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Molecular marking method for two mutation loci in 5' regulatory region of chicken MMP-11 gene and application in breeding of chicken sexual precocity traits

A technology of MMP-11 and molecular markers, applied in the field of molecular genetics, can solve problems affecting gene expression and transcription initiation, and achieve the effect of reducing feeding costs

Active Publication Date: 2019-09-06
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gene expression and transcription are regulated by the 5′ regulatory region sequence, and its base mutation usually affects the initiation of transcription, thereby affecting gene expression

Method used

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  • Molecular marking method for two mutation loci in 5' regulatory region of chicken MMP-11 gene and application in breeding of chicken sexual precocity traits
  • Molecular marking method for two mutation loci in 5' regulatory region of chicken MMP-11 gene and application in breeding of chicken sexual precocity traits
  • Molecular marking method for two mutation loci in 5' regulatory region of chicken MMP-11 gene and application in breeding of chicken sexual precocity traits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Cloning, sequencing, sequence alignment and polymorphic site analysis of chicken MMP-11 gene 5' regulatory region sequence

[0039] 1. Test material

[0040] Jining Hundred Day Chicken Genome (Jining Datang Hundred Day Chicken Breeding Farm), Recessive White Rock Chicken Genome (Shandong Jihua Poultry Breeding Co., Ltd.), Wenchang Chicken (Hainan Wenchang Chicken Breeding Co., Ltd.).

[0041] 2. Test method

[0042] 2.1 Primer design

[0043] The primer P-MMP-11 was designed according to the published red jungle fowl sequence (GenBank Accession NC_006102.5, Gene ID: 101751203). The jungle fowl sequence is specially designed.

[0044] 2.2 PCR amplification

[0045] Genome pools of 48 individuals of Jining Bairi Chicken, Recessive White Rock and Wenchang Chicken were randomly selected as templates, and PCR amplification was performed with primer P-MMP-11. The primers are shown in Table 1 (SEQ ID NO.1 and 2), the reaction system is 20 μL, including 1 μL gen...

Embodiment 2

[0051] Example 2: Association analysis of polymorphisms in the 5' regulatory region of chicken MMP-11 gene and age at first lay and egg production traits

[0052] 1. Test material

[0053] A total of 90 recessive white Roc chickens, 60 Wenchang chickens and 695 Jining hundred-day chickens with laying records were randomly selected. All of the above were randomly sampled, blood was collected from the wing vein, and the genome was extracted and stored at -20°C.

[0054] 2. Test method

[0055] 2.1 KASP method for genotyping SNP loci

[0056] The g.-889(C>T), g.-636(A>G) and g.-518(C>T) loci were typed by KASP method. KASP (KompetitiveAllele Specific PCR), that is, competitive allele-specific PCR. Design 3 primers for the SNP / Indel site, two forward specific primers and one reverse universal primer. The two forward primers correspond to two kinds of fluorescent signals. After PCR reaction, the fluorescence values ​​of the two kinds of fluorescence are finally detected. Deter...

Embodiment 3

[0079] Embodiment 3: chicken MMP-11 gene 5' regulatory region core promoter region screening

[0080] 1. Test material

[0081] Healthy Hai-Line brown chickens (3-5) at the peak laying period (42w) were randomly selected for the cultivation of follicular granulosa cells.

[0082] 2. Test method

[0083] 2.1 Construction of MMP-11 gene 5′ regulatory region deletion vector

[0084] Using the chicken genome DNA as a template, the 5′- GGAGCAGCACC TGGTAACT-3' is the upstream primer, named [MMP-11-(-3423 / +62)-F], with 5'- CTGCCTTTTGTTTCCCCCTC-3′is the downstream primer, named [MMP-11-(-3423 / +62)-R], amplifies the MMP-11 promoter region (GenBank AccessionNC_006102.5, Gene ID: 101751203)-3423~+62 The segment (3485 bp in total, the sequence shown in SEQ ID NO.13; the 3424th base "C" from the 5' end in the sequence is TSS.), named as F1. Among them, Kpn I restriction site and protective base are added to the 5' end of the upstream primer, and Xho I restriction site and protective ...

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Abstract

The invention discloses a molecular marking method for two mutation loci in a 5' regulatory region of a chicken MMP-11 gene and an application in breeding of chicken sexual precocity traits. Three mutation loci, namely g.-889 (C>T), g.-636 (A>G) and g.-518 (C>T) loci, are found out to exist in the 5' regulatory region of the chicken MMP-11 gene for the first time, wherein two SNP loci of g.-889 (C>T) and g.-636 (A>G) have significant correlation with the chicken laying starting day age and the egg laying number in the earlier stage of 30 weeks of age; the CA / CA genotype corresponds to the smaller laying starting day age and the higher egg laying number in the earlier stage, and the individual of the TG / TG genotype starts to lay late correspondingly, so that detection of the two molecular marker loci associated with sexual maturity traits is helpful for breeding sexual maturity variety / lines, beneficial help is provided for chicken breeding work, especially for local chicken breeds withlate sexual maturity, and the accurate and effective marker loci are also provided for customization of a molecular marker-assisted breeding chip.

Description

technical field [0001] The invention relates to the technical field of molecular genetics, in particular to a molecular marker method for two mutation sites in the 5' regulatory region of the chicken MMP-11 gene and its application in the breeding of chicken precocious puberty traits. Background technique [0002] Matrix metalloproteinases (MMPs) are a class of proteolytic enzymes dependent on zinc ions, which can degrade extracellular matrix, basement membrane, and interstitial matrix, and play an important role in the development of ovarian follicles. MMPs are involved in extracellular matrix (extracellular matrix) , ECM) is the major protease system for degradation and remodeling (Goldman et al., 2004). MMPs cooperate with their tissue inhibitory factor TIMP and other cytokines to play important regulatory roles in biological processes such as follicle growth, ovulation, atresia and degeneration, and have become a new field of reproductive system research (Littlepage et a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 康丽凡方涛姜运良陈秀萍侯立
Owner SHANDONG AGRICULTURAL UNIVERSITY
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