A PCR-based rapid genetic sex identification method for Scylla Paramamosain
A technology of simulating mud crab and an identification method, which is applied in the field of sex identification of marine crabs, can solve the problems of difficulty in accurately distinguishing male and female, complicated operation, long time consumption, etc., and achieves the effects of low cost, good repeatability and simple operation.
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Embodiment 1
[0031] 1. Primer Design
[0032] According to the known DNA sequence and female-specific SNP sites, a pair of control primers and three pairs of female-specific primers were designed using the primer design software Primer Premier 5.0. The nucleotide sequences of the control primers are:
[0033] Control-F: 5'GTTCTGCTTATCATAGTTATTGCCTTG 3';
[0034] Control-R: 5' CTGCCAGTGATTCAGTGACTTAGC 3';
[0035] The nucleotide sequence of the upstream primer in the three pairs of female-specific primers is:
[0036] Female-specific-F1: 5' C TTAGTATATCACAAC T ACATCAG G ATG T 3';
[0037] Female-specific-F2: 5'TTAGTATATCACAAC T ACATCAG GC TG T 3';
[0038] Female-specific-F3: 5'TTAGTATATCACAAC T ACATCGG G A G G T 3'
[0039] Bold and underlined letters are mismatched bases and corresponding sites.
[0040] Three pairs of female-specific primers share a downstream primer whose nucleotide sequence is
[0041]Female-specific-R1: 5'AAGATGCTTGCTGTCTCATTGGT 3'.
[0042] 2. S...
Embodiment 2
[0058] A method for rapid identification of the genetic sex of Scylla pseudocaveus based on PCR technology, such as figure 1 It mainly includes the following steps:
[0059] 1. Primer Design
[0060] According to the known DNA sequence and female-specific SNP sites, the primer design software Primer Premier 5.0 was used to design control primers and female-specific primers. The nucleotide sequence of the control primers was:
[0061] Control-F: 5'GTTCTGCTTATCATAGTTATTGCCTTG 3';
[0062] Control-R: 5' CTGCCAGTGATTCAGTGACTTAGC 3';
[0063] The nucleotide sequence of the female-specific primer is:
[0064] Female-specific-F: 5'CTTAGTATATCACAACTACATCAGGATGT 3';
[0065] Female-specific-R: 5'AAGATGCTTGCTGTCTCATTGGT 3'.
[0066] 2. Sampling and Genomic DNA Extraction
[0067] (1) Collect 24 adult crabs from Niutianyang Breeding Base in Shantou City, including 12 female crabs and 12 male crabs. The sexes were determined according to the morphological characteristics of the abdo...
Embodiment 3
[0077] Using the genomic DNA of 60 juvenile mud crabs (larvae 1st stage) of unknown gender as templates, steps 3-5 in the examples were repeated. The result is as image 3 shown, where M is DNA Marker, the band at 320bp is a female-specific band; 1, 3, 6, 14-16, 22, 24, 28, 30, 31, 33-36, 38, 40, 44, 48-51, 54 and 60 were identified as female crabs, and the rest were identified as male crabs.
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