Molecular marker, primer and application for genetic sex identification of Procambarus clarkii

Abstract

The present invention provides an Indel molecular marker for identifying the genetic sex of Procambarus clarkii, which is a 60-bp deletion sequence at the 18,720,238th base on chromosome NC_059608.1. After specific primer PCR amplification, differential products between male and female Procambarus clarkii can be obtained, so as to achieve the purpose of accurately identifying the sex of Procambarus clarkii. The present invention can be used to simply, quickly and stably identify the genetic sex of Procambarus clarkii, which is helpful for the development of the unisexual breeding technology of Procambarus clarkii, realize the unisexual culture of Procambarus clarkii, and can greatly increase the yield without increasing the breeding amount, thereby increasing the economic benefits of Procambarus clarkii culture.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to an Indel molecular marker, primer and application for identifying the genetic sex of Procambarus clarkii. Background Art

[0002] Shrimps and crabs have a relatively low evolutionary status and have primitive and complex sex determination mechanisms. Their sex determination and differentiation mainly include genetic sex determination (GSD) and environmental sex determination (ESD). Among them, GSD is mainly judged by heteromorphic sex chromosomes, such as male heterogametic XX / XY type and female heterogametic ZW / ZZ type. However, shrimps and crabs have a relatively large number of chromosomes (from more than 40 pairs to more than 120 pairs), and their morphology is small. At the same time, most of the chromosomes in the metaphase of mitosis are dot-shaped, and the centromeres are difficult to distinguish, which brings great difficulties to karyotype analysis.

[0003] Procambarus clarkii, commonly known as crayfish, belongs to Decapoda, Cambaridae, Procambarus. It is native to the southern United States and northern Mexico and was first introduced into China in the 1930s. It has now become an important aquaculture economic animal in China. Due to its fast growth rate, strong adaptability and high economic added value, the continuous increase in the market demand for Procambarus clarkii has brought an unprecedented aquaculture boom. The morphology of Procambarus clarkii has obvious sexual dimorphism. The meat content rate of female shrimps with the same weight is higher than that of male shrimps. Therefore, realizing the sex control of Procambarus clarkii and carrying out monosex culture can greatly increase the yield without increasing the aquaculture amount, and has great market prospects in production practice. Although there are relevant patent literatures and reports on the molecular markers for sex identification of Procambarus clarkii, this is still very limited for the research on sex control breeding of Procambarus clarkii, and more sex-specific molecular markers with a wider adaptation range still need to be developed. Summary of the Invention

[0004] The purpose of the present invention is to provide an Indel molecular marker for quickly and accurately identifying the sex of Procambarus clarkii.

[0005] Another purpose of the present invention is to provide a primer pair for detecting the above molecular marker and then identifying the sex of Procambarus clarkii.

[0006] Another purpose of the present invention is to provide the above detection method for identifying the sex of Procambarus clarkii.

[0007] Furthermore, the present invention also provides a kit and application for identifying the sex of Procambarus clarkii.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A molecular marker for identifying the genetic sex of Procambarus clarkii, which is a 60-bp insertion / deletion nucleotide sequence located at the 18,720,238th base on chromosome NC_059608.1 of Procambarus clarkii, as shown in SEQ ID NO.1. Females do not have the deletion fragment of SEQ ID NO.1 sequence, while male individuals have the deletion fragment of SEQ ID NO.1 sequence.

[0010] The present invention also provides a primer pair for detecting the above molecular marker and then identifying the sex of Procambarus clarkii. The forward primer F1 has a nucleotide sequence as shown in SEQ ID NO.2, and the reverse primer R1 has a nucleotide sequence as shown in SEQ ID NO.3.

[0011] A detection method for identifying the genetic sex of Procambarus clarkii using a molecular marker, and the specific method is as follows:

[0012] (1) Extract the genomic DNA to be tested;

[0013] (2) Using the genomic DNA to be tested as a template, perform PCR amplification with the above specific primers. The PCR reaction system is: Premix Taq (TakaRa Taq Version 2.0 plus one dye) 10 μL, forward and reverse primers (10 mM) 0.8 μL each, template DNA 0.4 μL, ddH 2 O 8 μL.

[0014] The PCR reaction program is: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 45 s, for 30 cycles; extension at 72 °C for 10 min;

[0015] (3) Judge the genetic sex of Procambarus clarkii according to the electrophoresis pattern. Individuals with only the 668-bp specific fragment are female Procambarus clarkii, and individuals with both 668-bp and 608-bp fragments are male Procambarus clarkii.

[0016] A kit for identifying the sex of Procambarus clarkii and its application, which contains the primer pair with nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3 above.

[0017] The present invention has the following beneficial effects:

[0018] The present invention can be used to simply, quickly and stably identify the genetic sex of Procambarus clarkii. Applying this Indel molecular marker to production practice can effectively identify the sex of Procambarus clarkii at different developmental stages, contribute to the development of the unisexual breeding technology of Procambarus clarkii, achieve the all-female culture of Procambarus clarkii, and greatly increase the yield without increasing the aquaculture volume, thereby increasing the economic benefits of Procambarus clarkii aquaculture. The operation of the present invention is simple, and it is also applicable to rapid sex identification of a large number of samples. At the same time, different bands can be detected in both female and male individuals, excluding the possibility of false negatives in the experiment and saving the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the DNA detection electrophoresis map of 12 male and 12 female individuals of Procambarus clarkii;

[0020] Figure 2 It is the verification electrophoresis map of the male-specific molecular marker of Procambarus clarkii in 24 individuals. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0022] 1. Primer design

[0023] By resequencing the genome of Procambarus clarkii and comparing the chromosomal sequences of females and males, a 60-bp polymorphic insertion / deletion was found on chromosome NC_059608.1. This insertion / deletion is located at positions 18720238-18720298, and the nucleotide sequence is shown as SEQ ID NO.1. Female individuals do not have the SEQ ID NO.1 sequence deletion fragment, while male individuals have the SEQ ID NO.1 sequence deletion fragment. Primers were designed on both sides of the above Indel locus. The forward primer is F1, and the nucleotide sequence is shown as SEQ ID NO.2. The reverse primer is R1, and the nucleotide sequence is shown as SEQ ID NO.3.

[0024] 2. DNA extraction

[0025] The genomic DNA of the Procambarus clarkii to be tested was extracted using the Ezup Column Animal Genomic DNA Extraction Kit from Sangon Biotech (Shanghai). The extracted DNA was verified by agarose gel electrophoresis to determine whether the extraction was successful. The results are as Figure 1 shown. The 12 female Procambarus clarkii and 12 male Procambarus clarkii for testing were all from the Procambarus clarkii aquaculture base of Shengfa Fishery Cooperative in Zhisheng Town, Rongchang District, Chongqing.

[0026] 3. PCR amplification to verify the sex of the tested Procambarus clarkii

[0027] Using the genomic DNA of the above-mentioned 12 female Procambarus clarkii and 12 male Procambarus clarkii as templates, PCR amplification was carried out using the above primer pairs to obtain PCR amplification products.

[0028] The PCR reaction system was: 10 μL of Premix Taq (TakaRa Taq Version 2.0 plus one dye), 0.8 μL each of forward and reverse primers (10 mM), 0.4 μL of template DNA, ddH 2 O 8 μL. The PCR reaction program was: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 45 s, for 30 cycles; extension at 72 °C for 10 min.

[0029] The PCR amplification products were subjected to agarose gel electrophoresis. Individuals with only the 668 bp specific fragment were female Procambarus clarkii, and individuals with two fragments of 668 bp and 608 bp had a deletion of the SEQ ID NO.1 sequence and were male Procambarus clarkii. The results of agarose gel electrophoresis were as Figure 2 shown.

[0030] The gender of the 24 tested Procambarus clarkii was verified by PCR amplification, and the accuracy rate was 100%, indicating that this molecular marker can accurately and efficiently identify the genetic gender of Procambarus clarkii, providing an effective way for the implementation of monosex culture of Procambarus clarkii.

Claims

1. A molecular marker for identifying the genetic sex of Procambarus clarkii, Characterized in that: The molecular marker is a nucleotide sequence with a 60-bp deletion at the 18,720,238th base on chromosome NC_059608.1 of Procambarus clarkii, as shown in SEQ ID NO.

1. Female individuals do not have the deletion fragment of the SEQ ID NO.1 sequence, while male individuals have the deletion fragment of the SEQ ID NO.1 sequence.

2. A primer pair for detecting the molecular marker as described in claim 1 and then identifying the sex of Procambarus clarkii, Characterized in that, The forward primer F1 of the primer pair has a nucleotide sequence as shown in SEQ ID NO.2, and the reverse primer R1 has a nucleotide sequence as shown in SEQ ID NO.

3.

3. A detection method for identifying the genetic sex of Procambarus clarkii using the primer pair as described in claim 2, Characterized in that: It includes extracting the genomic DNA to be tested, performing PCR amplification using the specific primers, and judging the genetic sex of Procambarus clarkii; the judgment of the genetic sex of Procambarus clarkii is that individuals with only the 668-bp specific fragment are female Procambarus clarkii, and individuals with both 668 bp and 608 bp fragments are male Procambarus clarkii.

4. A detection method for identifying the genetic sex of Procambarus clarkii using the primer pair as described in claim 2, Characterized in that, The specific method is as follows: (1) Extract the genomic DNA to be tested; (2)Using the genomic DNA to be tested as a template, perform PCR amplification with the specific primers. The PCR reaction system is as follows: The PCR reaction system is: 10 μL of Premix Taq of TakaRa Taq Version 2.0 plus one dye, 0.8 μL each of the forward and reverse primers at 10 mM, 0.4 μL of template DNA, ddH 2 O 8 μL; The PCR reaction procedure is: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 45 s, 30 cycles; extension at 72 °C for 10 min; (3) Judge the genetic sex of Procambarus clarkii according to the electrophoresis pattern. Individuals with only the 668-bp specific fragment are female Procambarus clarkii, and individuals with both 668 bp and 608 bp fragments are male Procambarus clarkii.

5. A kit for identifying the sex of Procambarus clarkii, Characterized in that: It contains the Indel molecular marker with the nucleotide sequence of SEQ ID NO.1 and the primer pair shown in SEQ ID NO.2 and SEQ ID NO.3.

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