CN109055571B - Specific primer of yellow fin spine porgy microsatellite marker and application - Google Patents
Specific primer of yellow fin spine porgy microsatellite marker and application Download PDFInfo
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Abstract
The invention discloses a specific primer marked by a microsatellite of a yellow fin acanthopagrus latus, which comprises 8 pairs of primers, namely AL15, AL20, AL51, AL01, AL37, AL18, AL14 and AL49, and the primers have high polymorphism and a PCR product is stable and reliable; the invention also discloses a microsatellite paternity test method of the yellow fin spine porgy, which establishes a paternity test molecular method of the yellow fin spine porgy for the first time, the method has accurate test and low cost, and the invention further discloses the application of the microsatellite marked specific primer of the yellow fin spine porgy in the paternity test of the yellow fin spine porgy.
Description
Technical Field
The invention belongs to the technical field of microsatellite markers, and particularly relates to a specific primer for a yellow fin sea bream microsatellite marker and application thereof.
Background
The yellow-fin spine sea bream (Acanthopagrus latus) belongs to Pisces of the class Dermatophagoides, Perciformes of the order Perciformes, Sparidae of the family Paciformae, Acanthopagrus of the genus Acanthopagrus, and is widely distributed in the red sea, Arabic, Indian ocean, Western pacific coast, Taiwan, Fujian, Guangdong, and Guangxi coast.
The yellow fin spine sea bream lives in the coastal sea area and the estuary and is an important economic fish due to omnivory. The culture cycle of the oplegnathus fasciatus is long, the market specification of about 5 can be reached only after one year, a half year or two years, and the enthusiasm of fishermen for culturing the oplegnathus fasciatus is greatly limited. In recent years, natural populations of the yellow fin spine porgy are seriously degraded, germplasm is seriously degraded and the like due to environmental pollution, over fishing and the like; meanwhile, the breeding germplasm of the sparus fasciatus is seriously degraded, the disease resistance is weakened, the breeding performance is reduced and the like due to the close breeding of the sparus fasciatus breeding population, the artificial breeding of small-sized parents and the like. The above problems seriously restrict the continuous and healthy development of the yellow fin spine porgy breeding industry. Therefore, the protection of germplasm resources and the improved variety breeding of the yellow fin acanthopagrus schlegelii of different geographical groups are urgent, and one of the important methods for the improved variety breeding is the family breeding. The family breeding can obtain a variety with excellent characters, simultaneously can accurately know pedigree information and effectively guide parent screening, so that the genetic characters can be continuously improved, the close-relative breeding is avoided, the breeding time is shortened, and the yield of the yellow fin spine porgy is improved.
In the research of fish genetic breeding, the sources of the fries are relatively disordered, and the fries with different sources and different qualities are difficult to distinguish only by adopting physical and morphological marks. Generally, the identification method of the co-pond mixed culture family of the aquatic animals is to perform electronic marker differentiation, but when large-scale group breeding or family breeding is performed, the price of the electronic marker is high, so that the method has certain limitation. The DNA molecular marker technology has the characteristics of abundant genetic information, quick and simple detection means, good repeatability and the like, so the DNA molecular marker technology is widely applied to aspects of molecular marker assisted breeding, pedigree identification and the like.
With the rapid development of sequencing technologies, a large number of DNA molecular markers such as Single Sequence Repeat (SSR) and Single-nucleotide polymorphisms (SNPs) are applied to aquatic animals, such as red sea bream (sparyrops edita), carp (Cyprinus carpio), large yellow croaker (Larimichthy crocea), and the like. The SSR markers and the SNPs can be used for determining the genetic relationship among individuals and establishing a correct pedigree relationship, so that the method is beneficial to screening and popularizing excellent breeding varieties and standardizing the offspring seed market of the yellow fin spine porgy in China. At present, a large number of SSR markers of the yellow fin spine porgy are developed by some scholars, but reports of applying the SSR markers to identification and research of the family of the yellow fin spine porgy are few, which is not beneficial to the process of fine variety breeding of the yellow fin spine porgy.
Disclosure of Invention
The invention aims to provide a specific primer of a yellow fin sea bream microsatellite marker, which has high polymorphism and stable and reliable PCR product.
The invention also aims to provide a microsatellite paternity test method of the yellow fin acanthopagrus latus, which has accurate test and low cost.
The last purpose of the invention is to provide the application of the specific primer marked by the yellow fin spine porgy microsatellite in paternity test of the yellow fin spine porgy.
The first object of the present invention is achieved by the following technical solutions: a specific primer marked by a microsatellite of a latus, which comprises 8 pairs of primers of AL15, AL20, AL51, AL01, AL37, AL18, AL14 and AL49, wherein:
the nucleotide sequence of the primer pair AL15 is shown as SEQ ID NO: 1 and SEQ ID NO: 2;
the nucleotide sequence of the primer pair AL20 is shown as SEQ ID NO: 3 and SEQ ID NO: 4;
the nucleotide sequence of the primer pair AL51 is shown as SEQ ID NO: 5 and SEQ ID NO: 6;
the nucleotide sequence of the primer pair AL01 is shown as SEQ ID NO: 7 and SEQ ID NO: 8 is shown in the figure;
the nucleotide sequence of the primer pair AL37 is shown as SEQ ID NO: 9 and SEQ ID NO: 10;
the nucleotide sequence of the primer pair AL18 is shown as SEQ ID NO: 11 and SEQ ID NO: 12;
the nucleotide sequence of the primer pair AL14 is shown as SEQ ID NO: 13 and SEQ ID NO: 14, shown in the figure;
the nucleotide sequence of the primer pair AL49 is shown as SEQ ID NO: 15 and SEQ ID NO: shown in 16.
The second object of the present invention is achieved by the following technical solutions: a microsatellite paternity test method of latus comprises the following steps:
(1) extracting DNA of the yellow fin spine porgy: collecting a fin ray and a filial generation whole fish of a yellow fin sea bream parent sample to be identified, and extracting genome DNA;
(2) polymorphic microsatellite marker screening and primer synthesis: screening and synthesizing the 8 pairs of primers, wherein the 8 pairs of primers are divided into three groups, wherein one group comprises AL15, AL20 and AL51, the second group comprises AL01, AL37 and AL18, the third group comprises AL14 and AL49, the 5 'ends of the forward primers of the first group and the second group are respectively modified by three different fluorescent groups, namely FAM, HEX and TAMRA, and the 5' ends of the forward primers of the third group are respectively modified by two different fluorescent groups, namely FAM and HEX, for PCR analysis;
(3) microsatellite locus genotyping: performing PCR amplification on the genomic DNA in the step (1) by respectively using the three groups of primers in the step (2) through a fluorescent PCR reaction, and then mixing amplification products;
(4) and carrying out genotyping on the mixed amplification product, analyzing the genotype of the parent and the genotype of the filial generation by using the genotyping result, and judging the parent and the filial generation of the filial generation individual.
In the microsatellite paternity test method of the yellow fin spine porgy:
in the fluorescent PCR reaction in the step (3), the total volume of each group of reaction systems is 10 mu L, and the method comprises the following steps: double distilled water 7.6. mu.L, 10 XPCR (TaKaRa) buffer 1. mu.L (containing Mg)2+) 0.15. mu.L of dNTPs with a concentration of 10mmol/L, 0.15. mu.L of rTaq DNA polymerase (TaKaRa), 0.3. mu.L of each of the forward and reverse primers, and 0.5. mu.L of genomic DNA with a concentration of 100 ng/. mu.L.
In the fluorescent PCR reaction in the step (3), the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 40min, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
In step (4), the mixed amplification product is preferably genotyped on an ABI3730XL gene analyzer, GS-500LIZ is used as an internal reference, and the genotype of the individual is read by GeneMapper V3.2 software.
In the step (4), the CERVUS3.0 software is preferably adopted to analyze the parental genotype and the offspring genotype and determine the parents of the offspring individuals.
By adopting the software CERVUS3.0, the allele frequency, the heterozygosity, the expected heterozygosity, the polymorphic information content, the average exclusion probability, Hardy-Weinberg balance, the invalid allele frequency and other information of parents and filial generations on each microsatellite locus can be calculated, so that the parents of filial generation individuals can be judged.
Further, the microsatellite paternity test method of the yellow fin sea bream provided by the invention comprises the following steps:
(1) DNA extraction of yellow fin spine sea bream
Selecting healthy and well-developed yellow fin sea bream as a parent to breed fries, collecting fin rays and whole fish from parent fish and fry samples respectively, storing the finrays and the whole fish in absolute ethyl alcohol for later use, adopting a magenta animal DNA extraction kit to operate, diluting the finrays and the whole fish to 100 ng/mu L after detecting the quality and the concentration, and storing the finrays and the whole fish at the temperature of minus 20 ℃ for later use;
(2) polymorphic microsatellite marker screening and primer synthesis
Selecting 8 pairs of microsatellite primers, and dividing the microsatellite primers into 3 groups according to the sizes of fragments, wherein the primers are AL15, AL20 and AL51, and the primers are AL01, AL37 and AL 18; the 5 'end of the forward primer is respectively modified by three different fluorescent groups of FAM, HEX and TAMRA for PCR analysis, the primers AL14 and AL49 form a group, and the 5' end of the forward primer is respectively modified by two different fluorescent groups of FAM and HEX for PCR analysis;
(3) microsatellite locus genotyping
Performing PCR amplification on DNA samples of parents and filial generations by adopting a fluorescent PCR reaction, and mixing amplification products according to the method for grouping the microsatellite primers selected in the step (2) to be used as samples for on-machine detection;
(4) the samples were typed on an ABI3730XL gene analyzer, GS-500 was used as an internal reference, the genotype of each sample was read with GeneMapper V3.2 software, and the parental and progeny genotypes were analyzed with CERVUS3.0 software to determine the parental parents of the progeny individuals.
The third objective of the present invention is achieved by the following technical solutions: the application of the specific primer marked by the yellow fin spine porgy microsatellite in paternity test of the yellow fin spine porgy.
Compared with the prior art, the invention has the following advantages:
(1) the invention establishes a paternity test platform on the yellow fin sea bream by using the fluorescent marked polymorphic microsatellite marker for the first time, and the test accuracy reaches more than 99 percent;
(2) the invention can quickly and effectively identify different families and sources of the yellow fin spine porgy, and provides a basis for the breeding, breeding and grouping and proliferation releasing evaluation of the yellow fin spine porgy;
(3) the invention combines according to the size of the microsatellite fragments and the different colors of the fluorescent markers, mixes 2 or 3 microsatellite locus products together for sample loading detection, improves the efficiency by 2 to 3 times compared with the common PCR detection method, and saves the cost and time;
(4) the microsatellite locus alleles selected by the invention have more number and high polymorphism, can be used for the group genetic structure, the genetic breeding evaluation, the paternity test and the like of the yellow fin spine porgy, and can greatly save the experiment cost;
(5) the invention adopts an SSR marking method, establishes a paternity test molecular method of the yellow fin spine porgy for the first time, and successfully applies the method to paternity test of the family of the yellow fin spine porgy to form an SSR paternity test molecular discrimination group with accurate identification and low price, thereby providing a convenient and efficient technical method for fine breed breeding and family management of the yellow fin spine porgy and promoting the healthy development of the yellow fin spine porgy industry.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments.
Example 1 screening and Synthesis of microsatellite-labeled specific primers of Sparus latus
Selecting a sequence containing a microsatellite marker from a yellow fin sea bream transcriptome library to design a primer, screening unigene containing a yellow fin sea bream microsatellite repetitive sequence through transcriptome sequencing, designing a microsatellite specificity primer from the sequence containing a microsatellite locus detected in the yellow fin sea bream transcriptome library, detecting polymorphism of the microsatellite specificity primer, finally selecting 8 pairs of primers with clear bands and high polymorphism as primers for family identification after screening, synthesizing the primers by Shanghai biological engineering Co., Ltd, modifying 5' ends of each pair of the forward primers of the microsatellite primers by three different fluorescent groups of FAM, HEX and TAMRA (see table 1), and synthesizing the primers by the Shanghai biological engineering Co., Ltd.
TABLE 1 microsatellite primer information for paternity test of sparus latus
Example 2 microsatellite paternity test method of sparus latus
(1) Establishment of relationship between parent and filial generation of yellow fin spine sea bream
The 112 tails of the parent fish of the oplegnathus fasciatus are derived from wild fingerlings caught in the sea of Yangjiang, Guangdong province, and are cultured in the experimental base of the research and development center of the south-China aquatic product research institute, south-China aquatic product science institute. The 393 caudate generation is from a mixed family of yellow fin acanthopagrus schlegelii obtained by artificial insemination in 2017 in 12 months. Collecting the fin ray and the whole fish of the parent and the offspring respectively, and storing in absolute ethyl alcohol for later use.
(2) Extraction of yellow fin spine sea bream parent and filial generation genome DNA
Using the magenta animal tissue DNA extraction kit, 20-50mg of tissue samples were processed into pieces as small as possible and transferred to 1.5mL centrifuge tubes. Add 550. mu.L Buffer MTL and 20. mu.L proteinase K and vortex to mix. Samples were digested at 55 ℃ for 3h with shaking or overnight. Add 5. mu.L RNase Solution to the digest and mix it by inversion. Standing at room temperature for 30-60min to digest RNA. 13000Xg for 3 min. Transfer the supernatant to a new 2.0mL centrifuge tube. Add 500. mu.L Buffer DL to the digest, vortex and mix for 20s, and water bath at 70 ℃ for 10 min. Add 500. mu.L of absolute ethanol to the digest and vortex for 20 s. Hipure gDNA Mini Column was loaded into a 2mL collection tube. The mixture was transferred to a column and centrifuged at 10000Xg for 1 min. The effluent was decanted, the column was returned to the collection tube, 500. mu.L of BufferGW1 was added to the column, and the column was centrifuged at 10000Xg for 1 min. The effluent was decanted, the column was returned to the collection tube, 650. mu.L of Buffer GW2 was added to the column, and the column was centrifuged at 10000Xg for 1 min. The effluent was decanted, the column was returned to the collection tube and centrifuged at 10000Xg for 2 min. The column was loaded into a new 1.5mL centrifuge tube. Add 30-200. mu.L of Buffer AE preheated to 55 ℃ to the center of the membrane of the column. Standing for 2min, centrifuging at 10000Xg for 1 min. The DNA binding column was discarded, the DNA concentration and quality were determined using a NanoDrop ND-1000 UV spectrophotometer, each DNA sample was diluted to 100 ng/. mu.L and the DNA was stored at-20 ℃ until use.
(3) Polymorphic microsatellite marker screening and reaction system
Selecting a sequence containing a microsatellite marker from a yellow fin sea bream transcriptome library to design a primer, and finally selecting 8 pairs of primers with clear bands and high polymorphism as the primer for family identification after screening.
The 5' end of each pair of forward primers of the microsatellite primers is modified by three different fluorophores of FAM, HEX and TAMRA respectively (see table 1), and the primers are synthesized by Shanghai biological engineering Co., Ltd.
The total volume of each PCR reaction system is 10 mu L: double distilled water 7.6. mu.L, 10 XPCR (TaKaRa) buffer 1. mu.L (containing Mg)2+) 0.15. mu.L of 10mmol/L dNTPs, 0.15. mu.L of rTaqDNA polymerase (TaKaRa), 0.3. mu.L of forward and reverse primers respectively, and 0.5. mu.L of 100 ng/. mu.L genomic DNA.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 40min, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃. The Tm values are shown in Table 1.
(4) Microsatellite locus genotyping and paternity testing
The amplified products were typed on an ABI3730XL Gene Analyzer, GS-500LIZ was used as an internal reference, and the genotype of the individuals was read using GeneMapper V3.2 software. The software CERVUS3.0 was used to calculate the allele frequencies, heterozygosity, desired heterozygosity, polymorphic information content, mean exclusion probability, Hardy-Weinberg equilibrium and null allele frequencies of the parents and progeny at each microsatellite locus (see Table 2).
TABLE 28 genetic diversity statistics and exclusion probabilities for microsatellite loci
Note: k is the number of alleles, Ho is the observed heterozygosity, HEFor expected heterozygosity PIC content, Excl1 unknown parental exclusion, Excl2 known single parental exclusion, HW hardweinberg equilibrium test, ND no test, x indicates very significant deviation, NS no significant deviation, f (null) indicates null allele frequency.
(5) Paternity test results
Under the condition that the sex of parents is unknown, by simulating 10000 offspring and 112 pairs of parents, when the confidence coefficient is 95%, the theoretical identification rate of 8 microsatellite loci reaches 99.1%, and only 4 offspring individuals are not successfully distributed to parents. According to the actual genotyping data of the yellow fin spine porgy filial generation, when the confidence coefficient is 95%, 353 pieces of filial generation can find the parents. Therefore, the practical identification rate of the present invention is 89.31%.
Therefore, the specific primer marked by the microsatellite of the yellow fin spine porgy can be applied to paternity test of the yellow fin spine porgy.
It should be understood that the above description is only for the purpose of illustrating the features of the present invention and not for the purpose of limiting the same, and variations in the technical fields corresponding thereto, which would be apparent to those skilled in the art from the present invention, should be considered to fall within the scope of the present invention.
Sequence listing
<110> research institute for aquatic products in south China sea
Specific primer of <120> yellow fin spine porgy microsatellite marker and application
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Claims (5)
1. A specific primer marked by a yellow fin acanthopagrus latus microsatellite is characterized in that: the specific primers comprise 8 pairs of primers, namely AL15, AL20, AL51, AL01, AL37, AL18, AL14 and AL49, wherein:
the nucleotide sequence of the primer pair AL15 is shown as SEQ ID NO: 1 and SEQ ID NO: 2;
the nucleotide sequence of the primer pair AL20 is shown as SEQ ID NO: 3 and SEQ ID NO: 4;
the nucleotide sequence of the primer pair AL51 is shown as SEQ ID NO: 5 and SEQ ID NO: 6;
the nucleotide sequence of the primer pair AL01 is shown as SEQ ID NO: 7 and SEQ ID NO: 8 is shown in the figure;
the nucleotide sequence of the primer pair AL37 is shown as SEQ ID NO: 9 and SEQ ID NO: 10;
the nucleotide sequence of the primer pair AL18 is shown as SEQ ID NO: 11 and SEQ ID NO: 12;
the nucleotide sequence of the primer pair AL14 is shown as SEQ ID NO: 13 and SEQ ID NO: 14, shown in the figure;
the nucleotide sequence of the primer pair AL49 is shown as SEQ ID NO: 15 and SEQ ID NO: shown in 16.
2. A microsatellite paternity test method of latus comprises the following steps:
(1) extracting DNA of the yellow fin spine porgy: collecting a fin ray and a filial generation whole fish of a yellow fin sea bream parent sample to be identified, and extracting genome DNA;
(2) polymorphic microsatellite marker screening and primer synthesis: screening and synthesizing 8 pairs of primers of claim 1, wherein the 8 pairs of primers are divided into three groups, one group comprises AL15, AL20 and AL51, the second group comprises AL01, AL37 and AL18, the third group comprises AL14 and AL49, the first group and the second group are respectively modified by three different fluorophores of FAM, HEX and TAMRA at the 5 'end of the forward primer, and the third group is respectively modified by two different fluorophores of FAM and HEX at the 5' end of the forward primer for PCR analysis;
(3) microsatellite locus genotyping: performing PCR amplification on the genomic DNA in the step (1) by respectively using the three groups of primers in the step (2) through a fluorescent PCR reaction, and then mixing amplification products;
(4) and carrying out genotyping on the mixed amplification product, analyzing the genotype of the parent and the genotype of the filial generation by using the genotyping result, and judging the parent and the filial generation of the filial generation individual.
3. The microsatellite paternity test method of latacanthopagrus latus according to claim 2, which is characterized in that: in the fluorescent PCR reaction in the step (3), the total volume of each group of reaction systems is 10 mu L, and the method comprises the following steps: 7.6 μ L of double distilled water containing Mg2+10 is not available1 μ L of PCR buffer, 0.15 μ L of dNTPs with a concentration of 10mmol/L, 0.15 μ L of rTaqDNA polymerase, 0.3 μ L of forward and reverse primers, respectively, and 0.5 μ L of genomic DNA with a concentration of 100ng/μ L.
4. The microsatellite paternity test method of latacanthopagrus latus according to claim 2, which is characterized in that: in the fluorescent PCR reaction in the step (3), the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 40min, and 30 cycles; extending for 10min at 72 ℃; storing at 4 ℃.
5. The use of the microsatellite marked specific primer of oplegnathus latus according to claim 1 for paternity test of oplegnathus latus.
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| CN111394478B (en) * | 2020-04-27 | 2022-06-17 | 西安理工大学 | PCR (polymerase chain reaction) microsatellite primer and method for paternity test of large yellow croaker by using same |
| CN112899282A (en) * | 2021-03-03 | 2021-06-04 | 中国水产科学研究院南海水产研究所 | SNP (single nucleotide polymorphism) site related to growth traits of yellow fin acanthopagrus latus, and screening method and application thereof |
| CN113667760B (en) * | 2021-07-06 | 2023-07-14 | 中山大学 | SSR (simple sequence repeat) marker primer and method for evaluating genetic diversity of sparus praecox population |
| CN114592070B (en) * | 2022-03-07 | 2023-10-17 | 珠海市现代农业发展中心(珠海市金湾区台湾农民创业园管理委员会、珠海市农渔业科研与推广中心) | Microsatellite family identification method and application of sparus flavescens |
| CN115820868B (en) * | 2022-07-27 | 2023-08-29 | 中国水产科学研究院南海水产研究所 | Polymorphic primer for amplifying SNP molecular markers of oplegnathus ruticosus and application thereof |
| CN115927662B (en) * | 2022-09-26 | 2024-01-05 | 中国水产科学研究院南海水产研究所 | Amplification primer of SNP locus marker combination of oplegnathus fasciatus and application thereof |
| CN116555433B (en) * | 2022-12-28 | 2024-03-19 | 中国水产科学研究院南海水产研究所 | Primer and kit for identifying Huang Qiji and flat sea bream and application |
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