CN104962634B - For detecting the kit of chicken body measurement trait and the molecular breeding method of chicken - Google Patents
For detecting the kit of chicken body measurement trait and the molecular breeding method of chicken Download PDFInfo
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Abstract
The invention discloses a kind of kit for detecting chicken body measurement trait and the molecular breeding method of chicken, belong to molecular genetics field.The present invention combines single primer extension reaction using the attached ionization time of flight mass spectrometry technology of MassARRAY system matrix assisted laser desorptions, to gu-shi chicken and An Ka chickens F2The single SNP site Genotyping detections of miRNA 1658 (rs16681031 C/G), and and F are carried out for resource population sample2Analysis is associated for resource population body measurement trait, result shows that the gene SNP SNPs of chicken miRNA 1658 are significantly associated with chicken body measurement trait, GG genotype is conducive to that the chest breadth of 4 week old chickens, pelvis are wide and Body steep length is significantly increased, assisted Selection and molecular genetic breeding mark as 4 week old chicken body measurement traits, can quickly set up the excellent chicken group of genetic resources.
Description
Technical field
The present invention relates to a kind of kit for detecting chicken body measurement trait, also relate to a kind of based on chicken miRNA-
The molecular breeding method of 1658 gene mononucleotide polymorphisms, belongs to molecular genetics field.
Background technology
With developing rapidly and the improvement of people's living standards for social economy, market to the demand of meat on the whole from
Preceding quantitative requirement has turned to the pursuit to quality flavor.Growth traits, body measurement trait, Meat Quality and Carcass Traits are current
The economic characters that poultry industry is most paid close attention to.Most economic characters belong to quantitative character, mainly by plurality of minor effect
Gene-determined, these genes have additive effect, dominant effect and an epistatic effect simultaneously, the hereditary basis of complex character be by
Influence what the inherent cause and environmental factor of the phenotype and the interaction between them were together decided on.Domestic high-quality indigenous chicken
The slow problem of the generally existing speed of growth, so as to have impact on economic benefit.Therefore, researcher is attempted by a large amount of for many years
Experiment find the candidate gene related to the related economic proterties of chicken, be that provided auxiliary is marked in terms of poultry molecule seed selection breeding
Selection.
Fine-variety breeding is to speed up using the GENERALIZATION OF MODERN BREEDING TECHNIQUE of molecular labeling and improve the important channel of population genetic quality.
First it is examination and detection and the closely related genetic marker of chicken economic characters on DNA level using molecular marker breeding, its
The secondary method for quick for being to set up its gene pleiomorphism, then realizes genetic marker assisted Selection and realizes early diagnosis choosing
Select.
Mononucleotide polymorphic (Single nucleotide polymorphism, SNP) refer in genomic level by
DNA sequence polymorphism caused by variation in single nucleotide acid.SNP can be in DNA, RNA and protein level influence gene
Function, be the heritable most common one kind of variation of the mankind, account for more than the 90% of known polymorphism.It is influential on gene outcome
The function of SNP is analyzed, and its meaning is very great.SNP diversity is always Application of Animal Genetic with the association analysis of economic characters
Learn the focus of research.With the discovery of miRNA and its going deep into for research, it is complicated that researcher has found that miRNA not only take part in animal
The molecular regulation process that trait phenotypes are formed, and its SNP changes also can result in miRNA dysfunctions, and then cause biology
The variation of phenotypic character.The related polymorphic Forming Mechanisms of miRNA include that insertion, missing, transposition, amplification and base are replaced.
The polymorphism of miRNA is the key factor for influenceing miRNA regulatory functions.During single miRNA abnormal expressions, may influence hundreds of
The expression of target gene, when some of which key protein expression quantity is reduced, can cause body physiological function abnormal and disease occurs.
Research finds that the polymorphism occurred on miRNA Primary products, precursor and ripe body can potentially influence hundreds of genes
Expression and path, so as to influence the function of miRNA extensively.
The detection method of SNP polymorphisms, it is most common to have single-strand conformation polymorphism technology (SSCP), PCR-RFLP and directly survey
Sequence technology etc..But SSCP is cumbersome, time-consuming, and influence factor is more, and there is Problem of False Negative in experimentation, so not
Preferable SNP detection means；Direct Sequencing technical costs is higher.Many research applications PCR-RFLP methods or PCR and poly- third
The method that acrylamide electrophoresis detection is combined.
The general principle that MassARRAY systems carry out SNP partings is with substance assistant laser desorpted ionized flight time matter
Spectrum (MALDI-TOF MS) technology combines single primer extension reaction, is realized by nucleic acid fragment molecular weight Direct Identification.
Primer sequence carries out multiplexed PCR amplification first after synthesis, dilution, to increase the template of single base extension, then enters again
Row single base extension, used in single base extension is ddNTP, therefore extends the automatic termination of reaction after a base.
Finally extension products are added on chip using full automatic point model machine, mass spectrum parting is carried out by MassARRAY.MASSARRAY
Detection of platform SNP has the advantage that：Accurately and reliably, directly molecular weight is detected, fluorescence labeling, gel electrophoresis is not related to
Deng, can just detect a difference for base, accuracy is high, and the probability that machine malfunctions in itself is very low, without verifying again, also without
Statistics repetition need to be designed.Flux is higher, goes for several to hundreds of detections of SNP site.Detection time is short, profit
Can just be completed to 380 detections of sample in 40min with Mass Spectrometer Method, and show testing result in real time.
The content of the invention
It is an object of the invention to provide a kind of kit for detecting chicken body measurement trait.
Meanwhile, the present invention also provides a kind of molecular breeding side based on chicken miRNA-1658 gene mononucleotide polymorphisms
Method.
In order to realize the above object the technical solution adopted in the present invention is：
Kit for detecting chicken body measurement trait, including：
Sense primer F PRimer：5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ' are (such as SEQ ID NO.1 institutes
Show),
Anti-sense primer R PRimer：5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ' are (such as SEQ ID NO.2 institutes
Show)；
Single base extension primer：5 '-CATCAACACCAACCCA-3 ' (as shown in SEQ ID NO.3).
Mentioned reagent box can also include：10×buffer、Mg2+, dNTP, Hotstar, iPLEX terminating reaction Mix, list
Base extends enzyme, SAP buffer, SAP enzyme, ddd H2O and DNA Marker etc..The addition of each component can be fitted in kit
Work as selection, the consumption of such as larger than 10 times.
A kind of molecular breeding method based on chicken miRNA-1658 gene mononucleotide polymorphisms, comprises the following steps：
1) chicken STb gene is extracted, adds primer to enter performing PCR amplification；
2) adding Single base extension primer carries out single base extension, obtains product；
3) after product is detected through flight mass spectrum, the male and female chicken individuals of GG genotype of selecting and remain set up target chicken group.
Step 1) in primer it is as follows：
Sense primer F PRimer：5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Anti-sense primer R PRimer：5’-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3’；
Step 2) in Single base extension primer be：5’-CATCAACACCAACCCA-3’.
Step 1) in PCR amplification reaction system be：Amount to 5.0 μ l；10 × buffer 0.5 μ l, 25mM Mg2+0.4 μ l,
0.1 μ l, 5U/ μ l Hotstar of 25mM dNTP 0.2 μ l, 10pmol/ μ l F Primer/R Primer each 0.5 μ l, 100ng/
μ l STb genes 1.0 μ l, ddd H2O 1.8μl。
Step 1) in PCR amplification response procedures be：95 DEG C of 2min of predegeneration；95℃30s；56℃30s；72 DEG C of 60s, 45
Individual circulation；72℃5min；25 DEG C of insulations.
Step 1) in PCR amplification it is latter as need add shrimp alkaline phosphotase (SAP) remove remaining dNTP, SAP enzymic digestions
It is this area routine techniques.In the present invention, SAP enzymic digestions system is：Amount to 2.0 μ l；SAP buffer 0.17 μ l, 1.7U/
μ l SAP enzymes 0.3 μ l, ddd H2O 1.53μl；SAP enzymic digestion programs are：37 DEG C of 40min, 85 DEG C of 5min, 25 DEG C of insulations.
Step 2) in the reaction system of single base extension be：Amount to 2.0 μ l；The μ l of 10 × buffer 0.2, react dense
It is the μ l of 1 × iPLEX terminating reactions Mix 0.2 to spend, and 7 μM of μ l of Single base extension primer 0.94, reaction density is the mono- alkali of 1 × iPLEX
Base extends enzyme 0.041 μ l, ddd H2O 0.619μl。
Step 2) in the response procedures of single base extension be：94 DEG C of 30s of predegeneration；40 circulations：94 DEG C of 5s, 52 DEG C
5s, 80 DEG C of 5s × 5 inner loops, 72 DEG C of 3min；25 DEG C of insulations.
Step 3) in product first use resin desalination, then point sample is in chip, chip matrix assisted laser desorption ionization electricity
From flight time mass spectrum detection.
Beneficial effects of the present invention：
The present invention is combined using the attached ionization time of flight mass spectrometry technology of MassARRAY system matrix assisted laser desorptions and singly drawn
Thing extension, to gu-shi chicken and An Ka chickens F2It is single miRNA-1658 (rs16681031 C/G) to be carried out for resource population sample
SNP site Genotyping is detected, and and F2Analysis is associated for resource population body measurement trait, as a result shows chicken miRNA-1658 bases
Because SNP SNPs are significantly associated with chicken body measurement trait, GG genotype be conducive to the chest breadth of 4 week old chickens, pelvis wide and
Body steep length is significantly increased, assisted Selection and the molecular genetic breeding mark as 4 week old chicken body measurement traits, can quickly be set up
The excellent chicken group of genetic resources.
Brief description of the drawings
Fig. 1 is the SNP sequencer maps of chicken miRNA-1658 genes；
Fig. 2 is the characterising mass spectrometry figure of SNP site different genotype.
Specific embodiment
Following embodiments are only described in further detail to the present invention, but do not constitute any limitation of the invention.
Embodiment 1
It is used to detect the kit of chicken body measurement trait in the present embodiment, including：
10pmol/ μ l sense primer F PRimer：5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
10pmol/ μ l anti-sense primer R PRimer：5’-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3’；
7 μM of Single base extension primers：5’-CATCAACACCAACCCA-3’.
Embodiment 2
It is used to detect the kit of chicken body measurement trait in the present embodiment, in addition to comprising following primer, also includes：10×
buffer、25mM Mg2+, 25mM dNTP, 5U/ μ l Hotstar, reaction density be 1 × iPLEX single base extension enzymes, SAP
Buffer, 1.7U/ μ l SAP enzymes, ddd H2O and DNA Marker；
10pmol/ μ l sense primer F PRimer：5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
10pmol/ μ l anti-sense primer R PRimer：5’-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3’；
7 μM of Single base extension primers：5’-CATCAACACCAACCCA-3’.
Test example
First, the detection of chicken miRNA-1658 gene mononucleotide polymorphisms, comprises the following steps：
1st, animal material：Gu-shi chicken and An Ka chickens.
Gu-shi chicken belongs to a kind of excellent local varieties of China's Huang chicken type, it centered on Gushi County, in outstanding life
Under state environment and rearing conditions, the self-assembling formation by long-term locking procreation.Gu-shi chicken is egg meat general purpose chicken breeds, with excellent
Proterties：One is resistance to thick feeding, and premunition is strong, and suitable field is herded and raised scattered；Two is fine and tender taste, and meat flavour is delicious, and soup is mellow, battalion
Support abundant, with stronger nourishing effects；Three to be that hen is laid eggs more, and egg is big, and egg white is thicker, yolk color depth, and eggshell is thick, resistance to storage
Fortune.
Peace card broiler chicken is one of excellent meat chicken kind in the world today, is also the most fast reddish yellow plumage of current country's speed of growth
Broiler chicken, with strong adaptability, it is resistance to stress, speed long is fast, the features such as the price of deed is high.
2nd, the cultivation of animal：
Gu-shi chicken-An Ka chicken resource populations used press the remote edge half sibs design establishments of F-2, test vertical 7 familys of building together,
Wherein rhombic system 4 is peace card chicken ♂ × gu-shi chicken ♀, and it is gu-shi chicken ♂ × peace card chicken ♀ that reciprocal cross is 3.F0Generation is simultaneous from egg meat
Pacified during card chicken is sheerly with type gu-shi chicken and meat type and choose breeder respectively, by male and female chicken 1:6 ratio combos are formed, it is desirable to selected public affairs
Hen has this varietal characteristic, and high egg production, body weight is medium, and blood lineage is pure, to ensure F1The generation individual heterozygosis in each site.
From the F of each family1Selected and remain in offspring a cock, by male and female 1:9 ratios mate with other family hens and produce F2Generation, it is desirable to
With with there is no affiliation, F between male and female chicken1The kind in generation is also dispersed in each family as far as possible with hen, choosing of being tried one's best during seed selection
Select appearance performance abundant, the individuality of heterozygosis is presented, it is ensured that F2Larger separation is produced for proterties.
3rd, method for breeding：
Chicken group feeding is supported and tests chicken house in Henan university, and later stage experiment is complete in Henan university poultry genetic improvement laboratory
Into.Each family mixes group, cage, free choice feeding, abundance drinking-water.Feeding to 12 week old, blood sampling is sampled.0~12 week old, week about
Every chicken carries out measurement and the record data of body measurement trait and growth traits.
4th, the extraction of genomic DNA and the mixed pond of structure
With gu-shi chicken-An Ka chicken sources groups F2 for chicken as material, every chicken jugular vein is drawn blood 5mL, EDTA anti-freezings
(10mmol/L Tris-Cl, 0.2mmol/L EDTA, PH 8.0), puts -80 DEG C of preservations.Using phenol chloroform method from F2Dai Zi
Genomic DNA is extracted in the individual preservation blood sample of source group, is detected with 1% agarose gel electrophoresis first, then used
The micro light splitting range meters of NanoDrop2000 determine the purity and concentration of DNA sample, and underproof DNA sample is extracted again.With
Machine chooses the mixed pond of 100 DNA sample constructed dnas of individuality, is screened.
5th, the prediction of SNP site is annotated with classification
By inquiring about miRNA-SNiPer (http://www.integratomics-time.com/miRNA-SNiPer/)
With dbSNP (http://www.ncbi.nlm.nih.gov/snp) database, with reference to http://www.mirbase.org/ numbers
Classification annotation is carried out to the SNP for screening according to storehouse (20.0).And the precursor sequence of corresponding miRNA is obtained from Genebank, lead to
Cross Blast and determine positions of the SNPs on miRNA, while analyzing positions of the miRNA on genome from NCBI.According to SNP
Position on miRNA is divided into the MIRSNP positioned at precursor, ripe body and germplasm area, and host is divided into according to the position on gene
Gene and nonhost gene.Annotation standard：SNP precursor, ripe bodies respectively positioned at miRNA etc., if having host gene.
6th, the screening primer of SNP
Purpose miRNA (500bp before and after including including precursor sequence) is downloaded in NCBI, 4 pairs of primers are designed.In F2Dai Zi
100 individual structure Polled DNAs are randomly selected in the colony of source, with DNA ponds as masterplate, is entered performing PCR with above-mentioned primer pair and is expanded.
PCR reaction systems are：Amount to 25.0 μ l；2 × Taq PCR Master Mix (MBI) 12.0 μ l, forward primer (10pmol/l)
1.0 μ l, the μ l of reverse primer (10pmol/l) 1.0, DNA profiling (100ng/ μ l) 0.5 μ l, ddH2O10.5μl.PCR response procedures：
94 DEG C of predegeneration 5min；94 DEG C of denaturation 30s, 60.2 DEG C of annealing 30s, 72 DEG C of extension 30s, 35 circulations；72 DEG C of extension 10min.
Whether PCR primer direct Sequencing (Shanghai life work), have intussusception peak to determine whether SNP according to sequencing peak.
Primer pair 1：
Sense primer：5 '-CACTGCTGTTTACAAATCTATTACACTG-3 ' (as shown in SEQ ID NO.4),
Anti-sense primer：5 '-TGACAAAGCTGCTTATAACTGCTTATAG-3 ' (as shown in SEQ ID NO.5)；
Primer pair 2：
Sense primer：5 '-CTTGTGAAGTTGCTCAGAGATTAAGTCAG-3 ' (as shown in SEQ ID NO.6),
Anti-sense primer：5 '-CAGCTGGCCTCACAGGATTTACTATG-3 ' (as shown in SEQ ID NO.7)；
Primer pair 3：
Sense primer：5 '-TGTGACAAAGCTGCTTATAACTGCTTATAG-3 ' (as shown in SEQ ID NO.8),
Anti-sense primer：5 '-TGTTTTTAATCTCGCCATTATACGCGCTGC-3 ' (as shown in SEQ ID NO.9)；
Primer pair 4：
Sense primer：5 '-GAGAGATCTTGGATTACAAG-3 ' (as shown in SEQ ID NO.10),
Anti-sense primer：5 '-GTTCGTAGATACAAAGTAAG-3 ' (as shown in SEQ ID NO.11).
7th, bioinformatic analysis chicken miRNA SNP
Using online software to http:SNP in //www.mirbase.org/ on the ripe miRNA of chicken is analyzed,
Using miRNA-SNiPer (http://www.integratomics-time.com/miRNA-SNiPer/) and dbSNP
(http://www.ncbi.nlm.nih.gov/snp) database, SNP ID are identified for rs16681031, miR-1658's
SNP site in prosoma, the 9th article of chromosome (Chr9).With reference to http://www.mirbase.org/ databases (20.0),
There are host gene, entitled ENSGALT00000016730, GMPS-201 and the intron of host gene in miR-1658SNP sites
13。
8th, the checkings of SNP present in gu-shi chicken-An Ka chickens colony
Using Polled DNA method, it is a pond that the individuality 100 of gu-shi chicken-An Ka chickens F2 is mixed, and 4 couple using synthesis draws
Thing is expanded, and by product direct Sequencing, sequencing result carries out snp analysis using DNA STAR, BioXM (2.0) and primer 5.0.
Have intussusception peak in sequencing result is the presence of mutational site, and the chicken miRNASNP of necessary being is verified by DNA ponds, is detected
MiRNA-1658 prosomas+60C/G is mutated, and the position on sequencer map is 133bp (see Fig. 1).
2nd, the association analysis of miRNA SNP and economic characters
1st, experimental animal
With experiment one.
2nd, the design of primer and synthesis
For the mutational site (i.e. rs16681031C/G) on miRNA-1658 genes maturation body complementary region, design and close
Into primer it is as follows：
Sense primer F PRimer：5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Anti-sense primer R PRimer：5’-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3’；
Single base extension primer：5’-CATCAACACCAACCCA-3’；
Annealing temperature Tm：46.7℃.
3rd, genotyping
Using Sequenom MassARRay time-of-flight mass spectrometry technologies, to F2Gene point is carried out for resource population Different Individual
Type, using MassARRay TypeR software analysis mass spectrograms.The process is complete by Beijing Co., Ltd of Hai Site clinical researches institute
Into.
Genome DNA sample after purification is quantitatively diluted to 100ng/ μ l, 1 μ l samples is added per hole on 384 orifice plates, so
Expanded according to following reaction system and program afterwards, reaction terminates to add shrimp alkaline phosphotase (SAP) to remove remaining dNTP, so
After carry out single base extension, after product is with resin desalination, through auto sample applicator point sample in the chip after chip, point sample
Detected with matrix solid-dispersion flight time mass spectrum.
A.PCR reaction systems and response procedures are set
PCR reaction systems are as shown in table 1 below：
The PCR overall reaction systems of table 1
Pcr amplification reaction program is as follows：
95 DEG C of 2min of predegeneration；95℃30s；56℃30s；72 DEG C of 60s, 45 circulations；72℃5min；25 DEG C of insulations.
B.SAP enzymic digestions are reacted
SAP enzymic digestion reaction systems are as shown in table 2 below：
The SAP enzymic digestions of table 2 are reacted
SAP enzymic digestions, digestion reaction program are carried out in PCR instrument：37℃40min；85℃5min；25 DEG C of insulations.
C. single base extension
Single base extension system is as shown in table 3 below：
The single base extension system of table 3
Single base extension, response procedures are carried out in PCR instrument：94 DEG C of 30s of predegeneration；40 circulations：94 DEG C of 5s, 52
DEG C 5s, 80 DEG C of 5s × 5 inner loops, 72 DEG C of 3min；25 DEG C of insulations.
D. resin desalination
Product resin desalination 20min, de-salting operation is：Add 16 μ l water to the corresponding aperture of extension product
It is interior, the rosin resin 6mg of cleaning is added in product, sealer, slow speed vertical rotation 20min makes resin be produced with reaction
Thing is fully contacted, then 3200r/min centrifugation 5min, resin is sunk to bottom hole portion.Using full automatic point model machine by the sample after desalination
Product are transferred on the chip of sequenom companies from 384 orifice plates, complete printing operation.
E. the flight mass spectrum detection of product
Using flight mass spectrum instrument Direct Analysis data, miRNA-1658 genes (rs16681031C/G) SNP site is carried out
Genotyping, obtains tri- kinds of genotype of GG, GC, CC, and the characterising mass spectrometry figure of different genotype is shown in Fig. 2.
4th, the frequency of polymorphism statistical analysis of chicken miRNA-1658 gene SNP sites
The analysis of Population genetic polymorphism：The genetic structure of colony is analyzed by Excel according to colony's genotype data, is estimated
Count the polymorphism information content (PIC) of gene, colony's heterozygosity (He) and effective number of allele (Ne).
Gene frequency refers to the relative ratios of a certain its allele of gene pairs in a colony, value 0~1 it
Between, i.e.,：
Same locus gene sum in gene frequency=certain gene number/colony.
Genotype frequency refers to the ratio of a certain genotype individuals Shuo Zhan colonies sum in colony, i.e.,：
Genotype frequency=a certain genotype individuals sum/measure colony sum.
To F2The site C of miRNA-1658 in sources group>The genotype frequency statistic analysis result of G see the table below 4.As a result
It has been shown that, in F2355 is CC genotype in sources group, and 298 is GC genotype, and 23 is GG genotype.CC, GC and GG base
Because type frequency is respectively 0.525,0.441 and 0.034.The frequency of C and G allele is respectively 0.746 and 0.254.
Chicken miRNA-1658 (+60bpC>G) mutational site is in moderate polymorphic 0.25<PIC<0.5, and have three kinds of genes
Type, the Population genetic polymorphism information parameter statistics in mutational site is polymorphism information content PIC (0.3072), heterozygosity He
(0.379), effective number of allele Ne (1.6104), therefore it is associated analysis with the related economic proterties of sources group.
The miRNA-1658 genes of table 4 are in F2For genotype frequency in resource population and gene frequency
5th, influence of the miRNA-1658SNP sites to miRNA secondary structures
Using Mfold online softwares, to miR-1658 (+60bp C>G) secondary structure of allele is predicted, SNP
Causing can value change.Wild type can be worth (- 34.6), and (- 36.1) can be worth after mutation, can be worth change (1.5), secondary structural change.
Positioned at miR-1658 (+60bpC>G the mutation on), produced in the secondary structure of its miRNA it is new expand structure, cause two grades
The change of the minimum free energy of structure, causes the change of secondary structural stability, indicates that these SNP sites may influence its miRNA
Process.
6th, the association analysis of the polymorphism of chicken miRNA-1658 gene SNP sites and chicken body measurement trait
Relation analysis model：It is related to the week old body measurement trait of chicken 4 using SPSS (20.0) software analysis gene SNP site
Property.Each trait data is ensured in normal distribution, recycles least-square analysis to Data correction；According to data characteristics, profit
With general linear model, as a result the difference between multi-variables analysis genotype effects and more each genotype of LSD multiple comparison graphs be shown in
Table 5 below.Model I is the relation analysis model of the loci polymorphism and 4 week old body measurement traits, i.e.,：
Model I：yijlnkm=μ+Gi+Sj+Rl+Pn+Hk+eijlnkm；
Wherein, yijlnkmIt is individual phenotypic number；μ is population mean；GiIt is genotype fixed effect；SjIt is sex-effects；Rl
It is Reciprocal effect；PlIt is family effect；HkIt is batch effect；eijlnkmIt is random error.
Variance analysis between the chicken miRNA-1658 gene SNP sites of table 5 and the week old body measurement trait of chicken 4
Note：Difference not significantly (P is represented with same letter>0.05), letter is different represents significant difference (P<0.05).
As shown in Table 5,4 week old chicken miRNA-1658SNP SNPs are wide significantly with chest breadth, Body steep length and pelvis
Association, and GG and GC, GG and CC significant difference are shown as, GG genotype is conducive to its chest breadth, pelvis wide and Body steep length is aobvious
Write increase (P<0.05).Therefore, the GG genotype in chicken miRNA-1658 gene SNP SNPs is chosen as 4 weeks
Assisted Selection and the molecular genetic breeding mark of age chicken body measurement trait.
This test example carries out the screening and checking in miRNASNP sites first, and with chicken complete genome DNA as template, design is drawn
Thing expands miRNA-1658 precursors and knows that chicken miRNA-1658 genes the 60th have C after interior purpose fragment, purifying sequencing
Or the nucleotide polymorphisms of G；Then design specific primer amplification contains the genetic fragment of pleomorphism site, and using MassARRAY systems
System matrix solid-dispersion ionization time of flight combines single primer extension reaction, to gu-shi chicken and An Ka chickens F2
The single SNP site Genotyping detections of miRNA-1658 (rs16681031C/G), and and F are carried out for resource population great amount of samples2Generation
Resource population body measurement trait is associated analysis, as a result shows chicken miRNA-1658 gene SNPs SNP and chicken body chi
Proterties is significantly associated, and GG genotype is conducive to that the chest breadth of 4 week old chickens, pelvis are wide and Body steep length significantly increases (P<0.05), by it
Marked as the assisted Selection and molecular genetic breeding of chicken body measurement trait, can quickly set up the excellent chicken group of genetic resources.The inspection
Survey method have accurately and reliably, rapid sensitive, it is cost-effective the features such as, can be used for the assisted Selection of chicken and molecular breeding, to carrying
The body measurement trait of chicken high has important effect.
Embodiment 3
Molecular breeding method based on chicken miRNA-1658 gene mononucleotide polymorphisms in the present embodiment, including following step
Suddenly：
1) extraction of STb gene and PCR are expanded
4 week old chicken peripheral blood DNAs are extracted using phenol chloroform isoamyl alcohol method, is dissolved in appropriate TE (pH8.0), -20 DEG C of preservations
It is standby, enter performing PCR using primers F Primer/R PRimer and expand, obtain amplified production；
Sense primer F PRimer：5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Anti-sense primer R PRimer：5’-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3’；
PCR amplification reaction system be：Amount to 5.0 μ l；10 × buffer 0.5 μ l, 25mM Mg2+0.4 μ l, 25mM
Each 0.5 μ l, the 100ng/ μ l of μ l, 10pmol/ μ l F Primer/R Primer of 0.1 μ l, 5U/ μ l Hotstar of dNTP 0.2 are total
DNA 1.0 μ l, ddd H2O 1.8μl；
PCR amplification response procedures be：95 DEG C of 2min of predegeneration；95℃30s；56℃30s；72 DEG C of 60s, 45 circulations；
72℃5min；25 DEG C of insulations；
2) SAP enzymic digestions
SAP enzymes, the remaining dNTP of digestion removal are added in amplified production；
SAP enzymic digestion systems are：Amount to 2.0 μ l；0.17 μ l, 1.7U/ μ l SAP enzymes of SAP buffer 0.3 μ l, ddd
H2O 1.53μl；
SAP enzymic digestion programs are：37 DEG C of 40min, 85 DEG C of 5min, 25 DEG C of insulations；
3) single base extension
Single base extension primer is added in product after SAP enzymic digestions, product is obtained；
Single base extension primer：5’-CATCAACACCAACCCA-3’；
The reaction system of single base extension is：Amount to 2.0 μ l；The μ l of 10 × buffer 0.2, reaction density be 1 ×
The μ l of iPLEX terminating reactions Mix 0.2,7 μM of μ l of Single base extension primer 0.94, reaction density is 1 × iPLEX single base extension enzymes
0.041 μ l, ddd H2O 0.619μl；
The response procedures of single base extension are：94 DEG C of 30s of predegeneration；40 circulations：94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C
5s × 5 inner loop, 72 DEG C of 3min；25 DEG C of insulations；
4) flight mass spectrum detection
After product is with resin desalination 20min, then it is transferred on chip with full automatic point model machine, using flight matter
Spectrometer device Direct Analysis data, carry out miRNA-1658 genes (rs16681031C/G) SNP site Genotyping, obtain GG,
Tri- kinds of genotype of GC, CC；
5) selected genes type a breed of chicken
The male and female chicken individuals of GG genotype of selecting and remain, set up target chicken group.
By the examination on DNA level and detection and the closely related molecular genetic marker of chicken body measurement trait, can be used for solid
The molecular marker assisted selection of the body measurement trait of beginning chicken and An Ka chickens, and then quickly set up the excellent gu-shi chicken of genetic resources and peace
Card chicken kind group.Select the male and female chicken individuals of GG genotype to reserve seed for planting, the accuracy of chicken body measurement trait selection can be increased, accelerate broiler chicken new
Breeding for quality process, effectively improves the production performance of chicken, obtains significant economic benefit.
Claims (6)
1. a kind of molecular breeding method based on chicken miRNA-1658 gene mononucleotide polymorphisms, it is characterised in that：Including with
Lower step：
1）Chicken STb gene is extracted, adds primer to enter performing PCR amplification；
2）Adding Single base extension primer carries out single base extension, obtains product；
3）After product is detected through flight mass spectrum, the male and female chicken individuals of GG genotype of selecting and remain set up target chicken group；
Step 1）Middle primer is as follows：
Sense primer F PRimer：5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Anti-sense primer R PRimer：5’-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3’；
Step 2）Middle Single base extension primer：5’-CATCAACACCAACCCA-3’.
2. molecular breeding method according to claim 1, it is characterised in that：Step 1）The reaction system of middle PCR amplification is：
Amount to 5.0 μ l；10 × buffer 0.5 μ l, 25mM Mg2+The μ of 0.4 μ l, 25mM dNTP, 0.1 μ l, 5U/ μ l Hotstar 0.2
Each 0.5 μ l, 100ng/ μ l STb genes 1.0 μ l, ddd H of l, 10pmol/ μ l F Primer/R Primer2O 1.8μl。
3. molecular breeding method according to claim 2, it is characterised in that：Step 1）The response procedures of middle PCR amplification are：
95 DEG C of 2min of predegeneration；95℃ 30s；56℃ 30s；72 DEG C of 60s, 45 circulations；72℃ 5min；25 DEG C of insulations.
4. molecular breeding method according to claim 1, it is characterised in that：Step 1）SAP enzymes are carried out after middle PCR amplifications to disappear
Change reaction, SAP enzymic digestion systems are：Amount to 2.0 μ l；0.17 μ l, 1.7U/ μ l SAP enzymes of SAP buffer 0.3 μ l, ddd
H2O 1.53μl；SAP enzymic digestion programs are：37 DEG C of 40min, 85 DEG C of 5min, 25 DEG C of insulations.
5. molecular breeding method according to claim 1, it is characterised in that：Step 2）The reaction of middle single base extension
System is：Amount to 2.0 μ l；The μ l of 10 × buffer 0.2, reaction density is the μ l of 1 × iPLEX terminating reactions Mix 0.2,7 μM of single alkali
The μ l of base extension primer 0.94, reaction density is 1 × iPLEX single base extension enzymes 0.041 μ l, ddd H2O 0.619μl。
6. molecular breeding method according to claim 5, it is characterised in that：Step 2）The reaction of middle single base extension
Program is：94 DEG C of 30s of predegeneration；40 circulations：94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s × 5 inner loops, 72 DEG C
3min；25 DEG C of insulations.
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| The chicken GGA-Mir-1658* Gene:Seed Region Polymorphisms,Frequency Distribution and Putative Targets;Li-Ying Geng等;《Journal of Animal and Veterinary Advances》;20111231;第10卷(第9期);第1187页左栏第3段 * |
| 鸡microRNA基因"seed"区4个SNPs的群体遗传分布;耿立英;《天津农业科学》;20111231;第17卷(第1期);第100-105页 * |
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