CN116656799A - Sex-specific DNA molecular marker for verasper variegatus and application thereof - Google Patents
Sex-specific DNA molecular marker for verasper variegatus and application thereof Download PDFInfo
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Abstract
According to the invention, two DNA fragments which are homologous to the Z, W chromosome and have the phase difference of 66bp are screened from the verasper variegatus genome sequence, and a verasper variegatus genetic sex identification method is established by designing primers according to flanking conserved sequences, so that the verasper variegatus genetic sex can be effectively distinguished. The marker has the characteristics that target strips can be amplified in male and female individuals and can be resolved by agarose electrophoresis, so that the time for accurately identifying the genetic sex of the verasper variegatus is greatly shortened, the marker is suitable for batch identification of the genetic sex of the verasper variegatus under the simple experimental condition of a farm, the detection cost is saved, and the working efficiency and the operability for detecting the genetic sex of the verasper variegatus in the farm in situ are improved. The method has important application value for controlling female sex proportion, family establishment, full-female offspring seed preparation and the like of the verasper variegatus culture population, can improve the verasper variegatus culture yield and economic benefit, and has important significance for promoting the high-quality sustainable development of the verasper variegatus culture industry.
Description
Technical Field
The invention belongs to the technical field of fish genetic sex identification and sex control in aquatic genetic breeding, and particularly relates to a verasper variegatus sex-specific DNA molecular marker and application thereof.
Background
Verasper variegatus (Verasper variegatus) (FishBase ID: 6607) belonging to the genus Verasper of the order Verasper, the family of the Verasperaceae. The verasper variegatus has tender meat, is white like jade, is rich in multiple vitamins and microelements, is one of the best food materials for making the sashimi, is deeply favored by consumers, is one of important rare sea water farmed fishes, and has important economic value.
Cultivation practices have shown that verasper variegatus has a pronounced sex-related growth binary, i.e. female fish grows at a significantly faster rate than male fish (Dou et al, 1995). The method has the characteristics of slow growth and small individuals of the verasper variegatus, greatly reduces the culture yield of the verasper variegatus, increases the culture cost, and restricts the sustainable development of the verasper variegatus culture industry. Therefore, developing the sex-specific molecular marker of the verasper variegatus, establishing a method for rapidly identifying the genetic sex of the verasper variegatus, improving the female proportion of the verasper variegatus breeding population through sex control, and even developing the verasper variegatus total female offspring seed, is an effective way for promoting the sustainable development of the verasper variegatus breeding industry.
The verasper variegatus sex-determining system is of the ZZ/ZW type, i.e. male is ZZ and female is ZW (Ma et., 2010). The verasper variegatus can produce Z-shaped sperm and two Z-shaped and W-shaped ova. Under natural conditions, normal ZZ male and ZW female offspring were obtained. Referring to the preparation method of other fish full-female fries (Xu et al, 2022), normal female fish (ZW) is treated by male hormone to obtain pseudo male fish (ZW), then the pseudo male fish (ZW) is hybridized with the normal female fish (ZW), WW (female) super female fish is obtained, and finally the super female fish (WW (female)) is hybridized with the normal male fish (ZZ (female)) to obtain the plaice full-female fries (ZW (female)). Therefore, the sex-specific molecular marker is developed, a rapid and effective sex identification method is established, ZZ male fish, ZW female fish, ZW pseudo male fish and WW super female fish are identified, the sex-specific molecular marker not only has important application value in the cultivation of the spotted halibut total female fingerlings, but also can provide important theoretical references for sex determination and sex chromosome evolution mechanism research of the spotted halibut.
In terms of sex-specific molecular marker screening and genetic sex identification of verasper variegatus, the yellow sea aquatic institute of China aquatic science institute has adopted an AFLP method to analyze and screen the genome of verasper variegatus to obtain female-specific AFLP markers of verasper variegatus. However, AFLP markers have their own disadvantages in practical applications. The AFLP marker cannot distinguish ZW females from WW superfemales due to the dominant genetic characteristics of the AFLP marker, and the AFLP marker only has a DNA band in female fishes, but the DNA band cannot be amplified in male fishes, so that false negatives possibly occur in actual identification, and misjudgment is generated on sex identification of ZZ males and ZW females. In addition, AFLP markers are expensive to apply, have high requirements on the purity of DNA and the quality of endonucleases, and are difficult to realize on-site rapid and accurate detection in farms. Therefore, the development of sex-specific DNA molecular markers for verasper variegatus and the establishment of a molecular method for rapid identification of the genetic sex of verasper variegatus are urgent demands for sex control research and production of high-female or full-female offspring.
Disclosure of Invention
The invention screens out a female specific DNA molecular marker of verasper variegatus, namely a female W chromosome specific DNA fragment, by carrying out whole genome re-sequencing and comparison analysis on the female and male fishes of verasper variegatus, and establishes a technical method capable of carrying out sex identification of the verasper variegatus in a farm by an agarose gel electrophoresis method.
The research firstly completes the whole genome re-sequencing of the 17-tail female fish and the 17-tail male fish of the verasper variegatus through an Illumina sequencing technology, then compares and analyzes the re-sequenced data of the female fish and the male fish through a bioinformatics method, and screens out the homologous difference DNA fragments of the W chromosome and the Z chromosome of the verasper variegatus, wherein the length of the DNA fragment on the W chromosome is 220bp, and the sequence is as follows (SEQ ID NO: 1):
TTCTCCATGCTTCCAATGGCTGATTTTACACTATCTAACTTGTGTTGAGAATTATCAAACTCAGCAAG TTCAGCAGCAAGACTTAGAAGTTGTTAAAAAGCTGAATTTGTCTGAAACGTTTTAAATATCAAGGTTTCTAAATGTAGCATTAGAACCCCATGTTCGAGAAACCAGGTATCATAAGAATTTTACGCAGTTCAGTGAGTCAGAGCTTTGGTCA；
the length of the homologous DNA fragment on the Z chromosome is 154bp, and the sequence is as follows (SEQ ID NO: 2):
TTCTCCATGCTTCCAATGGCTGAGTTTACACTATCTAACTTGTGTTGAGAATTATCAAATATCAAGGTTTCTAAATGTAGCATTAGAACCCCATGTTCGAGAAACCAGGTATCATAAGAATTTTACGTACTTCAGTGAGTCAGAGCTTTGGTCA。
wherein the DNA fragment of the W chromosome (SEQ ID NO: 1) is 66bp more than the fragment on the Z chromosome (SEQ ID NO: 2), the fragment is a specific DNA fragment of the W chromosome, and the nucleotide sequence is as follows:
AAACTCAGCAAGTTCAGCAGCAAGACTTAGAAGTTGTTAAAAAGCTGAATTTGTCTGAAACGTTTT(SEQ ID NO:3)。
according to the DNA sequence, PCR primers for identifying the genetic sex of the verasper variegatus are designed, and 2% agarose gel electrophoresis is adopted to detect PCR products, so that male and female individuals are judged, the genetic sex of the verasper variegatus is accurately identified, and the sequences of the upstream and downstream primers are respectively:
an upstream primer: 5'-TTCTCCATGCTTCCAATGGCT-3' (SEQ ID NO: 4),
A downstream primer: 5'-TGACCAAAGCTCTGACTCACT-3' (SEQ ID NO: 5);
the primers are used for rapid identification of the genetic sex of verasper variegatus, and mainly comprise the following steps:
extraction of verasper variegatus genome DNA, PCR amplification of DNA fragment and agarose gel electrophoresis detection of amplified product. It is characterized in that only DNA fragments with the size of 154bp are amplified in male individuals (ZZ); two DNA fragments of 154bp and 220bp, respectively, were amplified in female (ZW) individuals.
According to the invention, two DNA fragments which are homologous to the Z, W chromosome and have a 66bp difference are screened from the verasper variegatus genome sequence, and a primer is designed according to the flanking conserved sequence, so that the verasper variegatus genetic sex identification method is further established, and the verasper variegatus genetic sex can be rapidly, accurately and effectively distinguished. Because the marker can amplify target strips in male and female individuals and can be resolved by agarose electrophoresis, the time for accurately identifying the genetic sex of the verasper variegatus is greatly shortened, the marker is suitable for batch identification of the genetic sex of the verasper variegatus under the simple experimental condition of a farm, the detection cost is saved, and the working efficiency and the operability for detecting the genetic sex of the verasper variegatus in the farm in situ are improved. The method has important application value for controlling female sex proportion, family establishment, full-female offspring seed preparation and the like of the verasper variegatus culture population, can improve the verasper variegatus culture yield and economic benefit, and has important significance for promoting the high-quality sustainable development of the verasper variegatus culture industry.
Drawings
Fig. 1: the invention discloses a comparison chart of DNA fragment sequences of verasper variegatus Z, W chromosome, and primer positions are indicated by underlines; the Z chromosome deletion sequence is represented.
Fig. 2: female specific DNA marker screening 2% agarose gel electrophoresis result (4 female and 4 male) of the invention, M: DL 2000DNA Maker
Fig. 3: female specific DNA marker screening 2% agarose gel electrophoresis result (10 female and 10 male) of the invention, M: DL 2000DNA Maker
Fig. 4: the invention aims at sequence alignment of a band of interest for male and female amplification, F-L: female long strip, F-S: female short bands, M-S: male band, MF-F: upstream primer, MF-R: downstream primer
Fig. 5: the invention relates to a 2% agarose gel electrophoresis result diagram in sex identification of plaice at 117 tail in a culturing farm, M: DL 2000DNA Maker.
Detailed Description
The female specific DNA molecular markers and the rapid identification method of genetic sex of verasper variegatus according to the present invention will be described in detail with reference to the accompanying drawings.
Example 1: screening and verification of sex-specific DNA markers of verasper variegatus
The sex-specific DNA marker sequence used in the invention is derived from the complete genome sequence of verasper variegatus completed by the research team of the national institute of aquatic products, yellow sea, institute of aquatic products, and the institute of genome, chen Songlin. Firstly, comparing the male and female body weight sequencing data of the verasper variegatus with the whole genome sequence respectively by a bioinformatics method, thereby screening 359Z, W chromosome homologous differential DNA fragments.
Selecting 50-70bp differential DNA fragment, designing primers for its flanking 150bp sequence (figure 1), selecting 4 female and male verasper moseri genome DNA samples with known genetic sex, and PCR amplifying with primers (upstream primer: 5'-TTCTCCATGCTTCCAATGGCT-3', downstream primer: 5' -TGACCAAAGCTCTGACTCACT-3), PCR reaction system of 15 μl including 5.5 μl ddH 2 O；7.5μL Premix Taq TM (TaKaRa Taq TM Version 2.0plus dye); 0.5. Mu.L of SEQ ID NO:3 (10. Mu. Mol/L); 0.5. Mu.L of SEQ ID NO. 4 (10. Mu. Mol/L); 1.0. Mu.L of template DNA was well mixed by vortex centrifugation and PCR amplification was performed. The PCR amplification procedure was: denaturation (95 ℃ for 30 s), renaturation (57 ℃ for 30 s) and extension (72 ℃ for 60 s) are carried out for 35 cycles at 95 ℃ for 3min, extension at 72 ℃ for 7min and preservation at 4 ℃. The PCR products are subjected to 2% agarose gel electrophoresis for 30min, so that the difference of the bands of the male and female individuals can be distinguished.
Finally, a pair of primers with stable results was selected (FIG. 2). Then, amplification verification was performed using DNA samples of 20 individuals (10 each of female and male) according to the same amplification reaction conditions as described above, and the results are shown in FIG. 3. Then, the amplified bands of interest are used separatelyThe DNA gel recovery kit (Optimus ) is used for recovery, and after pClone007 vector is connected, the transformation into a competent cell is carried out, positive clones were picked and sent to the qing department for sequencing. Sequencing of the bands of interest confirmed the sequence of homologous differential DNA fragments on chromosome Z, W (fig. 4).
Wherein the length of the DNA fragment on the W chromosome is 220bp, and the sequence is as follows (SEQ ID NO: 1):
TTCTCCATGCTTCCAATGGCTGATTTTACACTATCTAACTTGTGTTGAGAATTATCAAACTCAGCAAG TTCAGCAGCAAGACTTAGAAGTTGTTAAAAAGCTGAATTTGTCTGAAACGTTTTAAATATCAAGGTTTCTAAATGTAGCATTAGAACCCCATGTTCGAGAAACCAGGTATCATAAGAATTTTACGCAGTTCAGTGAGTCAGAGCTTTGGTCA；
the length of the homologous DNA fragment on the Z chromosome is 154bp, and the sequence is as follows (SEQ ID NO: 2):
TTCTCCATGCTTCCAATGGCTGAGTTTACACTATCTAACTTGTGTTGAGAATTATCAAATATCAAGGTTTCTAAATGTAGCATTAGAACCCCATGTTCGAGAAACCAGGTATCATAAGAATTTTACGTACTTCAGTGAGTCAGAGCTTTGGTCA。
wherein the DNA fragment of the W chromosome (SEQ ID NO: 1) is 66bp more than the fragment on the Z chromosome (SEQ ID NO: 2), the fragment is a specific DNA fragment of the W chromosome, and the nucleotide sequence is as follows:
AAACTCAGCAAGTTCAGCAGCAAGACTTAGAAGTTGTTAAAAAGCTGAATTTGTCTGAAACGTTTT(SEQ ID NO:3)。
example 2: establishment and application of verasper variegatus genetic sex identification technology
1. Genetic sex identification of verasper variegatus under laboratory conditions
1.1 DNA extraction
The method comprises the steps of extracting the verasper variegatus fin DNA by using a marine organism genome DNA extraction kit (radix angelicae) and then identifying the integrity by 1% agarose gel electrophoresis, detecting the concentration by using an ultra-micro spectrophotometer P100 (Pulton, U.S.), adjusting the DNA concentration to 50 ng/. Mu.L, and freezing at-20 ℃ for later use.
1.2 PCR amplification and genetic sex determination
The genetic sex of verasper variegatus was detected by PCR amplification using female-specific DNA-labeled primers (upstream primer: 5'-TTCTCCATGCTTCCAATGGCT-3', downstream primer: 5' -TGACCAAAGCTCTGACTCACT-3) of verasper variegatus. 15. Mu.L of a PCR reaction system in which ddH was present 2 O 5.5μL；Premix Taq TM (TaKaRa Taq TM Version 2.0plus dye) 7.5 μl; 0.5. Mu.L of each of the upstream and downstream primers; template DNA 1.0. Mu.L. The PCR amplification procedure was: 3min at 95 ℃;95 ℃ for 30s, 57 ℃ for 30s, 72 ℃ for 60s,35 cycles; storing at 72deg.C for 7min and 4deg.C. 2% agarose gel electrophoresis, 120V,30 minutes, and recording the identification results under a gel imager. The two DNA bands of 220bp and 154bp can be clearly distinguished from each other, and only one DNA band of 154bp can be amplified by ZZ males, as shown in figure 3.
2. Identification of genetic sex of verasper variegatus under farm conditions
The developed DNA molecular markers and established genetic sex identification techniques are ultimately applied to actual breeding works such as sex control of verasper variegatus, which requires that the genetic sex identification works must be carried out and completed on site in the verasper variegatus farm.
2.1 preparation for field authentication
The method is characterized in that a simple laboratory is arranged in a farm, and a PCR instrument, a centrifuge, an electrophoresis instrument, a water bath, a microwave oven, a gel imager and various reagents, tools and consumables required by experiments are prepared.
2.2 collecting the fin of the verasper variegatus to be identified
The centrifuge tube and the label with the numbers are prepared in advance, and the numbers of the centrifuge tube and the label are in one-to-one correspondence. In a breeding workshop, a small number of fin strips of the verasper variegatus to be identified are collected on site one by one and placed in a numbering centrifuge tube. Placing the verasper variegatus with the fin into a bag, adding seawater, oxygenating and sealing, wherein the ratio of seawater to oxygen is 1:2, attaching a label corresponding to the number of the centrifuge tube, and placing the bag in a dark place for temporary storage.
2.3 extraction of genomic DNA from verasper variegatus
500. Mu.L of lysate and 15. Mu.L of proteinase K were added to each fin-loaded centrifuge tube and lysed in a 55℃water bath for 1 hour, during which time the tubes were gently shaken to accelerate the lysis. The lysed centrifuge tube was removed and 500 μl of phenol was added: chloroform: isoamyl alcohol is reversed and is gently shaken for 10min, and is centrifuged for 10min at 12,000 revolutions, 450 mu L of supernatant is taken and added into a centrifuge tube which is prepared in advance and is filled with 500 mu L of alcohol with corresponding number, and is gently shaken for 30 times, and is centrifuged for 5min at 12,000 revolutions, so that white DNA is precipitated at the bottom of the centrifuge tube. The centrifuge tube was decanted, the DNA pellet was dried and 100. Mu.LddH was added 2 O, vortex twice to dissolve the DNA sufficiently.
2.4, PCR amplification and sex identification of verasper variegatus
PCR amplification using the above female-specific DNA marker primer, comprising ddH 2 O 5.5μL；Premix Taq TM (TaKaRa Taq TM Version 2.0plus dye) 7.5 μl; 0.5. Mu.L of each of the upstream and downstream primers; template DNA 1.0. Mu.L. The PCR amplification procedure was: 3min at 95 ℃;95 ℃ for 30s, 57 ℃ for 30s, 72 ℃ for 60s,35 cycles; storing at 72deg.C for 7min and 4deg.C.
The PCR products were electrophoresed on a 2% agarose gel at 120V for 30 minutes, and the results were observed under a gel imager. One DNA band appeared in the male fish and two DNA bands appeared in the female fish (fig. 5).
According to the identification result, the halibut temporarily packed and stored can be classified into male and female, and placed into a corresponding culture pond. The whole process takes about 5 hours, so that the survival and subsequent normal cultivation of the verasper variegatus can be ensured.
Claims (10)
1. The sex-related DNA fragments on the plaice W and Z chromosomes are characterized in that the sequence of the DNA fragment on the plaice W chromosome is SEQ ID NO. 1, and the sequence of the DNA fragment on the plaice W chromosome is SEQ ID NO. 2.
2. The sex difference DNA fragment of the verasper variegatus W and Z chromosomes is characterized in that the sex difference DNA fragment is positioned on the W chromosome and has a nucleotide sequence of SEQ ID NO. 3.
3. A method of identifying sex of verasper variegatus by detecting the presence of the plaice W and Z chromosome sex differential DNA fragments of claim 2 in the amplified product.
4. The method of claim 3, wherein the method uses a primer pair having nucleotide sequences of SEQ ID NO. 4 and SEQ ID NO. 5 to PCR amplify the individual to be detected.
5. The method of claim 3, wherein the primer pair only amplifies a DNA fragment of 154bp in a male verasper variegatus individual; two DNA fragments of 154bp and 220bp, respectively, were amplified in female individuals.
6. A primer pair for identifying sex of verasper variegatus, wherein the primer pair is used to amplify a nucleic acid fragment comprising the sex-differentiated DNA fragments of chromosome W and Z of verasper variegatus of claim 2.
7. The primer pair of claim 6, wherein the primer pair has the nucleotide sequences of SEQ ID NO. 4 and SEQ ID NO. 5.
8. A PCR amplification test kit for identifying sex of verasper variegatus, wherein the reagent comprises the primer pair of claim 6.
9. The detection kit as claimed in claim 8, wherein the nucleotide sequences of the primer pair are SEQ ID NO. 4 and SEQ ID NO. 5.
10. The test kit of claim 8, wherein the kit comprises reagents for agarose gel electrophoresis detection.
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| CN118374586A (en) * | 2024-06-21 | 2024-07-23 | 中国水产科学研究院黄海水产研究所 | Sex identification method for verasper variegatus |
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| CN101429557A (en) * | 2008-11-26 | 2009-05-13 | 中国水产科学研究院黄海水产研究所 | Special molecular marker for sex of verasper variegates, and genetic sex identification method |
| CN113718043A (en) * | 2021-09-03 | 2021-11-30 | 广东海洋大学 | Specific molecular marker and primer for identifying genetic sex of plectropomus multiformis, and method and application thereof |
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| CN101429557A (en) * | 2008-11-26 | 2009-05-13 | 中国水产科学研究院黄海水产研究所 | Special molecular marker for sex of verasper variegates, and genetic sex identification method |
| CN113718043A (en) * | 2021-09-03 | 2021-11-30 | 广东海洋大学 | Specific molecular marker and primer for identifying genetic sex of plectropomus multiformis, and method and application thereof |
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| CN118374586A (en) * | 2024-06-21 | 2024-07-23 | 中国水产科学研究院黄海水产研究所 | Sex identification method for verasper variegatus |
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