Mitochondrial microsatellite marker primers for pigeon genetic diversity analysis and detection method

Abstract

The invention discloses mitochondrial microsatellite marker primers for pigeon genetic diversity analysis and a detection method, and belongs to the technical field of molecular genetics. According tothe invention, a microsatellite fluorescence labeling method is used for solving the problem that pigeon mitochondria cannot be accurately sequenced by using a conventional sequencing method due to complex sequences; and the mitochondrial marker and the microsatellite marker are organically combined together and are not influenced by paternal line sources, and the maternal ancestor can be tracedby a pair of primers; the molecular marker developed by the invention is good in polymorphism, large in allele number and accurate in genetic analysis data, can be used for genetic diversity evaluation and pedigree verification of pigeons, and provides a genetic basis for development and utilization of pigeon varieties.

Description

Technical field

[0001] The invention relates to the technical field of molecular genetics, and more specifically to a mitochondrial microsatellite marker primer used for pigeon genetic diversity analysis and a detection method.Background technique

[0002] With the improvement of residents' living standards, people's demand for high-quality and safe food is increasing. Pigeon meat has high protein content, low fat content and good safety. It is an ideal health food for human beings. It is known among folks that "one pigeon wins nine chickens". According to incomplete statistics, there are currently more than 60 million pairs of breeding pigeons in the country, with an annual output of more than 700 million pigeons, which accounts for more than 80% of the world's total.

[0003] Although the pigeon breeding industry has good economic benefits, the research work on its germplasm resources is still relatively weak, and the production and breeding system is extremely unsound. Most of them are sel...

Examples

Embodiment 1

[0026]Example 1 Research on genetic diversity of white king pigeon

[0027](1) Extract genomic DNA from 36 white king pigeon feathers;

[0028](2) Using the DNA extracted in step (1) as a template, synthesize forward and reverse primers, and label the 5'end of the forward primer with FAM fluorescent dye to perform PCR amplification reaction;

[0029]The sequence of the forward and reverse primers is as follows:

[0030]Forward primer: 5'-TTTCCTATGATTACCACTGG-3'; SEQ ID NO.1;

[0031]Reverse primer: 5'-TGTAAGCTACGGGGACG-3'; SEQ ID NO. 2;

[0032]The PCR reaction system is: 2×PCR Mix (Nanjing Novez Biotech Co., Ltd.) 25μL, 10μmol / L forward and reverse primers each 1μL, 50-100μg / ml template DNA 2μL, ultrapure water 21μL;

[0033]The PCR reaction program is: 95°C 5min; 95°C 30s, 59°C 30s, 72°C 60s, 35 cycles; 72°C 10min;

[0034](3) The PCR product is genotyped on the ABI 3730 analyzer;

[0035](4) Use Cervus3.0 software to analyze the number of alleles (NA), Observedheterozygosity (Observedheterozygosity, HO), E...

Embodiment 2

[0039]Example 2 Verification of Maternal Relationship of Silver King Pigeon

[0040](1) Extract the genomic DNA from the feathers of a litter of silver king pigeons (male pigeons, female pigeons, 2 squabs);

[0041](2) Using the DNA extracted in step (1) as a template, synthesize forward and reverse primers, and label the 5'end of the forward primer with FAM fluorescent dye to perform PCR amplification reaction;

[0042]The sequence of the forward and reverse primers is as follows:

[0043]Forward primer: 5'-TTTCCTATGATTACCACTGG-3'; SEQ ID NO.1;

[0044]Reverse primer: 5'-TGTAAGCTACGGGGACG-3'; SEQ ID NO. 2;

[0045]The PCR reaction system is: 2×PCR Mix (Nanjing Novez Biotech Co., Ltd.) 25μL, 10μmol / L forward and reverse primers each 1μL, 50-100μg / ml template DNA 2μL, ultrapure water 21μL;

[0046]The PCR reaction program is: 95°C 5min; 95°C 30s, 59°C 30s, 72°C 60s, 35 cycles; 72°C 10min;

[0047](3) The PCR product was genotyped on the ABI 3730 analyzer; the results showed that the bands of the two squabs ...

Embodiment 3

[0048]Example 3 Verification of Maternal Relationship of Taishen Pigeon

[0049](1) Extract the genomic DNA from the blood of a litter of Taishen pigeons (male pigeons, female pigeons, 2 young pigeons);

[0050](2) Using the DNA extracted in step (1) as a template, synthesize forward and reverse primers, and perform PCR amplification reaction;

[0051]The sequence of the forward and reverse primers is as follows:

[0052]Forward primer: 5'-TTTCCTATGATTACCACTGG-3'; SEQ ID NO.1;

[0053]Reverse primer: 5'-TGTAAGCTACGGGGACG-3'; SEQ ID NO. 2;

[0054]The PCR reaction system is: 2×PCR Mix (Nanjing Novez Biotech Co., Ltd.) 25μL, 10μmol / L forward and reverse primers each 1μL, 50-100μg / ml template DNA 2μL, ultrapure water 21μL;

[0055]The PCR reaction program is: 95°C 5min; 95°C 30s, 59°C 30s, 72°C 60s, 35 cycles; 72°C 10min;

[0056](3) The PCR products were subjected to agarose gel electrophoresis; the results showed that the bands of the two pigeons were consistent with the hens, but not consistent with the co...

Technical field

[0001] The invention relates to the technical field of molecular genetics, and more specifically to a mitochondrial microsatellite marker primer used for pigeon genetic diversity analysis and a detection method.Background technique

[0002] With the improvement of residents' living standards, people's demand for high-quality and safe food is increasing. Pigeon meat has high protein content, low fat content and good safety. It is an ideal health food for human beings. It is known among folks that "one pigeon wins nine chickens". According to incomplete statistics, there are currently more than 60 million pairs of breeding pigeons in the country, with an annual output of more than 700 million pigeons, which accounts for more than 80% of the world's total.

[0003] Although the pigeon breeding industry has good economic benefits, the research work on its germplasm resources is still relatively weak, and the production and breeding system is extremely unsound. Most of them are sel...

Examples

Embodiment 1

[0026]Example 1 Research on genetic diversity of white king pigeon

[0027](1) Extract genomic DNA from 36 white king pigeon feathers;

[0028](2) Using the DNA extracted in step (1) as a template, synthesize forward and reverse primers, and label the 5'end of the forward primer with FAM fluorescent dye to perform PCR amplification reaction;

[0029]The sequence of the forward and reverse primers is as follows:

[0030]Forward primer: 5'-TTTCCTATGATTACCACTGG-3'; SEQ ID NO.1;

[0031]Reverse primer: 5'-TGTAAGCTACGGGGACG-3'; SEQ ID NO. 2;

[0032]The PCR reaction system is: 2×PCR Mix (Nanjing Novez Biotech Co., Ltd.) 25μL, 10μmol / L forward and reverse primers each 1μL, 50-100μg / ml template DNA 2μL, ultrapure water 21μL;

[0033]The PCR reaction program is: 95°C 5min; 95°C 30s, 59°C 30s, 72°C 60s, 35 cycles; 72°C 10min;

[0034](3) The PCR product is genotyped on the ABI 3730 analyzer;

[0035](4) Use Cervus3.0 software to analyze the number of alleles (NA), Observedheterozygosity (Observedheterozygosity, HO), E...

Embodiment 2

[0039]Example 2 Verification of Maternal Relationship of Silver King Pigeon

[0040](1) Extract the genomic DNA from the feathers of a litter of silver king pigeons (male pigeons, female pigeons, 2 squabs);

[0041](2) Using the DNA extracted in step (1) as a template, synthesize forward and reverse primers, and label the 5'end of the forward primer with FAM fluorescent dye to perform PCR amplification reaction;

[0042]The sequence of the forward and reverse primers is as follows:

[0043]Forward primer: 5'-TTTCCTATGATTACCACTGG-3'; SEQ ID NO.1;

[0044]Reverse primer: 5'-TGTAAGCTACGGGGACG-3'; SEQ ID NO. 2;

[0045]The PCR reaction system is: 2×PCR Mix (Nanjing Novez Biotech Co., Ltd.) 25μL, 10μmol / L forward and reverse primers each 1μL, 50-100μg / ml template DNA 2μL, ultrapure water 21μL;

[0046]The PCR reaction program is: 95°C 5min; 95°C 30s, 59°C 30s, 72°C 60s, 35 cycles; 72°C 10min;

[0047](3) The PCR product was genotyped on the ABI 3730 analyzer; the results showed that the bands of the two squabs ...

Embodiment 3

[0048]Example 3 Verification of Maternal Relationship of Taishen Pigeon

[0049](1) Extract the genomic DNA from the blood of a litter of Taishen pigeons (male pigeons, female pigeons, 2 young pigeons);

[0050](2) Using the DNA extracted in step (1) as a template, synthesize forward and reverse primers, and perform PCR amplification reaction;

[0051]The sequence of the forward and reverse primers is as follows:

[0052]Forward primer: 5'-TTTCCTATGATTACCACTGG-3'; SEQ ID NO.1;

[0053]Reverse primer: 5'-TGTAAGCTACGGGGACG-3'; SEQ ID NO. 2;

[0054]The PCR reaction system is: 2×PCR Mix (Nanjing Novez Biotech Co., Ltd.) 25μL, 10μmol / L forward and reverse primers each 1μL, 50-100μg / ml template DNA 2μL, ultrapure water 21μL;

[0055]The PCR reaction program is: 95°C 5min; 95°C 30s, 59°C 30s, 72°C 60s, 35 cycles; 72°C 10min;

[0056](3) The PCR products were subjected to agarose gel electrophoresis; the results showed that the bands of the two pigeons were consistent with the hens, but not consistent with the co...

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