Specific molecular marker for acquiring genders of bighead carps and screening method and application thereof
A technology of molecular markers and screening methods, which is applied in the field of biology, can solve the problems of difficult identification of juvenile bighead carp, etc., and achieve the effects of low cost, simple operation, and overcoming cumbersome operations
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Embodiment 1
[0029] [Example 1] Identification of specific short sequences for male and bighead carp
[0030] This embodiment mainly includes the following steps, namely restriction enzyme digestion, ligation, amplification, recovery and analysis.
[0031] Digestion:
[0032] 2b-RAD library construction was carried out on 5 female and 5 male bighead carp with known genders cultured in this laboratory. The specific process is as follows (the following is the amount used for each individual):
[0033]
[0034] After mixing, the enzyme digestion reaction was performed at 37.0°C for 4 hours, and then incubated at 65°C for 20 minutes to inactivate the enzyme.
[0035] connect:
[0036] Sequencing adapter ligation. Ligate the above digested products with standard IlluMina sequencing adapters (slx-ad1 and slx-ad2) respectively, the reaction system (27.0μl):
[0037]
[0038] After mixing and centrifuging, place the ligation reaction at 16°C overnight (8h).
[0039] Amplification:
[0...
Embodiment 2
[0049] [Example 2] Whole Genome Sequencing of Male and Bighead Carp and Extension of Specific Short Sequence of Male and Bighead Carp
[0050] A) One of the five male and bighead carp was selected for small fragment library construction and used for IlluMina whole-genome sequencing. 48.2M PE150bp IlluMina original sequence was obtained by sequencing, and then the whole genome was assembled using SOAPdenovo v1.20;
[0051] B) Perform BLAST alignment search on the whole genome of the male and bighead carp-specific short sequence identified in Example 1, and obtain a long sequence with a length of 911bp, and its nucleic acid sequence is SEQ ID NO:2.
Embodiment 3
[0052] [Example 3] Design and verification of sex primers based on male and bighead carp-specific long sequences
[0053] A) Design a pair of gender primers according to SEQ ID NO:2, the sequence of which is:
[0054] ArS-9-1F: GCTCCTTACTCAGCAACT
[0055] ArS-9-1R: TCAGTAAACAGACGAGCA
[0056] B) select 30 tails from the bighead carp population of known sex of this laboratory culture, comprise 15 tails males and 15 are females, carry out PCR verification to its genome DNA sample, PCR reaction procedure is as follows:
[0057] PCR loading information
[0058]
[0059] PCR program settings
[0060]
[0061] The electrophoresis analysis method is as follows: electrophoresis the denatured amplification product with 10% non-denaturing polyacrylamide gel; electrophoresis conditions: voltage 230V, time 2 hours; then pass the electrophoresis product through ethidium bromide (EB) DNA bands were observed, photographed and analyzed by staining. The result is as figure 1 As sho...
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