Identification methods of different families of rock carp

Abstract

The invention discloses a method for identifying different families of rock carp, comprising the following steps: collecting fin rays of rock carp to be identified; extracting rock carp DNA from the fin rays; using the extracted DNA as a template, and performing PCR amplification on the DNA template using 10 pairs of microsatellite primers screened out in an experiment; performing electrophoresis detection on the amplified product, and then performing silver staining; performing digital analysis on the silver staining result, using genetic software to measure the genetic distance between individuals, drawing a UPGMA cluster analysis diagram according to the genetic distance, and distinguishing individuals of different families according to the cluster analysis results. The invention can quickly and accurately distinguish the families of rock carp, effectively prevent inbreeding, and has great application value in the selection and breeding of rock carp families.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and relates to a method for identifying different families of rock carp, and in particular to a method for identifying different families of rock carp by using microsatellite markers. Background Art

[0002] Rock carp (Procypris rabaudi), commonly known as black carp or rock carp, belongs to the genus Procypris, family Cyprinidae, order Cypriniformes. It is a unique fish in the upper reaches of the Yangtze River and an important economic fish. It is mainly distributed in the upper reaches of the Yangtze River and the middle and lower reaches of the Jinsha River. Rock carp is a species endemic to China. Its meat is tender and delicious, with a high meat content and no intramuscular spines. It is a famous and high-quality economic fish loved by people. Since the 1970s, its wild resources have decreased sharply and it has been listed as a vulnerable species and endangered fish. The wild population of rock carp was listed as a second-class protected animal in the "National Key Protected Wildlife List" in 2021. It is an important aquatic germplasm resource and genetic species gene bank in my country.

[0003] In recent years, with the increasing awareness of fish protection, the scale of artificial breeding and farming of rock carp has become larger and larger. At present, artificially bred rock carp face a problem, that is, the phenomenon of inbreeding has occurred in the farmed rock carp. As the number of artificially bred rock carp increases, but there is a lack of a complete artificial family management system, rock carp of different or the same family are mixed in artificial breeding. When the breeding season comes, it is difficult to tell whether the paired rock carp broodstock are from the same family, which leads to inbreeding. Therefore, it is of great theoretical and production significance to establish a family determination method for rock carp. Summary of the invention

[0004] In order to solve the above technical problems existing in the background technology, the present invention provides a method for identifying different families of rock carp, which helps to quickly and accurately identify different families of rock carp, effectively prevent inbreeding, and provide a theoretical basis for the breeding and genetic protection of rock carp.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A PCR primer for identifying rock carp, characterized in that: the PCR primer for identifying rock carp is a rock carp double PCR primer, the rock carp double PCR primer is 5 groups, and each group of rock carp double PCR primers includes two pairs of primer sequences.

[0007] The above-mentioned dual PCR primers for rock carp are:

[0008] Group I, including a YYL1 primer pair and a YYL2 primer pair; wherein: the forward primer F of YYL1 is CGAGTGCGATTGCTG, and the reverse primer R is GCGTCATAGGATGTAGTTT; the forward primer F of YYL2 is GCGTTACAATCACCT, and the reverse primer R is TGTCTGTTCTCGCTCT;

[0009] Group II includes a YYL3 primer pair and a YYL4 primer pair; wherein: the forward primer F of YYL3 is CGGCGTGATAACAATACAA, and the reverse primer R is AATGCCAGTGAAGACCG; the forward primer F of YYL4 is GAGTCTTTGCTGATAAA, and the reverse primer R is GAATTGTTGGCTTGG;

[0010] Group III, including YYL5 primer pair and YYL6 primer pair; wherein: the forward primer F of YYL5 is CCTTATTACTCGCACG, and the reverse primer R is AGCCATCAATACATCC; the forward primer F of YYL6 is GAACAAGGAAAGCG, and the reverse primer R is TGGGTGAATCAAAGA;

[0011] Group IV includes a YYL7 primer pair and a YYL8 primer pair; wherein the forward primer F of YYL7 is TCCAAGACTGAATAT, and the reverse primer R is AGTGCTTGACTACAA; the forward primer F of YYL8 is GCATGTTGCTAGGATA, and the reverse primer R is AGGCTTGCTGATAGG;

[0012] Group V includes YYL9 primer pair and YYL10 primer pair; wherein: the forward primer F of YYL9 is GCTTCCTGCCTACAT, and the reverse primer R is TATTCATCCTGGTCTC; the forward primer F of YYL10 is ATACTGTGGTTGACGG, and the reverse primer R is TCACGCTATCTTATCAT.

[0013] The PCR primers used to identify rock carp as described above are used to identify rock carp.

[0014] The PCR primers used to identify rock carp as described above are used to identify and distinguish different lineages of rock carp.

[0015] A method for identifying different families of rock carp based on PCR primers for identifying rock carp, characterized in that the identification method comprises the following steps:

[0016] 1) obtaining fin rays of rock carp to be identified, and extracting rock carp DNA from the fin rays of rock carp, wherein the rock carp to be identified are from at least two families, and each family includes at least two rock carp;

[0017] 2) using the DNA of rock carp obtained in step 1) as a template, and using the PCR primers for identifying rock carp as described above to perform PCR amplification to obtain an amplified product;

[0018] 3) electrophoresis and silver staining of the amplified product obtained in step 2) using polyacrylamide gel;

[0019] 4) Analyze the silver staining results in step 3) using genetic analysis software, measure the genetic distance between each individual of all the rock carp to be identified, draw a UPGMA cluster analysis diagram based on the genetic distance, and distinguish and identify the pedigrees of the rock carp to be identified based on the results shown in the UPGMA cluster analysis diagram.

[0020] The reaction system for PCR amplification in the above step 2) is 15ul, including: 10×PCR Buffer 1.5ul, 2.5mmol / L dNTP 0.5ul, 2.0mmol / LMgCl2 1.5ul, 0.5ul each of the forward primer F and reverse primer R of the PCR primers used to identify rock carp, 0.5ul of 5U / μL Taq enzyme, 2ul of DNA template, and 7ul of ultrapure water.

[0021] The reaction procedure of PCR amplification in the above step 2) is: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 35 cycles; finally extension at 72°C for 10 minutes and storage at 4°C.

[0022] The specific implementation method of the above step 3) is: the amplified product obtained in step 2) is subjected to electrophoresis and silver staining using 12% polyacrylamide gel.

[0023] The genetic analysis software in the above step 4) is MEGA software.

[0024] The beneficial effects of the present invention are:

[0025] The present invention can be applied to the production and breeding process of rock carp. The technology can be used to distinguish individuals of rock carp of different mixed families, which helps to prevent inbreeding and breeding. The present invention solves the problem of difficulty in distinguishing families in mixed breeding of rock carp in actual production. Mixed breeding of different families not only makes it difficult to evaluate the development of family offspring, but also a susceptible family can easily transmit diseases to other families. This method can identify the genetic distance between multiple families, and guide the selection of the two families with the farthest genetic distance for pairing and breeding in rock carp production, so as to produce better offspring. This method uses the first double PCR applied to rock carp, which saves more reagents and operation time compared to ordinary PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a polyacrylamide gel image of microsatellite primers obtained using 24 rock carp individuals in Example 2;

[0027] Figure 2 It is a UPGMA cluster analysis diagram drawn according to the genetic distance between individuals in Example 2;

[0028] Figure 3 This is a UPGMA cluster analysis diagram drawn according to the genetic distance between individuals in Example 3. DETAILED DESCRIPTION

[0029] The technical solution provided by the present invention is described in detail below in conjunction with embodiments:

[0030] Example 1 Extraction of DNA from fin rays of Cyprinus cyprinids

[0031] 1. Selection of rock carp: 24 rock carp were randomly selected from the Yibin rock carp breeding farm. It is known that the rock carp in the farm are a mixed group of many families.

[0032] 2. Extraction of DNA from parental rock carp: DNA from the fin ray of rock carp was extracted using a DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. The specific extraction method (i.e., the instruction manual of the DNA extraction kit) is as follows:

[0033] 1) Place the chopped fins in a 1.5 mL centrifuge tube, add 20 μL of Proteinase K solution, and mix well;

[0034] 2) Add 200 μL of buffer GB, mix thoroughly by inversion, place in a 70°C water bath for 10 min, and then centrifuge briefly;

[0035] 3) Add 200 μL of anhydrous ethanol, shake thoroughly for 15 seconds, and then centrifuge briefly;

[0036] 4) Place the adsorption column CB3 in a collection tube, then transfer all the solution obtained in step 3) to the adsorption column, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 back into the collection tube;

[0037] 5) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube;

[0038] 6) Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube;

[0039] 7) Repeat step 6);

[0040] 8) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for 5 min;

[0041] 9) Transfer the adsorption column CB3 into a clean 1.5 mL centrifuge tube, add 100 μL of elution buffer TE, place at room temperature for 10 min, and centrifuge at 12000 rpm for 2 min.

[0042] 10) Store the obtained DNA solution in a -20°C refrigerator for later use.

[0043] Example 2 Verification of Identification of Different Lineages of Rock Carp

[0044] Three families were selected from the Yibin rock carp breeding farm, and 8 offspring samples were randomly selected from each family (1-8 individuals were from the same family, 9-16 individuals were from the same family, and 17-24 individuals were from the same family). In this experiment, 5 sets of rock carp double PCR reaction systems were selected (the basic information of the 10 pairs of microsatellite primers used for rock carp is shown in Table 1) to perform PCR amplification on the selected 24 rock carp samples. The PCR amplification reaction system was 15ul: 10×PCR Buffer 1.5ul, 2.5mmol / L dNTP0.5ul, 2.0mmol / L MgCl21.5ul, 0.5ul of each of the upstream and downstream primers of the two pairs of primers, 0.5ul of 5U / μL Taq enzyme, 2ul of DNA template, and 7ul of ultrapure water. The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 45 seconds, for a total of 35 cycles; and finally extension at 72°C for 10 minutes and storage at 4°C.

[0045] The PCR products were separated by electrophoresis using 12% non-denaturing polyacrylamide gel and photographed after silver staining. Figure 1The genetic distance between each individual was calculated by MEGA software, and a UPGMA cluster analysis diagram was constructed. The results are shown in Figure 2 The results showed that individuals 1-8 clustered into the same branch, individuals 9-16 clustered into the same branch, and individuals 17-24 clustered into the same branch, proving that this method can accurately identify individuals from different families.

[0046] Table 1 Basic information of 10 pairs of microsatellite primers for Cyprinus cyprinid

[0047]

[0048]

[0049] Example 3 Identification of different families of rock carp

[0050] 24 rock carps were randomly selected from the Yibin rock carp breeding farm. It is known that the rock carps in the breeding farm are a mixed group of many families. The 5 groups of rock carp double PCR reaction systems of this method were used to PCR amplify the DNA of the 24 rock carp samples. The PCR amplification reaction system was 15ul: 10×PCR Buffer 1.5ul, 2.5mmol / L dNTP 0.5ul, 2.0mmol / LMgCl21.5ul, 0.5ul of the upstream and downstream primers of the two pairs of primers, 0.5ul of 5U / μL Taq enzyme, 2ul of DNA template, and 7ul of ultrapure water. The PCR amplification reaction program was: 94℃ pre-denaturation for 3 minutes; then 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 45 seconds, a total of 35 cycles; finally 72℃ extension for 10 minutes, and storage at 4℃.

[0051] The PCR products were separated by electrophoresis using 12% non-denaturing polyacrylamide gel and photographed after silver staining. The genetic distance between each individual was calculated using MEGA software, and a UPGMA cluster analysis diagram was constructed. The results are shown in Figure 3 As shown. The 24 rock carp were clustered into 7 large branches, samples 7, 19, 9, 3 and 21 were clustered into one branch, samples 1, 2, 8 and 23 were clustered into one branch, samples 12, 13, 20 and 24 were clustered into one branch, samples 11, 10 and 16 were clustered into one branch, samples 5, 6, 17 and 18 were clustered into one branch, sample 4 was a separate branch, and samples 22, 14 and 15 were clustered into one branch. It was preliminarily determined that the 24 rock carp came from 7 different families. The individuals distributed on different large branches were distantly related. When the rock carp were paired for breeding, parents of the same branch should be avoided from pairing and breeding with each other to avoid inbreeding.

Claims

1. A PCR primer for identifying rock carp, characterized in that: The PCR primers used for identifying rock carp are rock carp dual PCR primers, and the rock carp dual PCR primers are 5 groups, and each group of rock carp dual PCR primers includes two pairs of primer sequences; The dual PCR primers for rock carp are: Group I, including a YYL1 primer pair and a YYL2 primer pair; wherein: the forward primer F of YYL1 is CGAGTGCGATTGCTG, and the reverse primer R is GCGTCATAGGATGTAGTTT; the forward primer F of YYL2 is GCGTTACAATCACCT, and the reverse primer R is TGTCTGTTCTCGCTCT; Group II includes a YYL3 primer pair and a YYL4 primer pair; wherein: the forward primer F of YYL3 is CGGCGTGATAACAATACAA, and the reverse primer R is AATGCCAGTGAAGACCG; the forward primer F of YYL4 is GAGTCTTTGCTGATAAA, and the reverse primer R is GAATTGTTGGCTTGG; Group III, including YYL5 primer pair and YYL6 primer pair; wherein: the forward primer F of YYL5 is CCTTATTACTCGCACG, and the reverse primer R is AGCCATCAATACATCC; the forward primer F of YYL6 is GAACAAGGAAAGCG, and the reverse primer R is TGGGTGAATCAAAGA; Group IV includes a YYL7 primer pair and a YYL8 primer pair; wherein the forward primer F of YYL7 is TCCAAGACTGAATAT, and the reverse primer R is AGTGCTTGACTACAA; the forward primer F of YYL8 is GCATGTTGCTAGGATA, and the reverse primer R is AGGCTTGCTGATAGG; Group V includes YYL9 primer pair and YYL10 primer pair; wherein: the forward primer F of YYL9 is GCTTCCTGCCTACAT, and the reverse primer R is TATTCATCCTGGTCTC; the forward primer F of YYL10 is ATACTGTGGTTGACGG, and the reverse primer R is TCACGCTATCTTATCAT.

2. Use of the PCR primers for identifying rock carp as claimed in claim 1 in identifying rock carp.

3. Use of the PCR primers for identifying rock carp as claimed in claim 1 in identifying and distinguishing different families of rock carp.

4. A method for identifying different families of rock carp based on the PCR primers for identifying rock carp as claimed in claim 1, characterized in that: The identification method comprises the following steps: 1) obtaining fin rays of rock carp to be identified, and extracting rock carp DNA from the fin rays of rock carp, wherein the rock carp to be identified are from at least two families, and each family includes at least two rock carp; 2) using the DNA of rock carp obtained in step 1) as a template, and performing PCR amplification using the PCR primers for identifying rock carp as claimed in claim 1 to obtain an amplified product; 3) electrophoresis and silver staining of the amplified product obtained in step 2) using polyacrylamide gel; 4) Analyze the silver staining results in step 3) using genetic analysis software, measure the genetic distance between each individual of all the rock carp to be identified, draw a UPGMA cluster analysis diagram based on the genetic distance, and distinguish and identify the family of the rock carp to be identified based on the results shown in the UPGMA cluster analysis diagram.

5. The identification method according to claim 4, characterized in that: The reaction system for PCR amplification in step 2) is 15ul, including: 10×PCR Buffer 1.5ul, 2.5mmol / L dNTP 0.5ul, 2.0 mmol / L MgCl2 1.5ul, 0.5ul each of the forward primer F and reverse primer R of the two primer pairs in each set of rock carp double PCR primers, 0.5ul of 5 U / μL Taq enzyme, 2ul of DNA template, and 7ul of ultrapure water.

6. The identification method according to claim 5, characterized in that: The reaction procedure of PCR amplification in step 2) is: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 45 seconds, for a total of 35 cycles; and finally extension at 72°C for 10 minutes and storage at 4°C.

7. The identification method according to claim 6, characterized in that: The specific implementation method of step 3) is: electrophoresis and silver staining of the amplified product obtained in step 2) using 12% polyacrylamide gel.

8. The identification method according to any one of claims 4 to 7, characterized in that: The genetic analysis software in step 4) is MEGA software.

Images