CN105483243B - Method for identifying pure breed and hybrid of loach and paramisgurnus dabryanus - Google Patents
Method for identifying pure breed and hybrid of loach and paramisgurnus dabryanus Download PDFInfo
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Abstract
The invention discloses a method for identifying pure species and hybrid species of loaches and paramisgurnus dabryanus, which is characterized in that 4 unique microsatellite SSR markers (2N18, 2N19, 4N7 and 4N17) of the loaches and 2 unique microsatellite SSR markers (PD30 and PD50) of the paramisgurnus dabryanus are screened, and 1 known mitochondrial Marker (MP) capable of distinguishing the loaches and the paramisgurnus dabryanus and a method for carrying out ploidy detection on the loaches by using a fin strip are combined simultaneously, so that a platform capable of identifying the pure species and the hybrid species of the loaches and the paramisgurnus dabryanus is established. The method plays an important role in the breeding, crossbreeding and the like of the loaches and the paramisgurnus dabryanus.
Description
Technical Field
The invention relates to a method for identifying pure species and hybrid of loaches and paramisgurnus dabryanus, belonging to the technical field of aquatic organisms.
Background
Misgurni anguillicaudati (Misgurnus anguillicaudus) and Paramisgurnus dabryanus (Paramisgurus dabryanus) belong to the genus Misgurni and Paramisgurnus dabryanus respectively in the family Misgurni of the order Cyprinidae. The two kinds of loaches are widely distributed in China, and are distributed from south to north except western plateaus in China. Due to their high edible and medicinal value, they are increasingly favored by consumers. In recent years, with the increasing market demand, the wild resource amount of the two kinds of loaches is seriously reduced, so that the market price of the loaches is increased, and the market economic value of the loaches is not inconstant. At present, the breeding hot tide of loaches and paramisgurnus dabryanus is raised in China, and the demand of excellent loaches and paramisgurnus dabryanus fries is rapidly increased by large-scale production. However, loach germplasm resources in the current aquaculture market are mixed, and pure species and hybrids are difficult to distinguish. If the hybrid with low fertility is used as a parent, huge economic losses are directly caused to a breeding field from the breeding perspective. Meanwhile, the identification of pure species and hybrid species of the loaches and the paramisgurnus dabryanus is an important premise for carrying out the breeding and crossbreeding of the two kinds of loaches, which is directly related to the success of the breeding work.
Microsatellite markers, also known as Simple Sequence Repeats (SSRs), are one of the most widely used molecular markers to date. The application of the method is mainly divided into three directions: genetic diversity research, variety identification and genetic relationship identification, genetic linkage map construction and QTL positioning. Mitochondrial DNA markers, as a novel molecular tool, possess maternally inherited characteristics and lack the ability to repair and recombine. Loaches have various chromosome karyotypes in China, and diploid and tetraploid are mainly used. Therefore, ploidy identification is always an important content when loaches are bred, at present, people mostly utilize blood to carry out ploidy detection on the loaches, certain requirements are met on the specifications of the loaches, certain damage is caused to the loaches, and the problems can be well solved if the ploidy of the loaches can be judged through fin strips.
In recent years, the germplasm resources of domestic loaches are mixed, and a large number of loaches and paramisgurnus dabryanus hybrids appear in the market. At present, although methods for identifying loaches and paramisgurnus dabryanus through external forms, molecular markers and the like exist, no method for identifying pure species and hybrid species of the loaches and the paramisgurnus dabryanus exists. The invention aims to provide a method for identifying pure species and hybrid species of loaches and paramisgurnus dabryanus for breeding and cultivating excellent loach fries.
Disclosure of Invention
The invention provides a method capable of identifying pure species and hybrid of loaches and paramisgurnus dabryanus aiming at the current situation that the loach germplasm resources are mixed and the hybrid of the loaches and the paramisgurnus dabryanus appears in large quantity.
The invention comprises the following steps: screening 4 specific microsatellite SSR markers (2N18, 2N19, 4N7 and 4N17) of the loaches, 2 specific microsatellite SSR markers (PD30 and PD50) of the paramisgurnus dabryanus and 1 known mitochondrion Marker (MP) capable of distinguishing the loaches from the paramisgurnus dabryanus, designing primers of the above 7 markers, carrying out PCR amplification on genome DNA of a sample to be detected by using the primers, obtaining PCR amplification products and carrying out gel electrophoresis analysis. Meanwhile, the invention also uses the fin rays to carry out ploidy detection on the sample to be detected, and comprehensively analyzes the ploidy result and the gel electrophoresis result, thereby identifying pure species and hybrid species of the loaches and the paramisgurnus dabryanus.
The more detailed method is as follows:
(1) taking a sample to be detected, shearing part of tail fin tissues, dividing the tail fin tissues into two parts, and using one part of the tail fin tissues with the size of soybean for ploidy detection; the other part of the tail fin tissue is stored in a centrifugal tube of 1.5ml filled with 95% ethanol, the genome DNA is extracted by adopting an ammonium acetate/isoamylol method, then an ultraviolet spectrophotometer is used for detecting the DNA concentration, and the mass concentration of the sample is diluted to 100 ng/mu L and stored at minus 20 ℃ for standby;
(2) taking the tail fin tissue with the size of the soybeans in the step (1) as a sample, and performing ploidy detection by using a ploidy analyzer;
(3) performing PCR amplification by using the DNA in the step (1) as a template and 7 marked primers to obtain a PCR amplification product, and then performing gel electrophoresis analysis;
the names of the 7 markers and the primer sequences thereof are as follows:
(4) comprehensively analyzing the ploidy result obtained in the step (2) and the gel electrophoresis result obtained in the step (3), wherein in the diploid sample, only 4 samples marked with amplification products by SSR markers and mitochondria specific to loaches are diploid loaches female parent and diploid loaches male parent; only 2 samples with specific SSR marks of the paramisgurnus dabryanus and amplified products marked by mitochondria are the paramisgurnus dabryanus multiplied by the paramisgurnus dabryanus; under the condition that specific SSR (simple sequence repeat) markers of the loaches and the paramisgurnus dabryanus are amplified, a sample of which the amplification product of the mitochondrial marker is a band with the size of 285bp is diploid loach female multiplied by paramisgurnus dabryanus, and a sample of which the amplification product of the mitochondrial marker is a band with the size of 214bp is diploid loach female multiplied by diploid loach male; in the triploid samples, under the condition that specific SSR (simple sequence repeat) markers of loaches and paramisgurnus dabryanus are amplified, the sample of which the amplification product of the mitochondrial marker is a band with the size of 285bp is a tetraploid loach female parent multiplied by a paramisgurnus dabryanus loach male, and the sample of which the amplification product of the mitochondrial marker is a band with the size of 214bp is a paramisgurnus dabryanus multiplied by a tetraploid loach male; the tetraploid sample is tetraploid loach male parent and tetraploid loach male parent, and only 4 specific SSR markers and mitochondria of the loaches are marked with amplification products.
The ploidy detection method in the step (2) comprises the following steps: 200 μ L of the extract was applied to a soybean-sized tail fin tissue sample, cut several times with a blade, and then 800 μ L of the cell staining solution was added, and the sample was stained in the dark for 1min, followed by filtration through a 30 μm filter, and then the volume was fixed to 800 μ L with PBS to detect the ploidy using a ploidy analyzer.
The total volume of the PCR amplification in the step (3) is 10 mu L, wherein the total volume comprises 0.5 mu L of template DNA of L00 ng/mu L, 1.0 mu L of 10xBuffer, 0.2 mu L of 10mmol/L dNTPs, 0.1 mu L of Taq DNA polymerase of 0.5 IU/mu L, 10nmol/L forward and reverse SSR primers of 0.25 mu L respectively, and the balance of double distilled water.
The PCR amplification procedure in the step (3) is as follows: the PCR cycle parameters were: 5min at 94 ℃; then 30 cycles, each cycle comprising 94 ℃ for 1min, 30s at annealing temperature, 72 ℃ for 1 min; finally, extension is carried out for 5min at 72 ℃; then stored at 4 ℃.
And (3) carrying out gel electrophoresis analysis by using 1.0-2.0% MetaPhor agarose gel electrophoresis.
Compared with the prior art, the invention has the advantages that:
the method disclosed by the invention is established based on 4 specific microsatellite SSR markers (2N18, 2N19, 4N7 and 4N17) of the loaches, 2 specific microsatellite SSR markers (PD30 and PD50) of the paramisgurnus dabryanus, 1 known mitochondrion Marker (MP) capable of distinguishing the loaches from the paramisgurnus dabryanus and 1 method for detecting ploidy of the loaches by using the fin strips. By utilizing the method, pure species and hybrid species of the loaches and the paramisgurnus dabryanus can be efficiently and quickly identified, and an accurate and quick identification method is provided for solving the situation of disordered germplasm resources of the loach market at present.
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FIG. 1 is a ploidy test chart of a diploid loach inbred selected at random in example one.
FIG. 2 is the results of the PCR products electrophoresis detection of specific SSR marker 4N7 of Misgurni Anguillicaudati, wherein M is DNA Mark I, DD1-DD5 is diploid pure Misgurni Anguillicaudati, TT1-TT5 is tetraploid pure Misgurni Anguillicaudati, PP1-PP5 is Paramisgurnus dabryanus pure breed sample, TP1-TP5 is hybrid TP sample, and PT1-PT5 is hybrid PT sample.
FIG. 3 shows the results of the PCR products electrophoresis detection of specific SSR-labeled PD30 of Paramisgurnus dabryanus of example one, wherein M is DNA Mark I, DD1-DD5 is diploid pure loach sample, TT1-TT5 is tetraploid pure loach sample, and PP1-PP5 is Paramisgurnus dabryanus pure loach sample.
FIG. 4 shows the results of the PCR products electrophoresis detection of mitochondrial marker MP in the first example, wherein M is DNA MarkI, PD1-PD5 is a hybrid PD sample, and DP1-DP5 is a hybrid DP sample.
Detailed Description
The invention will be further illustrated with reference to specific examples.
The first embodiment is as follows:
the loaches and adult loaches of Paramisgurnus dabryanus used in this example were purchased from the Baishazhou aquatic product market in Wuhan, Hubei province, and diploid loaches and tetraploid loaches were identified using a ploidy analyzer. Mature female (25.1-48.3g) and male (11.3-28.4g) diploid loaches, tetraploid loaches and paramisgurnus dabryanus are selected as parents, and oxytocic drugs are injected. The injection dosage of female fish is LHRH-A2(20 mug/kg) and DOM (4mg/kg), the dosage of the male fish is halved, and the injection method is an intraperitoneal one-time injection method. Carrying out artificial insemination on the loaches after about 12h to obtain pure loaches and large-scale paramisgurnus loaches (diploid loaches female parent multiplied by diploid loaches male, DD; tetraploid loaches female multiplied by tetraploid loaches male, TT; large-scale paramisgurnus loaches female multiplied by large-scale paramisgurnus loaches male, PP) and four hybrids (diploid loaches female multiplied by large-scale paramisgurnus loaches male, DP; male parent of Paramisgurnus dabryanus, female parent of Paramisgurnus dabryanus and PD; tetraploid loaches are female, male and TP; and the male parent of the loach with the large scale is multiplied by the female parent of the tetraploid loach with the large scale. Pure seeds and hybrid seeds of the loaches and the paramisgurnus dabryanus are respectively put into a rectangular plastic water tank with the length of 1.0m multiplied by 0.5m for feeding. Randomly and respectively cutting 5 pure loaches (DD), 5 pure loach tetraploid loach (TT), 5 pure loach paramisgurnus dabryanus (PP) and four hybrids (DP, PD, TP and PT) of each 5Part of the tail fin structure of (1). Dividing the tail fin tissue of each fish into two parts, and using the tail fin with the size of soybean for ploidy detection; another portion of the tail fin tissue was stored in a 1.5ml centrifuge tube filled with 95% ethanol. Extracting genome DNA by referring to an improved ammonium acetate/isoamylol method, detecting the DNA concentration by using an ultraviolet spectrophotometer, and diluting the mass concentration of each sample to 100 ng/mu L, and storing at-20 ℃ for later use;
the ploidy detection method comprises the following steps: adding 200 μ L of cell nucleus extract on a soybean fin sample, cutting with a blade for several times, adding 800 μ L of cell staining solution, staining for 1min in a dark place, filtering with a 30 μm filter screen, and diluting to 800 μ L with PBS to obtain volume;
the extracted genomic DNA was used as a template, 7 labeled upstream and downstream primers (shown in Table 1) were designed for PCR amplification, and the total volume of the PCR reaction was 10. mu.L, containing 0.5. mu.L of template DNA of L00 ng/. mu.L and 1.0. mu.L of 10xBuffer (containing Mg)2+) 0.2 mu L of 10mmol/L dNTPs, 0.1 mu L of 0.5 IU/mu L Taq DNA polymerase, 0.25 mu L each of 10nmol/L forward and reverse SSR primers and the balance of double distilled water; the PCR amplification procedure was: the PCR cycle parameters were: 5min at 94 ℃; then 30 cycles, each cycle comprising 94 ℃ for 1min, 30s at annealing temperature (for each marker annealing temperature see table 1), 72 ℃ for 1 min; finally, extension is carried out for 5min at 72 ℃; then stored at 4 ℃. The PCR products were then analyzed by electrophoresis on a 1-2% MetaPhor agarose gel.
TABLE 1 tag names and their primer sequences and PCR annealing temperatures
The results of the ploidy obtained by the ploidy assay (FIG. 1) and the gel electrophoresis obtained by gel electrophoresis analysis of the PCR products (FIGS. 2-4) show: 5 DD pure-breed samples are diploid in ploidy detection results, and only 4 specific SSR markers and mitochondria of loaches are marked with amplification products; ploidy detection results of 5 TT pure samples are tetraploids, and only 4 specific SSR markers and mitochondrial markers of the loaches have amplification products; 5 PP pure-breed samples are diploid in ploidy detection results, and only 2 specific SSR markers and mitochondrial markers of paramisgurnus dabryanus are provided with amplification products; 5 DP samples are diploid in ploidy detection results, specific SSR markers of the loaches and the paramisgurnus dabryanus have amplification products, and amplification products marked by mitochondria are bands with the size of 285 bp; 5 PD samples are diploid in ploidy detection results, specific SSR markers of the loaches and the paramisgurnus dabryanus have amplification products, and amplification products of mitochondrial markers are strips with the size of 214 bp; the ploidy detection results of the 5 TP samples are triploid, the specific SSR markers of the loaches and the paramisgurnus dabryanus have amplification products, the amplification products of the mitochondrial markers are all bands with the size of 285bp, and the unique difference between the PT samples and the TP samples is that the amplification products of the mitochondrial markers are all bands with the size of 214 bp. The analysis result obtained by the identification method is completely consistent with the known background information (100 percent matching) of pure species and hybrid of the loaches and the paramisgurnus dabryanus, and the method is proved to be accurate and reliable.
Claims (5)
1. A method for identifying pure species and hybrid species of loaches and paramisgurnus dabryanus is characterized by comprising the following steps:
(1) taking a sample to be detected, shearing part of tail fin tissues, dividing the tail fin tissues into two parts, and using one part of the tail fin tissues with the size of soybean for ploidy detection; the other part of the tail fin tissue is stored in a 1.5mL centrifuge tube filled with 95% ethanol, the genome DNA is extracted by adopting an ammonium acetate/isoamylol method, then an ultraviolet spectrophotometer is used for detecting the DNA concentration, and the mass concentration of the sample is diluted to 100 ng/mu L and stored at-20 ℃ for later use;
(2) taking the tail fin tissue with the size of the soybeans in the step (1) as a sample, and performing ploidy detection by using a ploidy analyzer;
(3) performing PCR amplification by using the DNA in the step (1) as a template and 7 marked primers to obtain a PCR amplification product, and then performing gel electrophoresis analysis;
the names of the 7 markers and the primer sequences thereof are as follows:
(4) comprehensively analyzing the ploidy result obtained in the step (2) and the gel electrophoresis result obtained in the step (3), wherein in the diploid sample, only 4 samples marked with amplification products by SSR markers and mitochondria specific to loaches are diploid loaches female parent and diploid loaches male parent; only 2 samples with specific SSR marks of the paramisgurnus dabryanus and amplified products marked by mitochondria are the paramisgurnus dabryanus multiplied by the paramisgurnus dabryanus; under the condition that specific SSR (simple sequence repeat) markers of the loaches and the paramisgurnus dabryanus are amplified, a sample of which the amplification product of the mitochondrial marker is a band with the size of 285bp is diploid loach female multiplied by paramisgurnus dabryanus, and a sample of which the amplification product of the mitochondrial marker is a band with the size of 214bp is diploid loach female multiplied by diploid loach male; in the triploid samples, under the condition that specific SSR (simple sequence repeat) markers of loaches and paramisgurnus dabryanus are amplified, the sample of which the amplification product of the mitochondrial marker is a band with the size of 285bp is a tetraploid loach female parent multiplied by a paramisgurnus dabryanus loach male, and the sample of which the amplification product of the mitochondrial marker is a band with the size of 214bp is a paramisgurnus dabryanus multiplied by a tetraploid loach male; the tetraploid sample is tetraploid loach male parent and tetraploid loach male parent, and only 4 specific SSR markers and mitochondria of the loaches are marked with amplification products.
2. The method for identifying pure loach and paramisgurnus dabryanus as claimed in claim 1, wherein the ploidy detection method in step (2) comprises: 200 μ L of the extract was applied to a soybean-sized tail fin tissue sample, cut several times with a blade, and then 800 μ L of the cell staining solution was added, and the sample was stained in the dark for 1min, followed by filtration through a 30 μm filter, and then the volume was fixed to 800 μ L with PBS to detect the ploidy using a ploidy analyzer.
3. The method for identifying pure loach and paramisgurnus dabryanus as claimed in claim 1, wherein the total volume of PCR amplification in step (3) is 10 μ L, wherein the total volume comprises 0.5 μ L of template DNA of L00ng/μ L, 10xBuffer of 1.0 μ L, 10mmol/L dNTPs of 0.2 μ L, 0.5IU/μ L of SSR Taq DNA polymerase of 0.1 μ L, and 0.25 μ L of each of forward and reverse primers of 10nmol, and the balance is double distilled water.
4. The method for identifying pure loach and paramisgurnus dabryanus as claimed in claim 1, wherein the PCR amplification procedure in step (3) is: the PCR cycle parameters were: 5min at 94 ℃; then 30 cycles, each cycle comprising 94 ℃ for 1min, 30s at annealing temperature, 72 ℃ for 1 min; finally, extension is carried out for 5min at 72 ℃; then stored at 4 ℃.
5. The method of identifying pure loach and paramisgurnus dabryanus as claimed in claim 1, wherein: the gel electrophoresis analysis described in step (3) was performed on a 1-2% MetaPhor agarose gel.
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| CN106636204B (en) * | 2017-01-09 | 2019-08-23 | 华中农业大学 | A kind of albefaction Misgurnus auguillicaudatus breeding method that can stablize heredity |
| CN106957888A (en) * | 2017-04-11 | 2017-07-18 | 武汉百科金典生物科技有限公司 | A kind of method based on the ploidy analyser Rapid identification prelarva ploidy |
| CN107022630B (en) * | 2017-05-17 | 2019-09-10 | 华中农业大学 | A kind of loach Ploidy Identification primer and application based on microsatellite polymorphism |
| CN108841930B (en) * | 2018-06-15 | 2021-09-28 | 天津市水产研究所 | Paramisgurnus dabryanus microsatellite family identification method and application thereof |
| CN110663593A (en) * | 2019-10-28 | 2020-01-10 | 江西农业大学 | Construction method of hybrid loach with fast growth and good meat quality and suitable for paddy field cultivation |
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