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CN-121518670-B - SSR multiplex PCR primer for parent-child identification of grass carp and application thereof

CN121518670BCN 121518670 BCN121518670 BCN 121518670BCN-121518670-B

Abstract

The invention discloses an SSR multiplex PCR primer for paternity test of grass carp and application thereof, wherein the primer comprises 16 pairs of specific primers, and the base sequences of the 16 pairs of specific primers are respectively shown as SEQ ID NO. 1-32 in sequence. Also discloses a fish paternity test kit comprising the SSR multiplex PCR primer, an SSR multiplex fluorescence PCR method for the paternity test of grass carp and application of the SSR multiplex PCR primer, the kit or the method in the paternity test of grass carp. The invention can detect 16 sites at one time by combining 16 pairs of specific SSR primers, multiple fluorescence PCR and universal amplification primer technology, and the universal primers can be reused, so that the efficiency is improved and the cost is reduced compared with the simple single-site detection, microsatellite loci contained in the invention are 3-6 bases, the allele size is more accurate to judge and read, and the accuracy of genotype data is improved.

Inventors

  • GUO LIANG
  • QIN QINBO
  • PENG RUI
  • WANG YUHUAN

Assignees

  • 湖南师范大学

Dates

Publication Date
20260505
Application Date
20260119

Claims (10)

  1. 1. The SSR multiplex PCR primer for paternity test of grass carp is characterized by comprising 16 pairs of specific primers, wherein the 16 pairs of specific primers are respectively primer pairs G0-1, G0-2, G0-3, G0-4, G0-5, G0-6, G1-1, G1-2, G1-3, G1-4, G1-5, G1-6, G2-1, G2-2, G2-3 and G2-4, and the base sequences of the 16 pairs of specific primers are respectively shown as SEQ ID NO: 1-32 in sequence.
  2. 2. The SSR multiplex PCR primer for paternity test of grass carp according to claim 1, further comprising three fluorescent-labeled universal primers M13, PQE-F and RV3, wherein the base sequences of the universal primers M13, PQE-F and RV3 are respectively shown as SEQ ID NO. 33-35, the 5' end of the forward primer of the primer pair G0-1, G0-2, G0-3, G0-4, G0-5 and G0-6 is connected with the universal primer M13, the 5' end of the forward primer of the primer pair G1-1, G1-2, G1-3, G1-4, G1-5 and G1-6 is connected with the universal primer PQE-F, the 5' end of the forward primer of the primer pair G2-1, G2-2, G2-3 and G2-4 is connected with the universal primer RV3, the fluorescent label matched with the universal primer M13 is FAM, the fluorescent label matched with the forward primer of the forward primer is HEX 3 and the fluorescent label is matched with the fluorescent label of the HEX 3.
  3. 3. A grass carp paternity test kit comprising the SSR multiplex PCR primer of claim 1.
  4. 4. The grass carp paternity test kit according to claim 3, further comprising three fluorescent-labeled universal primers M13, PQE-F and RV3, wherein the base sequences of the universal primers M13, PQE-F and RV3 are respectively shown in SEQ ID NO. 33-35, the 5' ends of forward primers of the primer pairs G0-1, G0-2, G0-3, G0-4, G0-5 and G0-6 are connected with the universal primer M13, the 5' ends of forward primers of the primer pairs G1-1, G1-2, G1-3, G1-4, G1-5 and G1-6 are connected with the universal primer PQE-F, the 5' ends of forward primers of the primer pairs G2-1, G2-2, PQ2-3 and G2-4 are connected with the universal primer RV3, fluorescent labels matched with the universal primer M13 are FAM, fluorescent labels matched with the fluorescent labels of the forward primers G1-1, G1-2-3, G1-3 and G1-6 are connected with the fluorescent labels of the forward primers are HEX 3.
  5. 5. The SSR multiplex fluorescence PCR method for parent-child identification of grass carp is characterized by comprising the following steps of: (1) Extracting grass carp DNA, namely collecting a grass carp parent sample and a offspring sample, and extracting genome DNA; (2) Multiplex fluorescence PCR amplification, namely performing multiplex fluorescence PCR amplification on the genome DNA in the step (1) by using the SSR multiplex PCR primer in the claim 2 or the kit in the claim 4 to obtain an amplified product; (3) And (3) paternity test, namely genotyping the amplified product, analyzing the parent genotype and the offspring genotype by using the genotyping result, and judging the parent and the child of the offspring individual.
  6. 6. A SSR multiplex fluorescence PCR method for paternity test of grass carp according to claim 5, wherein the multiplex fluorescence PCR amplification reaction system in the step (2) is 25. Mu.L of 2X TAQ PCR MASTER Mix, 2. Mu.L of genomic DNA, 0.1. Mu.L of each forward primer having a concentration of 10. Mu.M, 0.4. Mu.L of each reverse primer having a concentration of 10. Mu.M, 2.4. Mu.L of each fluorescent-labeled universal primer M13 having a concentration of 10. Mu.M, 2.4. Mu.L of each fluorescent-labeled universal primer PQE-F having a concentration of 10. Mu.M, 1.6. Mu.L of fluorescent-labeled universal primer RV3 having a concentration of 10. Mu.M, 1.5. Mu.L of bovine serum albumin BSA, and the balance of the PCR amplification reaction system is supplemented with ultrapure water to 50. Mu.L.
  7. 7. A SSR multiplex fluorescence PCR method for paternity test of grass carp according to claim 5, wherein the multiplex fluorescence PCR amplification reaction procedure in the step (2) is 95 ℃ pre-denaturation for 5min, 95 ℃ 30s,60 ℃ 30s,72 ℃ 30s,27 cycles, 95 ℃ 30s,58 ℃ 30s,72 ℃ 30s,8 cycles, and finally 72 ℃ extension for 30min,4 ℃ preservation.
  8. 8. The SSR multiplex fluorescence PCR method for paternity test of grass carp according to claim 5, wherein the amplified product is genotyped on a genetic analyzer in step (3) to read the genotype of the individual.
  9. 9. Use of the SSR multiplex PCR primer of claim 1 or 2, the kit of claim 3 or 4 in paternity test of grass carp.
  10. 10. Use of the method of any one of claims 5-8 in paternity testing of grass carp.

Description

SSR multiplex PCR primer for parent-child identification of grass carp and application thereof Technical Field The invention belongs to the technical field of microsatellite markers, and particularly relates to an SSR multiplex PCR primer for parent-child identification of grass carp and application thereof. Background Grass carp (Ctenopharyngodon Idella) is a fish belonging to genus grass carp of family Cyprinus, and is called as four-big-family fish together with black carp, silver carp and bighead carp, and has a culture history of 1700 years, and after four-big-family fish were successfully artificially bred in 1958, full artificial culture is realized. Molecular markers are important tools for research and application such as population dynamic detection, germplasm resource identification, pedigree identification, proliferation and release effect evaluation and the like. Microsatellite (Microsatellite DNA) is also called as simple repeated sequence (Simple Sequence Repeats, SSR), which refers to a repeated sequence with a repeated unit of 2-6 bases, the sequences at two sides of the microsatellite are generally relatively conserved, and the microsatellite sequence as a molecular marker has the advantages of simple method, high information content of unit point, reliable result and low cost, and can be operated more simply and conveniently by combining multiple PCR and universal fluorescent marker primers. Microsatellite has high single-site allele polymorphism, mature technology and wide application in the fields of paternity test and the like, but has some problems, such as 3 groups of multiplex PCRs consisting of 13 sites are recorded in the patent of the invention (publication No. CN103757113A, publication No. 2014-04-30) issued in 2015, 5,4 and 4 microsatellite sites are respectively contained, and 13 sites are all 2 base repeats. The DNA polymerase has serious slipping phenomenon in the repeated region of 2 bases, so that a large number of fragments in the amplified product are increased or lack of bases, the amplified product is characterized in that the amplified product is in a peak type disorder and is difficult to read, and a higher error rate and a deletion rate are caused, and the problem that alleles are difficult to read at least at 5 positions exists in 13 sites because of the disorder peaks in the attached drawing of the patent. Disclosure of Invention The first aim of the invention is to provide SSR multiplex PCR primers for paternity test of grass carp, which can stably amplify specified SSR sites of 3-6 bases, and the amplified SSRs have the advantages of high polymorphism and clear peak type. The invention also aims at providing an SSR multiplex fluorescence PCR method for parent-offspring identification of grass carp, which has the advantages of accurate identification and low cost. The final object of the invention is to provide the application of the SSR multiplex PCR primer, the kit comprising the primer or the method in the paternity test of grass carp. The first object of the invention is realized by the following technical scheme that the SSR multiplex PCR primer for paternity test of grass carp comprises 16 pairs of specific primers, wherein the 16 pairs of specific primers are respectively primer pairs G0-1, G0-2, G0-3, G0-4, G0-5, G0-6, G1-1, G1-2, G1-3, G1-4, G1-5, G1-6, G2-1, G2-2, G2-3 and G2-4, and the base sequences of the 16 pairs of specific primers are respectively shown as SEQ ID NO. 1-32 in sequence. Further, three fluorescent-labeled universal primers M13, PQE-F and RV3 are included. In some preferred embodiments of the present invention, the forward primer of the primer pair G0-1, G0-2, G0-3, G0-4, G0-5, G0-6 is ligated to the universal primer M13 at the 5 'end thereof, the forward primer of the primer pair G1-1, G1-2, G1-3, G1-4, G1-5, G1-6 is ligated to the universal primer PQE-F at the 5' end thereof, and the forward primer of the primer pair G2-1, G2-2, G2-3, G2-4 is ligated to the universal primer RV3, but this is not a limitation of the present invention, and the universal primer may be selected according to the circumstances. The base sequences of the universal primers M13, PQEF and RV3 are respectively shown as SEQ ID NO. 33-35. In some preferred embodiments of the present invention, the fluorescent label matching the universal primer M13 is FAM, the fluorescent label matching the PQEF is HEX, the fluorescent label matching the RV3 is ROX, but the present invention is not limited thereto, and the fluorescent label may be selected according to the specific situation, and in theory, the fluorescent labels may be exchanged with each other. The invention also provides a grass carp paternity test kit which comprises the SSR multiplex PCR primer. Furthermore, the grass carp paternity test kit provided by the invention comprises three fluorescent-labeled universal primers M13, PQE-F and RV3 besides the SSR multiplex PCR primers. The second aim of the invention