阅读说明：本技术 鉴别黄河鲤遗传性别的分子标记、引物、试剂盒及应用 (Molecular marker, primer, kit and application for identifying genetic sex of yellow river carp ) 是由 殷战 翟刚 贾景怡 舒婷婷 贺江燕 娄气永 石闯 于 2021-03-15 设计创作，主要内容包括：本发明提供了一种鉴别黄河鲤遗传性别的分子标记、引物、试剂盒及应用。该鉴别黄河鲤遗传性别的分子标记为从黄河鲤基因组信息中筛选到的雄性特异序列,其存在于黄河鲤的雄性遗传性特征区域(即相当于性别遗传决定型Y的表征),而不出现于雌性黄河鲤基因组中(即对应于XX性别遗传型)。并针对该鉴别黄河鲤遗传性别的分子标记设计了相应的特异性引物和试剂盒,并基于上述引物和试剂盒,建立了用于黄河鲤遗传性别鉴定的PCR技术,可以快速、准确区分黄河鲤的遗传性别；该鉴别体系可用于对不同生长阶段和不同群体的黄河鲤性别鉴定和筛选,对研究黄河鲤性别决定和分化机制、实现其性别控制具有重要意义。(The invention provides a molecular marker, a primer, a kit and application for identifying the genetic sex of Cyprinus carpiod. The molecular marker for identifying the genetic sex of the yellow river carp is a male specific sequence screened from genome information of the yellow river carp, exists in a male genetic characteristic region of the yellow river carp (namely, the characteristic corresponding to the sex genetic determinant Y), and does not exist in a female yellow river carp genome (namely, the sex genetic determinant XX corresponds). Corresponding specific primers and a corresponding kit are designed aiming at the molecular marker for identifying the genetic sex of the yellow river carp, and a PCR (polymerase chain reaction) technology for identifying the genetic sex of the yellow river carp is established based on the primers and the kit, so that the genetic sex of the yellow river carp can be quickly and accurately distinguished; the identification system can be used for identifying and screening the sex of the yellow river carps in different growth stages and different groups, and has important significance for researching the sex determination and differentiation mechanism of the yellow river carps and realizing the sex control of the yellow river carps.)

1. A molecular marker for identifying the genetic sex of Cyprinus carpiod is characterized in that: the nucleotide sequence of the molecular marker for identifying the genetic sex of the cyprinus carpio is shown in SEQ ID NO. 1.

2. The molecular marker for identifying the genetic sex of yellow river carp according to claim 1, wherein: the molecular marker for identifying the genetic sex of the yellow river carp is a yellow river carp male specific sequence which exists in a male genetic characteristic region of the yellow river carp but is deleted in a female yellow river carp genome.

3. A primer for detecting the molecular marker for identifying the genetic sex of yellow river carp according to claim 1 or 2, characterized in that: the primer is specifically designed aiming at the nucleotide sequence of the molecular marker for identifying the genetic sex of the cyprinus carpio and comprises an upstream primer and a downstream primer;

the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;

the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

4. The primer of claim 3, wherein: the detection process of the primer comprises the following steps: carrying out PCR amplification on the genome DNA of the Cyprinus carpiod to be detected by using the primer, detecting the length of an amplified fragment, and identifying the genetic sex of the Cyprinus carpiod to be detected based on the length of the amplified fragment;

when the amplified fragment is a 1209bp strip, the yellow river carp to be detected contains a male genetic characteristic region with a nucleotide sequence shown in SEQ ID NO.1, and is identified as the yellow river carp with male genetic sex;

and when the amplified band does not appear, identifying the yellow river carp to be detected as the yellow river carp with female genetic sex.

5. The primer of claim 4, wherein: the PCR amplification system comprises: mu.L of DNA template at a concentration of 50 ng/. mu.L, 2. mu.L of 10 XBuffer, 1. mu.L of dNTP at a concentration of 2.5mM, 0.5. mu.L of each of the upstream and downstream primers at a concentration of 10. mu.M, and 0.3. mu.L of Taq enzyme at a concentration of 5 Units/. mu.L, and water was added to the mixture to make up to 20. mu.L.

6. The primer of claim 4, wherein: the procedure of PCR amplification is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃ and 30s at 72 ℃ for 38 cycles; extension was carried out at 72 ℃ for 5 min.

7. A kit for detecting the molecular marker for identifying the genetic sex of cyprinus carpio according to claim 1 or 2, wherein: the kit comprises the primer of any one of claims 3 to 6.

8. The kit of claim 7, wherein: the detection method of the kit comprises the following steps: carrying out PCR amplification on the genome DNA of the Cyprinus carpiod to be detected by using the kit, and detecting the length of an amplified fragment; identifying the genetic sex of the yellow river carp to be detected based on the length of the amplified fragment;

the PCR amplification system comprises: DNA template 1. mu.L at a concentration of 50 ng/. mu.L, 10 XBuffer 2. mu.L, dNTP 1. mu.L at a concentration of 2.5mM, upstream and downstream primers 0.5. mu.L each at a concentration of 10. mu.M, Taq enzyme 0.3. mu.L at a concentration of 5 Units/. mu.L, and water to 20. mu.L;

the procedure of PCR amplification is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃ and 30s at 72 ℃ for 38 cycles; extension was carried out at 72 ℃ for 5 min.

9. The kit of claim 8, wherein: based on the length of the amplified fragment, when the amplified fragment is a 1209bp strip, the yellow river carp to be detected contains a male genetic characteristic region with a nucleotide sequence shown in SEQ ID NO.1, and is identified as the yellow river carp with male genetic sex;

and when the amplified band does not appear, identifying the yellow river carp to be detected as the yellow river carp with female genetic sex.

10. Use of the molecular marker for identifying the genetic sex of cyprinus carpio according to claim 1 or 2, or the primer according to any one of claims 3 to 6, or the kit according to any one of claims 7 to 9 for identifying the genetic sex of cyprinus carpio.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a molecular marker, a primer, a kit and application for identifying the genetic sex of Cyprinus carpiod.

Background

The culture of aquatic animals such as fish becomes a main way for human beings to obtain animal protein, and the fish culture makes a vital contribution to the continuous supply of animal protein in the world. In fish species, there are widely sexual differences in individual size and external morphology, color characteristics, called the amphimorphism or gender dimorphism of fish. Many fishes show significant amphiprotic allotypes on important production traits such as individual size, and the economic value of the production traits is closely related to sex.

For example: the growth speed of female Cyprinus carpio is obviously faster than that of male Cyprinus carpio (especially after juvenile period), so that the development of monosomy breeding and monosomy culture technology of Cyprinus carpio has important economic value. Although individuals have size difference in sex, before gonad development and maturity, female and male cyprinus carpio are difficult to distinguish from external morphology, and embryo and larva stage female and male fish are difficult to distinguish from each other morphologically, which brings great obstruction to parthenocarpy breeding and related basic genetic researches on sex determination and differentiation mechanism of cyprinus carpio.

With the development of molecular biology techniques, molecular markers based on DNA polymorphisms were developed, such as: restriction Fragment Length Polymorphism (RFLP), random amplified polymorphic dna (rapd), Amplified Fragment Length Polymorphism (AFLP), microsatellite marker (microsatellite), and Single Nucleotide Polymorphism (SNP), among others. Sex-specific molecular markers have been developed for some fish, and different species have different genomic DNA sequence structures, so that the genetic sex of different species cannot be identified, and the sex-specific molecular markers can only be developed for different species. So far, no molecular marker for identifying the genetic sex of the yellow river carp is reported at home and abroad.

In view of the above, there is a need to design an improved molecular marker, primer, kit and application for identifying the genetic sex of cyprinus carpio to solve the above problems.

Disclosure of Invention

In order to solve the problem that the sex of male and female cyprinus carpio is difficult to identify and distinguish according to the appearance form of the existing morphological identification mode, the invention aims to provide a molecular marker, a primer, a kit and application for identifying the genetic sex of cyprinus carpio, which are beneficial to quickly and accurately identifying the genetic sex of cyprinus carpio and establish a foundation for developing the parthenocarpic breeding work of cyprinus carpio and the research on reproductive development and sex differentiation genetics.

In order to realize the aim, the invention provides a molecular marker for identifying the genetic sex of Cyprinus carpiod, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1.

As a further improvement of the invention, the molecular marker for identifying the genetic sex of the yellow river carp is a yellow river carp male specific sequence which exists in a male genetic characteristic region of the yellow river carp but is deleted in a genome of a female yellow river carp.

In order to realize the purpose, the invention also provides a primer for detecting the molecular marker for identifying the genetic sex of the cyprinus carpio. The primer is designed aiming at the nucleotide sequence of the molecular marker for identifying the genetic sex of the yellow river carp, and comprises an upstream primer and a downstream primer;

the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;

the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

As a further improvement of the invention, the detection process of the primer is as follows: carrying out PCR amplification on the genome DNA of the Cyprinus carpiod to be detected by using the primer, and detecting the length of an amplified fragment;

when the amplified fragment is a 1209bp strip, the yellow river carp to be detected contains a male genetic characteristic region with a nucleotide sequence shown in SEQ ID NO.1, and is identified as the yellow river carp with male genetic sex;

and when the amplified band does not appear, identifying the yellow river carp to be detected as the yellow river carp with female genetic sex.

As a further improvement of the invention, the PCR amplification system comprises: mu.L of DNA template at a concentration of 50 ng/. mu.L, 2. mu.L of 10 XBuffer, 1. mu.L of dNTP at a concentration of 2.5mM, 0.5. mu.L of each of the upstream and downstream primers at a concentration of 10. mu.M, and 0.3. mu.L of Taq enzyme at a concentration of 5 Units/. mu.L, and water was added to the mixture to make up to 20. mu.L.

As a further improvement of the invention, the PCR amplification procedure is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃ and 30s at 72 ℃ for 38 cycles; extension was carried out at 72 ℃ for 5 min.

In order to achieve the above object, the present invention further provides a kit for detecting the above molecular marker for identifying the genetic sex of cyprinus carpio, which comprises the above primer.

As a further improvement of the invention, the detection method of the kit comprises the following steps: carrying out PCR amplification on the genome DNA of the Cyprinus carpiod to be detected by using the kit, and detecting the length of an amplified fragment; identifying the genetic sex of the yellow river carp to be detected based on the length of the amplified fragment;

the PCR amplification system comprises: DNA template 1. mu.L at a concentration of 50 ng/. mu.L, 10 XBuffer 2. mu.L, dNTP 1. mu.L at a concentration of 2.5mM, upstream and downstream primers 0.5. mu.L each at a concentration of 10. mu.M, Taq enzyme 0.3. mu.L at a concentration of 5 Units/. mu.L, and water to 20. mu.L;

the procedure of PCR amplification is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃ and 30s at 72 ℃ for 38 cycles; extension was carried out at 72 ℃ for 5 min.

As a further improvement of the invention, based on the length of the amplified fragment, when the amplified fragment is a 1209bp strip, the yellow river carp to be detected contains a male genetic characteristic region with a nucleotide sequence shown in SEQ ID NO.1, and is identified as the yellow river carp with male genetic sex;

and when the amplified band does not appear, identifying the yellow river carp to be detected as the yellow river carp with female genetic sex.

In order to realize the purpose, the invention also provides the molecular marker for identifying the genetic sex of the cyprinus carpio, or the primer or the application of the kit in the identification of the genetic sex of the cyprinus carpio.

The invention has the beneficial effects that:

1. based on the characteristic that the yellow river carp is a diploid cultivated fish with a male heterozygosis genetic decision type (namely XX/XY sex genetic type), the molecular marker for identifying the genetic sex of the yellow river carp provided by the invention is a male specific sequence screened from genome information of the yellow river carp, exists in a male genetic characteristic region of the yellow river carp, but is deleted in a genome of a female yellow river carp; the invention also designs specific primers and a kit aiming at the molecular marker for identifying the genetic sex of the yellow river carp, establishes a PCR amplification technology for identifying the genetic sex of the yellow river carp based on the primers and the kit, and can quickly and accurately distinguish the genetic sex of the yellow river carp; the identification system can be used for identifying and screening the sex of the yellow river carps in different growth stages and different groups, and has important significance for researching the sex determination and differentiation mechanism of the yellow river carps and realizing the sex control of the yellow river carps; the technical defect that the gender of the yellow river carp is difficult to identify and distinguish according to the appearance form in the traditional morphological identification mode is effectively overcome.

2. The specific primer and the kit designed for detecting the molecular marker for identifying the genetic sex of the yellow river carp provided by the invention are beneficial to quickly and accurately identifying the genetic sex of the yellow river carp, and establish a basis for developing the parthenocarpic breeding work of the yellow river carp and the research of reproductive development and sex differentiation genetics; meanwhile, the preparation method of the primer and the kit is simple and controllable, the detection and identification result is accurate and reliable, and the primer and the kit are suitable for large-scale production and popularization and have great application prospects in the field of the identification of the genetic sex of the Cyprinus carpio.

Drawings

FIG. 1 is an electrophoresis chart of genetic sex determination of 12 sibling/non-sibling males (male) and 12 sibling/non-sibling females (female) in a cyprinus carpio breeding population by applying an upstream primer MYC-F and a downstream primer MYC-R provided by the invention (M represents DNA molecular weight standard DL2000 plus).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.

It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the aspects of the present invention are shown in the drawings, and other details not closely related to the present invention are omitted.

In addition, it is also to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention provides a molecular marker for identifying the genetic sex of Cyprinus carpiod, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1.

Preferably, the molecular marker for identifying the genetic sex of the yellow river carp is a yellow river carp male specific sequence which exists in a male genetic characteristic region of the yellow river carp and is deleted in a genome of a female yellow river carp.

In order to realize the purpose, the invention also provides a primer for detecting the molecular marker for identifying the genetic sex of the cyprinus carpio. The primer is designed aiming at the nucleotide sequence of the molecular marker for identifying the genetic sex of the yellow river carp, and comprises an upstream primer and a downstream primer;

the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 2;

the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3.

Preferably, the detection process of the primer is as follows: carrying out PCR amplification on the genome DNA of the Cyprinus carpiod to be detected by using the primer, and detecting the length of an amplified fragment;

when the amplified fragment is a 1209bp strip, the yellow river carp to be detected contains a male genetic characteristic region with a nucleotide sequence shown in SEQ ID NO.1, and is identified as the yellow river carp with male genetic sex;

and when the amplified band does not appear, identifying the yellow river carp to be detected as the yellow river carp with female genetic sex.

Preferably, the PCR amplification system is as follows: mu.L of DNA template at a concentration of 50 ng/. mu.L, 2. mu.L of 10 XBuffer, 1. mu.L of dNTP at a concentration of 2.5mM, 0.5. mu.L of each of the upstream and downstream primers at a concentration of 10. mu.M, and 0.3. mu.L of Taq enzyme at a concentration of 5 Units/. mu.L, and water was added to the mixture to make up to 20. mu.L.

Preferably, the procedure of PCR amplification is: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃ and 30s at 72 ℃ for 38 cycles; extension was carried out at 72 ℃ for 5 min.

The invention also provides a kit for detecting the molecular marker for identifying the genetic sex of the yellow river carp, which comprises the primer.

Preferably, the detection method of the kit comprises the following steps: carrying out PCR amplification on the genome DNA of the Cyprinus carpiod to be detected by using the kit, and detecting the length of an amplified fragment; identifying the genetic sex of the yellow river carp to be detected based on the length of the amplified fragment;

the PCR amplification system comprises: DNA template 1. mu.L at a concentration of 50 ng/. mu.L, 10 XBuffer 2. mu.L, dNTP 1. mu.L at a concentration of 2.5mM, upstream and downstream primers 0.5. mu.L each at a concentration of 10. mu.M, Taq enzyme 0.3. mu.L at a concentration of 5 Units/. mu.L, and water to 20. mu.L;

the procedure of PCR amplification is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃ and 30s at 72 ℃ for 38 cycles; extension was carried out at 72 ℃ for 5 min.

Preferably, based on the length of the amplified fragment, when the amplified fragment is a 1209bp band, the yellow river carp to be detected contains a male genetic characteristic region with a nucleotide sequence shown in SEQ ID NO.1, and is identified as the yellow river carp with male genetic sex;

and when the amplified band does not appear, identifying the yellow river carp to be detected as the yellow river carp with female genetic sex.

Example 1

The embodiment 1 of the invention provides a molecular marker for identifying the genetic sex of Cyprinus carpiod, and the nucleotide sequence of the molecular marker is shown as SEQ ID No. 1. The molecular marker for identifying the genetic sex of the yellow river carp is a yellow river carp male specific sequence which exists in a male yellow river carp genetic characteristic region but is deleted in a female yellow river carp genome.

In this embodiment, the screening process of the molecular marker for identifying the genetic sex of yellow river carp is as follows:

s1, yellow river carp genome sequencing: performing genome re-sequencing on DNA of 3-female and 3-male yellow river carp tail fin tissues, constructing a double-End genome DNA library, and performing Pair-End 150bp sequencing by using an Illumina Novaseq6000 sequencing platform. And (3) assembling the genome sequence by utilizing the software SOAPDenovo2 (V2.04) in a multi-Kmer assembling strategy, assembling the sequencing data into a scaffold sequence according to the overlap relation and the Pair-End relation of the sequencing data, and using the scaffold sequence as a reference genome sequence for subsequent analysis after GapCloser. Wherein, the Female genome (Female) is assembled after 3 cases of Female sequencing data are merged; male genomes (Male) were assembled after pooling of 3 Male sequencing data. And finally, respectively obtaining the reference genome sequences of the female and male of the yellow river carp.

S2, sequencing reads alignment: and comparing the double-ended sequencing sequence of the female sample and the double-ended sequencing sequence of the male sample to female and male reference genomes respectively by adopting bwa (V0.7.17-r 1188) sequence comparison software, adopting a mem comparison method and default parameters for comparison, adopting python analysis to screen sequencing reads, and screening the sequencing reads with correctly aligned double ends for subsequent analysis.

S3, male specific sequence detection: according to the result of the screened double-end sequence alignment, under the condition of a male reference genome, the sequence which is covered by all male samples but not covered by any female sample and a female mixed pool sample is counted to be a male specific sequence. Thus, a sequence is obtained and appears in 3-tailed male yellow river carps, but does not appear in 3-tailed female yellow river carps, the sequence is a realgar river carp specific short sequence, and the nucleic acid sequence is shown in SEQ ID NO. 1.

Example 2

The embodiment 2 of the invention provides a primer for detecting the molecular marker for identifying the genetic sex of the Cyprinus carpiod. Designing a specific primer aiming at the nucleotide sequence of the molecular marker for identifying the genetic sex of the cyprinus carpio, wherein the specific primer comprises an upstream primer and a downstream primer;

the nucleotide sequence of the upstream primer MYC-F is shown in SEQ ID NO. 2;

the nucleotide sequence of the downstream primer MYC-R is shown in SEQ ID NO. 3.

Specifically, the nucleotide sequence of the primer is shown as follows:

an upstream primer MYC-F: 5'-GAGCATCCACTGTCAACTT-3', respectively;

a downstream primer MYC-R: 5'-ACTCTTCCCAAACACTGATT-3' are provided.

Carrying out PCR amplification on the genome DNA of the yellow river carp to be detected by using the primer, detecting the length of an amplified fragment, and identifying the genetic sex of the yellow river carp to be detected based on the length of the amplified fragment, wherein the specific detection and identification process comprises the following steps:

p1, extracting the genome DNA of the yellow river carp to be detected: taking a tissue sample of the tail fin of the yellow river carp, extracting the DNA of the tail fin genome by using a high-salt method, detecting the concentration of the DNA by using an ultraviolet spectrophotometer, and storing the DNA in a refrigerator at the temperature of-20 ℃ for later use.

P2, PCR amplification of molecular specific marker primers (MYC-F/MYC-R):

the PCR amplification system comprises: mu.L of DNA template at a concentration of 50 ng/. mu.L, 2. mu.L of 10 XBuffer, 1. mu.L of dNTP at a concentration of 2.5mM, 0.5. mu.L of each of the upstream and downstream primers at a concentration of 10. mu.M, and 0.3. mu.L of Taq enzyme at a concentration of 5 Units/. mu.L, and water was added to the mixture to make up to 20. mu.L.

The procedure of PCR amplification is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃ and 30s at 72 ℃ for 38 cycles; extension was carried out at 72 ℃ for 5 min.

P3, carrying out PCR amplification on 12 sibling/non-sibling female yellow river carp genome DNA and 12 sibling/non-sibling male yellow river carp genome DNA by using the primer, the PCR amplification system and the amplification program.

P4, electrophoretic detection: mu.L of the PCR product was subjected to electrophoresis on 1.5% agarose gel (150V, 150mA, 10min), stained with EB dye, and detected by a gel imaging system, and the electropherogram is shown in FIG. 1.

When the amplified fragment is a 1209bp strip, the yellow river carp to be detected contains a male genetic characteristic region with a nucleotide sequence shown in SEQ ID NO.1, and is identified as the yellow river carp with male genetic sex;

and when the amplified band does not appear, identifying the yellow river carp to be detected as the yellow river carp with female genetic sex.

Example 3

The embodiment 3 of the invention provides a kit for detecting the molecular marker for identifying the genetic sex of the Cyprinus carpiod, which comprises the primer.

The detection method of the kit comprises the following steps: carrying out PCR amplification on the genome DNA of the Cyprinus carpiod to be detected by using the kit, and detecting the length of an amplified fragment; identifying the genetic sex of the yellow river carp to be detected based on the length of the amplified fragment, and the specific process comprises the following steps:

a1, extracting the genome DNA of the yellow river carp to be detected: taking a tissue sample of the tail fin of the yellow river carp, extracting the DNA of the tail fin genome by using a high-salt method, detecting the concentration of the DNA by using an ultraviolet spectrophotometer, and storing the DNA in a refrigerator at the temperature of-20 ℃ for later use.

A2, PCR amplification of molecular specific marker primers (MYC-F/MYC-R) in the kit:

the PCR amplification system comprises: mu.L of DNA template at a concentration of 50 ng/. mu.L, 2. mu.L of 10 XBuffer, 1. mu.L of dNTP at a concentration of 2.5mM, 0.5. mu.L of each of the upstream and downstream primers at a concentration of 10. mu.M, and 0.3. mu.L of Taq enzyme at a concentration of 5 Units/. mu.L, and water was added to the mixture to make up to 20. mu.L.

The procedure of PCR amplification is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃ and 30s at 72 ℃ for 38 cycles; extension was carried out at 72 ℃ for 5 min.

A3, carrying out PCR amplification on 12 sibling/non-sibling female yellow river carp genome DNA and 12 sibling/non-sibling male yellow river carp genome DNA by using the primer, the PCR amplification system and the amplification program.

A4, electrophoresis detection: the 8. mu. LPCR product was run through a 1.5% agarose gel (150V, 150mA, 10min), stained with EB dye, and examined by a gel imaging system, and the spectrum of the electrophoresis was shown in FIG. 1.

When the amplified fragment is a 1209bp strip, the yellow river carp to be detected contains a male genetic characteristic region with a nucleotide sequence shown in SEQ ID NO.1, and is identified as the yellow river carp with male genetic sex;

and when the amplified band does not appear, identifying the yellow river carp to be detected as the yellow river carp with female genetic sex.

In conclusion, the invention provides a molecular marker, a primer, a kit and application for identifying the genetic sex of Cyprinus carpiod. Based on the characteristics of the yellow river carp as a diploid cultured fish with a male heterozygosis genetic determinant (namely XX/XY sex genetic type), the molecular marker for identifying the genetic sex of the yellow river carp is a male specific sequence screened from the genome information of the yellow river carp, exists in a male genetic characteristic region of the yellow river carp (namely, a characteristic corresponding to the sex genetic determinant Y), and does not appear in a female yellow river carp genome (namely, corresponding to the XX sex genetic type). Corresponding specific primers and a corresponding kit are designed aiming at the molecular marker for identifying the genetic sex of the yellow river carp, and a PCR (polymerase chain reaction) technology for identifying the genetic sex of the yellow river carp is established based on the primers and the kit, so that the genetic sex of the yellow river carp can be quickly and accurately distinguished; the identification system can be used for identifying and screening the sex of the yellow river carps in different growth stages and different groups, and has important significance for researching the sex determination and differentiation mechanism of the yellow river carps and realizing the sex control of the yellow river carps.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the present invention.

SEQ ID NO.1

GAGCATCCACTGTCAACTTTTATCTGTGCTCAGCCCTTATTGTTTTCTCC AGTGAAAACATTAGCCTAACATTTAAATTTAAAGATAATTTTATCTAAA AATCTGAGTTGATGCAAAGGAGATTACATTATTCAACATATTATGACAT CTGATCAACATCTGTGAATCTGAGCTGATTTGACAGAAGAGTTGTGAA ATGTTATGATAACAAATCAAGAAAATATTTTTTACGATGCAATGACAG ACAGCATTCAGTCAATTAAACTGCTGTAATCAGAGTGAATGACAGACA CAGAAACAACATGTGGAGATATTGACCCAGCAGCTGTTTATGGCATTT ATAGAGTAAAGGAATTGTGTTTTTTTTATGTCACTTTGACTTATAGAAG TATATTTTGGTCTTTGCCCTCCTGAAGCCAGTGCTTCTTGTTTAAGTTCA GTATCCTATGCTATGAGTTATTAATAAAACGATTGACAGTGACCTTGTT GTTTGTGGTGCTGTGGACAAACTAGTGCTCTTTAAATCCTTATTATTGTT GTGTCCCTGCAGGAGCTGTATCGGATACAAGGCCCTGATGTGTTTTCAG GTTTGAAAATGTGTAATGCGCACCGTACTGCCTTTTTTTGTTTTATTTTT ATTTTGTTTTATGTTGTTTACCAAATCAGCTGTCCATTTGCATTTATACT TGTCTCTTCTCTTTTTTTCAGTTCTTTTAGTTCCTATTTCCTTCAGTGATT TAGTTGTTCATTATAAATGCTTAATTATCACTTAATCACACAGTGACAG GACACAGATCGAGAAAGATCACAACTCTGCTTAACATGTAATAACATT CACACATTGTGAAGAACCTTGAGATTAAATATTACTATTAATAGACAA ATGATTATAGTTCACTATGACAAATCACACAAACCAGTCCAGCTTGCA ATGAAATAATTCTATAAGTTAAACACTGTTGGATCAGCCTCATAAAAAT GCTGCCTGTTTAGACTTCTTTTGGAAAACTTTTTTTTTTTACCTTTTCAC CGAAACCTGCTTGACTTAAATAAATGAATGTGTTGAAACATTGAGTTTT AAGTCTTTTGTTTTTATGTATTCATATCATAAATATTTAATTAGCTGATC AGCACAGTCCGTAAGAGCTATAGGGCTGATCTCAGAAACATTGAGTTT ATTTTTAACATGAATCAGTGTTTGGGAAGAGT

SEQ ID NO.2

GAGCATCCACTGTCAACTT

SEQ ID NO.3

ACTCTTCCCAAACACTGATT。

Sequence listing

<110> institute of aquatic organisms of Chinese academy of sciences

<120> molecular marker, primer, kit and application for identifying genetic sex of Cyprinus carpiod

<141> 2021-03-15

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1209

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gagcatccac tgtcaacttt tatctgtgct cagcccttat tgttttctcc agtgaaaaca 60

ttagcctaac atttaaattt aaagataatt ttatctaaaa atctgagttg atgcaaagga 120

gattacatta ttcaacatat tatgacatct gatcaacatc tgtgaatctg agctgatttg 180

acagaagagt tgtgaaatgt tatgataaca aatcaagaaa atatttttta cgatgcaatg 240

acagacagca ttcagtcaat taaactgctg taatcagagt gaatgacaga cacagaaaca 300

acatgtggag atattgaccc agcagctgtt tatggcattt atagagtaaa ggaattgtgt 360

tttttttatg tcactttgac ttatagaagt atattttggt ctttgccctc ctgaagccag 420

tgcttcttgt ttaagttcag tatcctatgc tatgagttat taataaaacg attgacagtg 480

accttgttgt ttgtggtgct gtggacaaac tagtgctctt taaatcctta ttattgttgt 540

gtccctgcag gagctgtatc ggatacaagg ccctgatgtg ttttcaggtt tgaaaatgtg 600

taatgcgcac cgtactgcct ttttttgttt tatttttatt ttgttttatg ttgtttacca 660

aatcagctgt ccatttgcat ttatacttgt ctcttctctt tttttcagtt cttttagttc 720

ctatttcctt cagtgattta gttgttcatt ataaatgctt aattatcact taatcacaca 780

gtgacaggac acagatcgag aaagatcaca actctgctta acatgtaata acattcacac 840

attgtgaaga accttgagat taaatattac tattaataga caaatgatta tagttcacta 900

tgacaaatca cacaaaccag tccagcttgc aatgaaataa ttctataagt taaacactgt 960

tggatcagcc tcataaaaat gctgcctgtt tagacttctt ttggaaaact tttttttttt 1020

accttttcac cgaaacctgc ttgacttaaa taaatgaatg tgttgaaaca ttgagtttta 1080

agtcttttgt ttttatgtat tcatatcata aatatttaat tagctgatca gcacagtccg 1140

taagagctat agggctgatc tcagaaacat tgagtttatt tttaacatga atcagtgttt 1200

gggaagagt 1209

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gagcatccac tgtcaactt 19

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

actcttccca aacactgatt 20