 4; A described primer that is specific to the PinbD1b gene to the primer formed for two single stranded DNAs shown in sequence 9 in the sequence table and the sequence 10 to 5; A described primer that is specific to ω-secalin gene to the primer formed for two single stranded DNAs shown in sequence 11 in the sequence table and 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 such as Wang Liang, Mu Peiyuan, Xu Hongjun, Deng. Xinjiang wheat breed hmw glutenin subunit compositional analysis [J]. the 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. 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]. the 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 composition is carried out the multiplex PCR amplification.
The PCR reaction totally is 20 μ L, 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 pre-sex change 5min; 94 ℃ of sex change 45s, annealing temperature is from 62 ℃, and along with the carrying out of PCR reaction, 1 described 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 instrument, carries out electrophoresis with 1 * TAE at 2% sepharose, and the voltage during electrophoresis is 160V, and electrophoresis time is 30min, and electrophoresis finishes to take a picture through observing under gel imaging system 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 composition 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 and 89 (35) all obtains the band that size is about 250bp after above-mentioned multiplex PCR amplification, 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 explanation wheat breed (being) Kui winter No. 4 and flower 91-26 all 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 explanation wheat breed (being) stone winter is contained 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 is consistent with traditional substance PCR result.And the multiplex PCR amplified production carries out sequence alignment through reclaiming, checking order with target gene, is homologous sequence.By gene sequencing, further confirmed above-mentioned multiplex PCR result's accuracy.
Claims (5)
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, described hand-pulled noodles quality trait genes involved is Psy-A1 gene, Ppo-D1 gene, Ax2* gene, Pinb-D1b gene and ω-secalin gene, it is characterized in that: described multiple PCR primer is made up of group B group A and primer primer group; A described primer and primer being specific to described Ppo-D1 gene right by two primers that are specific to described Psy-A1 gene to group A is to forming; Described primer is right by a primer that is specific to described Ax2* gene to group B, is specific to the right and primer being specific to described ω-secalin gene of a primer of described Pinb-D1b gene to forming;
Described primer is among the group A, and two primers of the described Psy-A1 of being specific to gene are to being respectively two single stranded DNAs shown in the sequence 1 and sequence 2 are formed in the sequence table primer to 1, and the primer that two single stranded DNAs shown in sequence 3 and the sequence 4 are formed is to 2; A described primer that is specific to the Ppo-D1 gene to the primer formed for two single stranded DNAs shown in sequence 5 in the sequence table and the sequence 6 to 3;
Described primer is among the group B, a described primer that is specific to the Ax2* gene to the primer formed for two single stranded DNAs shown in sequence 7 in the sequence table and the sequence 8 to 4; A described primer that is specific to the Pinb-D1b gene to the primer formed for two single stranded DNAs shown in sequence 9 in the sequence table and the sequence 10 to 5; A described primer that is specific to ω-secalin gene to the primer formed for two single stranded DNAs shown in sequence 11 in the sequence table and the sequence 12 to 6;
Described primer uses at PCR reaction system moderate right two primers of each primer among the group A, and described primer is to 1, described primer to 2 and described primer be 1:4:1 to 3 mol ratios in the PCR reaction system; Described special primer uses at PCR reaction system moderate right two primers of each primer among the group B, and described primer is to 4, described primer to 5 and described primer be 1:1:1 to 6 mol ratios in the PCR reaction system, described wheat is a Winter Wheat in Xinjiang.
2. identify or test kit that assistant identification Winter Wheat in Xinjiang hand-pulled noodles to be measured quality trait is relevant that its primer is that the described primer of claim 1 is to group.
3. the preparation method of the described PCR test kit of claim 2, comprise the steps: with the described primer of claim 1 to right described 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.
4. identify or assistant identification Winter Wheat in Xinjiang to be measured contain any in Psy-A1a, Psy-A1b and these three kinds of genes of Psy-A1c, contain in Ppo-D1a and these two kinds of genes of Ppo-D1b any, and whether contain at least a method in these three kinds of genes of Ax2* gene, Pinb-D1b gene and ω-secalin gene, it is characterized in that:
Described method comprises the step of following (1) and (2):
Described (1) is following A) and B):
A) genomic dna with wheat to be measured is a template, with the described primer of claim 1 the described primer in the group is carried out the PCR reaction to group A, and the annealing temperature of described PCR reaction is 60 ℃;
B) genomic dna with wheat to be measured is a template, with the described primer of claim 1 primer described in the group is carried out the PCR reaction to group B, and the annealing temperature of described 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, as follows according to the PCR product determine described wheat to be measured contain any in Psy-A1a, Psy-A1b and these three kinds of genes of Psy-A1c, contain in Ppo-D1a and these two kinds of genes of Ppo-D1b anyly, and whether contain at least a in these three kinds of genes of Ax2* gene, Pinb-D1b gene and ω-secalin gene:
With described primer group A is carried out in the product of PCR reaction, if contain two dna fragmentations of size for 1686bp and 194bp simultaneously, described 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, described 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, described 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, described 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, described 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 described primer to group B, be the dna fragmentation of 1319bp if contain size, described wheat to be measured contains the Ax2* gene or the candidate is contained the Ax2* gene; Be not the dna fragmentation of 1319bp if do not contain size, described wheat to be measured does not contain the Ax2* gene or the candidate is not contained the Ax2* gene; Be the dna fragmentation of 250bp if contain size, described 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, described 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, described 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, described wheat to be measured do not contain ω-secalin gene or the candidate is not contained ω-secalin gene if do not contain size.
5. method of cultivating the hand-pulled noodles wheat breed, comprise employing by the method for claim 4 identify obtain following a)-e) at least a Winter Wheat in Xinjiang 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 the 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.
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| 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 |
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| Characterization of CLIMMYT bread wheats for high- and low-molecular weight glutenin subunits and other quality-related genes with SDS-PAGE,RP-HPLC and molecular markers;Liang Dan et al.;《Euphytica》;20101231;第172卷;表1，第236-237页 * |
| Development of two multiplex PCR assays targeting improvement of bread-making and noodle qualities in common wheat.;Zhang XK et al.;《Plant Breeding》;20081231;第127卷;第109-110、114页 * |
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