A kind of molecular identification primer and identification method of fast and slow feather phenotype of chicken
A technology of molecular identification and speed, which is applied in the field of molecular genetics, can solve the problems of long time for establishment of male and female matching lines, limited identification time, and unclear identification standards, etc., and achieves chicken blood processing and preservation conditions are simple, easy to operate, The effect of avoiding human error
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Embodiment 1
[0041] A molecular identification method for chicken fast and slow feather phenotype, comprising the following steps:
[0042] 1), template DNA extraction: usually extracted from chicken blood, of course, can also be extracted from other tissues;
[0043] 2), use the primers shown in SEQ ID NO:1 and SEQ ID NO:2, and carry out PCR amplification with the DNA extracted in 1) as a template;
[0044] 3), carrying out agarose gel electrophoresis to the product obtained by PCR amplification in 2);
[0045] 4). Analyze the electrophoresis results in 3).
Embodiment 2
[0047] 1. Primer preparation
[0048] The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., in which:
[0049] Forward primer 1305s: 5'CCTCCTTGCCAAACCTTA3';
[0050] Reverse primer 1305a: 5'ATCAGCCAGATCCGTCAG3'.
[0051] 2. The kit extracts chicken blood clot DNA steps:
[0052] The ClotBlood DNA Kit from Kangwei Century Biotechnology Co., Ltd. was selected for extraction, product number CW0545.
[0053] (1) Weigh 0.1-0.2 g of coagulated blood clot, place it in a centrifuge tube, add 550 μl Buffer GTL, and homogenize with an electric homogenizer. In order to fully crush the blood clot, small magnetic beads can be added for mechanical crushing.
[0054] (2) Add 20 μl of proteinase K and invert the sample upside down to fully mix the sample. Incubate overnight at 56°C in a shaker until all particles are completely dissolved.
[0055] (3) Add 200 μl BufferPP, vortex for 20 seconds, and incubate on ice for 3-5 minutes. Centrifuge at 13400×g for 3 minutes,...
Embodiment 3
[0080] 1. Primer design and preparation
[0081] Elferink et al. (2008) found that the slow feather K allele that controls feather speed is composed of tandem repeats with a total length of 176324bp, caused by partial duplication of two genes: PRLR gene and SPEF2 gene encoding sperm flagellin, including Exon 1-exon 11 and 558bp exon 12 of the PRLR gene and exon 1-exon 5 of the SPEF2 gene were identified; and primers were designed to amplify a 78bp long The breakpoint connects the sequence, but the fast feather chicken cannot amplify this sequence, so it can be used as a molecular detection method to distinguish fast and slow feathers. On the basis of the research results, primers were designed to amplify a breakpoint junction sequence containing 78bp and a length of 1305bp, which can only be amplified by slow-feathered chickens, but not by fast-feathered chickens, so that chickens can be distinguished The fast and slow feather type. The fragment is easy to amplify and has st...
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