CN114058713A - Acquisition method of Y chromosome sequence of Pseudobagrus ussuriensis - Google Patents
Acquisition method of Y chromosome sequence of Pseudobagrus ussuriensis Download PDFInfo
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- CN114058713A CN114058713A CN202111447846.6A CN202111447846A CN114058713A CN 114058713 A CN114058713 A CN 114058713A CN 202111447846 A CN202111447846 A CN 202111447846A CN 114058713 A CN114058713 A CN 114058713A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention relates to the technical field of aquaculture neutral chromosome analysis, and discloses a method for acquiring a pseudobagrus ussuriensis Y chromosome sequence, which comprises the following steps: step one, constructing interspecific cross groups, wherein the cross groups in the step one are obtained by artificial insemination of male pseudobagrus ussuriensis and female pseudobagrus ussuriensis; identifying male hybrid individuals, wherein the male hybrid individuals in the step two are identified by two methods of gonad characteristic observation and molecular marking; thirdly, sequencing and assembling the third-generation genome of the male hybrid individual, wherein the third-generation genome sequencing and assembling in the third step are implemented by adopting HiFi and Hi-C technologies; and step four, comparing the obtained product with female genome chromosome to obtain Y chromosome sequence. According to the method for acquiring the Y chromosome sequence of the Pseudobagrus ussuriensis, the time for acquiring the Y chromosome sequence under the prior art can be greatly shortened, the labor intensity is effectively reduced, the working efficiency is improved, and the sex control breeding process of the Pseudobagrus ussuriensis is powerfully promoted.
Description
Technical Field
The invention relates to the technical field of aquaculture neutral chromosome analysis, in particular to a method for acquiring a pseudobagrus ussuriensis Y chromosome sequence.
Background
Sex bimorph exists in fishes generally, and the growth rate of males is obviously higher than that of females in some fishes, and the growth rate of females is higher than that of males in some fishes. In fish breeding, if the sexual growth difference can be fully utilized to breed all-male or all-female strains, great promotion effect is generated for the improvement of the corresponding fish culture yield. In order to achieve the goal of sex-controlled breeding, it is necessary to know the sex determination mechanism of the target fish, and obtaining sex chromosome sequences is an important breakthrough for revealing the sex determination mechanism.
Currently, for the XX-XY sex-determining type of fish, the main methods for obtaining the Y chromosome sequence are: 1. treating gonad undifferentiated larva of XY genetic male with estrogen to reverse sex to pseudo female individual of physiological female; 2. breeding the XY pseudo female individuals to sexual maturity, and mating the XY pseudo female individuals with the XY normal male individuals to generate offspring; 3. developing a male specific molecular marker, and identifying YY supermale individuals from offspring by using the molecular marker; 4. the YY super-male individual is sequenced by utilizing a third-generation genome sequencing technology, and a Y chromosome sequence is obtained by comparing the YY super-male individual with the existing female genome.
Pseudobagrus ussuriensis (Pseudobagrus ussuriensis)Pseudobagrus ussuriensis) Is the fishes in the family of the Pseudobagrus distributed in the water system from Heilongjiang to Zhujiang in China. The fish is delicious in taste and rich in nutrition, is deeply loved by consumers, has low difficulty in breeding technology, few breeding diseases and continuously expanded breeding range, and is bred in more provinces and more countries at present. Pseudobagrus ussuriensis is XX-XY sex-determining fish, has very obvious sex characteristics, and has great male breeding potential because the size of a male individual can reach more than 3 times that of a female at the second age. However, current knowledge of the pseudobagrus ussuriensis sex determination mechanism is very limited and does not meet the need for all-male breeding.
Disclosure of Invention
Object of the Invention
The invention aims to provide a method for acquiring a pseudobagrus ussuriensis Y chromosome sequence, which can greatly shorten the time required for acquiring the Y chromosome sequence under the prior art and powerfully promote the pseudobagrus ussuriensis gender control breeding process.
Technical scheme
The invention provides a method for acquiring a pseudobagrus ussuriensis Y chromosome sequence, which comprises the following steps: step one, constructing an interspecific hybrid population: selecting male Pseudobagrus ussuriensis and male Pseudobagrus ussuriensis parents, and constructing a cross group through artificial spawning induction and insemination; step two, identifying male hybrid individuals: feeding 20 crossbreed individuals to 3 months old, dissecting and taking out gonads, observing the gonads by using a stereo microscope to perform physiological sex identification, performing genetic sex identification on each individual by using a pseudobagrus ussuriensis male specific molecular marker, and taking the individual which is identified as male by the two methods as a sample to be detected; thirdly, sequencing and assembling three generations of male hybrid individual genomes: extracting genome DNA from a sample to be detected, constructing a HiFi sequencing library, constructing a Hi-C cross-linked library by utilizing muscle tissues, and performing third-generation genome sequencing and chromosome level genome assembly; step four, obtaining a Y chromosome sequence by comparing the Y chromosome sequence with a female genome chromosome: and (3) comparing the chromosome sequence of the assembled male pseudobagrus ussuriensis with the existing pseudobagrus ussuriensis chromosome sequence to obtain a chromosome homologous to the X chromosome, namely a Y chromosome sequence.
Preferably, in the step one, the selection criteria of the pseudobagrus ussuriensis are as follows: the abdomen is enlarged, soft and elastic, the ovary outline can be seen by lying on the abdomen, the abdomen is free from injury, and the weight is more than 80 g; the selection criteria for male pseudobagrus ussuriensis were: the tip of the genitals is long and the end of the genitals is reddish, the constitution is strong, no injury is caused, and the weight is more than 200 g.
Preferably, in the step one, the artificial induced spawning is performed by means of artificial injection of an induced spawning agent.
Preferably, the oxytocin adopts 10-15 microgram/kg luteinizing hormone releasing hormone analogue A2（LRH-A2) 1000-2000 IU/kg of Human Chorionic Gonadotropin (HCG) and 3-6 mg/kg of Diospyrone (DOM).
Preferably, two needles of the oxytocic are coinjected into a pseudobagrus ussuriensis, the first needle injecting only LRH-a2The injection amount is 20% -30% of the total injection amount, and the injection allowance of the second needle is 11-14 hours later; male pseudobagrus ussuriensis was injected with a needle of the oxytocic at 60% of that of pseudobagrus ussuriensis.
Preferably, in the second step, the standard for identifying the physiological sex by observing the gonads is as follows: the gonad is flesh color or semitransparent white, is in two short thin linear structures, can not see granular cells under a stereoscopic microscope, and is male; the gonad is light yellow and has two long belt structures, granular cells can be seen under a stereoscopic microscope, and the gonad is female.
Preferably, the criteria for identifying genetic sex using a pseudobagrus ussuriensis male specific marker are: the male specific site can amplify a band which is male; the female is the one which can not amplify the band at the male specific site.
Preferably, in step three, the DNA used for the HiFi sequencing library construction is high-quality large-fragment DNA extracted by using a large-fragment DNA extraction kit.
Preferably, in step three, the Hi-C cross-linked library is constructed by using a muscle tissue sample which is not less than 1 g of fresh muscle tissue and is preserved by using liquid nitrogen before library construction.
Preferably, in step four, the pseudobagrus ussuriensis chromosome sequence used is obtained using Nanopore three generation sequencing technology.
Advantageous effects
(1) The method adopts male cross individuals of pseudobagrus ussuriensis and pseudobagrus ussuriensis as sequencing samples, Y chromosome is from the pseudobagrus ussuriensis, X chromosome is from the pseudobagrus ussuriensis, and the utilization of the distant cross individuals can effectively avoid the phenomenon that X and Y chromosome sequences are interfered with each other and cannot be correctly assembled due to the direct sequencing of the XY male individuals of the pseudobagrus ussuriensis, thereby ensuring the accuracy and reliability of the assembled Y chromosome sequences;
(2) the physiological sex and the genetic sex of the sample to be detected are identified simultaneously, so that the physiological sex and the genetic sex of the sample to be detected are consistent, and a pseudobagrus ussuriensis Y chromosome sequence can be obtained through sequencing and assembling;
(3) according to the invention, the YY super-male individual is not required to be constructed according to the prior art, so that the complicated operations of artificial sex transfer, artificial propagation again after feeding to sexual maturity, YY individual identification and the like are avoided, the time for obtaining the Y chromosome sequence is greatly shortened, the labor intensity is effectively reduced, and the working efficiency is improved.
In conclusion, the method for acquiring the pseudobagrus ussuriensis Y chromosome sequence can accurately and efficiently acquire the pseudobagrus ussuriensis Y chromosome sequence, greatly shorten the time required for acquiring the Y chromosome sequence under the prior art condition, effectively reduce the labor intensity for acquiring the Y chromosome sequence information, and powerfully promote the pseudobagrus ussuriensis gender control breeding process.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
A method for acquiring a Y chromosome sequence of Pseudobagrus ussuriensis comprises the following steps:
step one, constructing an interspecific hybrid population: selecting abdominal distension in 2021 and 5 monthsSoft and elastic, visible ovarian contour against the abdomen, no-injury pseudobagrus ussuriensis 6 tails, average weight 95 grams; selecting male parent with long and red genital tips and strong physique, and no injury, wherein the male parent has a pseudobagrus ussuriensis tail 3 and average body weight of 268 grams; the parents are selected and temporarily raised in a circular spawning pond with the diameter of 1.7 meters for 3 days. Injecting a first needle of oxytocic at 7:00 days 5 and 20 months 2021, and injecting 5 microgram/kilogram of LRH-A into each tail of Pseudobagrus ussuriensis2Male pseudobagrus ussuriensis did not inject. 5 months, 20 days, 19:00 injections of a second needle oxytocic, pseudobagrus per tail, according to LRH-A210 microgram + HCG 1500 units + DOM 5 mg/kg injection, Pseudobagrus ussuriensis per male tailed by LRH-A2The 6 microgram + HCG 900 units + DOM 3 mg/kg dose was injected, after which the male and female parent fish were returned to the spawning pond. Effect time was reached at 6:00 on day 21.5, and the pseudobagrus ussuriensis eggs were squeezed out into dry plastic pots, the male pseudobagrus ussuriensis spermary was removed, crushed and ground in a mortar, diluted with 0.65% physiological saline, and then mixed with the pseudobagrus ussuriensis eggs for artificial insemination. Fertilized eggs are adhered to boiled and disinfected palm pieces, and are incubated at 21 ℃ and water temperature for 5 months and 24 days at 5:00 for emergence of seedlings, so that about 2000 hybrid individuals are obtained.
Step two, identifying male hybrid individuals: feeding the hybrid individuals to 8 months and 24 days, randomly selecting 20 hybrid individuals, cutting off the abdomen of each individual by using a dissecting scissors, taking out the gonads, and then carrying out physiological sex identification by using a stereoscopic microscope, wherein the gonads of 12 individuals are meat-colored or semitransparent white, are two short thin linear structures, granular cells cannot be seen under the stereoscopic microscope, and are males; the gonads of 8 individuals are light yellow and have two long-banded structures, and granular cells can be seen under a stereoscopic microscope and are female. The DNA of the 20 individuals is extracted, PCR amplification and electrophoretic analysis are carried out by using a developed pseudobagrus ussuriensis male specific molecular marker (patent publication No. CN 106811540B), and the result shows that 12 individuals can amplify clear bands at male specific sites and are males; 8 individuals can not amplify a band at a male specific site and are female. The comparison result shows that the physiological sex and the genetic sex of 20 individuals are consistent.
Thirdly, sequencing and assembling three generations of male hybrid individual genomes: selecting the identified 1 male hybrid individuals, extracting the genomic DNA of the 1 male hybrid individuals by using a large fragment DNA extraction kit (product of Qiagen company, Germany), constructing a HiFi sequencing library according to the operation instruction of a HiFi library construction kit (product of PacBio company in the United states), and performing third-generation genome sequencing by using a PacBio platform. 1.5 g of fresh muscle tissue is stored in liquid nitrogen, 0.1 g of tissue is used for constructing a Hi-C crosslinking library by using a Hi-C library construction kit (product of Beijing Baimaike biology company) according to an operation instruction, and the Illumina Hiseq X Ten platform is used for high-throughput sequencing. Sequencing sequences the chromosome level genomic sequences of pseudobagrus ussuriensis containing 26 chromosomes and pseudobagrus ussuriensis containing 26 chromosomes were assembled separately using software.
Step four, obtaining a Y chromosome sequence by comparing the Y chromosome sequence with a female genome chromosome: the chromosome sequence of the male Pseudobagrus ussuriensis obtained by assembly was aligned with the sequence of the female chromosome obtained by the team using the Nanopore third generation sequencing technology, so that the chromosome 7 of the male Pseudobagrus ussuriensis was found to be homologous to the female X chromosome, i.e., the chromosome 7 of the male was the Y chromosome and the sequence length was 33.3 Mb.
It should be understood by those skilled in the art that the above embodiments are only for illustrating the present invention and are not to be used as a limitation of the present invention, and that changes and modifications to the above embodiments are within the scope of the claims of the present invention as long as they are within the spirit and scope of the present invention.
Claims (10)
1. A method for acquiring a Y chromosome sequence of pseudobagrus ussuriensis, which is characterized by comprising the following steps:
step one, constructing an interspecific hybrid population: selecting male Pseudobagrus ussuriensis and male Pseudobagrus ussuriensis parents, and constructing a cross group through artificial spawning induction and insemination;
step two, identifying male hybrid individuals: feeding 20 crossbreed individuals to 3 months old, dissecting and taking out gonads, observing the gonads by using a stereo microscope to perform physiological sex identification, performing genetic sex identification on each individual by using a pseudobagrus ussuriensis male specific molecular marker, and taking the individual which is identified as male by the two methods as a sample to be detected;
thirdly, sequencing and assembling three generations of male hybrid individual genomes: extracting genome DNA from a sample to be detected, constructing a HiFi sequencing library, constructing a Hi-C cross-linked library by utilizing muscle tissues, and performing third-generation genome sequencing and chromosome level genome assembly;
step four, obtaining a Y chromosome sequence by comparing the Y chromosome sequence with a female genome chromosome: and (3) comparing the chromosome sequence of the assembled male pseudobagrus ussuriensis with the existing pseudobagrus ussuriensis chromosome sequence to obtain a chromosome homologous to the X chromosome, namely a Y chromosome sequence.
2. The method for acquiring a pseudobagrus ussuriensis Y chromosome sequence according to claim 1, wherein in step one, the selection criteria of the pseudobagrus ussuriensis are as follows: the abdomen is enlarged, soft and elastic, the ovary outline can be seen by lying on the abdomen, the abdomen is free from injury, and the weight is more than 80 g;
the selection criteria for male pseudobagrus ussuriensis were: the tip of the genitals is long and the end of the genitals is reddish, the constitution is strong, no injury is caused, and the weight is more than 200 g.
3. The method for acquiring a pseudobagrus ussuriensis Y chromosome sequence according to claim 1, wherein in the step one, the artificial induced spawning is performed by means of artificial injection of an induced spawning agent.
4. The method for acquiring a pseudobagrus ussuriensis Y chromosome sequence according to claim 3, wherein the oxytocic agent is 10-15 microgram/kg of luteinizing hormone releasing hormone analogue a2（LRH-A2) 1000-2000 IU/kg of Human Chorionic Gonadotropin (HCG) and 3-6 mg/kg of Diospyrone (DOM).
5. The method of claim 4, wherein the sequence of the Y chromosome of Pseudobagrus ussuriensis is obtained byTwo needles of the oxytocic are co-injected into the female Pseudobagrus ussuriensis, and the first needle is only injected with LRH-A2The injection amount is 20% -30% of the total injection amount, and the injection allowance of the second needle is 11-14 hours later;
male pseudobagrus ussuriensis was injected with a needle of the oxytocic at 60% of that of pseudobagrus ussuriensis.
6. The method for acquiring a pseudobagrus ussuriensis Y chromosome sequence according to claim 1, wherein in step two, the criteria for identifying physiological sex by gonadal observation are: the gonad is flesh color or semitransparent white, is in two short thin linear structures, can not see granular cells under a stereoscopic microscope, and is male; the gonad is light yellow and has two long belt structures, granular cells can be seen under a stereoscopic microscope, and the gonad is female.
7. The method for acquiring a pseudobagrus ussuriensis Y chromosome sequence according to claim 1, wherein in step two, the criteria for identifying genetic sex using pseudobagrus ussuriensis male specific markers are: the male specific site can amplify a band which is male; the female is the one which can not amplify the band at the male specific site.
8. The method for acquiring a Y chromosome sequence of pseudobagrus ussuriensis according to claim 1, wherein in step three, the DNA used for constructing the HiFi sequencing library is high quality large fragment DNA extracted by using a large fragment DNA extraction kit.
9. The method for obtaining a pseudobagrus ussuriensis Y chromosome sequence according to claim 1, wherein in step three, the muscle tissue sample used for the construction of the Hi-C cross-linked library is not less than 1 g of fresh sample, and is preserved with liquid nitrogen before the construction of the library.
10. A method of acquisition of a pseudobagrus ussuriensis Y chromosome sequence according to any of the claims 1 to 9, wherein in step four the pseudobagrus ussuriensis Y chromosome sequences used were obtained using Nanopore three generation sequencing technology.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114464261A (en) * | 2022-04-12 | 2022-05-10 | 天津诺禾致源生物信息科技有限公司 | Method and apparatus for assembling elongated sex chromosomes |
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Cited By (2)
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| CN114464261A (en) * | 2022-04-12 | 2022-05-10 | 天津诺禾致源生物信息科技有限公司 | Method and apparatus for assembling elongated sex chromosomes |
| CN114464261B (en) * | 2022-04-12 | 2022-07-01 | 天津诺禾致源生物信息科技有限公司 | Method and apparatus for assembling extended sex chromosomes |
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