Molecular marker C42257 for rapidly identifying genetic sex of <i>Marsupenaeus japonicus </i>and applications thereof

Abstract

The present invention provides a molecular marker C42257 for rapidly identifying genetic sex of Marsupenaeus japonicus and applications thereof. The nucleotide sequence of the molecular marker C42257 is shown in SEQ ID NO:1. The nucleotide sequences of the primer pair of molecular marker C42257 used for detection are C42257F: GGGGGACAA ACAGAGACA (SEQ ID NO: 2) and C42257R: ATCGGGGTGGATTTAGAA (SEQ ID NO: 3), respectively. The molecular marker C42257 is not affected by the tissue specificity of M. japonicus and the environment, and the steps are simple and the results are obvious. The genomic DNA of M. japonicus and the primer pair are subjected to PCR reaction and electrophoresis detection. If a target band appears at 183 bp, it is a female M. japonicus. The molecular marker can also promote the establishment of the seedling breeding technology of all-female or high-female-ratio M. japonicus. Therefore, it has a broad application prospect.

Claims

1. A method for identifying the genetic sex of Marsupenaeus japonicus, comprising the following steps: (1) synthesizing a primer pair C42257F and C42257R having the following nucleotide sequences: C42257F: GGGGGACAAACAGAGACA (SEQ ID NO:2); C42257R: ATCGGGGTGGATTTAGAA (SEQ ID NO:3); (2) extracting genomic DNA from Marsupenaeus japonicus samples; (3) performing PCR amplification with the primer pair C42257F and C42257R using the genomic DNA extracted in step (2) as a template, to obtain PCR amplification products; and (4) performing agarose gel electrophoresis on the PCR amplification product and observing the electrophoresis maps; if a target band of amplification appears at 183 bp in the electrophoresis map, Penaeus japonicus is female, and if no target band of amplification appears at 183 bp in the electrophoresis map, Penaeus japonicus is male.

2. The method according to claim 1, wherein the PCR amplification reaction conditions in the step (3) are as follows: 94° C., 3 min; 94° C., 30 s, 55° C., 30 s, 72° C., 30 s, repeating 32 cycles; 72° C., 10 min.

3. The method according to claim 1, wherein the PCR amplification reaction system in the step (3) is: 1 μL of genomic DNA, 5.0 μL of Mix1, 0.2 μL of each of upstream and downstream primers, and sterilized water added to 10 μL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The sole FIGURE shows female-specific marker map of M. japonicus, wherein, M: standard molecular weight; 1-10 and 21-30 are for female shrimps; 11-20 and 31-40 are for male shrimps.

DETAILED DESCRIPTION

(2) The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. The test methods used in the following examples are conventional unless otherwise specified; the materials and reagents used, unless otherwise specified, are all commercially available reagents and materials.

Example 1

(3) 1. DNA Extraction

(4) The DNA of M. japonicus was extracted by Novogene, with 30 males and 30 females.

(5) 2. Construction of DNA Mixing Pool

(6) The individual DNAs were mixed in equal ratio, to prepare a male and female DNA mixing pool with a concentration of 292.8 ng/μL to 363.3 ng/μL. Each mixing pool contained 30 individuals, which would be used for subsequent experiments.

(7) 3. Primer Design

(8) Through the comparative genomics and bioinformatics analysis methods, sex-specific segments were screened, and then sex markers were screened and verified from the sex-specific segments. Using the Primer Premier 5.0 software, primers were designed for sex candidate Contig, the marker DNA sequence (as shown in SEQ ID No. 1, No. C42257) was screened, and the detection primer for this sequence was designed.

(9) The Design Criteria for Primers:

(10) 1) Primer annealing temperature: 50° C. to 60° C.

(11) 2) Avoid the formation of stable dimers and hairpin structures for primers and between primers.

(12) The information or primers used in this Example is shown in Table 1:

(13) TABLE-US-00002 TABLE 1 Primer Information Product Site Primers Use Primer Information Length (bp) C42257F Amplification GGGGGACAAACAGAGACA 183 (SEQ ID NO:2) C42257R primer ATCGGGGTGGATTTAGAA (SEQ ID NO:3)

(14) 4. PCR Amplification of Mixed Template and Electrophoresis

(15) PCR amplification was performed with TransGen Biotech's HiFi enzyme using mixed DNA as a template. The PCR reaction procedure was as follows: 94° C., 3 min; 94° C., 30 s, 55° C., 30 s, 72° C., 30 s, 32 cycles; 72° C., 10 min. The PCR system was as follows:

(16) TABLE-US-00003 Reagent Amount DNA template 1.0 μL Mix 1 5.0 μL Primer (upstream and downstream) Each 0.2 μL Sterilized water Added to 10.0 μL

(17) The mixed template PCR product was subjected to 1% agarose gel electrophoresis. The electrophoresis maps showed that the no target band was amplified in the male mixed DNA pool, while the target band was amplified in the female mixed pool.

(18) 5. PCR Amplification and Electrophoresis of Male and Female Individuals

(19) The tissue DNA of 40 M. japonicus individuals was extracted as a template for PCR. The PCR primers were C42257F: GGGGGACAAACAGAGACA (SEQ ID NO:2); C42257R: ATCGGGGTGGATTTAGAA (SEQ ID NO:3); the PCR system and PCR amplification reaction procedures were the same as above. After amplification, the individual template PCR products obtained were subjected to 1% agarose gel electrophoresis. The electrophoresis map showed (the sole FIGURE) that, no target band was amplified in all tested male individuals, and the target band was amplified at 183 bp in all tested female individuals.

(20) The results showed that, the molecular marker C42257 obtained in the present invention can accurately identify the genetic sex of M. japonicus. The steps were simply described as follows: extracting the genomic DNA of tissues of M. japonicus, and performing PCR amplification reaction with the amplification primers C42257F and C42257R of molecular marker using the extracted genomic DNA as a template; performing agarose gel electrophoresis on the PCR amplification product. If a target band of amplification appeared at 183 bp in the electrophoresis map, the sex of M. japonicus was female, and if no target band of amplification appeared at 183 bp in the electrophoresis map, the sex of M. japonicus was male. The molecular marker C42257 can also be used for seedling breeding of all-female or high-female-ratio M. japonicus, to facilitate the aquaculture and development of M. japonicus.

(21) The above embodiments are only used to illustrate the technical solutions of the present invention rather than limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can make modifications to the technical solutions recorded in the foregoing embodiments or make equivalent replacement on some of the technical features; and these modifications or replacements shall not deviate from the spirit and scope of the technical solutions claimed by the present invention.