CN104962634A - Kit used for detecting body size traits of chicken and molecular breeding method of chicken - Google Patents

Kit used for detecting body size traits of chicken and molecular breeding method of chicken Download PDF

Info

Publication number
CN104962634A
CN104962634A CN201510389964.4A CN201510389964A CN104962634A CN 104962634 A CN104962634 A CN 104962634A CN 201510389964 A CN201510389964 A CN 201510389964A CN 104962634 A CN104962634 A CN 104962634A
Authority
CN
China
Prior art keywords
chicken
primer
gene
mirna
breeding method
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201510389964.4A
Other languages
Chinese (zh)
Other versions
CN104962634B (en
Inventor
石建州
赵金兵
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanyang Normal University
Original Assignee
Nanyang Normal University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanyang Normal University filed Critical Nanyang Normal University
Priority to CN201510389964.4A priority Critical patent/CN104962634B/en
Publication of CN104962634A publication Critical patent/CN104962634A/en
Application granted granted Critical
Publication of CN104962634B publication Critical patent/CN104962634B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kit used for detecting body size traits of chicken and a molecular breeding method of the chicken and belongs to the field of molecular genetics. MassARRAY system matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and a single-primer extension reaction are combined, miRNA (micro ribonucleic acid)-1658 (rs16681031 C/G) single-SNP (single nucleotide polymorphism) site genotyping detection on F2 generation resource population samples of Gushi chicken and Anak chicken is performed, and correlation analysis with the size traits of F2 generation resource populations is performed. Results show that the chicken miRNA-1658 gene SNP (single nucleotide polymorphism) is obviously correlated with the body size traits of the chicken; a GG gene facilitates obvious increasing of chest breadth, crista breadth and body slanting length of a four-week-year-old chick; chicken flocks with excellent genetic resources can be quickly built by taking the GG gene as assistant selection of the body size trait of the four-week-year-old chick and as a molecular genetic breeding marker of the four-week-year-old chick.

Description

For the molecular breeding method of the test kit and chicken that detect chicken body measurement trait
Technical field
The present invention relates to a kind of test kit for detecting chicken body measurement trait, also relating to a kind of molecular breeding method based on chicken miRNA-1658 gene mononucleotide polymorphism simultaneously, belonging to molecular genetics field.
Background technology
Develop the raising with people's living standard rapidly along with socioeconomic, market to the demand of meat on the whole from before quantitative requirement turned to pursuit to quality flavor.Growth traits, body measurement trait, Meat Quality and Carcass Traits are the economic characters that current poultry industry is paid close attention to most.Most economic characters belong to quantitative character, mainly determined by numerous minor gene, these genes have additive effect, dominant effect and epistatic effect simultaneously, and the hereditary basis of complex character jointly determines by affecting the inherited genetic factors of this phenotype and environmental factors and the interaction between them.Domestic high-quality indigenous chicken ubiquity speed of growth problem slowly, thus have impact on economic benefit.Therefore, researchist attempts to find the candidate gene relevant to the related economic proterties of chicken, for poultry molecule seed selection breeding aspect provides aid mark to select by a large amount of tests for many years.
The GENERALIZATION OF MODERN BREEDING TECHNIQUE of application molecule marker is the important channel of accelerating fine-variety breeding and improving population genetic quality.First application molecular marker breeding is examination and detection and the closely-related genetic marker of chicken economic characters on DNA level, is secondly the method for quick setting up its gene pleiomorphism, then realizes genetic marker assisted Selection and realize early diagnosis selecting.
Mononucleotide polymorphic (Single nucleotide polymorphism, SNP) refers to the DNA sequence polymorphism caused by the variation of single core thuja acid in genomic level.SNP can affect the function of gene at DNA, RNA and protein level, be that the mankind can the modal one of heritable variation, account for more than 90% of known polymorphism.Analyze the function of the influential SNP of gene product, its meaning is very great.The association analysis of SNP diversity and economic characters is the focus that Application of Animal Genetic is studied always.Along with the discovery of miRNA and going deep into of research thereof, investigator finds that miRNA not only take part in the molecular regulation process of animal complex character phenotype formation, and its SNP change also may cause miRNA dysfunction, and then causes the variation of biological phenotype proterties.MiRNA is correlated with, and polymorphic Forming Mechanism comprises insertion, disappearance, transposition, amplification and base are replaced.The polymorphism of miRNA is the important factor affecting miRNA regulatory function.During single miRNA abnormal expression, the expression of hundreds of target gene may be affected, when wherein some key protein expression amount reduces, body physiological function exception and disease can be caused to occur.Research finds, the expression of the hundreds of gene of impact that the polymorphism occurred on miRNA Primary product, precursor and ripe body can be potential and path, thus extensively affects the function of miRNA.
The detection method of SNP polymorphism, modal have single-strand conformation polymorphism technology (SSCP), PCR-RFLP and direct Sequencing technology etc.But SSCP complex operation, length consuming time, influence factor is many, and there is Problem of False Negative in experimentation, so and nonideal SNP detection means; Direct Sequencing technical costs is higher.Many research applications PCR-RFLP method or PCR detect with polyacrylamide gel electrophoresis the method combined.
The ultimate principle that MassARRAY system carries out SNP somatotype is with Matrix-assisted laser desorption ionization (MALDI-TOF MS) combine with technique list primer extension reaction, by realizing nucleic acid fragment molecular weight Direct Identification.First primer sequence carries out multiplexed PCR amplification after synthesis, dilution, and to increase the template of single base extension, and then carry out single base extension, used in single base extension is ddNTP, and after therefore extending a base, reaction stops automatically.Finally utilized by extension products full automatic point model machine to be added on chip, carry out mass spectrum somatotype by MassARRAY.MASSARRAY detection of platform SNP has following advantage: accurately and reliably, directly molecular weight is detected, do not relate to fluorescent mark, gel electrophoresis etc., just can detect the difference of a base, accuracy is high, the probability that machine itself is made mistakes is very low, without the need to again verifying, also repeats without the need to designing statistics.Flux is higher, goes for several detection to a hundreds of SNP site.Detection time is short, utilizes mass spectrometric detection in 40min, just can complete detection to 380 samples, and shows detected result in real time.
Summary of the invention
The object of this invention is to provide a kind of test kit for detecting chicken body measurement trait.
Meanwhile, the present invention also provides a kind of molecular breeding method based on chicken miRNA-1658 gene mononucleotide polymorphism.
In order to realize above object, the technical solution adopted in the present invention is:
For detecting the test kit of chicken body measurement trait, comprising:
Upstream primer F PRimer:5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ' (as shown in SEQ ID NO.1),
Downstream primer R PRimer:5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ' (as shown in SEQ ID NO.2);
Single-basic extension primer: 5 '-CATCAACACCAACCCA-3 ' (as shown in SEQ ID NO.3).
Mentioned reagent box can also comprise: 10 × buffer, Mg 2+, dNTP, Hotstar, iPLEX termination reaction Mix, single-basic extension enzyme, SAP buffer, SAP enzyme, ddd H 2o and DNA Marker etc.In test kit, the addition of each component can suitably be chosen, as being greater than the consumption of 10 times.
Based on a molecular breeding method for chicken miRNA-1658 gene mononucleotide polymorphism, comprise the following steps:
1) extract chicken STb gene, add primer and carry out pcr amplification;
2) add single-basic extension primer and carry out single base extension, obtain reaction product;
3) reaction product is after flight mass spectrum detects, and the genotypic male and female chicken individuals of the GG that selects and remain, sets up target chicken group.
Step 1) in primer as follows:
Upstream primer F PRimer:5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Downstream primer R PRimer:5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ';
Step 2) in single-basic extension primer be: 5 '-CATCAACACCAACCCA-3 '.
Step 1) in the reaction system of pcr amplification be: amount to 5.0 μ l; 10 × buffer 0.5 μ l, 25mM Mg 2+0.4 μ l, 25mM dNTP 0.1 μ l, 5U/ μ l Hotstar 0.2 μ l, 10pmol/ μ l F Primer/R Primer each 0.5 μ l, 100ng/ μ l STb gene 1.0 μ l, ddd H 2o 1.8 μ l.
Step 1) in the response procedures of pcr amplification be: denaturation 95 DEG C of 2min; 95 DEG C of 30s; 56 DEG C of 30s; 72 DEG C of 60s, 45 circulations; 72 DEG C of 5min; 25 DEG C of insulations.
Step 1) in generally need add shrimp alkaline phosphotase (SAP) after pcr amplification to remove remaining dNTP, SAP enzymic digestion be this area routine techniques.In the present invention, SAP enzymic digestion system is: amount to 2.0 μ l; SAP buffer 0.17 μ l, 1.7U/ μ l SAP enzyme 0.3 μ l, ddd H 2o 1.53 μ l; SAP enzymic digestion program is: 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; 10 × buffer 0.2 μ l, reaction density is 1 × iPLEX termination reaction Mix 0.2 μ l, 7 μMs of single-basic extension primer 0.94 μ l, and reaction density is 1 × iPLEX single-basic extension enzyme 0.041 μ l, ddd H 2o 0.619 μ l.
Step 2) in the response procedures of single base extension be: denaturation 94 DEG C of 30s; 40 circulations: 94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s × 5 internal recycling, 72 DEG C of 3min; 25 DEG C of insulations.
Step 3) middle reaction product first uses resin desalination, then point sample is in chip, and chip matrix solid-dispersion flight time mass spectrum detects.
Beneficial effect of the present invention:
The present invention adopts MassARRAY system matrix assisted laser desorption attached ionization time of flight mass spectrometry combine with technique list primer extension reaction, to gu-shi chicken and An Ka chicken F 2miRNA-1658 (rs16681031 C/G) single SNP site genotype tests is carried out for resource population sample, and and F 2association analysis is carried out for resource population body measurement trait, result shows that chicken miRNA-1658 gene SNP single nucleotide polymorphism significantly associates with chicken body measurement trait, GG genotype is conducive to the wide and Body steep length of the chest breadth of chicken in 4 week age, pelvis and enlarges markedly, it can be used as assisted Selection and the molecular genetic breeding mark of chicken body measurement trait in 4 week age, the chicken group that genetic resources is excellent can be set up fast.
Accompanying drawing explanation
Fig. 1 is the SNP sequencer map of chicken miRNA-1658 gene;
Fig. 2 is the characterising mass spectrometry figure of SNP site different genotype.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Embodiment 1
For detecting the test kit of chicken body measurement trait in the present embodiment, comprising:
10pmol/ μ l upstream primer F PRimer:5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
10pmol/ μ l downstream primer R PRimer:5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ';
7 μMs of single-basic extension primer: 5 '-CATCAACACCAACCCA-3 '.
Embodiment 2
For detecting the test kit of chicken body measurement trait in the present embodiment, except comprising following primer, also comprise: 10 × buffer, 25mM Mg 2+, 25mM dNTP, 5U/ μ l Hotstar, reaction density be 1 × iPLEX single-basic extension enzyme, SAP buffer, 1.7U/ μ l SAP enzyme, ddd H 2o and DNA Marker;
10pmol/ μ l upstream primer F PRimer:5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
10pmol/ μ l downstream primer R PRimer:5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ';
7 μMs of single-basic extension primer: 5 '-CATCAACACCAACCCA-3 '.
Test example
One, the detection of chicken miRNA-1658 gene mononucleotide polymorphism, comprises the following steps:
1, animal material: gu-shi chicken and An Ka chicken.
Gu-shi chicken belongs to the excellent local variety of one of the yellow chicken type of China, and it is centered by Gushi County, under outstanding ecotope and rearing conditions, and the self-assembling formation through long-term locking procreation.Gu-shi chicken is egg meat general purpose chicken breeds, has excellent proterties: one is crude feed tolerance, and disease resistance is strong, and suitable field is herded and raised scattered; Two is fine and tender taste, and meat flavour is delicious, and soup juice is mellow, nutritious, has stronger nourishing effects; Three to be that hen is laid eggs more, and egg is large, and egg white is thicker, yolk color depth, and eggshell is thick, storage tolerance.
Peace card broiler chicken is one of excellent meat chicken kind in the world today, is also the fastest red yellow-feathered broiler of the current domestic speed of growth, has strong adaptability, resistance to stress, long speed is fast, price of deed high.
2, the cultivation of animal:
Gu-shi chicken-An Ka chicken resource population used presses the establishment of F-2 edge half sibs far away design, tests vertical 7 familys of building together, and wherein orthorhombic system 4 is peace card chicken ♂ × gu-shi chicken ♀, and reciprocal cross is 3 is that gu-shi chicken ♂ × peace blocks chicken ♀.F 0in generation, chooses kind of a chicken respectively from egg meat dual-purpose type gu-shi chicken and meat type peace card chicken pure lines, and form in male and female chicken 1:6 ratio combo, require that selected male and female chicken has this varietal characteristic, egg productivity is high, and body weight is medium, and blood lineage is pure, to ensure F 1the heterozygosis of generation individuality in each site.From the F of each family 1to select and remain in offspring a cock, produce F in male and female 1:9 ratio and other family hen mating 2in generation, require and join between male and female chicken there is no sibship, F 1the kind in generation is also dispersed in each family with hen as far as possible, selects appearance performance abundant as far as possible, present the individuality of heterozygosis, ensure F during seed selection 2larger separation is produced for proterties.
3, method for breeding:
Chicken group feeding is supported in Henan university test chicken house, and later stage test completes at Henan university poultry genetic improvement laboratory.Each family mixes group, raises in cages, free choice feeding, sufficient drinking-water.Feed to 12 week age, blood sampling sampling.In 0 ~ 12 week age, every chicken all carries out the measurement of body measurement trait and growth traits and record data week about.
4, genomic dna extraction and build mixed pond
With gu-shi chicken-An Ka chicken sources group F2 for chicken for material, every chicken jugular vein blood drawing 5mL, EDTA anti-freezing (10mmol/L Tris-Cl, 0.2mmol/L EDTA, PH 8.0), puts-80 DEG C of preservations.Adopt phenol chloroform extraction method from F 2preserve in blood sample for resource population individuality and extract genomic dna, first detect with the agarose gel electrophoresis of 1%, then use the measurement of NanoDrop2000 trace light splitting range to determine purity and the concentration of DNA sample, the extracting again of underproof DNA sample.The individual DNA sample constructed dna of random selecting 100 mixes pond, screens.
5, the predict and classification annotation of SNP site
By inquiry miRNA-SNiPer (http://www.integratomics-time.com/miRNA-SNiPer/) and dbSNP (http://www.ncbi.nlm.nih.gov/snp) database, in conjunction with http://www.mirbase.org/ database (20.0), classification annotation is carried out to the SNP screened.And from the precursor sequence that Genebank obtains corresponding miRNA, determine the position of SNPs on miRNA by Blast, analyze the position of miRNA on genome from NCBI simultaneously.Be divided into the MIRSNP being positioned at precursor, ripe Ti Hezhongzhi district according to the position of SNP on miRNA, be divided into host gene and nonhost gene according to the position on gene.Annotation standard: SNP lays respectively at the precursor of miRNA, ripe body etc., whether has host gene.
6, the screening primer of SNP
Download object miRNA (comprising the front and back 500bp of precursor sequence) at NCBI, design 4 pairs of primers.At F 2build Polled DNA for random selecting in sources group 100 individuality, with DNA pond for masterplate, carry out pcr amplification with above-mentioned primer pair.PCR reaction system is: amount to 25.0 μ l; 2 × Taq PCR Master Mix (MBI) 12.0 μ l, forward primer (10pmol/l) 1.0 μ l, reverse primer (10pmol/l) 1.0 μ l, DNA profiling (100ng/ μ l) 0.5 μ l, ddH 2o10.5 μ l.PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 60.2 DEG C of annealing 30s, 72 DEG C extend 30s, 35 circulations; 72 DEG C extend 10min.
Whether PCR primer direct Sequencing (the raw work in Shanghai), have intussusception peak to determine whether SNP according to order-checking peak.
Primer pair 1:
Upstream primer: 5 '-CACTGCTGTTTACAAATCTATTACACTG-3 ' (as shown in SEQ ID NO.4),
Downstream primer: 5 '-TGACAAAGCTGCTTATAACTGCTTATAG-3 ' (as shown in SEQ ID NO.5);
Primer pair 2:
Upstream primer: 5 '-CTTGTGAAGTTGCTCAGAGATTAAGTCAG-3 ' (as shown in SEQ ID NO.6),
Downstream primer: 5 '-CAGCTGGCCTCACAGGATTTACTATG-3 ' (as shown in SEQ ID NO.7);
Primer pair 3:
Upstream primer: 5 '-TGTGACAAAGCTGCTTATAACTGCTTATAG-3 ' (as shown in SEQ ID NO.8),
Downstream primer: 5 '-TGTTTTTAATCTCGCCATTATACGCGCTGC-3 ' (as shown in SEQ ID NO.9);
Primer pair 4:
Upstream primer: 5 '-GAGAGATCTTGGATTACAAG-3 ' (as shown in SEQ ID NO.10),
Downstream primer: 5 '-GTTCGTAGATACAAAGTAAG-3 ' (as shown in SEQ ID NO.11).
7, bioinformatic analysis chicken miRNA SNP
Online software is utilized to analyze the SNP on the miRNA of chicken maturation in http://www.mirbase.org/, utilize miRNA-SNiPer (http://www.integratomics-time.com/miRNA-SNiPer/) and dbSNP (http://www.ncbi.nlm.nih.gov/snp) database, identifying SNP ID is rs16681031, the SNP site of miR-1658 in prosoma, the 9th article of karyomit(e) (Chr9).In conjunction with http://www.mirbase.org/ database (20.0), there is host gene in miR-1658SNP site, and the name of host gene is called ENSGALT00000016730, GMPS-201 and intron 13.
8, the SNP checking existed in gu-shi chicken-An Ka chicken colony
Adopt Polled DNA method, it is a pond that the individuality 100 of gu-shi chicken-An Ka chicken F2 mixes, and utilizes 4 pairs of primer amplifications of synthesis, and by product direct Sequencing, sequencing result uses DNA STAR, BioXM (2.0) and primer 5.0 to carry out snp analysis.Have in sequencing result intussusception peak for there is mutational site, by DNA pond checking necessary being chicken miRNASNP, detect that miRNA-1658 prosoma+60C/G suddenlys change, the position on sequencer map is 133bp (see Fig. 1).
Two, the association analysis of miRNA SNP and economic characters
1, experimental animal
With test one.
2, the Design and synthesis of primer
For the mutational site (i.e. rs16681031C/G) in the complementary district of the ripe body of miRNA-1658 gene, the primer of Design and synthesis is as follows:
Upstream primer F PRimer:5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Downstream primer R PRimer:5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ';
Single-basic extension primer: 5 '-CATCAACACCAACCCA-3 ';
Annealing temperature Tm:46.7 DEG C.
3, gene type assay
Adopt Sequenom MassARRay time-of-fight mass spectrometry technology, to F 2gene type is carried out, application MassARRay TypeR software analysis mass spectrum for resource population Different Individual.This process is completed by Beijing company limited of Hai Site clinical study institute.
Genome DNA sample after purifying is quantitatively diluted for 100ng/ μ l, every hole adds 1 μ l sample on 384 orifice plates, then according to reaction system below and program amplification, reaction end adds shrimp alkaline phosphotase (SAP) and removes remaining dNTP, then single base extension is carried out, reaction product is with after resin desalination, and through auto sample applicator point sample in chip, the matrix solid-dispersion flight time mass spectrum of the chip after point sample detects.
A.PCR reaction system and response procedures are arranged
PCR reaction system is as shown in table 1 below:
Table 1 PCR total reaction system
Pcr amplification reaction program is as follows:
Denaturation 95 DEG C of 2min; 95 DEG C of 30s; 56 DEG C of 30s; 72 DEG C of 60s, 45 circulations; 72 DEG C of 5min; 25 DEG C of insulations.
B.SAP enzymic digestion is reacted
SAP enzymic digestion reaction system is as shown in table 2 below:
Table 2 SAP enzymic digestion is reacted
SAP enzymic digestion is carried out, digestion reaction program: 37 DEG C of 40min in PCR instrument; 85 DEG C of 5min; 25 DEG C of insulations.
C. single base extension
Single base extension system is as shown in table 3 below:
Table 3 single base extension system
Single base extension is carried out, response procedures: denaturation 94 DEG C of 30s in PCR instrument; 40 circulations: 94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s × 5 internal recycling, 72 DEG C of 3min; 25 DEG C of insulations.
D. resin desalination
Reaction product resin desalination 20min, de-salting operation is: add 16 μ l water in the corresponding aperture of extension product, clean Gum Rosin 6mg is joined in reaction product, sealer, slow speed vertical rotates 20min, resin is fully contacted with reaction product, the more centrifugal 5min of 3200r/min, resin is sunk to bottom hole.Utilize full automatic point model machine to be transferred to the chip of sequenom company from 384 orifice plates by the sample after desalination, complete printing operation.
E. the flight mass spectrum of reaction product detects
Utilize flight mass spectrum instrument direct analysis data, carry out miRNA-1658 gene (rs16681031C/G) SNP site gene type, obtain GG, GC, CC tri-kinds of genotype, the characterising mass spectrometry figure of different genotype is shown in Fig. 2.
4, the frequency of polymorphism statistical study of chicken miRNA-1658 gene SNP site
The analysis of Population genetic polymorphism: the genetic construction being analyzed colony according to colony's genotype data by Excel, estimates polymorphism information content (PIC), colony's heterozygosity (He) and the effective number of allele (Ne) of gene.
Gene frequency refers to its allelic relative ratios of a certain gene pairs in a colony, value between 0 ~ 1, that is:
Same locus gene sum in gene frequency=certain gene number/colony.
Genotype frequency refers to the ratio of a certain genotype individuals Shuo Zhan colony sum in colony, that is:
Genotype frequency=a certain genotype individuals sum/this mensuration colony sum.
To F 2in sources group, the genotype frequency statistic analysis result of the site C>G of miRNA-1658 sees the following form 4.Result shows, at F 2in sources group, 355 is CC genotype, and 298 is GC genotype, and 23 is GG genotype.CC, GC and GG genotype frequency is respectively 0.525,0.441 and 0.034.The allelic frequency of C and G 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 genotype, 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 carries out association analysis with the related economic proterties of sources group.
Table 4 miRNA-1658 gene is at F 2for genotype frequency in resource population and gene frequency
5, miRNA-1658SNP site is on the impact of miRNA secondary structure
Use the online software of Mfold, predict miR-1658 (+60bp C>G) allelic secondary structure, SNP causes and can value change.Wild-type can be worth (-34.6), can be worth (-36.1) after sudden change, can be worth change (1.5), secondary structural change.Be positioned at the sudden change on miR-1658 (+60bpC>G), new structure of expanding is produced in the secondary structure of its miRNA, cause the change of the minimum free energy of secondary structure, cause the change of secondary structural stability, indicate that these SNP site may affect the course of processing of its miRNA.
6, the polymorphism of chicken miRNA-1658 gene SNP site and the association analysis of chicken body measurement trait
Relation analysis model: the dependency utilizing SPSS (20.0) software analysis gene SNP site and chicken body measurement trait in 4 week age.Guarantee that each trait data is normal distribution, recycling least-square analysis is to Data correction; According to data characteristics, utilize general linear model, the difference between multivariate analysis genotype effects and the more each genotype of LSD multiple comparison graph, the results are shown in following table 5.Model I be this loci polymorphism and 4 week age body measurement trait relation analysis model, that is:
Model I：y ijlnkm＝μ+G i+S j+R l+P n+H k+e ijlnkm；
Wherein, y ijlnkmfor individual phenotypic number; μ is population mean; G ifor genotype fixed effect; S jfor sex-effects; R lfor Reciprocal effect; P lfor family effect; H kfor a batch effect; e ijlnkmfor random error.
Variance analysis between table 5 chicken miRNA-1658 gene SNP site and chicken body measurement trait in 4 week age
Note: have same letter and represent difference not significantly (P>0.05), letter is different represents significant difference (P<0.05).
As shown in Table 5,4 week age, chicken miRNA-1658SNP single nucleotide polymorphism significantly to associate with chest breadth, Body steep length and pelvis are wide, and all show as GG and GC, GG and CC significant difference, GG genotype is conducive to the wide and Body steep length of its chest breadth, pelvis and enlarges markedly (P<0.05).Therefore, choose GG genotype in chicken miRNA-1658 gene SNP single nucleotide polymorphism as 4 week age chicken body measurement trait assisted Selection and molecular genetic breeding mark.
First this test example carries out screening and the checking in miRNASNP site, with chicken complete genome DNA for template, design primer amplification miRNA-1658 precursor, in interior object fragment, knows that chicken miRNA-1658 gene the 60th exists the nucleotide polymorphisms of C or G after purifying order-checking; Then design the gene fragment of specific primer amplification containing pleomorphism site, and utilize MassARRAY system matrix assisted laser desorption attached ionization time of flight mass spectrometry combine with technique list primer extension reaction, to gu-shi chicken and An Ka chicken F 2the single SNP site genotype tests of miRNA-1658 (rs16681031C/G) is carried out for resource population great amount of samples, and and F 2association analysis is carried out for resource population body measurement trait, result shows that chicken miRNA-1658 gene SNP single nucleotide polymorphism significantly associates with chicken body measurement trait, GG genotype is conducive to the wide and Body steep length of the chest breadth of chicken in 4 week age, pelvis and enlarges markedly (P<0.05), it can be used as assisted Selection and the molecular genetic breeding mark of chicken body measurement trait, the chicken group that genetic resources is excellent can be set up fast.This detection method has accurately and reliably, rapid sensitive, the feature such as cost-saving, can be used for assisted Selection and the molecular breeding of chicken, has important effect to the body measurement trait improving chicken.
Embodiment 3
Based on the molecular breeding method of chicken miRNA-1658 gene mononucleotide polymorphism in the present embodiment, comprise the following steps:
1) extraction of STb gene and pcr amplification
Adopt phenol chloroform isoamyl alcohol method to extract chicken peripheral blood DNA in 4 week age, be dissolved in appropriate TE (pH8.0) ,-20 DEG C save backup, and utilize primers F Primer/R PRimer to carry out pcr amplification, obtain amplified production;
Upstream primer F PRimer:5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Downstream primer R PRimer:5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ';
The reaction system of pcr amplification is: amount to 5.0 μ l; 10 × buffer 0.5 μ l, 25mM Mg 2+0.4 μ l, 25mM dNTP0.1 μ l, 5U/ μ l Hotstar 0.2 μ l, 10pmol/ μ l F Primer/R Primer each 0.5 μ l, 100ng/ μ l STb gene 1.0 μ l, ddd H 2o 1.8 μ l;
The response procedures of pcr amplification is: denaturation 95 DEG C of 2min; 95 DEG C of 30s; 56 DEG C of 30s; 72 DEG C of 60s, 45 circulations; 72 DEG C of 5min; 25 DEG C of insulations;
2) SAP enzymic digestion
In amplified production, add SAP enzyme, remaining dNTP is removed in digestion;
SAP enzymic digestion system is: amount to 2.0 μ l; SAP buffer 0.17 μ l, 1.7U/ μ l SAP enzyme 0.3 μ l, ddd H 2o1.53 μ l;
SAP enzymic digestion program is: 37 DEG C of 40min, 85 DEG C of 5min, 25 DEG C of insulations;
3) single base extension
Add single-basic extension primer in product after SAP enzymic digestion, obtain reaction product;
Single-basic extension primer: 5 '-CATCAACACCAACCCA-3 ';
The reaction system of single base extension is: amount to 2.0 μ l; 10 × buffer 0.2 μ l, reaction density is 1 × iPLEX termination reaction Mix 0.2 μ l, 7 μMs of single-basic extension primer 0.94 μ l, and reaction density is 1 × iPLEX single-basic extension enzyme 0.041 μ l, ddd H 2o 0.619 μ l;
The response procedures of single base extension is: denaturation 94 DEG C of 30s; 40 circulations: 94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s × 5 internal recycling, 72 DEG C of 3min; 25 DEG C of insulations;
4) flight mass spectrum detects
Reaction product is with after resin desalination 20min, transferred on chip with full automatic point model machine again, utilize flight mass spectrum instrument direct analysis data, carry out miRNA-1658 gene (rs16681031C/G) SNP site gene type, obtain GG, GC, CC tri-kinds of genotype;
5) selected genes type a breed of chicken
The genotypic male and female chicken individuals of the GG that selects and remain, sets up target chicken group.
By examination on DNA level and detection and the closely-related molecular genetic marker of chicken body measurement trait, can be used for the molecular marker assisted selection of the body measurement trait of gu-shi chicken and An Ka chicken, and then set up the excellent gu-shi chicken of genetic resources and An Ka chicken population fast.Select the genotypic male and female chicken individuals of GG to reserve seed for planting, the accuracy that chicken body measurement trait is selected can be increased, accelerate broiler chicken breeding of new varieties process, effectively improve the production performance of chicken, obtain significant economic benefit.

Claims (7)

1., for detecting the test kit of chicken body measurement trait, it is characterized in that: test kit comprises:
Upstream primer F PRimer:5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Downstream primer R PRimer:5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ';
Single-basic extension primer: 5 '-CATCAACACCAACCCA-3 '.
2. based on a molecular breeding method for chicken miRNA-1658 gene mononucleotide polymorphism, it is characterized in that: comprise the following steps:
1) extract chicken STb gene, add primer and carry out pcr amplification;
2) add single-basic extension primer and carry out single base extension, obtain reaction product;
3) reaction product is after flight mass spectrum detects, and the genotypic male and female chicken individuals of the GG that selects and remain, sets up target chicken group;
Step 1) in primer as follows:
Upstream primer F PRimer:5 '-ACGTTGGATGTTCTGCCATACCAGTGTGTG-3 ',
Downstream primer R PRimer:5 '-ACGTTGGATGACTCCTCCAGCTGCTGCTTC-3 ';
Step 2) middle single-basic extension primer: 5 '-CATCAACACCAACCCA-3 '.
3. molecular breeding method according to claim 2, is characterized in that: step 1) in the reaction system of pcr amplification be: amount to 5.0 μ l; 10 × buffer 0.5 μ l, 25mM Mg 2+0.4 μ l, 25mM dNTP 0.1 μ l, 5U/ μ l Hotstar0.2 μ l, 10pmol/ μ l F Primer/R Primer each 0.5 μ l, 100ng/ μ l STb gene 1.0 μ l, ddd H 2o 1.8 μ l.
4. molecular breeding method according to claim 3, is characterized in that: step 1) in the response procedures of pcr amplification be: denaturation 95 DEG C of 2min; 95 DEG C of 30s; 56 DEG C of 30s; 72 DEG C of 60s, 45 circulations; 72 DEG C of 5min; 25 DEG C of insulations.
5. molecular breeding method according to claim 2, is characterized in that: step 1) in carry out SAP enzymic digestion reaction after pcr amplification, SAP enzymic digestion system is: amount to 2.0 μ l; SAP buffer 0.17 μ l, 1.7U/ μ l SAP enzyme 0.3 μ l, ddd H 2o 1.53 μ l; SAP enzymic digestion program is: 37 DEG C of 40min, 85 DEG C of 5min, 25 DEG C of insulations.
6. molecular breeding method according to claim 2, is characterized in that: step 2) in the reaction system of single base extension be: amount to 2.0 μ l; 10 × buffer 0.2 μ l, reaction density is 1 × iPLEX termination reaction Mix 0.2 μ l, 7 μMs of single-basic extension primer 0.94 μ l, and reaction density is 1 × iPLEX single-basic extension enzyme 0.041 μ l, ddd H 2o 0.619 μ l.
7. molecular breeding method according to claim 6, is characterized in that: step 2) in the response procedures of single base extension be: denaturation 94 DEG C of 30s; 40 circulations: 94 DEG C of 5s, 52 DEG C of 5s, 80 DEG C of 5s × 5 internal recycling, 72 DEG C of 3min; 25 DEG C of insulations.
CN201510389964.4A 2015-07-02 2015-07-02 For detecting the kit of chicken body measurement trait and the molecular breeding method of chicken Expired - Fee Related CN104962634B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510389964.4A CN104962634B (en) 2015-07-02 2015-07-02 For detecting the kit of chicken body measurement trait and the molecular breeding method of chicken

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510389964.4A CN104962634B (en) 2015-07-02 2015-07-02 For detecting the kit of chicken body measurement trait and the molecular breeding method of chicken

Publications (2)

Publication Number Publication Date
CN104962634A true CN104962634A (en) 2015-10-07
CN104962634B CN104962634B (en) 2017-06-30

Family

ID=54216719

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510389964.4A Expired - Fee Related CN104962634B (en) 2015-07-02 2015-07-02 For detecting the kit of chicken body measurement trait and the molecular breeding method of chicken

Country Status (1)

Country Link
CN (1) CN104962634B (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109207608A (en) * 2018-10-17 2019-01-15 佛山科学技术学院 One kind SNP relevant to chicken body measurement trait and its application
CN109609660A (en) * 2018-12-24 2019-04-12 郑州华之源医学检验实验室有限公司 A kind of individual identification system, detection method and its application
CN111910008A (en) * 2020-08-21 2020-11-10 云南农业大学 Molecular marker related to chicken growth and development and application thereof
CN111910009A (en) * 2020-08-21 2020-11-10 云南农业大学 Molecular marker influencing chicken bursal disease index and application thereof
CN111926086A (en) * 2020-08-21 2020-11-13 云南农业大学 Molecular marker influencing oblique growth of chicken body and application thereof
CN113151509A (en) * 2021-06-04 2021-07-23 河南农业大学 Molecular marker related to chicken serum alkaline phosphatase level, detection primer, kit and application

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293904A (en) * 2014-04-08 2015-01-21 河南农业大学 Detection kit and detection method for chicken miRNA-1606 gene polymorphism

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293904A (en) * 2014-04-08 2015-01-21 河南农业大学 Detection kit and detection method for chicken miRNA-1606 gene polymorphism

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI-YING GENG等: "The chicken GGA-Mir-1658* Gene:Seed Region Polymorphisms,Frequency Distribution and Putative Targets", 《JOURNAL OF ANIMAL AND VETERINARY ADVANCES》 *
耿立英: "鸡microRNA基因"seed"区4个SNPs的群体遗传分布", 《天津农业科学》 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109207608A (en) * 2018-10-17 2019-01-15 佛山科学技术学院 One kind SNP relevant to chicken body measurement trait and its application
CN109609660A (en) * 2018-12-24 2019-04-12 郑州华之源医学检验实验室有限公司 A kind of individual identification system, detection method and its application
CN111910008A (en) * 2020-08-21 2020-11-10 云南农业大学 Molecular marker related to chicken growth and development and application thereof
CN111910009A (en) * 2020-08-21 2020-11-10 云南农业大学 Molecular marker influencing chicken bursal disease index and application thereof
CN111926086A (en) * 2020-08-21 2020-11-13 云南农业大学 Molecular marker influencing oblique growth of chicken body and application thereof
CN111910009B (en) * 2020-08-21 2022-05-31 云南农业大学 Molecular marker influencing chicken bursal disease index and application thereof
CN111910008B (en) * 2020-08-21 2022-05-31 云南农业大学 Molecular marker related to chicken growth and development and application thereof
CN111926086B (en) * 2020-08-21 2022-05-31 云南农业大学 Molecular marker influencing oblique growth of chicken body and application thereof
CN113151509A (en) * 2021-06-04 2021-07-23 河南农业大学 Molecular marker related to chicken serum alkaline phosphatase level, detection primer, kit and application
CN113151509B (en) * 2021-06-04 2023-09-05 河南农业大学 Molecular marker related to chicken serum alkaline phosphatase level, detection primer, kit and application

Also Published As

Publication number Publication date
CN104962634B (en) 2017-06-30

Similar Documents

Publication Publication Date Title
CN104962634A (en) Kit used for detecting body size traits of chicken and molecular breeding method of chicken
CN108220408B (en) Grain-saving green-shin recessive white feather broiler new strain breeding method
KR102062452B1 (en) Genetic maker for parentage and thereod in Turbot
KR102077917B1 (en) Genetic maker for parentage and thereof in Olive flounder
CN108676897B (en) SNP marker influencing daily gain traits of pigs and application thereof
KR101213217B1 (en) SNP Markers Associated with Meat Quantity and Beef Quality in Hanwoo
CN106906303B (en) SNP marker influencing pork quality traits and application thereof
CN104823919B (en) The breeding method of southeast of Guizhou Province Fructus Foeniculi chicken recessive white feather new lines and application
CN111926085B (en) Molecular marker influencing chicken muscle brightness and application thereof
CN109554486A (en) SNP marker relevant to grass carp character and its application
CN111910008A (en) Molecular marker related to chicken growth and development and application thereof
CN104711339B (en) The authentication method of the dominant white feather genes of one breeder
CN104894289B (en) For detecting the kit of chicken growth traits and the molecular breeding method of chicken
CN111926086A (en) Molecular marker influencing oblique growth of chicken body and application thereof
JP2019041695A (en) Quantitative trait loci involved in shearing force value of chicken breast meat and methods of use thereof
CN110079613B (en) Molecular marker of heat stress tolerance of Holstein cattle and detection method
CN103710427B (en) Single nucleotide polymorphism, detection method and application of chicken gene
CN116179714B (en) Molecular marker related to chicken slaughtering and meat quality characteristics and breeding method of high-quality slaughtering and processing type novel variety
CN107937568B (en) Application and method of PRLR gene
CN114480669B (en) Molecular marker related to chicken breast muscle color traits and application thereof
CN114736972A (en) Reagent for evaluating growth-related traits of eleutheronema tetradactylum
KR102111238B1 (en) Microsatellite marker composition for analysis Epinephelus bruneus genes and method of analysis using the same
CN114438221A (en) SNP molecular marker related to black porgy growth traits and application thereof
CN111910009A (en) Molecular marker influencing chicken bursal disease index and application thereof
KR102527604B1 (en) Genetic marker for parentage and thereof in Tribolodon hakonensis.

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20170630

Termination date: 20200702

CF01 Termination of patent right due to non-payment of annual fee