CN113897440B - Molecular marker for rapidly identifying sex of large yellow croaker in daiqu and application - Google Patents

Molecular marker for rapidly identifying sex of large yellow croaker in daiqu and application Download PDF

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CN113897440B
CN113897440B CN202111276425.1A CN202111276425A CN113897440B CN 113897440 B CN113897440 B CN 113897440B CN 202111276425 A CN202111276425 A CN 202111276425A CN 113897440 B CN113897440 B CN 113897440B
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large yellow
yellow croaker
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CN113897440A (en
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谢庆平
牛宝龙
楼宝
蔚敏
何雪
詹炜
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Zhejiang Academy of Agricultural Sciences
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Abstract

The invention discloses a molecular marker for rapidly identifying the sex of large yellow croaker and application thereof, belonging to the technical field of molecular identification. The invention provides a molecular marker for rapidly identifying the sex of the large yellow croaker in the dai on one hand, and provides application and an application method of the molecular marker on the other hand. According to the invention, the DNA of the large yellow croaker sample to be detected is extracted, PCR amplification is carried out by matching with the primer of the sex specific molecular marker, and then agarose gel electrophoresis detection is carried out, so that the sex of the large yellow croaker can be accurately judged, and compared with the prior art, the sex detection accuracy of the large yellow croaker is 100%, and the sex detection can be carried out on the same day, and the repeatability is high. By the method, a technical foundation is laid for sex control breeding and parthenolization breeding of daiqu large yellow croaker.

Description

Molecular marker for rapidly identifying sex of large yellow croaker in daiqu and application
Technical Field
The invention belongs to the technical field of molecular identification, and particularly relates to a molecular marker for rapidly identifying sex of large yellow croaker in the wild and application thereof.
Background
Large yellow croaker is an important economic fish in China, is one of four marine products in China, is distributed in the northwest pacific ocean area, comprises China, japan, korea and Vietnam coast, and is mainly distributed in the south of yellow sea and the east sea in China. In recent years, the large yellow croaker industry has developed rapidly through long-term artificial breeding and artificial cultivation. Based on the segmentation and body type measurement characteristics, there have been studies to divide the geographic population of large yellow croakers into three groups of daiqu, min Yue Dong and Sal Ammoniacus. Lv Siyang, daiqu ocean and cat ocean, etc. spawning groups belong to daiqu families; the Guangdong nationality of Min includes oviposition groups such as Chan Jing Yang, nan Ao and Shan Yuan; the sal ammoniac state group only has sal ammoniac oviposition population. At present, researches on the genetic background of large yellow croakers are gradually enriched, and related researches comprise researches on chromosome karyotypes, isozymes, genetic diversity levels of different groups and the like of cultivated and wild large yellow croakers by domestic and foreign scholars, but the researches are mostly carried out on Minyudong nationality which is one of 3 large yellow croaker geographical groups along the coast of China. And the research result also shows that the chromosome karyotype of the Min Yue Dong large yellow croaker is different from that of the daiqu large yellow croaker.
As shown by the prior study, the large yellow croaker is a fish with a XX/XY sex determination system, and as the large yellow croaker is sexually mature, female and male individuals have obvious sex two-state in terms of growth, female abdomen is raised, body is bulked, male is slimmer, and female weight is obviously higher than male. Therefore, the sex control breeding and the parthenolization breeding of the large yellow croaker are developed, and the large yellow croaker has higher market potential. The development and application of the genetic sex molecular marker are the basis of sex control breeding, and the preparation of XX pseudo-male parent fish can be realized by combining methods of gynogenesis, induction of exogenous androgens and the like on the basis of the genetic sex molecular marker, so that XX total female fish is further obtained. On the other hand, through genetic sex molecular markers and by matching with exogenous estrogen induction, XY pseudo-female fish can be screened, and the XY pseudo-female fish and XY male fish are mated, so that YY super-male parent fish can be screened, and XY total male fish can be further obtained. It can be seen that genetic sex molecular markers are very important technical means in sex control breeding.
At present, research results on molecular markers of genetic sex of large yellow croakers are mainly concentrated in Min Yue Dong group and have been applied. However, the application effect of the molecular marker in large yellow croaker in dai is not 100% discrimination, because different geographical populations have differentiated in shape and sex determination site selection. In recent years, two new aquatic products of large yellow croaker, namely 'Donghai No. 1' and 'hengdai No. 1', are announced, the boosting effect on industry is obvious, and the yield is increased year by year, so that the development of a genetic sex molecular marker of large yellow croaker in the Daighur family is a technical problem to be solved urgently.
Chinese patent publication Nos. CN107236814A and CN108424958B disclose a molecular marker for identifying the genetic sex of large yellow croaker and application thereof, and a SNP marker related to the genetic sex of large yellow croaker and primers and application thereof, wherein the two markers represent the polymorphism of the insertion/deletion length of a nucleotide sequence and the SNP marker related to the sex are closely related to the sex of large yellow croaker through the genetic molecular marker, and can be used for identifying the sex of the female and male of large yellow croaker. However, the sex molecular marker is mainly applied to large yellow croakers in Mindong nationality, so that the method has obvious population specificity and is not suitable for sex identification of large yellow croakers in daiqu nationality, especially a new large yellow croaker variety 'Dai No. 1'.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to design and provide a molecular marker for rapidly identifying the sex of large yellow croaker in the dai and a technical scheme for application. According to the technical scheme, the DNA of the large yellow croaker sample to be detected is extracted, PCR amplification is carried out by matching with the primer of the sex specific molecular marker, and then agarose gel electrophoresis detection is carried out, so that the sex of the large yellow croaker can be accurately judged, the current detection accuracy is 100%, and the result can be obtained in the same day. Lays a technical foundation for sex control breeding and parthenolization breeding of large yellow croaker in daiqu.
The invention is realized by the following technical scheme:
the invention provides a molecular marker for rapidly identifying the sex of large yellow croaker in the dai, wherein the sequence of a forward primer F of the molecular marker is shown as SEQ ID No.1, and the sequence of a reverse primer R is shown as SEQ ID No. 2.
In another aspect, the invention provides the use of molecular markers for rapid identification of the sex of large yellow croaker of the dai family.
The invention also provides a method for rapidly identifying the sex of the large yellow croaker of the dai by utilizing the molecular marker, which comprises the following steps:
1) Extracting DNA of large yellow croaker of daiqu family;
2) And (3) carrying out PCR amplification by using the DNA in the step (1) as a template and using a molecular marker, detecting by agarose gel electrophoresis after the PCR amplification is finished, wherein if the amplified product only contains 500bp bands, the amplified product is a female individual, and if the amplified product contains two 500bp bands and 390bp bands, the amplified product is a male individual.
Further, the PCR reaction system is as follows: 2XPCR Taq mix 10. Mu.L, F and R primers each 0.5. Mu.L, 50 ng/. Mu.L DNA 1. Mu.L, double distilled water 8. Mu.L; the amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94℃for 30 seconds, annealing at 61℃for 30 seconds, elongation at 72℃for 30 seconds, 35 cycles; extension was carried out at 72℃for 10 minutes.
According to the invention, the DNA of the large yellow croaker sample to be detected is extracted, PCR amplification is carried out by matching with the primer of the sex specific molecular marker, and then agarose gel electrophoresis detection is carried out, so that the sex of the large yellow croaker can be accurately judged, and compared with the prior art, the sex detection accuracy of the large yellow croaker is 100%, and the sex detection can be carried out on the same day, and the repeatability is high. By the method, a technical foundation is laid for sex control breeding and parthenolization breeding of daiqu large yellow croaker.
Drawings
FIG. 1 shows the result of molecular marker agarose gel electrophoresis specific to the sex of large yellow croaker in daiqu family;
FIG. 2 shows the sequencing result of the sex-specific molecular marker PCR product of Daqu yellow croaker.
Detailed Description
The invention is further described below in connection with specific examples to provide a better understanding of the present invention.
Examples: application and application method of molecular marker
1. Experimental materials
30 empty barrels containing sand filtered water, label paper, a water absorbing towel, surgical scissors, a surgical tray, forceps, 75% sterilized alcohol cotton balls, 95% alcohol, a 1.5mL centrifuge tube, an anesthetic for MS222 fish, an animal tissue genome DNA extraction kit, a pipette gun, double distilled water, a Nanodrop 2000/One ultra-micro ultraviolet-visible light spectrophotometer, a 2XPCRTaqmix, a specific sex marker primer SEQ ID No.1SEQ ID No.2, a PCR instrument, agarose gel, a gel recovery kit, a pMD19-T cloning vector connection kit, an electrophoresis instrument and an ultraviolet gel imaging system are prepared. Wherein, the animal tissue genome DNA extraction kit, the glue recovery kit product and the pMD19-T cloning vector connection kit follow the guiding steps of the kit.
2. Sample selection
1) Selecting 20 healthy individuals of a new variety of dai-1 of sexually mature large yellow croaker, wherein 1-10 are female fish and 11-20 are male fish, and placing the female fish and the male fish in sand filtered water with MS222 anesthetic solution, wherein the concentration of MS222 is 20mg/L; after anesthesia is effective, fishing out the fish tail fin by using a fishing net to a water absorption towel, gently wiping the fish tail fin, drying the water in the fish tail fin, and shearing the fish tail fin to 0.3-1 cm 2 Sequentially placing the tail fins with the sizes into centrifuge tubes which are provided with 1mL of 95% alcohol and correspond to the numbers 1-20, and placing the centrifuge tubes in a refrigerator at-20 ℃ for standby; and (3) putting the large yellow croaker with the tail fins into a corresponding numbered cultivation barrel with sand filtered water, changing water for feeding normally every day, wherein the anesthesia effect is that the fish body is kept still, the reaction is slow, only the tail part slightly swings, and the large yellow croaker cannot escape when touched by hands.
3. Gene DNA extraction
Taking out a large yellow croaker fin sample stored in a refrigerator at-20 ℃, performing surgical shearing and crushing, and extracting DNA by using an animal tissue genome DNA extraction kit; the DNA concentration and OD of the extracted DNA with the number of 1-20 are measured by a nanodrop one ultra-micro ultraviolet-visible spectrophotometer (Thermo Fisher) 260 /OD 280 Values were obtained and the integrity of the DNA was checked by agarose gel electrophoresis at 1%. 1 to 20 are high in quality and OD 260 /OD 280 Between 1.8 and 2.0, the DNA main band is obvious and has no serious degradation), a 200 mu L centrifuge tube with the number of 1 to 20 is additionally taken, the extracted DNA sample with the corresponding number is added with a proper amount of double distilled water to be diluted to 50 ng/mu L, the concentration of working solution is obtained, and the rest DNA stock solution is put into a refrigerator with the temperature of minus 20 ℃ for standby;
3. PCR amplification and gel electrophoresis detection
Preparing a PCR reaction solution: according to the daiqu yellow croaker male-female specific molecular marker primer developed by the patent:
SEQ ID No.1：F5’-TATTTCTACAGAAGCCCAGCACAG-3’，
SEQ ID No.2：R5’-GTCCTCCTTGTACTCATCAAGG-3’，
preparing a PCR reaction solution for diluted 1-20 DNA samples, wherein the PCR reaction is 20 mu L system: wherein 2XPCR Taq mix 10. Mu.L, F and R primers each 0.5. Mu.L, 50 ng/. Mu.L DNA 1. Mu.L, double distilled water 8. Mu.L); placing the prepared PCR reaction liquid with the number of 1-20 in a PCR instrument, and setting the amplification program to 94 ℃ for pre-denaturation for 5 minutes; denaturation at 94 ℃ for 30 seconds, annealing at 61 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, 35 cycles; extending at 72 ℃ for 10 minutes; cooling to 10 ℃ for standby;
4. analysis of results
Electrophoresis detection is carried out on the obtained PCR product by using 1.2% agarose gel, a gel imaging system photographs, and the photographing result shows that: the fish with the above numbers are judged to be female when the brighter single 500bp band appears in lanes 1-10, and the two brighter bands with 390bp and 500bp appear in the remaining lanes 11-20, respectively, so that the fish with the above numbers are judged to be male, and the fish with the above numbers is consistent with the sampling result. As shown in FIG. 1, females represent females, males represent males, and M represents DNA molecular weight markers. And then marking the male and female genetic sexes of each single cultured large yellow croaker according to the number. The accuracy of identification of sex molecular markers of the large yellow croaker in the invention is 100%.
5. Sequencing analysis
The three bands of 500bp and 500bp of male and 390bp of female in the PCR electrophoresis result obtained in FIG. 1 are respectively cut, the cut gel is recovered by a gel recovery kit to obtain recovered product DNA fragments, the recovered product DNA fragments of 500bp and 500bp of male and 390bp are respectively connected into a pMD19-T vector, subclones are introduced into DH5 alpha escherichia coli, sequencing is carried out after culturing, the sequencing result is shown in FIG. 2, F represents female, M-L represents male 500bp band, and M-S represents male 390bp band.
Sequence listing
<110> academy of agricultural sciences in Zhejiang province
<120> a molecular marker for rapidly identifying sex of large yellow croaker and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Primer (Primer)
<400> 1
tatttctaca gaagcccagc acag 24
<210> 2
<211> 22
<212> DNA
<213> Primer (Primer)
<400> 2
gtcctccttg tactcatcaa gg 22

Claims (2)

1. The application of the primer group in rapid identification of the sex of large yellow croaker is characterized in that the sequence of the forward primer F of the primer group is shown as SEQ ID No.1, the sequence of the reverse primer R is shown as SEQ ID No.2,
SEQ ID No.1：5’-TATTTCTACAGAAGCCCAGCACAG-3’，
SEQ ID No.2：5’-GTCCTCCTTGTACTCATCAAGG-3’；
and taking the DNA of the large yellow croaker in the daiqu family as a template, carrying out PCR amplification by using the primer group, detecting by agarose gel electrophoresis after the PCR amplification is finished, if the amplified product only contains 500bp bands, determining the amplified product as female individuals, and if the amplified product contains 500bp and 390bp bands, determining the amplified product as male individuals.
2. The use according to claim 1, characterized in that the PCR reaction system is: 2XPCR Taq mix 10. Mu.L, F and R primers each 0.5. Mu.L, 50 ng/. Mu.L DNA 1. Mu.L, double distilled water 8. Mu.L; the amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94℃for 30 seconds, annealing at 61℃for 30 seconds, elongation at 72℃for 30 seconds, 35 cycles; extension was carried out at 72℃for 10 minutes.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1966723A (en) * 2006-11-28 2007-05-23 厦门大学 Determination primer and method for yellow croaker in different population
CN108048576A (en) * 2017-12-15 2018-05-18 浙江海洋大学 Differentiate method of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1966723A (en) * 2006-11-28 2007-05-23 厦门大学 Determination primer and method for yellow croaker in different population
CN108048576A (en) * 2017-12-15 2018-05-18 浙江海洋大学 Differentiate method of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea and application thereof

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