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CN-121992091-A - Molecular marker for sex identification of siniperca chuatsi and application thereof

CN121992091ACN 121992091 ACN121992091 ACN 121992091ACN-121992091-A

Abstract

The invention discloses a siniperca chuatsi sex identification molecular marker and application thereof, wherein the molecular marker comprises a DNA fragment shown as SEQ ID NO.1 and a DNA fragment shown as SEQ ID NO.2, wherein the siniperca chuatsi sex is female when the 647 th position of the DNA fragment shown as SEQ ID NO.1 is C base, the siniperca chuatsi sex is male when the 647 th position of the DNA fragment shown as SEQ ID NO.1 is C base and the 249 th position of the DNA fragment shown as SEQ ID NO.2 is A base, and the molecular marker detection kit and application thereof in siniperca chuatsi genotyping, siniperca chuatsi genetic sex identification or siniperca chuatsi auxiliary breeding are disclosed.

Inventors

  • HAN CHONG
  • CAI GUOJUN
  • LI QIANG
  • LI WEIBIN
  • LIN SHENGYUE
  • CHEN WEIJIAN
  • LI SIXUN
  • DENG BINHUA
  • WU MENGMENG
  • DENG ZIYAN

Assignees

  • 广州大学

Dates

Publication Date
20260508
Application Date
20260408

Claims (10)

  1. 1. A siniperca chuatsi sex identification molecular marker is characterized by comprising a DNA fragment shown as SEQ ID NO.1 and a DNA fragment shown as SEQ ID NO.2, wherein when the 647 th bit of the DNA fragment shown as SEQ ID NO.1 is a C base, the siniperca chuatsi sex is female, and when the 647 th bit of the DNA fragment shown as SEQ ID NO.1 is a C base and the 249 th bit of the DNA fragment shown as SEQ ID NO.2 is an A base, the siniperca chuatsi sex is male.
  2. 2. A primer for amplifying the siniperca chuatsi sex determination molecular marker according to claim 1, wherein the primer comprises a reverse primer X, a reverse primer Y and a forward primer, the sequence of the reverse primer X is shown as SEQ ID NO.3, the sequence of the reverse primer Y is shown as SEQ ID NO.4, and the sequence of the forward primer is shown as SEQ ID NO. 5.
  3. 3. The primer of claim 2, wherein the reverse primer X and reverse primer Y comprise a tag sequence of a fluorescent group, and wherein the fluorescent groups of the reverse primer X and reverse primer Y are different.
  4. 4. A primer according to claim 3 wherein the tag sequence of the fluorophore is located at the 5' end of the reverse primer X and reverse primer Y.
  5. 5. The primer of claim 4 wherein the fluorophore is FAM, HEX, VIC, TAMRA, ROX, texas-Red, CY5, MGB, BHQ1, BHQ2 or BHQ3.
  6. 6. A kit for sex determination of siniperca chuatsi, comprising the primer of any one of claims 2-5.
  7. 7. The kit of claim 6, further comprising a DNA polymerase, dntps, and MgCl 2 .
  8. 8. The siniperca chuatsi sex identification method is characterized by comprising the following steps of: (1) Extracting genome DNA of siniperca chuatsi to be detected; (2) Performing PCR amplification using the primer according to any one of claims 2 to 5 or the kit according to claim 6 or 7 using the genomic DNA obtained in step (1) as a template; (3) And (3) analyzing PCR amplification products, and identifying the sex of siniperca chuatsi according to the genotype of the molecular marker in the genome of siniperca chuatsi to be detected.
  9. 9. The method according to claim 8, wherein in the step (3), when the individual to be tested detects that only the 647 th base of the DNA fragment shown in SEQ ID NO.1 is C base, the siniperca chuatsi sex is female, and when the individual to be tested detects that the 647 th base of the DNA fragment shown in SEQ ID NO.1 and the 249 th base of the DNA fragment shown in SEQ ID NO.2 are both C base and A base, the siniperca chuatsi sex is male.
  10. 10. The application of the reagent for detecting the molecular marker in claim 1, the primer in any one of claims 2-5 and the kit in claim 6 or 7 in siniperca chuatsi genotyping, siniperca chuatsi genetic sex identification or siniperca chuatsi auxiliary breeding.

Description

Molecular marker for sex identification of siniperca chuatsi and application thereof Technical Field The invention belongs to the technical field of molecular biology, and particularly relates to a siniperca chuatsi sex identification molecular marker and application thereof. Background Mandarin fish (SINIPERCA CHUATSI) is used as an important freshwater aquaculture economic fish in China, and the aquaculture industry has a remarkable scale. Recent researches clearly reveal that the siniperca chuatsi has obvious sex growth sex two-state property that female individuals grow faster before sexual maturity, and male individuals show stronger overwintering adaptability and disease resistance after sexual maturity. Therefore, in the cultivation practice, if sex can be accurately identified in an early stage, and sex control breeding is carried out on the basis, the growth performance, stress resistance and overall economic benefit of a cultivation population can be expected to be obviously improved. However, siniperca chuatsi belongs to fish lacking in obvious external sex characteristics in morphology, sex is difficult to distinguish by naked eyes in juvenile stage, traditional sex identification is dependent on anatomical or histological observation, and the methods are destructive and cannot be applied in early culture. With the development of molecular biology technology, a molecular identification method based on sex-specific genetic markers has become a key way for realizing early and nondestructive sex identification of fish. The development of the sex-specific molecular marker aiming at siniperca chuatsi and the establishment of a corresponding typing system provide a key technical support for early sex identification of the fingerling. Disclosure of Invention The invention aims to provide a molecular marker for identifying the sex of siniperca chuatsi, a primer for amplifying the molecular marker and a kit, and the primer and the kit can rapidly and accurately realize nondestructive identification of the sex of siniperca chuatsi. The invention also aims at providing a siniperca chuatsi sex identification method. The invention finally aims at providing an application of the reagent, the primer, the kit or the method for detecting the sex of siniperca chuatsi in genotyping siniperca chuatsi genes or siniperca chuatsi sex identification or siniperca chuatsi auxiliary breeding. The first aim of the invention can be achieved by adopting the following technical scheme that the molecular marker comprises a DNA fragment shown as SEQ ID NO.1 and a DNA fragment shown as SEQ ID NO.2, wherein when the 647 th bit of the DNA fragment shown as SEQ ID NO.1 is a C base, the siniperca chuatsi sex is female, and when the 647 th bit of the DNA fragment shown as SEQ ID NO.1 is a C base, and when the 249 th bit of the DNA fragment shown as SEQ ID NO.2 is an A base, the siniperca chuatsi sex is male. The invention also discloses a primer for amplifying the siniperca chuatsi sex determination molecular marker, which comprises a reverse primer X, a reverse primer Y and a forward primer, wherein the sequence of the reverse primer X is shown as SEQ ID NO.3, the sequence of the reverse primer Y is shown as SEQ ID NO.4, and the sequence of the forward primer is shown as SEQ ID NO. 5. Wherein: The nucleotide sequence of the reverse primer X is as follows: 5’-ACAGTAATCAAGGCGTGAGT-3’; the nucleotide sequence of the reverse primer Y is as follows: 5’-ACAGTAATCAAGGCGTGAGG-3’。 The nucleotide sequence of the forward primer is as follows: 5’-CATTGCAGTCCACACAGAAG-3’。 preferably, the reverse primer X and the reverse primer Y contain tag sequences of fluorescent groups, and the fluorescent groups of the reverse primer X and the reverse primer Y are different. Preferably, the tag sequence of the fluorescent group is located at the 5' end of the reverse primer X and the reverse primer Y. Preferably, the fluorophore is FAM, HEX, VIC, TAMRA, ROX, texas-Red, CY5, MGB, BHQ1, BHQ2 or BHQ3. In some embodiments of the present invention, the 5' ends of the reverse primer X and the reverse primer Y are respectively connected to different fluorescent group universal tag sequences, and the two tag sequences can be respectively complementary paired with the universal reporting primer with the HEX and FAM fluorescent groups, so as to correspond to the HEX and FAM fluorescent detection channels. The invention further discloses a kit for identifying the sex of siniperca chuatsi, which comprises the primer. Preferably, the kit further comprises a DNA polymerase, dntps and MgCl 2. In some embodiments of the invention, the kit further comprises TRET CASSETTE fluorescent primers and a ROX internal reference dye. The second aim of the invention can be achieved by the following technical scheme that the siniperca chuatsi sex identification method comprises the following steps: (1) Extracting genome DNA of siniperca chuatsi to be detected; (2) Taking the genome DNA