CN116064759A - Molecular marker, primer group, kit, method and application for identifying sex of pelteobagrus vachelli - Google Patents
Molecular marker, primer group, kit, method and application for identifying sex of pelteobagrus vachelli Download PDFInfo
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Abstract
The application relates to the technical field of sex detection of pelteobagrus vachelli, and discloses a molecular marker, a primer group, a kit and a method for identifying the sex of pelteobagrus vachelli. The molecular marker is a differential sequence of a nucleotide sequence shown in SEQ ID NO.1 relative to a nucleotide sequence shown in SEQ ID NO.1 derived from an X chromosome genome of pelteobagrus vachelli and a nucleotide sequence shown in SEQ ID NO.2 derived from a Y chromosome genome of pelteobagrus vachelli, wherein the differential sequence is shown in SEQ ID NO.3, and the differential sequence is a specific sequence of the Y chromosome genome of pelteobagrus vachelli. The identification primer, the kit and the method provided by the embodiment of the application are not influenced by the development period and the environment of individuals, the sex identification is simple, convenient and quick, the requirement on samples is low, the accuracy of identification results is high, and therefore the defects that other methods are complex in operation, long in time consumption and the like are overcome, and the method is particularly suitable for quick identification of large samples.
Description
Technical Field
The application relates to the technical field of sex detection of pelteobagrus vachelli, in particular to a molecular marker, a primer group, a kit, a method and application for identifying the sex of pelteobagrus vachelli.
Background
Pelteobagrus vachelli (Pelteobagrus vachelli) and Pelteobagrus fulvidraco (P.fulvidraco) belong to the genus Pelteobagrus of the order of Cladoniotidis, are benthic small-sized fishes, and are widely distributed in natural waters such as rivers, lakes, reservoirs and the like in China. The pelteobagrus fulvidraco has delicious fish meat, less intramuscular thorns and high nutritional value, is deeply favored by consumers and markets, and becomes one of the most rapid special fish for pond culture development in the last ten years. The species of pelteobagrus have obvious difference of male and female growth, under the same culture condition, the growth speed of male pelteobagrus fulvidraco of one age is about 30 percent faster than that of female cuttlefish of the same age, the male cuttlefish of two ages can grow to 150-200 g, the female cuttlefish of the same age is only 50-75 g, and the difference of male and female cuttlefish of the same age is close to 3 times; also, pelteobagrus vachelli has similar phenomena. According to the growth difference of pelteobagrus fulvidraco, chinese scholars develop a sex molecular marker-assisted all-male fish cultivation technical route, and cultivate a new variety of 'all-male No. 1', thereby remarkably improving the yield and economic benefit of pelteobagrus fulvidraco. However, the parent super-male breeding line is degraded after multi-generation selfing, the selfing line shows a weakening trend in the aspects of growth, disease resistance and the like, and at the moment, the variety improvement of the all-male pelteobagrus fulvidraco is required or other novel pelteobagrus fulvidraco varieties are developed. Crossbreeding is one of the most classical methods in genetic breeding, and pelteobagrus vachelli has low market acceptance due to appearance and other reasons, but has the advantages of high growth speed, large individual, excellent stress resistance and close relationship with pelteobagrus vachelli, and can be selected and bred as a hybrid male parent. The research shows that the morphological characteristics of the hybrid pelteobagrus fulvidraco (pelteobagrus fulvidraco P.vachelli) are similar to those of pelteobagrus fulvidraco, and the growth speed is superior to that of pelteobagrus fulvidraco. The method for crossbreeding is adopted by the university of Chinese agricultural pelteobagrus fulvidraco germplasm resource and variety improvement team, the pelteobagrus fulvidraco caught in Liang Zihu water domain and bred through continuous 3 generations is selected as a female parent, the pelteobagrus vachelli caught in Yangtze river section and bred through continuous 2 generations is selected as a male parent, and F1 generation is obtained through artificial hybridization, namely the hybrid pelteobagrus fulvidraco 'yellow you No. 1'.
The breeding practice proves that the hybrid pelteobagrus fulvidraco No.1 has the advantages of quick growth, fierce feeding and transportation resistance, but the male and female individuals still have larger growth difference, and the growth speed of the second-age male fish is about 50% faster than that of the sibling female fish, which indicates that the hybrid pelteobagrus fulvidraco still has the necessary and potential of full male breeding. However, the molecular biological method such as molecular marker is adopted to assist the all-male breeding, so that the breeding efficiency and success rate can be greatly improved. However, pelteobagrus vachelli still has no clear sex determination mechanism report, whether a heterogenic sex chromosome exists or not has not been determined, and the genetic sex of the pelteobagrus vachelli cannot be identified from the cytology perspective.
Disclosure of Invention
The application provides a molecular marker, a primer pair, a kit, a method and application of the male-female difference of pelteobagrus vachelli, and solves one of the technical problems to a certain extent.
Therefore, the embodiment of the application at least discloses the following technical scheme:
in a first aspect, an embodiment of the application discloses a molecular marker of a male-female difference of pelteobagrus vachelli, wherein the molecular marker is a difference sequence of a nucleotide sequence shown in SEQ ID NO.1 relative to a nucleotide sequence shown in SEQ ID NO.2 derived from an X chromosome genome of pelteobagrus vachelli, the difference sequence is shown in SEQ ID NO.3, and the difference sequence is a specific sequence of a Y chromosome genome of pelteobagrus vachelli.
In a second aspect, the examples disclose primer pairs that base pair with a DNA molecule as set forth in SEQ ID NO.1 and/or a DNA molecule as set forth in SEQ ID NO.2, the primer pairs comprising DNA molecules having the nucleotide sequences as set forth in SEQ ID NO. 4-5.
In a third aspect, the embodiment of the application discloses a kit for identifying the sex of pelteobagrus vachelli, which comprises the primer pair in the second aspect.
In a fourth aspect, an embodiment of the present application discloses a method for identifying the sex of pelteobagrus vachelli, which includes the following steps:
taking a DNA sample of pelteobagrus vachelli to be detected;
performing a PCR reaction using the primer set according to claim 2 using the DNA as a template;
detecting the obtained PCR reaction product to carry out gel electrophoresis, and confirming the sex genotype of the pelteobagrus vachelli according to the band of the gel electrophoresis.
In a fifth aspect, an embodiment of the present application discloses a breeding method of a full male hybrid pelteobagrus fulvidraco, including:
carrying out estrogen induction on juvenile pelteobagrus vachelli, and screening out individuals which are successfully reversed, namely pseudofemale pelteobagrus vachelli; the physiological phenotype of the pseudofemale pelteobagrus vachelli is female, and the sex genotype is XY;
mating the pseudofemale pelteobagrus vachelli with male pelteobagrus vachelli, and screening the mated offspring to obtain the supermale pelteobagrus vachelli; the sex genotype of the pelteobagrus vachelli male fish is XY, and the sex genotype of the pelteobagrus vachelli male fish is YY; and
mating the super-male fish of the pelteobagrus vachelli with the female fish of the pelteobagrus fulvidraco to obtain offspring, namely the fully-male hybrid pelteobagrus fulvidraco, wherein the sex genotype of the female fish of the pelteobagrus fulvidraco is XX;
wherein the step of "screening" comprises the method of the fourth aspect.
In a sixth aspect, an embodiment of the present application discloses a breeding method of a full male hybrid pelteobagrus fulvidraco, including:
carrying out estrogen induction reversion on the super-male fish of the pelteobagrus vachelli, and screening to obtain pseudo-female fish of the pelteobagrus vachelli; the sex genotypes of the super-male fish of the pelteobagrus vachelli and the pseudo-female fish of the pelteobagrus vachelli are YY, and the physiological phenotype of the pseudo-female fish of the pelteobagrus vachelli is female;
mating the pseudofemale pelteobagrus vachelli with the super-male pelteobagrus vachelli to obtain the super-male offspring of pelteobagrus vachelli, wherein the sex genotype of the super-male pelteobagrus vachelli is YY; and
hybridizing the offspring of the super-male pelteobagrus vachelli with a full-female pelteobagrus fulvidraco mating line to obtain the full-male hybridized pelteobagrus fulvidraco; the full female mating line of the pelteobagrus fulvidraco is a breeding fish with a mating offspring of XX pseudo-male fish and XX female fish obtained by carrying out trazoic inversion by a height of Wen Lian as full female, and the genotype of the full female mating line of the pelteobagrus fulvidraco is XX;
wherein the step of "screening" comprises the method of the fourth aspect.
In a seventh aspect, an embodiment of the present application discloses a method for developing a molecular marker according to the first aspect, which is characterized in that the method includes:
extracting DNA from 20 male and female pelteobagrus vachelli, respectively carrying out whole genome library construction and double-end sequencing by using a DNB-T7 sequencing platform, wherein the sequencing quantity is not lower than 15G, and the average sequencing depth is not lower than 20X;
comparing the sequencing data after quality control to a pelteobagrus vachelli reference genome by using a mem algorithm of BWA;
analyzing the comparison result by using a GATK variation detection flow to obtain a difference sequence comprising single nucleotide polymorphism sites and indels of small fragments;
firstly, filtering out the differential sequences with the sequencing depth lower than 5, and then screening the differential sequences according to the standards of homozygosity of all female samples and heterozygosity of male samples, wherein the obtained differential sequences are the differential sequences with male specificity.
In an eighth aspect, an embodiment of the application discloses application of the molecular marker in the first aspect, the primer pair in the second aspect or the kit in the third aspect in the total male breeding of pelteobagrus vachelli.
Compared with the prior art, the application has at least one of the following beneficial effects:
according to the molecular marker, the primer, the kit and the method provided by the embodiment of the application, genomic DNA can be used as a template, sex identification can be carried out on pelteobagrus vachelli through simple PCR and electrophoresis results, the sex genotype of pelteobagrus vachelli can be determined according to the electrophoresis results, and the time for accurately identifying the genetic sex of pelteobagrus vachelli is shortened. The detection method is suitable for identifying the genetic sex of pelteobagrus vachelli in a simple environment of a farm, and detection time and cost are saved. The method for detecting the genetic sex of the pelteobagrus vachelli has important significance and application value for breeding parent pelteobagrus vachelli, establishing families and controlling the sex ratio of the female and male of the breeding group and promoting the sustainable healthy development of pelteobagrus vachelli breeding industry.
The identification primer, the kit and the method provided by the embodiment of the application are not influenced by the development period and the environment of individuals, the sex identification is simple, convenient and quick, the requirement on samples is low, the accuracy of identification results is high, and therefore the defects that other methods are complex in operation, long in time consumption and the like are overcome, and the method is particularly suitable for quick identification of large samples. The identification primer, the kit and the method provided by the embodiment of the application are low in cost, simple in operation, rapid and accurate, and suitable for popularization and application.
Drawings
Fig. 1 is a schematic diagram of a development flow of a molecular marker according to an embodiment of the present application.
FIG. 2 is a schematic diagram showing pairing of a primer pair and a target fragment according to an embodiment of the present application.
Fig. 3 is a gel electrophoresis chart of the genetic sex of pelteobagrus vachelli provided in the embodiment of the present application.
Fig. 4 is a schematic diagram of a total male breeding process of pelteobagrus vachelli provided in an embodiment of the present application.
Fig. 5 is a schematic diagram of a total male breeding process of pelteobagrus vachelli provided in an embodiment of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In order to establish a method for rapidly identifying pelteobagrus vachelli, the time for breeding parent pelteobagrus vachelli, establishing a family and controlling the sex ratio of the female and male of a breeding population is shortened, the cost is reduced, the inventor of the application screens DNA fragments (SEQ ID NO. 3) of the female and male differences of pelteobagrus vachelli through bioinformatics analysis, and establishes a pelteobagrus vachelli genetic sex identification method, and the genetic sex of pelteobagrus vachelli can be rapidly and accurately identified by adopting the method.
As shown in fig. 1, in some screening examples of DNA fragments with male and female differences of pelteobagrus vachelli, 20 pieces of DNA from male and female pelteobagrus vachelli were extracted, and whole genome library construction and double-ended sequencing were performed respectively using a DNB-T7 sequencing platform, with a sequencing amount of not less than 15G and a sequencing average depth of not less than 20X. And comparing the sequencing data after quality control with a pelteobagrus vachelli reference genome by using a mem algorithm of BWA. And analyzing the comparison result by using a GATK variation detection flow to obtain a difference sequence comprising single nucleotide polymorphism sites and insertion deletion of small fragments. Firstly, filtering out the differential sequences with the sequencing depth lower than 5, and then screening the differential sequences according to the standards of homozygosity of all female samples and heterozygosity of male samples, wherein the obtained differential sequences are the differential sequences with male specificity. The nucleotide sequence of the difference sequence is shown as SEQ ID NO. 3.
To verify the possibility of using the differential sequence (shown as SEQ ID NO: 3) of male and female pelteobagrus vachelli as a molecular marker for sex identification of pelteobagrus vachelli, as shown in FIG. 2, the present example also discloses a primer pair comprising an upstream primer and a downstream primer for amplifying the sequences shown as SEQ ID NO.1 and/or 2. The primer pair comprises DNA molecules with nucleotide sequences shown as SEQ ID NO. 4-5.
Furthermore, the embodiment of the application also discloses a kit for sex identification, which comprises the primer set. Preferably, the kit further comprises a DNA polymerase, mg 2+ One or more of dNTPs, PCR Buffer, and sterile water. Which is a kind ofThe DNA polymerase is selected from the group consisting of DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV and DNA polymerase V, such as Taq polymerase or rTap enzyme.
Based on the sex identification kit, the sex of pelteobagrus vachelli can be identified, and the detection principle is that the primer group and/or the kit are used for carrying out PCR amplification on a genomic DNA sample of pelteobagrus vachelli, and the amplification products are identified. Specifically, the embodiment of the application also discloses a method for identifying the sex of pelteobagrus vachelli, which comprises the following steps:
(1) Taking a DNA sample of pelteobagrus vachelli to be detected;
(2) Taking the DNA sample as a template, and adopting a primer pair as shown in SEQ ID NO. 4-5 to carry out PCR reaction;
(3) Detecting the obtained PCR reaction product to carry out gel electrophoresis, and confirming the sex genotype of the pelteobagrus vachelli according to the band of the gel electrophoresis.
In some embodiments, the DNA sample of pelteobagrus vachelli to be detected in step (1) can be any DNA sample extracted from the tissue or blood of pelteobagrus vachelli, such as from its fin. In this step, a DNA sample thereof may be extracted by a conventional CTAB (cetyltrimethylammonium bromide) method or a magnetic bead method.
In one embodiment of step (1), the method comprises the following specific steps:
1) Sample collection: taking 17 tails of 5-month-old pelteobagrus vachelli sex reversal individuals, respectively dripping Bouin's fixative to fix gonads after dissection, and observing gonad morphology to determine physiological gender.
2) Tissue DNA extraction: animal tissue DNA extraction kit (MagMAX Ultra 2.0 multiple sample DNA extraction kit, thermo Fisher Scientific) was used.
3) Taking 10-50 mg of fin tissue, shearing, adding 200 μl of tissue digestion liquid GHA and 20 μl of proteinase K (20 mg/ml), and digesting in water bath at 65deg.C for 30min until tissue fragments are completely digested. If the digestion is completed and fragments remain, the impurities are removed by centrifugation at 12000rpm for 1 min, and the supernatant is left.
4) After completion of the digestion, the mixture was cooled to room temperature, 20. Mu.l of RNase enzyme was added thereto, and the mixture was subjected to water bath at 55℃for 10 minutes, followed by centrifugation again to suck the supernatant.
5) Adding 300 μl of the lysate GHB, shaking uniformly, and mixing in a water bath at 75deg.C for 15 min for 3 times (3-5 times).
6) After 5 minutes at room temperature, 350. Mu.l of isopropanol was added and mixed by shaking for 10 seconds.
7) Add 30. Mu.l of magnetic bead suspension G, mix for 1 min with shaking every 3 min, and rest for 9 min.
8) The centrifuge tube was placed on a magnetic rack for 30 seconds and the liquid was aspirated.
9) Mu.l of buffer GDA (ethanol) was added and mixed by shaking for 30 seconds.
10 Place the centrifuge tube on a magnetic rack for 30 seconds to suck the liquid.
11 Repeating steps 9) to 10).
12 700. Mu.l of the rinse PWD (with ethanol) was added and mixed by shaking for 30 seconds.
13 Place the centrifuge tube on a magnetic rack for 30 seconds to suck the liquid.
14 Repeating steps 12) to 13).
15 Placing the centrifuge tube on a magnetic rack, and airing for 10-15 minutes at room temperature.
16 Taking the centrifuge tube off the magnetic rack, adding 100-200 μl of elution buffer TB, shaking uniformly, placing in 56 deg.C water bath for 10 min, and mixing for 3 times (3-5 times).
17 Placing the centrifuge tube on a magnetic rack for 2 minutes, carefully transferring the DNA solution into a new centrifuge tube after the magnetic beads are completely adsorbed, and preserving at 4 ℃.
18 Measuring the DNA concentration, running 1% agarose gel to check the integrity of the extracted genomic DNA. The DNA concentration was adjusted to 50 ng/. Mu.l.
In some embodiments, the reaction system of the PCR reaction described in step (2) comprises, in 10. Mu.l, 5. Mu.l of 2 XTaqMastermix, 0.5. Mu.l of forward and reverse primer (10. Mu.M), 1. Mu.l of DNA template, 3. Mu.l of ddH 2 O。
In some embodiments, in the reaction procedure of the PCR reaction described in step (2), the mixture is centrifuged slightly and immediately placed on a PCR instrument to perform amplification. The reaction procedure generally includes: pre-denaturing at 95 ℃ for 3-5min, and entering a cyclic amplification stage: the temperature is between 95 ℃ and 40s and 54 ℃ and between 30s and 72 ℃ and 30s, the cycle is 30 to 35 times, and the temperature is kept at 72 ℃ for 7 minutes.
In some embodiments, in the step (3), 3% agarose Gel is prepared according to the size of the product, the PCR product is mixed with Gel-red dye solution and spotted, electrophoresis is performed for about 30min at 200V and 200A, and the Gel is taken out and placed on an ultraviolet photo-adhesive instrument to observe the banding condition.
In some embodiments, in the step (3), through PCR amplification and gel electrophoresis, if only one 259bp band is displayed in the gel electrophoresis, the sex genotype of the pelteobagrus vachelli is XX;
if only one 310bp band is displayed in the gel electrophoresis, the sex genotype of the pelteobagrus vachelli is YY;
if a 259bp band and a 310bp band are displayed in the gel electrophoresis, the sex genotype of the pelteobagrus vachelli is XY.
In one embodiment, the results of PCR amplification and agarose gel electrophoresis are shown in FIG. 3, wherein the first position from left to right is marker (DL 2000 plus), and the 2 nd to 8 are female fish of pelteobagrus vachelli (XX), a strip is amplified, and the strip size is 259bp; 9-15 are pseudofemale fish (XY) of pelteobagrus vachelli, two strips are amplified, and the sizes of the strips are respectively 255 bp and 310bp; the 16 th to 18 th are the male fish (XY) of pelteobagrus vachelli, two strips are amplified, and the sizes of the strips are 319 bp and 310bp respectively. Therefore, the molecular marker identification result provided by the embodiment of the application is completely matched with the anatomical gonad morphology observation result, so that the identification success rate is 100%, the genetic sex of pelteobagrus vachelli can be accurately identified, and the primer group, the kit and the sex identification method provided by the embodiment of the application can be used for accurately identifying the sex of pelteobagrus vachelli.
Based on this, as shown in fig. 4, the embodiment of the present application further provides a breeding method of the all male hybrid pelteobagrus fulvidraco, including:
carrying out estrogen induction on juvenile pelteobagrus vachelli, and screening out individuals which are successfully reversed, namely pseudofemale pelteobagrus vachelli; the physiological phenotype of the pseudofemale pelteobagrus vachelli is female, and the sex genotype is XY;
mating the pseudofemale pelteobagrus vachelli with male pelteobagrus vachelli, and screening the mated offspring to obtain the supermale pelteobagrus vachelli; the sex genotype of the pelteobagrus vachelli male fish is XY, and the sex genotype of the pelteobagrus vachelli male fish is YY; and
mating the super-male fish of the pelteobagrus vachelli with the female fish of the pelteobagrus fulvidraco to obtain offspring, namely the fully-male hybrid pelteobagrus fulvidraco, wherein the sex genotype of the female fish of the pelteobagrus fulvidraco is XX;
the step of screening includes the primer group, the kit and the sex identification method provided by the embodiment. Therefore, the screening process of each step can be rapidly simplified by the method, the crossbreeding process is shortened, and the actual breeding success rate is improved.
In addition, as shown in fig. 5, the embodiment of the application also provides a breeding method of the all-male hybrid pelteobagrus fulvidraco, which comprises the following steps:
carrying out estrogen induction reversion on the super-male fish of the pelteobagrus vachelli, and screening to obtain pseudo-female fish of the pelteobagrus vachelli; the sex genotypes of the super-male fish of the pelteobagrus vachelli and the pseudo-female fish of the pelteobagrus vachelli are YY, and the physiological phenotype of the pseudo-female fish of the pelteobagrus vachelli is female;
mating the pseudofemale pelteobagrus vachelli with the super-male pelteobagrus vachelli to obtain the super-male offspring of pelteobagrus vachelli, wherein the sex genotype of the super-male pelteobagrus vachelli is YY; and
hybridizing the offspring of the super-male pelteobagrus vachelli with a full-female pelteobagrus fulvidraco mating line to obtain the full-male hybridized pelteobagrus fulvidraco; the full female mating line of the pelteobagrus fulvidraco is a breeding fish with a mating offspring of XX pseudo-male fish and XX female fish obtained by carrying out trazoic inversion by a height of Wen Lian as full female, and the genotype of the full female mating line of the pelteobagrus fulvidraco is XX;
the step of screening includes the primer group, the kit and the sex identification method provided by the embodiment. Therefore, the screening process of each step can be rapidly simplified by the method, the crossbreeding process is shortened, and the actual breeding success rate is improved. In this embodiment, the construction method of the pelteobagrus fulvidraco total female mating line refers to "Yang Xiong, qingqing Han, ying Liu, shii Wang, jinhu Yang, wei Jiang, jingqi Hu, jian Chen, pei Li, jie Mei; biotechnological manipulation of the transition from genetic to temperature-dependent sex determination to obtain high quality neomale in Aquaculture [ J ] Aquaculture 560, (2022): 738471".
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (10)
1. The molecular marker is a differential sequence of a nucleotide sequence shown in SEQ ID NO.1 relative to a nucleotide sequence shown in SEQ ID NO.1 derived from an X chromosome genome of pelteobagrus vachelli and a nucleotide sequence shown in SEQ ID NO.2 derived from a Y chromosome genome of pelteobagrus vachelli, wherein the differential sequence is shown in SEQ ID NO.3, and is a specific sequence of the Y chromosome genome of pelteobagrus vachelli.
2. A primer pair, which base pairs with a DNA molecule as shown in SEQ ID NO.1 and/or a DNA molecule as shown in SEQ ID NO.2, comprising a DNA molecule having a nucleotide sequence as shown in SEQ ID NO. 4-5.
3. The kit for identifying the sex of pelteobagrus vachelli is characterized by comprising the primer set as claimed in claim 2.
4. The kit of claim 3, further comprising a DNA polymerase, mg 2+ dNTP, PCRBuffer and sterile water.
5. The method for identifying the sex genotype of pelteobagrus vachelli is characterized by comprising the following steps:
taking a DNA sample of pelteobagrus vachelli to be detected;
performing a PCR reaction using the primer set according to claim 2 using the DNA as a template;
detecting the obtained PCR reaction product to carry out gel electrophoresis, and confirming the sex genotype of the pelteobagrus vachelli according to the band of the gel electrophoresis.
6. The method according to claim 5, wherein if only one 259bp band is displayed in the gel electrophoresis, the sex genotype of pelteobagrus vachelli is XX;
if only one 310bp band is displayed in the gel electrophoresis, the sex genotype of the pelteobagrus vachelli is YY;
if a 259bp band and a 310bp band are displayed in the gel electrophoresis, the sex genotype of the pelteobagrus vachelli is XY.
7. The breeding method of the all-male hybrid pelteobagrus fulvidraco is characterized by comprising the following steps of:
carrying out estrogen induction on juvenile pelteobagrus vachelli, and screening out individuals which are successfully reversed, namely pseudofemale pelteobagrus vachelli; the physiological phenotype of the pseudofemale pelteobagrus vachelli is female, and the sex genotype is XY;
mating the pseudofemale pelteobagrus vachelli with male pelteobagrus vachelli, and screening the mated offspring to obtain the supermale pelteobagrus vachelli; the sex genotype of the pelteobagrus vachelli male fish is XY, and the sex genotype of the pelteobagrus vachelli male fish is YY; and
mating the super-male fish of the pelteobagrus vachelli with the female fish of the pelteobagrus fulvidraco to obtain offspring, namely the fully-male hybrid pelteobagrus fulvidraco, wherein the sex genotype of the female fish of the pelteobagrus fulvidraco is XX;
wherein the step of "screening" comprises the method of claim 5 and/or 6.
8. The breeding method of the all-male hybrid pelteobagrus fulvidraco is characterized by comprising the following steps of:
carrying out estrogen induction reversion on the super-male fish of the pelteobagrus vachelli, and screening to obtain pseudo-female fish of the pelteobagrus vachelli; the sex genotypes of the super-male fish of the pelteobagrus vachelli and the pseudo-female fish of the pelteobagrus vachelli are YY, and the physiological phenotype of the pseudo-female fish of the pelteobagrus vachelli is female;
mating the pseudofemale pelteobagrus vachelli with the super-male pelteobagrus vachelli to obtain the super-male offspring of pelteobagrus vachelli, wherein the sex genotype of the super-male pelteobagrus vachelli is YY; and
hybridizing the offspring of the super-male pelteobagrus vachelli with a full-female pelteobagrus fulvidraco mating line to obtain the full-male hybridized pelteobagrus fulvidraco; the full female mating line of the pelteobagrus fulvidraco is a breeding fish with a mating offspring of XX pseudo-male fish and XX female fish obtained by carrying out trazoic inversion by a height of Wen Lian as full female, and the genotype of the full female mating line of the pelteobagrus fulvidraco is XX;
wherein the step of "screening" comprises the method of claim 5 and/or 6.
9. A method of developing a molecular marker as defined in claim 1, comprising:
extracting DNA from 20 male and female pelteobagrus vachelli, respectively carrying out whole genome library construction and double-end sequencing by using a DNB-T7 sequencing platform, wherein the sequencing quantity is not lower than 15G, and the average sequencing depth is not lower than 20X;
comparing the sequencing data after quality control to a pelteobagrus vachelli reference genome by using a mem algorithm of BWA;
analyzing the comparison result by using a GATK variation detection flow to obtain a difference sequence comprising single nucleotide polymorphism sites and indels of small fragments;
firstly, filtering out the differential sequences with the sequencing depth lower than 5, and then screening the differential sequences according to the standards of homozygosity of all female samples and heterozygosity of male samples, wherein the obtained differential sequences are the differential sequences with male specificity.
10. Use of the molecular marker of claim 1, the primer pair of claim 2, or the kit of claim 3 or 4 in the holter breeding of pelteobagrus vachelli.
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