CN112553347A - Development method of bighead carp sex identification molecular marker by taking Gapdh gene as reference - Google Patents

Development method of bighead carp sex identification molecular marker by taking Gapdh gene as reference Download PDF

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CN112553347A
CN112553347A CN202011583565.9A CN202011583565A CN112553347A CN 112553347 A CN112553347 A CN 112553347A CN 202011583565 A CN202011583565 A CN 202011583565A CN 112553347 A CN112553347 A CN 112553347A
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周颖
童金苟
俞小牧
廖德杰
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Abstract

The invention discloses a bighead carp gender identification molecular marker development method taking a Gapdh gene as a reference, belonging to the field of fish genetics. The invention discovers a specific sequence (SEQ ID NO.2) only existing in a male bighead genome sequence, and a primer designed based on the specific sequence can be used for identifying the bighead sex. The Gapdh gene is taken from the bighead genome as a reference design primer, and the double PCR amplification is carried out with a primer for identifying the bighead gender, and the genetic gender of the bighead can be quickly and accurately identified by using agarose electrophoresis. The molecular marker developed by the invention has strong specificity and can accurately identify the sex of the bighead carp. The Gapdh gene is used as an internal reference gene to carry out identification through double PCR, so that gender judgment errors caused by DNA degradation, sample adding errors or PCR amplification failure and the like are avoided. The method has the advantages of rapidness, simplicity, convenience, high accuracy and the like, and can be used for accurately identifying the genetic sex of the bighead carp in a large batch.

Description

Development method of bighead carp sex identification molecular marker by taking Gapdh gene as reference
Technical Field
The invention belongs to the field of fish genetics, relates to a method for developing a molecular marker for fish sex identification, and particularly relates to a method for developing a bighead carp sex identification molecular marker by taking a Gapdh gene as a reference.
Background
Fish, the most primitive vertebrate, is a large group of animals whose sex determination shows primitiveness, complexity and diversity, with a rapid shift to amphoterism in the history of biological evolution. The primordial sex of fish sex determination is shown in that fish do not develop obvious sex chromosomes and definite sex determining genes like high-grade mammals, most fish do not form obvious sex chromosomes and the sex determining genes are not clear, and the fish with sex chromosomes accounts for only about 10% (176 out of 1700 species). Fish determined by genetic factors are mainly classified into two types, i.e., XX/XY type and ZW/ZZ type. Males of the first type of fish contain sex-determining regions or sex chromosomes, while in the second type of fish, the sex-determining regions or sex chromosomes are located in the genome of the female fish. According to literature reports, most fish have no visually distinguishable sex chromosomes (Mei J, Gui jf. genetic basic and biological management of sexal dimorphism and sexed determination in fish China 2015,58:124) that physically image mammals, and many fish have difficulty in breeding sex-reversed populations under laboratory conditions, making the development of sex-specific molecular markers the most promising method for studying fish sex determination mechanisms. Sex determination primers are designed for fishes with XX/XY sex determination mechanism, and ideally, no amplification band exists in a female sample, and a specific band exists in a male sample. However, since there is no amplified band in the sample, and there is a possibility that it is caused by DNA degradation, sample addition error, PCR amplification failure, etc., it is necessary to introduce an internal reference into the sex-specific identification marker for double PCR amplification, and it is possible to eliminate judgment errors caused by amplification failure, etc.
Bighead carp (Hypophthalmichthys nobilis) is one of four major Chinese fishes and is an important economically cultured fish in China. According to the statistics of the world Food and Agriculture Organization (FAO) in 2018, the worldwide culture yield of bighead carp reaches 314 ten thousand tons, wherein the culture yield of China accounts for more than 90% of the total culture yield of the world. With the improvement of the living standard of residents in China, the demand for aquatic products such as fish meat, fish heads and the like and high-quality protein is more and more increased, which further stimulates the development of the bighead carp breeding industry in China. However, the sexual maturity time of bighead carp is long (4-5 years or longer), which brings great inconvenience to the selection of breeding, and how to select a suitable breeding strategy and improve the breeding efficiency of bighead carp is a great concern for the breeding workers. In order to identify the sex of a target breeding parent in advance before the breeding season comes, or to perform sex-related screening and breeding at the early growth stage of bighead carp, development of a specific molecular marker for the sex of bighead carp, which has high accuracy, is simple and rapid, is urgently needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for developing a sex marker by taking a bighead carp Gapdh gene sequence as a reference, which not only can accurately identify the sex of the bighead carp, but also avoids sex judgment errors caused by DNA degradation, sample adding errors or PCR amplification failure and the like. The invention lays a technical foundation for identifying the genetic sex of bighead carp accurately in large batch. .
The purpose of the invention is realized by the following technical scheme:
a molecular marker for identifying the sex of bighead carp is a part or all of DNA molecule with a sequence shown as SEQ ID NO. 2.
The primer for identifying the sex of the bighead carp is a primer for amplifying the molecular marker. In a further embodiment, the primer is preferably:
HynS-4F：CCAATGGCGAGTTTCAC，
HynS-4R：CGCAAGGCAGTGTTTCTA。
by using the pair of primers to amplify the bighead carp sample, a specificity band of 697bp can be amplified by the male bighead carp, and no amplification band exists in the female bighead carp.
The molecular marker or the primer has the application of identifying the sex of the bighead carp.
A method for identifying the sex of bighead carp, comprising the steps of: extracting genome DNA of the bighead carp with the sex to be identified, and performing PCR amplification by using the primers, wherein the bighead carp with the amplified molecular marker fragment is a male bighead carp, and the bighead carp without the amplified molecular marker fragment is a female bighead carp.
A method for identifying the sex of bighead carp, comprising the steps of: extracting genome DNA of bighead to be sex identified, carrying out double PCR amplification by using the primer and the internal reference gene amplification primer, amplifying molecular marker fragments and bighead of the internal reference gene fragments to be male bighead, and amplifying bighead of only the internal reference gene fragments to be female bighead.
The reference gene is preferably Gapdh gene (a partial sequence of the reference gene is shown as SEQ ID NO. 3); in a further embodiment, the amplification primers for the Gapdh gene are preferably:
HynGapdh-3 F：GGGAAGTTTGCGTATTTG，
HynGapdh-3 R：CAAAGATAGATGAGCGACAA。
using the pair of Gapdh gene amplification primers, 466bp specific bands can be amplified by both male and female bighead, and can be used for determining whether PCR amplification is successful or not.
In the double PCR amplification system, the concentration ratio of the primer for identifying the bighead carp gender to the internal reference gene amplification primer is 3: 1.
A kit for identifying the sex of bighead carp, which comprises the primer for identifying the sex of bighead carp; can also comprise an internal reference gene amplification primer; further may comprise PCR reagents.
Compared with the prior art, the invention has the following advantages and beneficial effects: the molecular marker developed by the invention has strong specificity and can accurately identify the sex of the bighead carp. The Gapdh gene is used as an internal reference gene to carry out identification through double PCR, so that gender judgment errors caused by DNA degradation, sample adding errors or PCR amplification failure and the like are avoided. The method has the advantages of rapidness, simplicity, convenience, high accuracy and the like, and can be used for accurately identifying the genetic sex of the bighead carp in a large batch.
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FIG. 1 is a graph showing the results of the accuracy verification of the primer HynS-4 using bighead of known sex. In the figure, 13 lanes from left to right, female bighead strips are shown as 1-6, male bighead strips are shown as 7-12, DNA marker is shown as 13, and a specificity strip of 697bp can be amplified by the male bighead strips.
FIG. 2 is a graph showing the results of validation of amplification in combination with primer HynS-4 in a bighead of known sex using the Gapdh gene as a reference. In the figure, 12 lanes from left to right, 1-6 are female bighead, 7-12 are male bighead, and the 466bp bands of the Gapdh gene can be amplified from both the female bighead and the male bighead, and the 697bp specific band can be amplified from the male bighead.
Detailed Description
The invention utilizes BSA XI endonuclease to excavate a short sequence specific to a male bighead through simplified genome sequencing, carries out sequence extension in the whole genome sequence of the male bighead, designs a bighead sex specific PCR primer, simultaneously designs a PCR primer of a Gapdh gene sequence as an internal reference, successfully and accurately identifies the sex of the bighead through double PCR amplification through primer proportion and amplification condition optimization, and simultaneously avoids sex judgment errors caused by DNA degradation, sample adding errors or PCR amplification failure.
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 development of molecular markers for sex determination of bighead carp
5-female 5-male bighead carp of known sex cultivated in the laboratory is used as a sample, tail fins are cut for extracting genome DNA (gDNA), a sequencing library is constructed by using BSA XI endonuclease according to a 2b-RAD method, and the sequencing library is programmed on a computer after being qualified. After filtering the sequencing data, sex-specific sequence identification was performed using Stacks v1.31 to obtain a specific sequence that was present in all but not in 5-tailed male bighead.
(1) Enzyme digestion:
the extracted DNA was subjected to 2b-RAD library construction using the endonuclease XI BSA in the following steps: (the amount used per individual is as follows)
gDNA 5.0μL
BSA XI(1U/μL)5.0μL
CutSmart Buffer 0.5μL
After mixing, the mixture was incubated at 37.0 ℃ for 1 hour.
(2) Connecting:
first, a connection joint is prepared. For linker 1, 10. mu.M of 5Ill-NNN and an equal volume of 10. mu.M of anti-Ill primer were mixed and stored at-20 ℃ until use. For linker 2, 10. mu.M of 3Ill-NNN and an equal volume of 10. mu.M of anti-Ill primer were mixed and stored at-20 ℃ until use.
Figure BDA0002866442290000031
Figure BDA0002866442290000041
mu.L of the mixture was added to the reaction product of the first step and incubated at 4 ℃ for 8 hours for further use.
(3) Amplification:
the following mixtures were prepared for each individual
Figure BDA0002866442290000042
Putting the mixture into a PCR instrument, and setting program parameters of the PCR instrument as follows: pre-denaturation at 70 ℃ for 30 seconds, (denaturation at 95 ℃ for 30 seconds, annealing at 65 ℃ for 2 minutes, and extension at 72 ℃ for 30 seconds) for 16 cycles; the PCR product was stored at 4 ℃.
The linker and primer sequences used for the above-described sequencing library construction according to the 2b-RAD method are shown in Table 1 below.
TABLE 12 b-RAD sequencing library construction of linker and primer sequences
Figure BDA0002866442290000043
Figure BDA0002866442290000051
(4) Recovering, sequencing and identifying:
the PCR products were electrophoresed on a 2% agarose gel, the voltage was set at 220V, and the electrophoresis time was 20 minutes. The gel after electrophoresis was stained with 1% Ethidium Bromide (EB) solution, each gel was scanned with a JS-a380 (shanghai bacon) gel imager and stored by photographing, and finally the target band (about 170 bp) was cut off, DNA was recovered using e.z.n.a company D2501-02 kit, and the recovered product was directly sequenced using ILLUMINA HiSeq 2500.
Filtering FASTQ format sequences generated by sequencing, performing site reconstruction and sex-specific site identification by using Stacks v1.31 software, and finally obtaining a male bighead carp specific short sequence which exists in 5-tailed male bighead trees but does not exist in 5-tailed female bighead trees. The specific short sequence is shown in SEQ ID NO. 1.
BLAST comparison is carried out on the obtained male bighead specific short sequence in a whole genome to obtain a long sequence with the length of 830 bp. Based on the long sequence, a pair of primers (shown in figure 1) capable of distinguishing genetic sex of bighead carp by 100% is obtained by designing primers for PCR amplification, and the name of the primers is HynS-4. The designed primer sequences are shown in Table 2.
TABLE 2 Bighead gender Mark primer information Table
Figure BDA0002866442290000052
Example 2Gapdh reference sequence primer design and primer concentration optimization
A primer function reference for amplifying the bighead housekeeping gene Gapdh is designed to eliminate the influence of factors such as degradation, sample addition errors and the like, and the designed primer sequences are shown in Table 3.
TABLE 3 primer information table for bighead carp Gapdh gene
Figure BDA0002866442290000053
The Gapdh gene is used as a reference, and the dual PCR amplification is carried out in the same PCR reaction system with the bighead carp sex identification specific primer. Due to the differences in amplification efficiency between the different primers, optimization of the concentration ratio of the sex specific primer to the Gapdh primer is required. In a 25 mu L amplification system, when the concentration ratio of the primer HynS-4 to the primer Gapdh-3 is 3:1, the amplification bands of the primer HynS-4 and the primer Gapdh-3 are clear, and the identification effect is optimal. The method comprises the steps of selecting 12 bighead carp groups of known sex from the laboratory culture, wherein 6 males and 6 females are selected, carrying out PCR verification on genome DNA samples of the bighead carp groups, and the results (figure 1 and figure 2) show that the sex of the bighead carp can be accurately identified.
The PCR amplification system and conditions were as follows:
the following mixtures were prepared for each individual:
Figure BDA0002866442290000061
putting the mixture into a PCR instrument, and setting program parameters of the PCR instrument as follows: pre-denaturation at 94 ℃ for 30 seconds, (denaturation at 94 ℃ for 30 seconds, annealing at 50 ℃ for 35 seconds, and extension at 72 ℃ for 40 seconds) for 36 cycles, and final extension at 72 ℃ for 10 minutes.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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<120> development method of bighead carp sex identification molecular marker by using Gapdh gene as reference
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tcactgtaat ggagatcacg tgtcacacac ctg 33
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atcatgtgca caaacgcgag caacacaaca gccaatggcg agtttcacca atttatgctg 60
aatttaacta atatattttt aaactccgtt acacatacac acacacgtac acacacaatc 120
tcacagatct cacctcattg tgtcctcact gtaatggaga tcacgtgtca cacacctgtc 180
agtctcttct cacagtcttg tctttcaggt ttagctaccg agtgaagatg gggggaggtg 240
caccttcctc cccatgagtg cctccagcaa acggcaactt caaaaagtta aataaaaaac 300
attctcctct tttaaatgtt tataaaataa tttataaagt ataatttata aaacaactca 360
catttttcag agagcagaga agatgtagtt tttactcggt gtggaagtga cttcttgcat 420
ctttgcttat ttcaaagggg taattttgtc acatctagag atcgcactgc aatcgactcg 480
gctgaaacaa tgcccgtatt ttacttcttt gcaatagtcc cacggtcgcg atatgctgag 540
ggtcgtagca ttttctcaca tgttctcaac aatagtattc tttgacactc agcattccat 600
agcatcacca gcggtctttc gagaggttcc cgtgaacgtc tgatttcgtg ccatagctgt 660
gacaccggct attggattca atcccgctgg tgcagcagac agctatccag tagaaacact 720
gccttgcgga tgttacccag tgatcgtgta atatcgattg ttttaaatgt acaataggtg 780
tattcagtag acgtatatga cttcaaacaa gaatcgcatt catgattttc 830
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gtcagagttg actgaactac atcacataga agtgtcttgt gatatttact caaaagtgga 180
atgttctgtt tggggaagtt tgcgtatttg atgtccttta atttttttgc ttcccctttc 240
tatgatccaa acctattctg atgcttcctg ttttagcaag cttactggta tggccttccg 300
tgtccccacc cccaatgtgt ctgtggtgga tctgactgtc cgccttgaga aacctgtgag 360
tttattttac aaaccttcat gataaaaatg gtttcagttg aacaacctca atttgacttt 420
tgttggctct tcatgtctca tctgttgact acaggccaag tatgatgata tcaagaaagt 480
ggtcaaggct gcagctgatg gacccatgaa aggcattctg ggatacacag agcaccaagt 540
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ttgtgtgtgc aggttgtgtc cactgacttc aacggggatt gtcgctcatc tatctttgat 660
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gccttcaagg gctcctagaa aatgtgtacc tcagtgacac gtcgtaatac ttaactcccg 780
tcttgagcaa acccaaatga ctacatatta attcaaagta cagcatgtaa aacttgcagg 840
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Claims (10)

1. A molecular marker for identifying the sex of bighead carp is characterized in that: the molecular marker is part or all of a DNA molecule with a sequence shown as SEQ ID NO. 2.
2. A primer for identifying the sex of bighead carp is characterized in that: the primer is a primer for amplifying the molecular marker in claim 1.
3. The primer according to claim 2, characterized in that: the primer is as follows:
HynS-4F：CCAATGGCGAGTTTCAC，
HynS-4R：CGCAAGGCAGTGTTTCTA。
4. use of the molecular marker of claim 1 or the primer of any one of claims 2-3 for identifying the sex of bighead carp.
5. A method for identifying the sex of bighead carp, comprising the steps of: the method comprises the following steps: extracting genome DNA of bighead carp with sex to be identified, carrying out PCR amplification by using the primer of claim 2 or 3, wherein bighead carp with amplified molecular marker fragment is male bighead carp, and bighead carp without amplified molecular marker fragment is female bighead carp.
6. A method for identifying the sex of bighead carp, comprising the steps of: the method comprises the following steps: extracting genome DNA of bighead carp with sex to be identified, carrying out double PCR amplification by using the primer of claim 2 or 3 and the internal reference gene amplification primer, wherein bighead carp with amplified molecular marker fragment and internal reference gene fragment is male bighead, and bighead carp with amplified internal reference gene fragment is female bighead.
7. The method of claim 6, wherein: the method comprises the following steps: the reference gene is Gapdh gene.
8. The method of claim 7, wherein: the amplification primers of the internal reference gene are as follows:
HynGapdh-3 F：GGGAAGTTTGCGTATTTG，
HynGapdh-3 R：CAAAGATAGATGAGCGACAA。
9. the method of claim 7, wherein: in the double PCR amplification system, the concentration ratio of the primer of claim 3 to the internal reference gene amplification primer is 3: 1.
10. A kit for identifying the sex of bighead carp is characterized in that: comprising the primer of claim 2 or 3.
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