CN103540670A - PCR (polymerase chain reaction) system for identifying genes related to color and luster of wheat flour - Google Patents

PCR (polymerase chain reaction) system for identifying genes related to color and luster of wheat flour Download PDF

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CN103540670A
CN103540670A CN201310507057.6A CN201310507057A CN103540670A CN 103540670 A CN103540670 A CN 103540670A CN 201310507057 A CN201310507057 A CN 201310507057A CN 103540670 A CN103540670 A CN 103540670A
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primer pair
tazds
sequence
psy
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张晓科
张钰玉
蒋雷
王宪国
刘芳军
王晓龙
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Northwest A&F University
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Abstract

The invention discloses a PCR (polymerase chain reaction) system for identifying or assisting to identify genes related to color and luster of wheat flour and an application thereof. Primer pairs of the provided PCR system are a primer pair A and a primer pair B, which comprise at least one primer pair containing the primer pair A, wherein the primer pair A comprises a primer pair 1 (sequence 1 and sequence 2), a primer pair 2 (sequence 3 and sequence 4), and a primer pair 3 (sequence 5 and sequence 6), and the primer pair B comprises a primer pair 4 (sequence 7 and sequence 8) and a primer pair 5 (sequence 9 and sequence 10). The primer pairs do not suppress mutually and mismatch, a group of genes is identified at the same temperature through PCR once, the result is reliable, the specificity is strong, and less time is spent. According to the PCR system, the method can rapidly evaluate the color and luster quality of the wheat flour more comprehensively, and provides a reference for color and luster quality and breeding of wheat.

Description

A kind of PCR system for the identification of whole meal flour color and luster genes involved
Technical field
The present invention relates to a kind of for the identification of or PCR system and the application thereof of assistant identification flour color and luster genes involved, particularly a kind of for the identification of or the flour color and luster genes involved of assistant identification Winter Wheat in Xinjiang to be measured if the multiple PCR primer of Psy-B1 and TaZds-A1 is to group, test kit and application thereof.
Background technology
Wheat flour-made food take as one of the most important staple food of China people ，Wei China the people that wheaten food is main area provides energy derive more than 50-80%.Along with social life level improves constantly, people are more and more higher to the requirement of wheat flour product property, and quality breeding also more and more comes into one's own.China, as producing country and the country of consumption of wheat maximum, is often only for producing the amount of wheat of noodles and accounts for 40% of wheat aggregate consumption.And the traditional boiling class flour-made foods of China such as noodles, steamed bun, dumpling, color and luster is more subject to China human consumer favor more in vain.Therefore in the sensory evaluation system of ， China tradition flour products, the color and luster of flour products accounts for very large ratio, and scoring is higher more in vain.Yet, the current average whiteness of flour lower (74.8) of the main cultivation wheat breed of China, the requirement of person to Flour whiteness (more than 80.0) that can not meet market and consumption.Before 2005, most of milling plants meet the requirement of human consumer to Flour whiteness, in flour, add benzoyl peroxide (BPO).But BPO can destroy nutritive substance and human health is constituted a threat to, in the wheat-flour standard of the up-to-date revision of country, clearly provide against and use BPO.Therefore work out breeding objective, select the wheat that nature is white significant.Meanwhile, jonquilleous flour also has certain consumption, as yellow alkaline noodle, Japanese noodle, spaghetti etc. are also popular.Therefore,, according to different processing objects, need respectively seed selection to be applicable to the new variety of wheat of the high whiteness of processing or glassy yellow flour.
Except environment, whitening agent, processing mode and cultivation step etc., the formation of whole meal flour and noodle color is mainly subject to the impact of the genetic material such as colors and redox enzymes.Colors mainly comprises yellow pigment (Yellow pigment, YP) and brown pigments (Brown pigment), yellow pigment is mainly comprised of carotenoid (Carotenoid) and flavonoid (Flavonoid), oxidases in flour mainly comprises polyphenoloxidase (Polyphenol oxidase, PPO), lipoxidase (Lipoxygenase, LOX) and peroxidase (Peroxidase, POD).YP content, LOX and PPO activity have material impact to the color and luster of flour and flour products.The relation conefficient of YP content and dough Huang degree is up to 0.8-0.9; LOX can form superoxide by catalysis polyunsaturated fatty acid oxygenation, and the oxyradical that reaction discharges is further oxidized pigment molecular, and flour is bleached; PPO can finally become oxygenate quinone by catalysis list aldehydes matter in the presence of oxygen molecule, generates brown material, determines the 50-70% of color and luster brown stain in flour and flour products processing and storage process.When needs nature white wheat, should select the wheat breed (being) containing low YP content gene type, high LOX active gene type and low PPO active gene type.
Multiplex PCR be a kind of efficiently, detection method cheaply, become the focus of research.Although reported the multiplex PCR system that several covers are relevant to whole meal flour Color gene, but only relate to Ppo-A1 and the Ppo-D1 site of PPO active gene, or the Psy-A1 site of PSY active gene, yet there are no and detect the Psy-B1 site of PSY active gene, TaZds-A1 and the TaZds-D1 site of ZDS active gene, and the report of the multiplex PCR system in LOX active gene TaLOX-B1 site.
Summary of the invention
The object of this invention is to provide a kind of for the identification of or PCR system and the application thereof of assistant identification flour color and luster genes involved, particularly a kind of for the identification of or the flour color and luster genes involved of assistant identification Winter Wheat in Xinjiang to be measured if the multiple PCR primer of Psy-B1 and TaZds-A1 is to group, test kit and application thereof.
Provided by the present invention for the identification of or the multiple PCR primer of assistant identification wheat relevant coefficient evaluation in quality selection to be measured trait related gene to group, be following a1) or primer pair group a2):
A1) the primer pair group being formed by primer pair group A and primer pair group B;
A2) primer pair group A;
Described flour color and luster genes involved is Psy-B1 gene, TaZds-A1 gene, TaZds-D1 gene and Ppo-A1 gene.
Described primer pair group A is by two primer pairs that are specific to described Psy-B1 gene, and a primer pair that is specific to described TaZds-A1 gene forms; Concrete, described in be specific to described Psy-B1 gene two primer pairs be respectively the primer pair 1 that in sequence table, two single stranded DNAs shown in sequence 1 and sequence 2 form, and the primer pair 2 that forms of two single stranded DNAs shown in sequence 3 and sequence 4; A described primer pair that is specific to described TaZds-A1 gene is the primer pair 3 that in sequence table, two single stranded DNAs shown in sequence 5 and sequence 6 form;
Described primer pair group B is by a primer pair that is specific to described TaZds-D1 gene, and a primer pair that is specific to described Ppo-A1 gene forms; Concrete, described in be specific to described TaZds-D1 gene a primer pair be the primer pair 4 that in sequence table, two single stranded DNAs shown in sequence 7 and sequence 8 form; A described primer pair that is specific to described Ppo-A1 gene is the primer pair 5 that in sequence table, two single stranded DNAs shown in sequence 9 and sequence 10 form.
Wherein, sequence 1 is comprised of 21 Nucleotide; Sequence 2 is comprised of 20 Nucleotide; Sequence 3 is comprised of 21 Nucleotide; Sequence 4 is comprised of 20 Nucleotide; Sequence 5 is comprised of 21 Nucleotide; Sequence 6 is comprised of 24 Nucleotide; Sequence 7 is comprised of 21 Nucleotide; Sequence 8 is comprised of 23 Nucleotide; Sequence 9 is comprised of 21 Nucleotide; Sequence 10 is comprised of 20 Nucleotide.
In the present invention, described Psy-B1 gene relates to three allelotrope, is respectively Psy-B1a, Psy-B1b and Psy-B1c; Described TaZds-A1 gene relates to two allelotrope, is respectively TaZds-A1a and TaZds-A1b; Described TaZds-D1 gene relates to two allelotrope, is respectively TaZds-D1a and TaZds-D1b; Described Ppo-A1 gene relates to two allelotrope, is respectively Ppo-A1a and Ppo-A1b.
In described primer pair group A, two primers of each primer pair are used at PCR reaction system moderate, and described primer pair 1, and described primer pair 2 and the mol ratio of described primer pair 3 in PCR reaction system are 6:6:5; In described primer pair group B, two primers of each primer pair are used at PCR reaction system moderate, and described primer pair 4 and the mol ratio of described primer pair 5 in PCR reaction system are 12:6.
Concrete, in described primer pair group A, the final concentration of two DNA single chains shown in the sequence 1 in described primer pair 1 and sequence 2 in described PCR reaction system is 0.3 μ M; The final concentration of two DNA single chains shown in sequence 3 in described primer pair 2 and sequence 4 in described PCR reaction system is 0.3 μ M; The final concentration of two DNA single chains shown in sequence 5 in described primer pair 3 and sequence 6 in described PCR reaction system is 0.25 μ M.In described primer pair group B, the final concentration of two DNA single chains shown in the sequence 7 in described primer pair 4 and sequence 8 in described PCR reaction system is 0.6 μ M; The final concentration of two DNA single chains shown in sequence 9 in described primer pair 5 and sequence 10 in described PCR reaction system is 0.3 μ M.
The test kit that contains described primer pair group also belongs to protection scope of the present invention.
The preparation method of described primer pair group also belongs to protection scope of the present invention.
The preparation method of described primer pair group, specifically can comprise the step that described two single stranded DNAs of each primer pair in described primer pair group are packed separately respectively.
The preparation method of described test kit also belongs to protection scope of the present invention.
The preparation method of described test kit, specifically can comprise the steps: described two single stranded DNAs of each primer pair in described primer pair group respectively separately after packing, be packaged in same reagent box with at least one in following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
Following a1) or a2) method also belongs to protection scope of the present invention:
A1) evaluation or assistant identification wheat to be measured contain in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c any, contain in these two kinds of genes of TaZds-A1a and TaZds-A1b any, contain in these two kinds of genes of TaZds-D1a and TaZds-D1b anyly, and contain in Ppo-A1a gene and these two kinds of genes of Ppo-A1b gene any;
A2) identify or assistant identification wheat to be measured contains in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c anyly, and contain in TaZds-A1a and these two kinds of genes of TaZds-A1b any;
Described a1) method comprises the step of following (1) and (2):
Described (1) is following b1) and b2):
B1) take the genomic dna of wheat to be measured is template, with the described primer pair group A in described primer pair group, carries out PCR reaction, and the annealing temperature of described PCR reaction is 59 ℃;
In the present invention, the program of described PCR reaction is specially: 95 ℃ of denaturation 5min; 95 ℃ of sex change 45s, 59 ℃ of annealing 45s, 72 ℃ are extended 30s, and amplified reaction carries out 35 circulations; 72 ℃ are extended 10min; 4 ℃, insulation finishes.
B2) take the genomic dna of wheat to be measured is template, with primer pair group B described in described primer pair group, carries out PCR reaction, and the annealing temperature of described PCR reaction is 62 ℃;
In the present invention, the program of described PCR reaction is specially: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 70s, and amplified reaction carries out 34 circulations; 72 ℃ are extended 5min; 4 ℃, insulation finishes.
(2) size of the PCR product that detecting step (1) obtains, according to PCR product, determine that described wheat to be measured contains in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c as follows any, contain in these two kinds of genes of TaZds-A1a and TaZds-A1b any, contain in these two kinds of genes of TaZds-D1a and TaZds-D1b anyly, and contain in Ppo-A1a gene and these two kinds of genes of Ppo-A1b gene any:
With described primer pair group A, carrying out in the product of PCR reaction, is the DNA fragmentation of 151bp if contain size, and described wheat to be measured contains Psy-B1a gene or candidate is contained Psy-B1a gene; If containing size is the DNA fragmentation of 156bp, described wheat to be measured contains Psy-B1b gene or candidate is contained Psy-B1b gene; If containing size is the DNA fragmentation of 428bp, described wheat to be measured contains Psy-B1c gene or candidate is contained Psy-B1c gene; If containing size is the DNA fragmentation of 183bp, described wheat to be measured contains TaZds-A1a gene or candidate is contained TaZds-A1a gene; If containing size is the DNA fragmentation of 179bp, described wheat to be measured contains TaZds-A1b gene or candidate is contained TaZds-A1b gene;
In the product that carries out PCR reaction with described primer pair group B, if do not contain size, be the DNA fragmentation of 981bp, described wheat to be measured contains TaZds-D1a gene or candidate is contained TaZds-D1a gene; If containing size is the DNA fragmentation of 981bp, described wheat to be measured contains TaZds-D1b gene or candidate is contained TaZds-D1b gene; If containing size is the DNA fragmentation of 685bp, described wheat to be measured contains Ppo-A1a gene or candidate is contained Ppo-A1a gene; If containing size is the DNA fragmentation of 876bp, described wheat to be measured contains Ppo-A1b gene or candidate is contained Ppo-A1b gene;
Described a2) method comprises the step of following (3) and (4):
(3) take the genomic dna of wheat to be measured is template, with the described primer pair group A in described primer pair group, carries out PCR reaction, and the annealing temperature of described PCR reaction is 59 ℃;
In the present invention, the program of described PCR reaction is specially: 95 ℃ of denaturation 5min; 95 ℃ of sex change 45s, 59 ℃ of annealing 45s, 72 ℃ are extended 30s, and amplified reaction carries out 35 circulations; 72 ℃ are extended 10min; 4 ℃, insulation finishes.
(4) size of the PCR product that detecting step (3) obtains, according to PCR product, determine that described wheat to be measured contains in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c as follows any, and contain in TaZds-A1a and these two kinds of genes of TaZds-A1b any:
With described primer pair group A, carrying out in the product of PCR reaction, is the DNA fragmentation of 151bp if contain size, and described wheat to be measured contains Psy-B1a gene or candidate is contained Psy-B1a gene; If containing size is the DNA fragmentation of 156bp, described wheat to be measured contains Psy-B1b gene or candidate is contained Psy-B1b gene; If containing size is the DNA fragmentation of 428bp, described wheat to be measured contains Psy-B1c gene or candidate is contained Psy-B1c gene; If containing size is the DNA fragmentation of 183bp, described wheat to be measured contains TaZds-A1a gene or candidate is contained TaZds-A1a gene; If containing size is the DNA fragmentation of 179bp, described wheat to be measured contains TaZds-A1b gene or candidate is contained TaZds-A1b gene.
In aforesaid method, the nucleotide sequence of the DNA fragmentation that described size is 151bp is specifically as shown in sequence in sequence table 11; Described size is that the nucleotide sequence of DNA fragmentation of 156bp is specifically as shown in sequence in sequence table 12; Described size is that the nucleotide sequence of DNA fragmentation of 428bp is specifically as shown in sequence in sequence table 13; Described size is that the nucleotide sequence of DNA fragmentation of 183bp is specifically as shown in sequence in sequence table 14; Described size is that the nucleotide sequence of DNA fragmentation of 179bp is specifically as shown in sequence in sequence table 15; Described size is that the nucleotide sequence of DNA fragmentation of 981bp is specifically as shown in sequence in sequence table 16; Described size is that the nucleotide sequence of DNA fragmentation of 685bp is specifically as shown in sequence in sequence table 17; Described size is that the nucleotide sequence of DNA fragmentation of 876bp is specifically as shown in sequence in sequence table 18.
In one embodiment of the invention, with the described primer pair group A in described primer pair group, carry out in the reaction system of PCR reaction, amount as the genomic dna of the described wheat to be measured of template is 100-200ng, and the final concentration of dNTPs is 200 μ M, and the amount of archaeal dna polymerase is 1.5U.PCR response procedures is specially: 95 ℃ of denaturation 5min; 95 ℃ of sex change 45s, 59 ℃ of annealing 45s, 72 ℃ are extended 30s, and amplified reaction carries out 35 circulations; 72 ℃ are extended 10min; 4 ℃, insulation finishes.
In one embodiment of the invention, with the described primer pair group B in described primer pair group, carrying out in the reaction system of PCR reaction, is 100-200ng as the amount of the genomic dna of the described wheat to be measured of template, and the final concentration of dNTPs is 200 μ M.PCR response procedures is specially: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 70s, and amplified reaction carries out 34 circulations; 72 ℃ are extended 5min; 4 ℃, insulation finishes.
Another object of the present invention is to provide a kind of method of cultivating the wheat breed of Flour whiteness raising.
The method of the wheat breed that cultivation Flour whiteness provided by the present invention improves, comprise adopt following a)-d) at least one wheat as parent, carry out the step of breeding:
A) contain or candidate is contained Psy-B1b gene;
B) contain or candidate is contained TaZds-A1a gene;
C) contain or candidate is contained TaZds-D1b gene;
D) contain or candidate is contained Ppo-A1b gene.
From numerous wheat breeds, select meet a)-d) in the method for wheat of at least one, be above-mentioned " identify or assistant identification wheat to be measured contains in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c any; contain in these two kinds of genes of TaZds-A1a and TaZds-A1b any; contain in these two kinds of genes of TaZds-D1a and TaZds-D1b any, and contain in Ppo-A1a gene and these two kinds of genes of Ppo-A1b gene any " method.Selected wheat meet above-mentioned a)-d) item number in four is more, represents that selected wheat is more suitable as the parent who adopts while cultivating the wheat breed that Flour whiteness improves.
In the present invention, described wheat to be measured is specially at least one in following wheat breed (being): peaceful winter No. 6, all wheats 16, all wheats 17, western agriculture 979, western agriculture 88, Shan 354, littlely lay down 54, Xuzhou 25, western agriculture 6028, western agriculture 8727 and Xifeng 27.
Between each primer pair of primer pair group A provided by the present invention or primer pair group B, there is not mutual restraining effect and mispairing, the reliable results of detection kind (being), reproducible, cost is low.Primer pair group A provided by the present invention or primer pair group B all select same annealing temperature, have realized the discriminating that uniform temp PCR completes one group of gene, and high specificity, and testing process is consuming time short.The invention provides a kind of method of more fully Fast Evaluation wheat Color Quality, and provide reference for wheat color and luster quality breeding.
Accompanying drawing explanation
Fig. 1 is the pcr amplification result of 11 parts of wheat breeds (being) flour color and luster genes involved Psy-B1 and TaZds-A1 in embodiment 1.Wherein, twice repetition of A1 and A2() be the single PCR detected result of genotype of TaZds-A1 gene; Twice repetition of B1 and B2() and B3 and twice repetition of B4() be the single PCR detected result of genotype of Psy-B1 gene; C is multiplex PCR amplification.Wherein, 1: the peaceful winter No. 6; 2: all wheats 16; 3: all wheats 17; 4: western agriculture 979; 5: western agriculture 88; 6: Shan 354; 7: littlely lay down 54; 8: Xuzhou 25; 9: western agriculture 6028; 10: western agriculture 8727; 11: Xifeng 27.
Fig. 2 is the pcr amplification result of 11 parts of wheat breeds (being) flour color and luster genes involved TaZds-D1 and Ppo-A1 in embodiment 2.Wherein, twice repetition of A1 and A2() be the single PCR detected result of genotype of TaZds-D1 gene; Twice repetition of B1 and B2() be the single PCR detected result of genotype of Ppo-A1 gene; C is multiplex PCR amplification.Wherein, M: standard molecular weight DL2000; 1: the peaceful winter No. 6; 2: all wheats 16; 3: all wheats 17; 4: western agriculture 979; 5: western agriculture 88; 6: Shan 354; 7: littlely lay down 54; 8: Xuzhou 25; 9: western agriculture 6028; 10: western agriculture 8727; 11: Xifeng 27.
Fig. 3 is the multiplex PCR amplification of 7 parts of known type wheat breeds (being) flour color and luster genes involved Psy-B1 and TaZds-A1 in comparative example 1.Wherein, 1,8: all wheats 17; 2: all wheats 16; 3: the peaceful winter No. 6; 4: western agriculture 88; 5: littlely lay down 54; 6: western agriculture 979; 7: western agriculture 6028.
Fig. 4 is the multiplex PCR amplification of 7 parts of known type wheat breeds (being) flour color and luster genes involved TaZds-D1 and Ppo-A1 in comparative example 2.Wherein, 1,3: the peaceful winter No. 6; 2: western agriculture 979; 4: western agriculture 88; 5: all wheats 16; 6: all wheats 17; 7: Shan 354; 8,9: Xuzhou 25.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The multiplex PCR of embodiment 1, whole meal flour color and luster genes involved Psy-B1 and TaZds-A1 detects
The multiple PCR primer of the present embodiment is to group by two primer pairs that are specific to Psy-B1 gene, and the primer pair that is specific to TaZds-A1 gene forms; Wherein, two primer pairs that are specific to Psy-B1 gene described in are respectively the primer pair 1 that in sequence table, two single stranded DNAs shown in sequence 1 and sequence 2 form, and the primer pair 2 that forms of two single stranded DNAs shown in sequence 3 and sequence 4; A primer pair of the described TaZds-A1 of being specific to gene is the primer pair 3 that in sequence table, two single stranded DNAs shown in sequence 5 and sequence 6 form.
The multiple PCR primer of the present embodiment can detect 5 kinds of genotype Psy-B1a, Psy-B1b, Psy-B1c, TaZds-A1a and TaZds-A1b of Psy-B1 and two gene locuss of TaZds-A1 to group simultaneously.The material that contains Psy-B1a gene, pcr amplification goes out 151bp(sequence 11) a band; The material that contains Psy-B1b gene, pcr amplification goes out 156bp(sequence 12) a band; The material that contains Psy-B1c gene, pcr amplification goes out 428bp(sequence 13) a band; The material that contains TaZds-A1a gene, pcr amplification goes out 183bp(sequence 14) a band; The material that contains TaZds-A1b gene, pcr amplification goes out 179bp(sequence 15) a band.
One, the preparation of wheat cdna group
Adopt CTAB method to carry out the extraction of genomic dna to 11 parts of wheat breeds (being), obtain the genomic dna corresponding to 11 parts of wheat breeds (being), as the template of hand-pulled noodles color and luster genes involved Psy-B1 and the amplification of TaZds-A1 multiplex PCR.Wherein, 11 parts of wheat breeds (being) be peaceful winter No. 6, all wheats 16, all wheats 17, western agriculture 979, western agriculture 88, Shan 354, littlely lay down 54, Xuzhou 25, western agriculture 6028, western agriculture 8727 and Xifeng 27.The Psy-B1 of these 11 wheat breeds (being) and the genotype of TaZds-A1 gene refer to table 1.
Wherein, western agriculture 979, western agriculture 88, Shan 354, littlely lay down 54, the genotype of the Psy-B1 gene of Xuzhou 25, western agriculture 6028 and Xi Nong 8727 referring to " Ye Shi. the allelic variation of Shaanxi wheat grain PPO active gene and YP content gene detects and distributional analysis. Xibei Univ. of Agricultural & Forest Science & Technology; 2010, Master's thesis " literary composition; Shan 354, littlely lay down 54, the genotype of the TaZds-A1 gene in Xuzhou 25 referring to " Dong Changhai. clone and the function labeling development of common wheat Kernel yellow pigment genes involved. Agricultural University Of Hebei,, Master's thesis in 2011 " literary composition.
Meanwhile, the present inventor all identifies through the single PCR at least twice each site the genotype of the Psy-B1 of above 11 wheat breeds (being) and TaZds-A1 gene, specific as follows:
(1) genotype detection of the TaZds-A1 gene of above 11 wheat breeds (being) referring to " Dong Changhai. clone and the function labeling development of common wheat Kernel yellow pigment genes involved. Agricultural University Of Hebei,, Master's thesis in 2011 " literary composition carries out.The wheat cdna group DNA of take is template, adopts YP2A-1 labeled primer (sequence 5/ sequence 6 in table 2) to carry out single pcr amplification.Result shows: it is the object band of 179bp that peaceful winter of wheat breed (being) No. 6, all wheats 16, western agriculture 88, Xuzhou 25, western agriculture 8727 and Xifeng 27 obtain size through pcr amplification, and the genotype that shows its TaZds-A1 gene is TaZds-A1b; Week wheat 17, western agriculture 979, Shan 354, little lay down 54 and Xi Nong 6028 through pcr amplification, to obtain size be the object band of 183bp, the genotype that shows its TaZds-A1 gene is TaZds-A1a.Experiment repeats at least twice, and result is consistent.See A1 and A2 in Fig. 1.
(2) genotype detection of the Psy-B1 gene of above 11 wheat breeds (being) referring to " Ye Shi. the allelic variation of Shaanxi wheat grain PPO active gene and YP content gene detects and distributional analysis. Xibei Univ. of Agricultural & Forest Science & Technology; 2010, Master's thesis " literary composition carries out.The wheat cdna group DNA of take is template, adopts respectively YP7B-1 labeled primer (sequence 1/ sequence 2 in table 2), YP7B-2 labeled primer (sequence 3/ sequence 4 in table 2) to carry out single pcr amplification.Result shows: peaceful winter of wheat breed (being) No. 6, all wheats 17, western agriculture 6028 and Xifeng 27 adopt sequence 1/ sequences 2 to carry out pcr amplification to obtain size be the object band of 151bp, the genotype that shows its Psy-B1 gene is Psy-B1a; The all wheats 16 of wheat breed (being), western agriculture 979, western agriculture 88, little lay down 54 and Xi Nong 8727 adopt sequence 1/ sequences 2 to carry out pcr amplification to obtain size be the object band of 156bp, the genotype that shows its Psy-B1 gene is Psy-B1b; 354He Xuzhou, wheat breed (being) Shan 25 adopt sequence 3/ sequences 4 to carry out pcr amplification to obtain size be the object band of 428bp, the genotype that shows its Psy-B1 gene is Psy-B1c.Experiment repeats at least twice, and result is consistent.See B1 in Fig. 1, B2 and B3, B4.
The genotype of 11 parts of wheat breeds of table 1 (being) flour color and luster genes involved Psy-B1 and TaZds-A1
Figure BDA0000401303250000081
Two, multiplex PCR amplification wheat breed (being) flour color and luster genes involved Psy-B1 and TaZds-A1
The genomic dna of 11 parts of wheat breeds (being) of preparing with above-mentioned step 1 is respectively template, utilizes the primer pair composition in table 2 to carry out multiplex PCR amplification.
PCR reaction is totally 20 μ L, wherein, DNA profiling 100-200ng, the final concentration of dNTPs in the total system of reaction is 200 μ M, Taq archaeal dna polymerase 1.5U, the consumption that each primer reacts in total system at PCR is: two DNA single chains shown in the sequence 1 in primer pair 1 and sequence 2 react consumption in total system at PCR and are the final concentration of 6pmol(in reaction system and are 0.3 μ M); Two DNA single chains shown in sequence 3 in primer pair 2 and sequence 4 react consumption in total system at PCR and are the final concentration of 6pmol(in reaction system and are 0.3 μ M); Two DNA single chains shown in sequence 5 in primer pair 3 and sequence 6 react consumption in total system at PCR and are the final concentration of 5pmol(in reaction system and are 0.25 μ M).
PCR response procedures is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 45s, 59 ℃ of annealing 45s, 72 ℃ are extended 30s, and amplified reaction carries out 35 circulations; 72 ℃ are extended 10min; 4 ℃, insulation finishes.
Pcr amplification carries out on 6% denaturing polyacrylamide gel, and voltage during electrophoresis is 2000V, and electrophoresis time is 1h.
Table 2 flour color and luster genes involved Psy-B1 and TaZds-A1 amplimer sequence information
Figure BDA0000401303250000091
Note: the genotype that phenotype is low YP is beneficial to the raising of whole meal flour whiteness.
Result decision method: be the DNA fragmentation of 151bp (sequence 11) if contain size in PCR product, described wheat to be measured contains Psy-B1a gene; If containing size in PCR product is the DNA fragmentation of 156bp (sequence 12), described wheat to be measured contains Psy-B1b gene; If containing size in PCR product is the DNA fragmentation of 428bp (sequence 13), described wheat to be measured contains Psy-B1c gene; If containing size in PCR product is the DNA fragmentation of 183bp (sequence 14), described wheat to be measured contains TaZds-A1a gene; If containing size in PCR product is the DNA fragmentation of 179bp (sequence 15), described wheat to be measured contains TaZds-A1b gene.
Three, the multiplex PCR amplification of wheat breed flour color and luster genes involved Psy-B1 and TaZds-A1
Electrophoresis result is as shown in C in Fig. 1:
Peaceful winter of wheat breed (being) No. 6, after above-mentioned multiplex PCR amplification, obtains size for two bands of 151bp and 179bp, and the peaceful winter of this explanation wheat breed (being) is contained Psy-B1a and TaZds-A1b gene for No. 6;
The all wheats 16 of wheat breed (being), after above-mentioned multiplex PCR amplification, obtain size for two bands of 156bp and 179bp, and all wheats 16 of this explanation wheat breed (being) contain Psy-B1b and TaZds-A1b gene;
The all wheats 17 of wheat breed (being), after above-mentioned multiplex PCR amplification, obtain size for two bands of 151bp and 183bp, and all wheats 17 of this explanation wheat breed (being) contain Psy-B1a and TaZds-A1a gene;
The western agriculture 979 of wheat breed (being), after above-mentioned multiplex PCR amplification, obtains size for two bands of 156bp and 183bp, and the western agriculture 979 of this explanation wheat breed (being) contains Psy-B1b and TaZds-A1a gene;
The western agriculture 88 of wheat breed (being), after above-mentioned multiplex PCR amplification, obtains size for two bands of 156bp and 179bp, and the western agriculture 88 of this explanation wheat breed (being) contains Psy-B1b and TaZds-A1b gene;
Wheat breed (being) Shan 354, after above-mentioned multiplex PCR amplification, obtains size for two bands of 428bp and 183bp, and Psy-B1c and TaZds-A1a gene are contained in this explanation wheat breed (being) Shan 354;
Wheat breed (being) is little lays down 54 after above-mentioned multiplex PCR amplification, obtains size for two bands of 156bp and 183bp, and this explanation wheat breed (being) is little lays down and 54 contain Psy-B1b and TaZds-A1a gene;
Wheat breed (being) Xuzhou 25, after above-mentioned multiplex PCR amplification, obtains size for two bands of 428bp and 179bp, and Psy-B1c and TaZds-A1b gene are contained in this explanation wheat breed (being) Xuzhou 25;
The western agriculture 6028 of wheat breed (being), after above-mentioned multiplex PCR amplification, obtains size for two bands of 151bp and 183bp, and the western agriculture 6028 of this explanation wheat breed (being) contains Psy-B1a and TaZds-A1a gene;
The western agriculture 8727 of wheat breed (being), after above-mentioned multiplex PCR amplification, obtains size for two bands of 156bp and 179bp, and the western agriculture 8727 of this explanation wheat breed (being) contains Psy-B1b and TaZds-A1b gene.
Wheat breed (being) Xifeng 27, after above-mentioned multiplex PCR amplification, obtains size for two bands of 151bp and 179bp, and Psy-B1a and TaZds-A1b gene are contained in this explanation wheat breed (being) Xifeng 27.
The above results and table 1 are in full accord, the present inventor further will check order after each amplified band recovery of above each wheat breed (being), find that the nucleotide sequence that each wheat breed (being) amplification gained size is the DNA fragmentation of 151bp is sequence 11, the nucleotide sequence that amplification gained size is the DNA fragmentation of 156bp is sequence 12, the nucleotide sequence that amplification gained size is the DNA fragmentation of 428bp is sequence 13, the nucleotide sequence that amplification gained size is the DNA fragmentation of 183bp is sequence 14, the nucleotide sequence that amplification gained size is the DNA fragmentation of 179bp is sequence 15, this has further confirmed the accuracy of above-mentioned multiplex PCR result.The multiple PCR primer of this explanation the present embodiment to group can be simultaneously, detect fast and accurately 5 kinds of genotype Psy-B1a, Psy-B1b, Psy-B1c, TaZds-A1a and TaZds-A1b in whole meal flour color and luster genes involved Psy-B1 and two sites of TaZds-A1.This will be conducive to select and contain Psy-B1b and the genotypic wheat breed of TaZds-A1a or flour simultaneously, carry out hand-pulled noodles color and luster genetic improvement or evaluation.
The multiplex PCR of embodiment 2, whole meal flour color and luster genes involved TaZds-D1 and Ppo-A1 detects
The multiple PCR primer of the present embodiment is to group by a primer pair that is specific to TaZds-D1 gene, and the primer pair that is specific to Ppo-A1 gene forms; Wherein, a primer pair that is specific to TaZds-D1 gene described in is the primer pair 4 that in sequence table, two single stranded DNAs shown in sequence 7 and sequence 8 form; A primer pair of the described Ppo-A1 of being specific to gene is the primer pair 5 that in sequence table, two single stranded DNAs shown in sequence 9 and sequence 10 form.
The multiple PCR primer of the present embodiment can detect 4 kinds of genotype TaZds-D1a, TaZds-D1b, Ppo-A1a and Ppo-A1b of TaZds-D1 and two gene locuss of Ppo-A1 to group simultaneously.The material that contains TaZds-D1a gene, pcr amplification does not go out 981bp(sequence 16) a band; The material that contains TaZds-D1b gene, pcr amplification goes out 981bp(sequence 16) a band; The material that contains Ppo-A1a gene, pcr amplification goes out 685bp(sequence 17) a band; The material that contains Ppo-A1b gene, pcr amplification goes out 876bp(sequence 18) a band.
One, the preparation of wheat cdna group
Adopt CTAB method to carry out the extraction of genomic dna to 11 parts of wheat breeds (being), obtain the genomic dna corresponding to 11 parts of wheat breeds (being), as the template of hand-pulled noodles color and luster genes involved TaZds-D1 and the amplification of Ppo-A1 multiplex PCR.Wherein, 11 parts of wheat breeds (being) be peaceful winter No. 6, all wheats 16, all wheats 17, western agriculture 979, western agriculture 88, Shan 354, littlely lay down 54, Xuzhou 25, western agriculture 6028, western agriculture 8727 and Xifeng 27.The TaZds-D1 of these 11 wheat breeds (being) and the genotype of Ppo-A1 gene refer to table 3.
Wherein, western agriculture 979, western agriculture 88, Shan 354, littlely lay down 54, the genotype of the Ppo-A1 gene of Xuzhou 25, western agriculture 6028 and Xi Nong 8727 referring to " Ye Shi. the allelic variation of Shaanxi wheat grain PPO active gene and YP content gene detects and distributional analysis. Xibei Univ. of Agricultural & Forest Science & Technology; 2010, Master's thesis " literary composition.
Meanwhile, the present inventor all identifies through the single PCR at least twice each site the genotype of the TaZds-D1 of above 11 wheat breeds (being) and Ppo-A1 gene, specific as follows:
(1) genotype detection of the TaZds-D1 gene of above 11 wheat breeds (being) referring to " Zhang Caiying. the QTL of the main yield and quality proterties of wheat identifies and the exploitation of ZDS functional label. Agricultural University Of Hebei,, Ph D dissertation in 2010 " literary composition carries out.The wheat cdna group DNA of take is template, adopts YP2D-1 labeled primer (sequence 7/ sequence 8 in table 4) to carry out single pcr amplification.Result shows: all wheats 16 of wheat breed (being), all wheats 17, western agriculture 979, western agriculture 88, Shan 354, littlely lay down 54, through pcr amplification, all not obtain size be the object band of 981bp for Xuzhou 25, western agriculture 6028 and Xi Nong 8727, the genotype that shows its TaZds-D1 gene is TaZds-D1a.It is the object band of 981bp that peaceful winter of wheat breed (being) No. 6 and Xifeng 27 obtain size through pcr amplification, and the genotype that shows its TaZds-D1 gene is TaZds-D1b.Experiment repeats at least twice, and result is consistent.See A1 and A2 in Fig. 2.
(2) genotype detection of the Ppo-A1 gene of above 11 wheat breeds (being) referring to " Ye Shi. the allelic variation of Shaanxi wheat grain PPO active gene and YP content gene detects and distributional analysis. Xibei Univ. of Agricultural & Forest Science & Technology; 2010, Master's thesis " literary composition carries out.The wheat cdna group DNA of take is template, adopts PPO18 labeled primer (sequence 9/ sequence 10 in table 4) to carry out single pcr amplification.Result shows: it is the object band of 685bp that peaceful winter of wheat breed (being) No. 6, all wheats 16, all wheats 17,354He Xuzhou 25, Shan obtain size through pcr amplification, and the genotype that shows its Ppo-A1 gene is Ppo-A1a; The western agriculture 979 of wheat breed (being), western agriculture 88, littlely lay down 54, through pcr amplification, to obtain size be the object band of 876bp for western agriculture 6028, western agriculture 8727 and Xifeng 27, the genotype that shows its Ppo-A1 gene is Ppo-A1b.Experiment repeats at least twice, and result is consistent.See B1 and B2 in Fig. 2.
The genotype of 11 parts of wheat breeds of table 3 (being) flour color and luster genes involved TaZds-D1 and Ppo-A1
Figure BDA0000401303250000111
Figure BDA0000401303250000121
Two, multiplex PCR amplification wheat breed (being) flour color and luster genes involved TaZds-D1 and Ppo-A1
The genomic dna of 11 parts of wheat breeds (being) of preparing with above-mentioned step 1 is respectively template, utilizes the primer pair composition in table 4 to carry out multiplex PCR amplification.
PCR reaction is totally 20 μ L, wherein, DNA profiling 100-200ng, MasterMix(contains archaeal dna polymerase, PCR reaction buffer, 4 kinds of dNTP) to make the concentration of every kind of dNTP in the PCR reaction system of 20 μ L be 200 μ M to (Kang Wei century bio tech ltd, Beijing) 10 μ L(), the consumption that each primer reacts in total system at PCR is: two DNA single chains shown in the sequence 7 in primer pair 4 and sequence 8 react consumption in total system at PCR and are the final concentration of 12pmol(in reaction system and are 0.6 μ M); Two DNA single chains shown in sequence 9 in primer pair 5 and sequence 10 react consumption in total system at PCR and are the final concentration of 6pmol(in reaction system and are 0.3 μ M).
PCR response procedures is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 70s, and amplified reaction carries out 34 circulations; 72 ℃ are extended 5min; 4 ℃, insulation finishes.
Pcr amplification carries out on 2% sepharose, and voltage during electrophoresis is 100V, and electrophoresis time is 1.5h.
Table 4 flour color and luster genes involved TaZds-D1 and Ppo-A1 amplimer sequence information
Figure BDA0000401303250000122
Note: the genotype that phenotype is low YP is beneficial to the raising of whole meal flour whiteness, and phenotype is the raising that low PPO genotype is beneficial to whole meal flour whiteness.
Result decision method: be the DNA fragmentation of 981bp if do not contain size in PCR product, described wheat to be measured contains TaZds-D1a gene; If containing size in PCR product is the DNA fragmentation of 981bp (sequence 16), described wheat to be measured contains TaZds-D1b gene; If containing size in PCR product is the DNA fragmentation of 685bp (sequence 17), described wheat to be measured contains Ppo-A1a gene; If containing size in PCR product is the DNA fragmentation of 876bp, described wheat to be measured contains Ppo-A1b gene (sequence 18).
Three, the multiplex PCR amplification of wheat breed flour color and luster genes involved TaZds-D1 and Ppo-A1
Electrophoresis result is as shown in C in Fig. 2:
Peaceful winter of wheat breed (being) No. 6, after above-mentioned multiplex PCR amplification, obtains size for two bands of 981bp and 685bp, and the peaceful winter of this explanation wheat breed (being) is contained TaZds-D1b and Ppo-A1a gene for No. 6;
The all wheats 16 of wheat breed (being) are after above-mentioned multiplex PCR amplification, and obtaining size is a band of 685bp (not obtaining size is the band of 981bp), and all wheats 16 of this explanation wheat breed (being) contain TaZds-D1a and Ppo-A1a gene;
The all wheats 17 of wheat breed (being) are after above-mentioned multiplex PCR amplification, and obtaining size is a band of 685bp (not obtaining size is the band of 981bp), and all wheats 17 of this explanation wheat breed (being) contain TaZds-D1a and Ppo-A1a gene;
The western agriculture 979 of wheat breed (being) is after above-mentioned multiplex PCR amplification, and obtaining size is a band of 876bp (not obtaining size is the band of 981bp), and the western agriculture 979 of this explanation wheat breed (being) contains TaZds-D1a and Ppo-A1b gene;
The western agriculture 88 of wheat breed (being) is after above-mentioned multiplex PCR amplification, and obtaining size is a band of 876bp (not obtaining size is the band of 981bp), and the western agriculture 88 of this explanation wheat breed (being) contains TaZds-D1a and Ppo-A1b gene;
Wheat breed (being) Shan 354 is after above-mentioned multiplex PCR amplification, and obtaining size is a band of 685bp (not obtaining size is the band of 981bp), and TaZds-D1a and Ppo-A1a gene are contained in this explanation wheat breed (being) Shan 354;
Wheat breed (being) is little lays down 54 after above-mentioned multiplex PCR amplification, and obtaining size is a band of 876bp (not obtaining size is the band of 981bp), and this illustrates that little the laying down of wheat breed (being) 54 contains TaZds-D1a and Ppo-A1b gene;
Wheat breed (being) Xuzhou 25 is after above-mentioned multiplex PCR amplification, and obtaining size is a band of 685bp (not obtaining size is the band of 981bp), and TaZds-D1a and Ppo-A1a gene are contained in this explanation wheat breed (being) Xuzhou 25;
The western agriculture 6028 of wheat breed (being) is after above-mentioned multiplex PCR amplification, and obtaining size is a band of 876bp (not obtaining size is the band of 981bp), and the western agriculture 6028 of this explanation wheat breed (being) contains TaZds-D1a and Ppo-A1b gene;
The western agriculture 8727 of wheat breed (being) is after above-mentioned multiplex PCR amplification, and obtaining size is a band of 876bp (not obtaining size is the band of 981bp), and the western agriculture 8727 of this explanation wheat breed (being) contains TaZds-D1a and Ppo-A1b gene.
Wheat breed (being) Xifeng 27, after above-mentioned multiplex PCR amplification, obtains size for two bands of 981bp and 876bp, and TaZds-D1b and Ppo-A1b gene are contained in this explanation wheat breed (being) Xifeng 27.
The above results and table 3 are in full accord, the present inventor further will check order after each amplified band recovery of above each wheat breed (being), find that the nucleotide sequence that each wheat breed (being) amplification gained size is the DNA fragmentation of 981bp is sequence 16, the nucleotide sequence that amplification gained size is the DNA fragmentation of 685bp is sequence 17, the nucleotide sequence that amplification gained size is the DNA fragmentation of 876bp is sequence 18, and this has further confirmed the accuracy of above-mentioned multiplex PCR result.The multiple PCR primer of this explanation the present embodiment to group can be simultaneously, detect fast and accurately 4 kinds of genotype TaZds-D1a, TaZds-D1b, Ppo-A1a and Ppo-A1b in whole meal flour color and luster genes involved TaZds-D1 and two sites of Ppo-A1.This will be conducive to select and contain TaZds-D1b and the genotypic wheat breed of Ppo-A1b or flour simultaneously, carry out hand-pulled noodles color and luster genetic improvement or evaluation.
The multiplex PCR of comparative example 1, whole meal flour color and luster genes involved Psy-B1 and TaZds-A1 detects
In this comparative example, genomic dna with the wherein wheat breed of 7 parts of known types shown in table 1 (all wheats 17, all wheats 16, peaceful winter No. 6, western agriculture 88, littlely lay down 54, western agriculture 979 and Xi Nong 6028) is respectively template, the primer pair group that utilization is comprised of YP7B-1 and two primer pairs corresponding to YP2A-1 mark (in Table 2, i.e. primer pair 1 and primer pair 3) is carried out multiplex PCR amplification.
PCR reaction is totally 20 μ L, wherein, DNA profiling 100-200ng, the final concentration of dNTPs in the total system of reaction is 160 μ M, Taq archaeal dna polymerase 1.5U, the consumption that each primer reacts in total system at PCR is: two DNA single chains shown in the sequence 1 in primer pair 1 and sequence 2 react content in total system at PCR and are the final concentration of 8pmol(in reaction system and are 0.4 μ M); Two DNA single chains shown in sequence 5 in primer pair 3 and sequence 6 react content in total system at PCR and are the final concentration of 8pmol(in reaction system and are 0.4 μ M).
PCR response procedures is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 45s, 61 ℃ of annealing 45s, 72 ℃ are extended 30s, and amplified reaction carries out 37 circulations; 72 ℃ are extended 10min; 4 ℃, insulation finishes.
Pcr amplification carries out on 6% denaturing polyacrylamide gel, and voltage during electrophoresis is 2000V, and electrophoresis time is 1h.
Electrophoresis result as shown in Figure 3, utilize aforesaid method can one-time detection to go out 4 kinds of genotype Psy-B1a, Psy-B1b, TaZds-A1a and TaZds-A1b of Psy-B1 and two gene locuss of TaZds-A1, each kind goes out to be with result consistent with single PCR result, but band a little less than, result is undesirable, needs further system to be debugged.
The multiplex PCR of comparative example 2, whole meal flour color and luster genes involved TaZds-D1 and Ppo-A1 detects
In this comparative example, genomic dna with the wherein wheat breed of 7 parts of known types shown in table 3 (peaceful winter No. 6, western agriculture 979, western agriculture 88, all wheats 16, all wheats 17,354He Xuzhou, Shan 25) is respectively template, the primer pair group that utilization is comprised of YP2D-1 and two primer pairs corresponding to PPO18 mark (in Table 4, i.e. primer pair 4 and primer pair 5) is carried out multiplex PCR amplification.
PCR reaction is totally 20 μ L, wherein, DNA profiling 100-200ng, MasterMix(contains archaeal dna polymerase, PCR reaction buffer, 4 kinds of dNTP) to make the concentration of every kind of dNTP in the PCR reaction system of 20 μ L be 200 μ M to (Kang Wei century bio tech ltd, Beijing) 10 μ L(), the consumption that each primer reacts in total system at PCR is: two DNA single chains shown in the sequence 7 in primer pair 4 and sequence 8 react content in total system at PCR and are the final concentration of 8pmol(in reaction system and are 0.4 μ M); Two DNA single chains shown in sequence 9 in primer pair 5 and sequence 10 react content in total system at PCR and are the final concentration of 8pmol(in reaction system and are 0.4 μ M).
PCR response procedures is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 64 ℃ of annealing 30s, 72 ℃ are extended 30s, and amplified reaction carries out 32 circulations; 72 ℃ are extended 5min; 4 ℃, insulation finishes.
Pcr amplification carries out on 2% sepharose, and voltage during electrophoresis is 100V, and electrophoresis time is 1.5h.
As shown in Figure 4, for the wheat breed that contains TaZds-D1b gene (being), the object band of the 981bp that it amplifies only mays be seen indistinctly at (white arrow place in Fig. 4) electrophoresis result, and result is undesirable, need further debug.
Figure IDA0000401303330000011
Figure IDA0000401303330000021
Figure IDA0000401303330000031
Figure IDA0000401303330000041
Figure IDA0000401303330000051
Figure IDA0000401303330000061
Figure IDA0000401303330000071
Figure IDA0000401303330000081
Figure IDA0000401303330000091
Figure IDA0000401303330000101

Claims (9)

  1. For the identification of or the multiple PCR primer of assistant identification whole meal flour color and luster to be measured genes involved to group, described flour color and luster genes involved is Psy-B1 gene, TaZds-A1 gene, TaZds-D1 gene and Ppo-A1 gene, it is characterized in that: described multiple PCR primer is comprised of primer pair group A and primer pair group B group; Described primer pair group A is by two primer pairs that are specific to described Psy-B1 gene, and a primer pair that is specific to described TaZds-A1 gene forms; Described primer pair group B is by a primer pair that is specific to described TaZds-D1 gene, and a primer pair that is specific to described Ppo-A1 gene forms;
    In described primer pair group A, described in be specific to described Psy-B1 gene two primer pairs be respectively the primer pair 1 that in sequence table, two single stranded DNAs shown in sequence 1 and sequence 2 form, and the primer pair 2 that forms of two single stranded DNAs shown in sequence 3 and sequence 4; A described primer pair that is specific to described TaZds-A1 gene is the primer pair 3 that in sequence table, two single stranded DNAs shown in sequence 5 and sequence 6 form;
    In described primer pair group B, described in be specific to described TaZds-D1 gene a primer pair be the primer pair 4 that in sequence table, two single stranded DNAs shown in sequence 7 and sequence 8 form; A described primer pair that is specific to described Ppo-A1 gene is the primer pair 5 that in sequence table, two single stranded DNAs shown in sequence 9 and sequence 10 form.
  2. For the identification of or the multiple PCR primer of assistant identification whole meal flour color and luster to be measured genes involved to group, be the described primer pair group A in claim 1.
  3. 3. primer pair group according to claim 1 and 2, it is characterized in that: in described primer pair group A, two primers of each primer pair are used at PCR reaction system moderate, and described primer pair 1, described primer pair 2 and the mol ratio of described primer pair 3 in PCR reaction system are 6:6:5; Described special primer is used at PCR reaction system moderate two primers of each primer pair in group B, and described primer pair 4 and the mol ratio of described primer pair 5 in PCR reaction system are 12:6.
  4. 4. the test kit that contains arbitrary described primer pair group in claim 1-3.
  5. 5. the preparation method of arbitrary described primer pair group in claim 1-3, is characterized in that: described preparation method comprises the step that described two single stranded DNAs of each primer pair in arbitrary described primer pair group in claim 1-3 are packed separately respectively.
  6. 6. the preparation method of test kit described in claim 4, comprise the steps: described two single stranded DNAs of each primer pair in arbitrary described primer pair group in claim 1-3 respectively separately after packing, be packaged in same reagent box with at least one in following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
  7. Following a1) or method a2) 7.:
    A1) evaluation or assistant identification wheat to be measured contain in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c any, contain in these two kinds of genes of TaZds-A1a and TaZds-A1b any, contain in these two kinds of genes of TaZds-D1a and TaZds-D1b anyly, and contain in Ppo-A1a and these two kinds of genes of Ppo-A1b any;
    A2) identify or assistant identification wheat to be measured contains in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c anyly, and contain in TaZds-A1a and these two kinds of genes of TaZds-A1b any;
    It is characterized in that: method described a1) comprises the step of following (1) and (2):
    Described (1) is following b1) and b2):
    B1) take the genomic dna of wheat to be measured is template, with the described primer pair group A in arbitrary described primer pair group in claim 1-3, carries out PCR reaction, and the annealing temperature of described PCR reaction is 59 ℃;
    B2) take the genomic dna of wheat to be measured is template, with the B of primer pair group described in primer pair group described in claim 1 or 3, carries out PCR reaction, and the annealing temperature of described PCR reaction is 62 ℃;
    (2) size of the PCR product that detecting step (1) obtains, according to PCR product, determine that described wheat to be measured contains in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c as follows any, contain in these two kinds of genes of TaZds-A1a and TaZds-A1b any, contain in these two kinds of genes of TaZds-D1a and TaZds-D1b anyly, and contain in Ppo-A1a and these two kinds of genes of Ppo-A1b any:
    With described primer pair group A, carrying out in the product of PCR reaction, is the DNA fragmentation of 151bp if contain size, and described wheat to be measured contains Psy-B1a gene or candidate is contained Psy-B1a gene; If containing size is the DNA fragmentation of 156bp, described wheat to be measured contains Psy-B1b gene or candidate is contained Psy-B1b gene; If containing size is the DNA fragmentation of 428bp, described wheat to be measured contains Psy-B1c gene or candidate is contained Psy-B1c gene; If containing size is the DNA fragmentation of 183bp, described wheat to be measured contains TaZds-A1a gene or candidate is contained TaZds-A1a gene; If containing size is the DNA fragmentation of 179bp, described wheat to be measured contains TaZds-A1b gene or candidate is contained TaZds-A1b gene;
    In the product that carries out PCR reaction with described primer pair group B, if do not contain size, be the DNA fragmentation of 981bp, described wheat to be measured contains TaZds-D1a gene or candidate is contained TaZds-D1a gene; If containing size is the DNA fragmentation of 981bp, described wheat to be measured contains TaZds-D1b gene or candidate is contained TaZds-D1b gene; If containing size is the DNA fragmentation of 685bp, described wheat to be measured contains Ppo-A1a gene or candidate is contained Ppo-A1a gene; If containing size is the DNA fragmentation of 876bp, described wheat to be measured contains Ppo-A1b gene or candidate is contained Ppo-A1b gene;
    Described a2) method comprises the step of following (3) and (4):
    (3) take the genomic dna of wheat to be measured is template, with the described primer pair group A in arbitrary described primer pair group in claim 1-3, carries out PCR reaction, and the annealing temperature of described PCR reaction is 59 ℃;
    (4) size of the PCR product that detecting step (3) obtains, according to PCR product, determine that described wheat to be measured contains in Psy-B1a, Psy-B1b and these three kinds of genes of Psy-B1c as follows any, and contain in TaZds-A1a and these two kinds of genes of TaZds-A1b any:
    With described primer pair group A, carrying out in the product of PCR reaction, is the DNA fragmentation of 151bp if contain size, and described wheat to be measured contains Psy-B1a gene or candidate is contained Psy-B1a gene; If containing size is the DNA fragmentation of 156bp, described wheat to be measured contains Psy-B1b gene or candidate is contained Psy-B1b gene; If containing size is the DNA fragmentation of 428bp, described wheat to be measured contains Psy-B1c gene or candidate is contained Psy-B1c gene; If containing size is the DNA fragmentation of 183bp, described wheat to be measured contains TaZds-A1a gene or candidate is contained TaZds-A1a gene; If containing size is the DNA fragmentation of 179bp, described wheat to be measured contains TaZds-A1b gene or candidate is contained TaZds-A1b gene.
  8. 8. method according to claim 7, is characterized in that: the nucleotide sequence of the DNA fragmentation that described size is 151bp is as shown in sequence in sequence table 11; Described size is that the nucleotide sequence of DNA fragmentation of 156bp is as shown in sequence in sequence table 12; Described size is that the nucleotide sequence of DNA fragmentation of 428bp is as shown in sequence in sequence table 13; Described size is that the nucleotide sequence of DNA fragmentation of 183bp is as shown in sequence in sequence table 14; Described size is that the nucleotide sequence of DNA fragmentation of 179bp is as shown in sequence in sequence table 15; Described size is that the nucleotide sequence of DNA fragmentation of 981bp is as shown in sequence in sequence table 16; Described size is that the nucleotide sequence of DNA fragmentation of 685bp is as shown in sequence in sequence table 17; Described size is that the nucleotide sequence of DNA fragmentation of 876bp is as shown in sequence in sequence table 18.
  9. 9. cultivate a method for the wheat breed that Flour whiteness improves, comprise adopt by the method for claim 7 identify obtain following a)-d) at least one wheat as parent, carry out the step of breeding:
    A) contain or candidate is contained Psy-B1b gene;
    B) contain or candidate is contained TaZds-A1a gene;
    C) contain or candidate is contained TaZds-D1b gene;
    D) contain or candidate is contained Ppo-A1b gene.
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