CN109182535A - A kind of screening technique of chicken breeding material - Google Patents
A kind of screening technique of chicken breeding material Download PDFInfo
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- CN109182535A CN109182535A CN201811102191.7A CN201811102191A CN109182535A CN 109182535 A CN109182535 A CN 109182535A CN 201811102191 A CN201811102191 A CN 201811102191A CN 109182535 A CN109182535 A CN 109182535A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention discloses a kind of screening techniques of chicken breeding material, belong to agriculture Animal Breeding field.The screening technique of the chicken breeding material, includes the following steps: step 1: the extraction of genomic DNA；Step 2: microsatellite marker and PCR reaction；Step 3:mtDNA PCR reaction and sequencing；Step 4: statistical analysis；Step 5: breeding selection of materials.The present invention carries out genetic diversity and Genetic Distance Analysis to breeding material using molecular biotechnology, it selects genetic diversity to enrich, guarantee have the individual of abundant hereditary information as breeding material, reduce the blindness and risk of breeding, reduce breeding time, breeding efficiency is improved, breeding cost is reduced.
Description
Technical field
The present invention relates to a kind of screening techniques of chicken breeding material, belong to agriculture Animal Breeding field.
Background technique
The Animal Breeding material of the prior art screens, often by appearance traits, pedigree and performance test etc.
Information carries out.The above method is based primarily upon livestock and poultry phenotypic number to assess Animal Breeding material, cannot really reflect poultry comprehensively
The characteristic of fowl kind, has some limitations and unpredictability, and it is long to assess the time, at high cost.
In recent years, with the development of molecular biology technology, scientific research personnel is by constructing livestock and poultry species from gene level
Molecular genetic linkage map assesses livestock and poultry species resource.But stock assessment is separated with the selection of breeding material, often basis
Phenotype carries out the selection of breeding material, and not using assessment result as scientific basis.Therefore, the selection of breeding material exists quasi-
Really the defects of rate is low, breeding time is long, at high cost, and the kind selected there may be genetic diversity it is not abundant enough or
There is the possibility of inbreeding, causes the new varieties production performance cultivated not high.
Microsatellite is also known as simple repeated sequence (Simple Sequence Repeat, SSR) or short-movie section repetitive sequence
(Short Tandem Repeat, STR) is the one kind formed by the base of 1-6bp as core repetitive unit, tandem sequence repeats
DNA sequence dna.Since microsatellite is extensive and is relatively evenly distributed in genome (about 30-50kb there is a microsatellite DNA),
Have the characteristics that relatively rich state property, information content it is big, in codominance, meet mendelian inheritance, be widely used in even
Lock map construction, population genetic diversity analysis, cultivars and strains selection and foundation and different lines between affiliation circle
Fixed, individual identification and paternity identification etc..
Mitochondrial genomes are also mitochondrial DNA (mt DNA, Mitochondrial DNA).MtDNA structure and bacterium
DNA is similar, at double-stranded circular.The design feature of mitochondrial genomes is also identical as prokaryotes.MtDNA can be with absolute coding line
Some protein in plastochondria are extranuclear heredity substances.Genome is that cell carries life-information DNA and its protein complex
General name.
Chicken mitochondrial genomes generally in 16785bp or so, are closed hoop duplex structure, independently can replicate and turn
Record.Fgs encoder area (13 protein coding genes, 2 ribosomal RNA genes, 22 transfer RNA bases including 37 genes
Cause) and 1 control zone (area D-loop).The area D-loop is most important noncoding region on mtDNA, and evolutionary rate is more compared with code area
It fastly, is the ideal molecular labeling for being suitble to the closer group of affiliation to carry out genetic variation and genetic differentiation research.The area D-loop part is complete
Portion's sequence has been widely used in the genetic diversity and phyletic evolution research of animal monoid.
Currently, not yet occurring analyzing using microsatellite marker combination mtDNA D-loop sequence to carry out chicken breeding material
The relevant report of screening.Therefore, it is necessary to a kind of screening technique of new chicken breeding material be provided, to solve the prior art not
Foot.
Summary of the invention
Present invention aims to solve the deficiencies of the prior art, and provides a kind of a kind of screening techniques of chicken breeding material.The present invention
Genetic diversity and Genetic Distance Analysis are carried out to breeding material using molecular biotechnology, genetic diversity is selected to enrich, protect
Card has the individual of abundant hereditary information as breeding material, reduces the blindness and risk of breeding, reduces breeding time, and raising is educated
Kind efficiency, reduces breeding cost.
The technical scheme to solve the above technical problems is that a kind of screening technique of chicken breeding material, including such as
Lower step:
Step 1: the extraction of genomic DNA
Extract the genomic DNA in sample to be tested blood；
Step 2: microsatellite marker and PCR reaction
Select microsatellite number be MCW0123, ADL0112, MCW0014, MCW0034, MCW0103, MCW0295,
MCW0078、MCW0222、MCW0098、MCW01111、MCW0037、MCW0248、LEI0166、ADL0268、MCW0216、
18 pairs of microsatellite locus of MCW0020, MCW0206, MCW0183 carry out PCR- fluorescent marker-semi-automatic genome respectively and sweep
It retouches, obtains genome scanning figure；
Step 3:mtDNA PCR reaction and sequencing
The specific primer of mtDNA PCR reaction includes upstream primer shown in SEQ ID NO.1 and SEQ ID NO.2 institute
The downstream primer shown；And 5 ' end FAM fluorescent markers of upstream primer；
SEQ ID NO.1 (upstream primer): 5 '-ACCCATTATATGTATACGGGCATTAA-3 ',
SEQ ID NO.2 (downstream primer): 5 '-AGTTATGCATGGGATGTGCCTGACCGAG-3 '；
The genomic DNA extracted using step 1 carries out PCR amplification as template, using above-mentioned specific primer；By PCR amplification
Product carries out agarose gel electrophoresis, if the electrophoresis result of pcr amplification product and target fragment are in the same size, recovery purifying,
PCR product sequencing is carried out, sequencing sequence is obtained；
Step 4: statistical analysis
The genome scanning figure obtained according to step 2 counts 18 pairs of microsatellite locus using GenePOP32 biosoftware
Number of alleles Na, effective number of allele Ne, gene frequency P, observation heterozygosity Ho, expected heterozygosity He, calculate
Polymorphism information content PIC out；Calculate genetic distance；
MtDNA D-loop sequence: made with accession number in ncbi database for the Hongyuan chicken mt DNA complete sequence of GU261695
For reference sequences, the sequencing sequence that step 3 obtains is sheared and compared with MEGA software, searches variant sites, then use
5.0 software of DNASP calculates haplotype number, haplotype diversity and nucleotide diversity, finally uses Network software building
Meta network figure；
It assesses the genetic structure between chicken kind: utilizing genetic distance, construct UPGMA phylogenetic tree or NJ phylogenetic tree
And/or the analysis of population grouping and clustering is carried out to individual using the Bayesian clustering method of STRUCTURE software；
Step 5: breeding selection of materials
According to the statistic analysis result of step 4, select polymorphism information content PIC > 0.5, number of alleles Na be 5 with
Group of the upper and genetic distance greater than 0.1 or more or individual, the highest group of meta network figure moderate purity are as chicken breeding element
Material.
The principle of the present invention explanation:
In step 1 of the invention, the genomic DNA in sample to be tested blood is extracted, is extracted using DNA kit.It is above-mentioned
DNA kit is purchased from Beijing CoWin Bioscience Co., Ltd., model CW0540-version04202010-2.2.
In step 2 of the invention, 18 pairs of chicken microsatellite locus of selection have that number of alleles is more, polymorphism content is rich
Rich feature, and it is good to show as polymorphism, high specificity and not chain between each other.PCR- fluorescent marker-semi-automatic gene
Group scanning is carried out in Invitrogen (Shanghai) Trading Co., Ltd..
In step 3 of the invention, 5 ' end FAM fluorescent markers of upstream primer, convenient for the base of subsequent PCR product segment
Because of scanning.The primer of above-mentioned 5 '-FAM of band label, is synthesized by Invitrogen (Shanghai) Trading Co., Ltd..Sequencing is in Beijing Hua Nuo
Time Technology Co., Ltd carries out.
In step 4 of the invention, population grouping is carried out to individual using the Bayesian clustering method of STRUCTURE software
Clustering is to more intuitively show the genetic variation and genetic differentiation difference between each breeding material.It can be with by NetWork software
Component meta network figure (median network), this network may include all most brief beams, and can real sequence
Information (such as position in homogeney site, mutantional hotspot and differentiate Haplogroup), between clustering cluster interior joint
Distance is closer, their haplotype is more close.For large sample, by identifying parallel evolution, it is possible to reduce network is answered
Polygamy.And by NetWork software, the mode of mtDNA Control region sequence nucleotide replacement can be investigated.
In step 5 of the invention, polymorphism information content PIC is the ideal indicator for measuring allele segment polymorphism.When
When seat PIC>0.5, which is that height is polymorphic, and as 0.25<PIC<0.5, which is moderate polymorphic, when PIC<0.25
When, which is low polymorphic.Genetic distance refers to the degree of the gene difference of different population or inter-species, and with certain number
Value is measured.Genetic distance is the basis for studying species genetic diversity, reflects the system differentiation of studied group.
The present invention selects polymorphism information content PIC > 0.5, and number of alleles Na is 5 or more and genetic distance is greater than 0.1
The high group of above group or individual, meta network figure moderate purity is because of population genetic diversity as chicken breeding material
Abundant, hereditary information amount entrained by kind is big, and inbreeding is just not present greatly in genetic distance, and going out in production reality can table
Reveal better heterosis, hybrid vigor, meets the needs of merit and production performance.
Based on the above technical solution, the present invention can also be improved as follows.
Further, in step 2, the reaction system of the PCR is calculated as with 15 μ L: template DNA 50ng, 1.0 μ of upstream primer
Mol/L, 1.0 μm of ol/L of downstream primer, 2 × Taq PCR Master Mix7.5 μ L, ddH2O supplies 15 μ L.
Be using above-mentioned further beneficial effect: 2 × Taq MasterMix is by Taq DNA Polymerase, Mg2 +, dNTPs, PCR stabilizer and reinforcing agent composition premix system.Pre-assigned PCR mixed liquor make to operate it is simpler quick,
And human error and pollution can be reduced to the maximum extent.
Above-mentioned 2 × Taq PCR Master Mix is purchased from Beijing CoWin Bioscience Co., Ltd., model
CW0716M。
Further, in step 2, the response procedures of the PCR are as follows: 95 DEG C of initial denaturation 5min；Subsequently enter circulation, 95 DEG C of changes
Property 30s, 50-65 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 circulation；Later, 72 DEG C of extension 5min；4 DEG C of preservations.
Further, in step 3, the reaction system of the PCR is calculated as with 25 μ L: template 80ng-100ng, upstream primer 1.0
μm ol/L, 1.0 μm of ol/L of downstream primer, 2 × Taq PCR Master Mix 13 μ L, ddH2O supplies 25 μ L.
Further, in step 3, the response procedures of the PCR are as follows: 95 DEG C of initial denaturation 5min；Subsequently enter circulation, 95 DEG C of changes
Property 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 circulation；Later, 72 DEG C of extension 5min；4 DEG C of preservations.
Further, in step 2 and step 3, the mass percent of the Ago-Gel is 1.5%.
Further, in step 4, the calculation formula of the polymorphism information content PIC are as follows:
In formula, pi、pjIth and jth gene frequency respectively in group, j=i+1, n is on a certain locus etc.
Position gene number.
The beneficial effects of the present invention are:
It is breeding material 1. the present invention is according to the assessment result combination breeding objective of resource of animal and birds come Animal Breeding material
Selection provide science accurate basic data, this method can realize livestock and poultry Seedling selection, shorten the generation inteval, accelerate breeding into
Journey.
2. the present invention objectively screens breeding material by science, the accuracy of selection is improved, is guaranteed in breeding material
Hereditary information.
3. the present invention can reduce breeding time, reduce cost, breeding efficiency is improved.Because the breeding material of selection is with rich
Rich hereditary information, the next generation cultivated will have corresponding hereditary information and trait expression, reach breeding objective, reduce
Invalid experiment, reduces time generation inteval, to reduce breeding time, reduces breeding cost, improves breeding efficiency.
Detailed description of the invention
Fig. 1 is the network structure of Guangxi Sanhuang chicken kind.CM represents Yulin 1 in figure, and ZS represents Yulin 2, and JL represents Nanning,
GD represents Cenxi allusion Sanhuang chicken, and A represents East Asia pedigree, and B represents Southeast Asia pedigree, and C represents the peculiar pedigree in East Asia, and E represents Europe
U.S. commercial chicken pedigree, F represent the peculiar pedigree in Yunnan and adjoining area, and H represents Haplogroup (haplotype).
Fig. 2 is the NJ phylogenetic tree of 6, Guangxi Sanhuang chicken strain.In figure, NN represents Nanning Sanhuang chicken, and XY represents Yulin
Industrial Sanhuang chicken, RX represent Beiliu City Rong County Sanhuang chicken, and BB represents Yulin Bobai Sanhuang chicken, and XD represents He Prefecture letter Sanhuang chickens, GD
Represent Cenxi allusion Sanhuang chicken.
Fig. 3 is the network structure of Guangxi Local chicken kind.In figure, TY represents Sanhuang chicken, and XY represents rosy clouds pharynx chicken, and GX represents wide
Western Gallus domesticlus brisson, MA represent numb chicken, and ND represents Nandan precious jade chicken, and LS represents Long Sheng Salted chicken.RE represents Red Jungle-fowl, and A represents East Asia pedigree,
B represents Southeast Asia pedigree, and C represents the peculiar pedigree in East Asia, and E represents American-European commercial chicken pedigree.
Fig. 4 is the phylogenetic tree of Guangxi Local chicken kind and external chicken kind.In figure, TY represents Sanhuang chicken, and XY represents rosy clouds pharynx
Chicken, MA represent numb chicken, and ND represents Nandan precious jade chicken, and GX represents Guangxi Gallus domesticlus brisson, and LS represents Long Sheng Salted chicken, and AA represents Ai Weiyin chicken, RM
Represent Luo Man chicken.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
The selection of embodiment 1: Gui Feng bis- yellow chicken breeding material
The screening technique of the osmanthus phoenix No. two yellow chicken breeding materials of the present embodiment, includes the following steps:
Step 1: the extraction of genomic DNA
Nanning Sanhuang chicken (NN) 60, Yulin industrial Sanhuang chicken (XY) 60, Beiliu City appearance of the sample from Guangxi Sanhuang chicken
County's Sanhuang chicken (RX) 60, Yulin Bobai Sanhuang chicken (BB) 60, He Prefecture letter all Sanhuang chicken (XD) 60, Cenxi allusion Sanhuang chicken
(GD) 60 are waited 6 strains, totally 360 samples.
It is extracted using DNA kit, extracts the genomic DNA in above-mentioned sample to be tested blood.
Step 2: microsatellite marker
Select microsatellite number be MCW0123, ADL0112, MCW0014, MCW0034, MCW0103, MCW0295,
MCW0078、MCW0222、MCW0098、MCW01111、MCW0037、MCW0248、LEI0166、ADL0268、MCW0216、
18 pairs of microsatellite locus of MCW0020, MCW0206, MCW0183 carry out PCR- fluorescent marker-semi-automatic genome respectively and sweep
It retouches, obtains genome scanning figure.
The reaction system of the PCR is calculated as with 15 μ L: template DNA 50ng, upstream primer 1.0 μm of ol/L, downstream primers
1.0 μm of ol/L, 2 × Taq PCR Master Mix7.5 μ L, ddH2O supplies 15 μ L.The response procedures of the PCR are as follows: 95 DEG C pre-
It is denaturalized 5min；Circulation is subsequently entered, 95 DEG C of denaturation 30s, 50-65 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 recycle；Later,
72 DEG C of extension 5min；4 DEG C of preservations.
Step 3:mtDNA PCR reaction and sequencing
The specific primer of mtDNA PCR reaction includes upstream primer shown in SEQ ID NO.1 and SEQ ID NO.2 institute
The downstream primer shown；And 5 ' end FAM fluorescent markers of upstream primer；
SEQ ID NO.1 (upstream primer): 5 '-ACCCATTATATGTATACGGGCATTAA-3 ',
SEQ ID NO.2 (downstream primer): 5 '-AGTTATGCATGGGATGTGCCTGACCGAG-3 '；
The genomic DNA extracted using step 1 carries out PCR amplification as template, using above-mentioned specific primer；
The reaction system of the PCR is calculated as with 25 μ L: template 80ng-100ng, upstream primer 1.0 μm of ol/L, downstream primers
1.0 μm of ol/L, 2 × Taq PCR Master Mix 13 μ L, ddH2O supplies 25 μ L.The response procedures of the PCR are as follows: 95 DEG C pre-
It is denaturalized 5min；Circulation is subsequently entered, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 recycle；Later, 72
DEG C extend 5min；4 DEG C of preservations.
Pcr amplification product is subjected to the agarose gel electrophoresis that mass percent is 1.5%, if the electricity of pcr amplification product
Result of swimming DNA fragmentation corresponding with above-mentioned primer is in the same size, then recovery purifying, carries out PCR product sequencing, obtains sequencing sequence
Column.
Step 4: statistical analysis
The genome scanning figure obtained according to step 2 counts 18 pairs of microsatellite locus using GenePOP32 biosoftware
Number of alleles Na, effective number of allele Ne, gene frequency P, observation heterozygosity Ho, expected heterozygosity He, calculate
Polymorphism information content PIC out；Calculate genetic distance.
Wherein, the calculation formula of polymorphism information content PIC are as follows:
In formula, pi、pjIth and jth gene frequency respectively in group, j=i+1, n is on a certain locus etc.
Position gene number.
1 Sanhuang chicken difference conservation field Polymorphism of Microsatellite Markers of table
Note: in table 1, CM represents Yulin 1, and ZS represents Yulin 2, and JL represents Nanning, and GD represents Cenxi.
MtDNA D-loop sequence: made with accession number in ncbi database for the Hongyuan chicken mt DNA complete sequence of GU261695
For reference sequences, the sequencing sequence that step 3 obtains is sheared and compared with MEGA software, searches variant sites, then use
5.0 software of DNASP calculates haplotype number, haplotype diversity and nucleotide diversity, finally uses Network software building
Meta network figure.The network structure of Guangxi Sanhuang chicken kind, as a result as shown in Figure 1.
It should be noted that alphabetical A, B, C, E, F are pedigree of the forefathers by each family chicken of letter designation in Fig. 1.According to
The phylogenetic tree that Liu (2006) and Miao (2013) et al. are constructed based on family chicken mitochondria full-length genome, determines world's family chicken
And its sibling species Hongyuan chicken can be divided into 11 pedigree such as A, B, C, D, E, F, G, H, I, W and X.Wherein, A pedigree and B pedigree are East Asia
The generally existing pedigree with south east asia, C pedigree are the peculiar pedigree in East Asia, and E pedigree is American-European commercial chicken pedigree, and F is Yunnan
And adjoining area (such as Guangxi China, Vietnam etc.) peculiar pedigree.
It assesses the genetic structure between chicken kind: utilizing genetic distance, the NJ system of building 6, Guangxi Sanhuang chicken strain occurs
Tree, as shown in Figure 2.As shown in Figure 2, Yulin Bobai Sanhuang chicken (BB), Beiliu City Rong County Sanhuang chicken (RX) and the industrial Sanhuang chicken of Yulin
(XY) gather for one kind, He Prefecture letter all Sanhuang chicken (XD), Cenxis allusion Sanhuang chicken (GD) are gathered for cluster；Nanning Sanhuang chicken (NN) is independent
Gather for one kind.Wherein, Nanning Sanhuang chicken (NN) and the industrial Sanhuang chicken of Yulin (XY) distance are farthest, and hereditary variation is maximum.
Step 5: breeding selection of materials
According to the statistic analysis result of step 4, select polymorphism information content PIC > 0.5, number of alleles Na be 5 with
Group of the upper and genetic distance greater than 0.1 or more is as chicken breeding material.
According to variety evaluation and testing result, the Nanning Sanhuang chicken that genetic diversity is abundant, genetic distance is farthest is screened
(NN) and the industrial Sanhuang chicken of Yulin (XY) is osmanthus phoenix No. two yellow chicken breeding materials.
The Sanhuang chicken cultivated using above-mentioned osmanthus phoenix No. two yellow chicken breeding materials, maintains the appearance traits and meat of Sanhuang chicken
The good characteristic of matter, while its production performance is improved, for egg production up to 175, feed-weight ratio reaches 3.6:1.
Embodiment 2: the selection of Jinling broiler breeding material
The screening technique of the Jinling broiler breeding material of the present embodiment, includes the following steps:
Step 1: the extraction of genomic DNA
Sample comes from Guangxi Sanhuang chicken (TY) 60, and rosy clouds swallow chicken (XY) 60, numb chicken (MA) 60, Nandan precious jade chicken (ND) 60
A, Guangxi Gallus domesticlus brisson (GX) 60, dragon wins Salted chicken (LS) 60, Ai Weiyin chicken (AA) 50, Luo Man chicken (RM) 50, totally 8 product
460 samples of kind.
It is extracted using DNA kit, extracts the genomic DNA in above-mentioned sample to be tested blood.
Step 2: microsatellite marker
Select microsatellite number be MCW0123, ADL0112, MCW0014, MCW0034, MCW0103, MCW0295,
MCW0078、MCW0222、MCW0098、MCW01111、MCW0037、MCW0248、LEI0166、ADL0268、MCW0216、
18 pairs of microsatellite locus of MCW0020, MCW0206, MCW0183 carry out PCR- fluorescent marker-semi-automatic genome respectively and sweep
It retouches, obtains genome scanning figure.
The reaction system of the PCR is calculated as with 15 μ L: template DNA 50ng, upstream primer 1.0 μm of ol/L, downstream primers
1.0 μm of ol/L, 2 × Taq PCR Master Mix 7.5 μ L, ddH2O supplies 15 μ L.The response procedures of the PCR are as follows: 95 DEG C
Initial denaturation 5min；Circulation is subsequently entered, 95 DEG C of denaturation 30s, 50-65 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 recycle；It
Afterwards, 72 DEG C of extension 5min；4 DEG C of preservations.
Step 3:mtDNA PCR reaction and sequencing
The specific primer of mtDNA PCR reaction includes upstream primer shown in SEQ ID NO.1 and SEQ ID NO.2 institute
The downstream primer shown；And 5 ' end FAM fluorescent markers of upstream primer；
SEQ ID NO.1 (upstream primer): 5 '-ACCCATTATATGTATACGGGCATTAA-3 ',
SEQ ID NO.2 (downstream primer): 5 '-AGTTATGCATGGGATGTGCCTGACCGAG-3 '；
The genomic DNA extracted using step 1 carries out PCR amplification as template, using above-mentioned specific primer；
The reaction system of the PCR is calculated as with 25 μ L: template 80ng-100ng, upstream primer 1.0 μm of ol/L, downstream primers
1.0 μm of ol/L, 2 × Taq PCR Master Mix 13 μ L, ddH2O supplies 25 μ L.The response procedures of the PCR are as follows: 95 DEG C pre-
It is denaturalized 5min；Circulation is subsequently entered, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 recycle；Later, 72
DEG C extend 5min；4 DEG C of preservations.
Pcr amplification product is subjected to the agarose gel electrophoresis that mass percent is 1.5%, if the electricity of pcr amplification product
Result of swimming DNA fragmentation corresponding with above-mentioned primer is in the same size, then recovery purifying, carries out PCR product sequencing and carries out PCR product survey
Sequence obtains sequencing sequence.
Step 4: statistical analysis
The genome scanning figure obtained according to step 2 counts 18 pairs of microsatellite locus using GenePOP32 biosoftware
Number of alleles Na, effective number of allele Ne, gene frequency P, observation heterozygosity Ho, expected heterozygosity He, calculate
Polymorphism information content PIC out；Calculate genetic distance.
Wherein, the calculation formula of polymorphism information content PIC are as follows:
In formula, pi、pjIth and jth gene frequency respectively in group, j=i+1, n is on a certain locus etc.
Position gene number.
The results are shown in Table 2.
The polymorphism of 2 Guangxi Local chicken kind of table
Note: in table 2, TY represents Sanhuang chicken, and XY represents rosy clouds pharynx chicken, and MA represents numb chicken, and ND represents Nandan precious jade chicken, and GX represents wide
Western Gallus domesticlus brisson, LS represent Long Sheng Salted chicken, and AA represents Ai Weiyin chicken, and RM represents Luo Man chicken.
As can be seen from Table 2,18 microsatellite markers show higher polymorphism.144 allele are detected altogether, respectively
Site be averaged number of alleles be 8 ± 3.43, average effective allele be 3.59 ± 1.30.Single locus is at most up to 16
A allele (MCW0034), least is 3 (MCW0103)；And effective number of allele from 1.66 (MCW0098) to
5.91 (MCW0034) are differed.The observation heterozygosity (Ho) of 18 microsatellite locus is from 0.2478 (MCW0222) to 0.7756
(MCW0111) etc., mean value is 0.5605 ± 0.1673；It is expected that heterozygosity (HE) is from 0.3998 (MCW0098) to 0.8322
(MCW0034) etc., mean value is 0.6798 ± 0.1329.
MtDNA D-loop sequence: made with accession number in ncbi database for the Hongyuan chicken mt DNA complete sequence of GU261695
For reference sequences, the sequencing sequence that step 3 obtains is sheared and compared with MEGA software, searches variant sites, then use
5.0 software of DNASP calculates haplotype number, haplotype diversity and nucleotide diversity, finally uses Network software building
Meta network figure.As a result as shown in Figure 3.
It assesses the genetic structure between chicken kind: individual being carried out using the Bayesian clustering method of STRUCTURE software
The analysis of population grouping and clustering.As a result as shown in Figure 4.
Step 5: breeding selection of materials
According to the statistic analysis result of step 4, select polymorphism information content PIC > 0.5, number of alleles Na be 5 with
On, the highest group of meta network figure moderate purity is as chicken breeding material.
According to variety evaluation and testing result, numb chicken polymorphism information content highest, the intrusion by adventive gene is small, because
This selects breeding material of the numb chicken as Jinling broiler.
The Jinling broiler cultivated using the breeding material of above-mentioned Jinling broiler, it is strong to maintain numb chicken premunition, with good meat quality
The advantages of, while chicken group's feather color is consistent, the uniformity is up to 80% or more.
The selection of 1 osmanthus phoenix of comparative example No. two yellow chicken breeding materials
The selection of osmanthus phoenix No. two yellow chicken breeding materials of the prior art only selects outside chicken individuals figure in standby chicken group
Looks meet the chicken of this varietal characteristic as breeding material, and the chicken separation cultivated is serious, offspring individuals hair color, weight differences
Greatly, production performance fails to reach breeding goal.
The selection of 2 Nanjing fiber crops chicken breeding material of comparative example
The selection of Nanjing fiber crops chicken breeding material of the prior art, only selection chicken individuals appearance traits accord in standby chicken group
The chicken of this varietal characteristic is closed as breeding material, the chicken separation cultivated is serious, and offspring individuals hair color, weight differences are big, raw
Performance is produced to fail to reach breeding goal.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of screening technique of chicken breeding material, which comprises the steps of:
Step 1: the extraction of genomic DNA
Extract the genomic DNA in sample to be tested blood；
Step 2: microsatellite marker and PCR reaction
Select microsatellite number be MCW0123, ADL0112, MCW0014, MCW0034, MCW0103, MCW0295, MCW0078,
MCW0222、MCW0098、MCW01111、MCW0037、MCW0248、LEI0166、ADL0268、MCW0216、MCW0020、
18 pairs of microsatellite locus of MCW0206, MCW0183 carry out PCR- fluorescent marker-semi-automatic genome scanning respectively, obtain base
Because of a group scanning figure；
Step 3:mtDNA PCR reaction and sequencing
The specific primer of mtDNA PCR reaction includes shown in upstream primer shown in SEQ ID NO.1 and SEQ ID NO.2
Downstream primer；And 5 ' end FAM fluorescent markers of upstream primer；
SEQ ID NO.1 (upstream primer): 5 '-ACCCATTATATGTATACGGGCATTAA-3 ',
SEQ ID NO.2 (downstream primer): 5 '-AGTTATGCATGGGATGTGCCTGACCGAG-3 '；
The genomic DNA extracted using step 1 carries out PCR amplification as template, using above-mentioned specific primer；By pcr amplification product
Agarose gel electrophoresis is carried out, if the electrophoresis result of pcr amplification product and target fragment are in the same size, recovery purifying is carried out
PCR product sequencing, obtains sequencing sequence；
Step 4: statistical analysis
The genome scanning figure obtained according to step 2, using GenePOP32 biosoftware, count 18 pairs of microsatellite locus etc.
Position gene number Na, effective number of allele Ne, gene frequency P, observation heterozygosity Ho, expected heterozygosity He, calculate more
Polymorphism information content PIC；Calculate genetic distance；
MtDNA D-loop sequence: using in ncbi database accession number for GU261695 Hongyuan chicken mt DNA complete sequence as ginseng
Sequence is examined, the sequencing sequence that step 3 obtains is sheared and compared with MEGA software, searches variant sites, then use DNASP
5.0 softwares calculate haplotype number, haplotype diversity and nucleotide diversity, finally use Network software building intermediary net
Network figure；
Assess the genetic structure between chicken kind: utilizing genetic distance, construct UPGMA phylogenetic tree or NJ phylogenetic tree and/
Or the analysis of population grouping and clustering is carried out to individual using the Bayesian clustering method of STRUCTURE software；
Step 5: breeding selection of materials
According to the statistic analysis result of step 4, select polymorphism information content PIC > 0.5, number of alleles Na be 5 or more and
Group of the genetic distance greater than 0.1 or more or individual, the highest group of meta network figure moderate purity are as chicken breeding material.
2. a kind of screening technique of chicken breeding material according to claim 1, which is characterized in that in step 2, the PCR
Reaction system be calculated as with 15 μ L: template DNA 50ng, 1.0 μm of ol/L of upstream primer, downstream primer 1.0 μm of ol/L, 2 × Taq
PCR Master Mix 7.5 μ L, ddH2O supplies 15 μ L.
3. a kind of screening technique of chicken breeding material according to claim 1, which is characterized in that in step 2, the PCR
Response procedures are as follows: 95 DEG C of initial denaturation 5min；Circulation is subsequently entered, 95 DEG C of denaturation 30s, 50-65 DEG C of annealing 30s, 72 DEG C extend
30s, totally 35 recycle；Later, 72 DEG C of extension 5min；4 DEG C of preservations.
4. a kind of screening technique of chicken breeding material according to claim 1, which is characterized in that in step 3, the PCR
Reaction system be calculated as with 25 μ L: template 80ng-100ng, 1.0 μm of ol/L of upstream primer, 1.0 μm of ol/L of downstream primer, 2 ×
Taq PCR Master Mix 13 μ L, ddH2O supplies 25 μ L.
5. a kind of screening technique of chicken breeding material according to claim 1, which is characterized in that in step 3, the PCR
Response procedures are as follows: 95 DEG C of initial denaturation 5min；Subsequently enter circulation, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s,
Totally 35 circulations；Later, 72 DEG C of extension 5min；4 DEG C of preservations.
6. a kind of screening technique of chicken breeding material according to claim 1, which is characterized in that in step 2 and step 3,
The mass percent of the Ago-Gel is 1.5%.
7. a kind of screening technique of chicken breeding material according to claim 1, which is characterized in that described polymorphic in step 4
The calculation formula of information content PIC are as follows:
In formula, pi、pjIth and jth gene frequency respectively in group, j=i+1, n are equipotential base on a certain locus
Factor.
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