Molecular marker method of two mutation sites in the 5′ regulatory region of chicken mmp13 gene and its application in chicken breeding

Abstract

The invention relates to the field of molecular genetics, in particular to a molecular marker method for two mutation sites of a chicken MMP13 gene 5' control region and application of the molecular marker method in chicken breeding. It is found by the inventor that six mutation sites exist in the chicken MMP13 5' control region, namely, -1719 (T>C), -1661 (C>A), -1356 (G>A), -1128 (A>G), -1094 (C>A) and -1079 (T>C); linkage disequilibrium analysis is performed through the SHEsis online software, and it is found through the result that the sites -1719 and -1661, the sites -1356 and -1128 and the sites -1094 and -1079 are respectively in a completely-linked state in White Recessive Rock. The method is easy, convenient and fast to implement, beneficial for breeding chicken breeds laying eggs early at a high yield and capable of providing favorable help for the marker-assisted breeding work.

Description

technical field

[0001] The invention relates to the field of molecular genetics, in particular to a molecular marker method for two mutation sites in the 5' regulatory region of chicken MMP13 gene and its application in breeding. Background technique

[0002] Extracellular matrix metalloproteinases (Matrix metalloproteinases, MMPs) and their related endogenous inhibitors together constitute the MMP system, which is involved in the regulation of extracellular matrix (Extracellular matrix, ECM) remodeling, such as cell proliferation, granulosa cell differentiation, and ovarian vascularization. process of formation and degradation. MMP13, also known as collagenase 3, is able to cleave fibrillar and non-fibrillar collagen. It can catalyze the key domain of the triple helix of fibrillar collagen, resulting in changes in the stability and solubility of collagen. Balbin et al (1996) and Huang et al (1999) studies have shown that MMP13 is involved in the regulation of the estrus c...

Examples

Embodiment 1

[0017] Example 1 Sequence Alignment and Polymorphic Site Analysis of Chicken MMP13 5' Regulatory Region

[0018] 1. Test material

[0019] Recessive White Roc chicken (Shandong Jihua Poultry Breeding Co., Ltd.) was randomly sampled, blood was collected from the wing vein, and the genome was extracted and stored at -20°C.

[0020] 2. Test method

[0021] 2.1 Primer design

[0022] The primer P-MMP13 was designed according to the published red jungle fowl sequence (GenBank Accession NC_006088.3) (see Table 1 and Seq ID No: 1 and 2 for its sequence). It is specially designed according to the sequence of the red jungle fowl registered in the database.

[0023] 2.2PCR amplification

[0024] The genomes of 36 recessive White Roc chickens were randomly selected and amplified by PCR with primer P-MMP13. The primers are shown in Table 1. The reaction system is 20 μL, including 1 μL of genomic DNA (50-100ng), 2 μL of 10×Ex-buffer, 1.6 μL of dNTPs (2.5mMeach, TaKaRa), 0.4 μL of upst...

Embodiment 2

[0031] Example 2 Association analysis of chicken MMP13 5'regulatory region polymorphism and age at first lay and egg production

[0032] 1 test material

[0033] Randomly selected 37 Jining hundred-day chickens (Jining Datang Hundred-day Chicken Breeding Farm), 53 Wenchang chickens (Hainan Wenchang Chicken Breeding Company), 46 Wenshang Reed Chickens (Wenshang County, Jining City, Shandong Province), 45 Hailan brown chickens (Shandong Taian Hailan Brown Breeding Co., Ltd.) and 510 recessive White Rock chickens with egg production records (Shandong Jihua Poultry Breeding Co., Ltd.). All of the above were randomly sampled, blood was collected from the wing vein, and the genome was extracted and stored at -20°C.

[0034] 2 test method

[0035] 2.1 PCR amplification

[0036] Using the genome as a template, PCR amplification was performed. The primers are shown in Table 1. The reaction system is 40 μL, including 2 μL of genomic DNA (50-100ng), 4 μL of 10×Ex-buffer, 3.2 μL of dN...

Embodiment 3

[0081] Example 3 Effect of chicken MMP13 5' regulatory region polymorphic site on gene expression

[0082] 1 test material

[0083] Random sampling of healthy Hailan brown chickens (3-5) in the peak egg production period of the farm in Linxi Village, Tai'an City

[0084] 2 test method

[0085] 2.1 Construction of luciferase expression vector with polymorphic site mutation in MMP13 5′ regulatory region

[0086] 1) In the recessive White Lock population, two individuals whose -1356, -1128, -1094 and -1079 sites in the MMP13 5' regulatory region were wild-type and mutant were selected, and their DNA was used as a template to obtain wt-MMP13 and The mut-MMP13 sequence, the length of the amplified fragment is 1863bp, and the primer sequences are shown in Table 12 (Seq ID No.4 and 5).

[0087] Table 12 constructs luciferase vector amplification primer sequence

[0088]

[0089] 2) PCR amplification

[0090] The high-fidelity enzyme Prime STAR GXL was used for the amplificati...

technical field

[0001] The invention relates to the field of molecular genetics, in particular to a molecular marker method for two mutation sites in the 5' regulatory region of chicken MMP13 gene and its application in breeding. Background technique

[0002] Extracellular matrix metalloproteinases (Matrix metalloproteinases, MMPs) and their related endogenous inhibitors together constitute the MMP system, which is involved in the regulation of extracellular matrix (Extracellular matrix, ECM) remodeling, such as cell proliferation, granulosa cell differentiation, and ovarian vascularization. process of formation and degradation. MMP13, also known as collagenase 3, is able to cleave fibrillar and non-fibrillar collagen. It can catalyze the key domain of the triple helix of fibrillar collagen, resulting in changes in the stability and solubility of collagen. Balbin et al (1996) and Huang et al (1999) studies have shown that MMP13 is involved in the regulation of the estrus c...

Examples

Embodiment 1

[0017] Example 1 Sequence Alignment and Polymorphic Site Analysis of Chicken MMP13 5' Regulatory Region

[0018] 1. Test material

[0019] Recessive White Roc chicken (Shandong Jihua Poultry Breeding Co., Ltd.) was randomly sampled, blood was collected from the wing vein, and the genome was extracted and stored at -20°C.

[0020] 2. Test method

[0021] 2.1 Primer design

[0022] The primer P-MMP13 was designed according to the published red jungle fowl sequence (GenBank Accession NC_006088.3) (see Table 1 and Seq ID No: 1 and 2 for its sequence). It is specially designed according to the sequence of the red jungle fowl registered in the database.

[0023] 2.2PCR amplification

[0024] The genomes of 36 recessive White Roc chickens were randomly selected and amplified by PCR with primer P-MMP13. The primers are shown in Table 1. The reaction system is 20 μL, including 1 μL of genomic DNA (50-100ng), 2 μL of 10×Ex-buffer, 1.6 μL of dNTPs (2.5mMeach, TaKaRa), 0.4 μL of upst...

Embodiment 2

[0031] Example 2 Association analysis of chicken MMP13 5'regulatory region polymorphism and age at first lay and egg production

[0032] 1 test material

[0033] Randomly selected 37 Jining hundred-day chickens (Jining Datang Hundred-day Chicken Breeding Farm), 53 Wenchang chickens (Hainan Wenchang Chicken Breeding Company), 46 Wenshang Reed Chickens (Wenshang County, Jining City, Shandong Province), 45 Hailan brown chickens (Shandong Taian Hailan Brown Breeding Co., Ltd.) and 510 recessive White Rock chickens with egg production records (Shandong Jihua Poultry Breeding Co., Ltd.). All of the above were randomly sampled, blood was collected from the wing vein, and the genome was extracted and stored at -20°C.

[0034] 2 test method

[0035] 2.1 PCR amplification

[0036] Using the genome as a template, PCR amplification was performed. The primers are shown in Table 1. The reaction system is 40 μL, including 2 μL of genomic DNA (50-100ng), 4 μL of 10×Ex-buffer, 3.2 μL of dN...

Embodiment 3

[0081] Example 3 Effect of chicken MMP13 5' regulatory region polymorphic site on gene expression

[0082] 1 test material

[0083] Random sampling of healthy Hailan brown chickens (3-5) in the peak egg production period of the farm in Linxi Village, Tai'an City

[0084] 2 test method

[0085] 2.1 Construction of luciferase expression vector with polymorphic site mutation in MMP13 5′ regulatory region

[0086] 1) In the recessive White Lock population, two individuals whose -1356, -1128, -1094 and -1079 sites in the MMP13 5' regulatory region were wild-type and mutant were selected, and their DNA was used as a template to obtain wt-MMP13 and The mut-MMP13 sequence, the length of the amplified fragment is 1863bp, and the primer sequences are shown in Table 12 (Seq ID No.4 and 5).

[0087] Table 12 constructs luciferase vector amplification primer sequence

[0088]

[0089] 2) PCR amplification

[0090] The high-fidelity enzyme Prime STAR GXL was used for the amplificati...

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