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Development method and application of siniperca chuatsi sex specific marker

A specific marker and gender technology, applied in the field of fish genetics, can solve the problems of inability to breed sex-reversed populations, great difficulty, etc., and achieve the effect of saving time and economic costs and being easy to operate

Pending Publication Date: 2021-02-12
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the first two methods are quite difficult in fish sex identification, mainly because most fish have not yet formed visually distinguishable sex chromosomes like mammals; Unable to breed sex reversal groups

Method used

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  • Development method and application of siniperca chuatsi sex specific marker
  • Development method and application of siniperca chuatsi sex specific marker
  • Development method and application of siniperca chuatsi sex specific marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] [Example 1] Identification of the specific short sequence of male mandarin fish

[0026] This embodiment mainly includes the following steps, namely restriction enzyme digestion; ligation, amplification and recovery.

[0027] Digestion:

[0028] The male mandarin fish is sex-specific short. The 5 female and 5 male mandarin mandarin fish with known genders bred in this laboratory are used to build a 2b-RAD library. The specific process is as follows: (the following is the amount used for each individual)

[0029] gDNA 5.0ul,

[0030] BSA XI (1U / ul) 0.5ul,

[0031] CutSmart Buffer 0.5ul

[0032] After mixing, it was incubated at 37.0°C for 1 hour.

[0033] connect:

[0034] Prepare the connectors first. For linker 1, 10uM P1(3β) and an equal volume of 10uM anti-P primer were mixed and stored at -20°C for later use. For linker 2, 10uM P2(5β) and an equal volume of 10uM anti-P primer were mixed and stored at -20°C for later use.

[0035]

[0036] Add 20 ul of the...

Embodiment 2

[0045] [Example 2] Genome sequencing of male mandarin fish and extension of specific short sequence of mandarin mandarin fish

[0046] 1. Select one of the 5 male mandarin fish for whole genome sequencing, and then use SOAPDENOVO v1.20 for whole genome assembly

[0047] 2. The specific short sequence of male mandarin fish identified in Example 1 was searched by BLAST in the whole genome, and a long sequence with a length of 1226bp was obtained, and its nucleic acid sequence was SEQ ID NO.2.

Embodiment 3

[0048] [Example 3] Primer design and performance verification based on the specific long sequence of male mandarin fish

[0049] PCR primers were designed on the basis of this long sequence, and a pair of specific marker primers ( figure 1 ), the names are ScSex1-11-1 and ScSex1-11-2, and the sequences are shown in Table 1.

[0050] Table 1 Information table of specific marker primers for male mandarin fish

[0051]

[0052] (A) select some tails to carry out PCR verification from the mandarin fish population of known sex of this laboratory culture, experimental condition is as follows:

[0053] The total PCR amplification reaction system is 12.5 μL:

[0054]

[0055]

[0056] The PCR reaction conditions are as follows:

[0057]

[0058] Amplify for 32 cycles, the effect is the best.

[0059] The result is as figure 1 As shown, among them, picture a shows 12 ScSex1-11-1 verifications, including 6 females and 6 males; picture b shows 16 ScSex1-11-2 verification...

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Abstract

The invention provides a development method and the application of a siniperca chuatsi sex specific marker. The development method comprises the following steps: (1) obtaining male and female siniperca chuatsi specific short sequences as shown in SEQ ID NO.1 by utilizing a high-throughput sequencing method based on a 2b RAD technology; (2) obtaining a male siniperca chuatsi whole genome sequence by using a high-throughput de novo sequencing method; and (3) searching the male siniperca chuatsi specific short sequence obtained in the step (1) in the male siniperca chuatsi whole genome sequence obtained in the step (2) to obtain a longer male siniperca chuatsi specific sequence, namely SEQ ID NO.2, and performing later primer design to be applied to genetic sex identification of siniperca chuatsi. The method has the advantages of quickness, directness, simplicity, convenience and the like, and sex molecular identification can be performed on a large number of samples.

Description

technical field [0001] The invention relates to the field of fish genetics, in particular to a development method and application of a mandarin fish sex-specific marker. Background technique [0002] The study of sex determination in fish has great theoretical significance and application value. It has attracted the attention of scientists for many years and is one of the hotspots in fish research. Fish, as the most primitive vertebrates, is a large group in the history of biological evolution that has rapidly changed to bisexual reproduction, and its sex determination shows primitiveness, complexity and diversity (Kikuchi K, Hamaguchi S. Novel sex-determining genes in fish and sex chromosome evolution. Dev Dyn, 2013, 242:339–353). The primitiveness of fish sex determination is manifested in the fact that fishes do not evolve obvious sex chromosomes and definite sex-determining genes like higher mammals, and most fish have not yet formed obvious sex chromosomes or sex-deter...

Claims

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Application Information

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IPC IPC(8): C12Q1/6879C12Q1/6858C12Q1/6809C12N15/11G16B20/30G16B30/10
CPCC12Q1/6879C12Q1/6858C12Q1/6809G16B20/30G16B30/10C12Q2600/156C12Q2600/124C12Q2531/113C12Q2535/122
Inventor 周颖童金苟俞小牧廖德杰刘雪丽付北德
Owner INST OF AQUATIC LIFE ACAD SINICA
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