CN113667760B - SSR (simple sequence repeat) marker primer and method for evaluating genetic diversity of sparus praecox population - Google Patents
SSR (simple sequence repeat) marker primer and method for evaluating genetic diversity of sparus praecox population Download PDFInfo
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Abstract
The invention discloses an SSR (simple sequence repeat) marker primer for evaluating genetic diversity of a oplegnathus fasciatus population, which comprises 8 pairs of primers 1-8, wherein the nucleotide sequences of the primers are respectively shown in SEQ ID NO:1 to 16. The invention also discloses a method for evaluating genetic diversity of the sparus flavescens population, which comprises the steps of extracting RNA from sparus flavescens tissues, sequencing and SSR mining, taking genomic DNA of the sparus flavescens in a plurality of areas as a material, and verifying the developed SSR marker primers to obtain 8 pairs of excellent SSR molecular marker primers, wherein all the SSR markers obtained by the method can be specifically amplified, and have the advantages of cross-species universality, high polymorphism, co-dominance, easiness in detection and the like. The SSR molecular marker can be used in the fields of identification of the germplasm of the yellow-fin sea bream, analysis of genetic diversity and the like, and simultaneously provides a theoretical basis for investigation, development and protection of germplasm resources of the yellow-fin sea bream in the future.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an SSR (simple sequence repeat) marker primer for evaluating genetic diversity of a yellow fin sea bream population, which is obtained based on transcriptome sequencing of the yellow fin sea bream, a method and application of the SSR marker primer in analysis of genetic diversity of the population.
Background
The yellow-fin sea bream (Acanthopagrus latus) belongs to the weever shape fish of the genus acanthus of the family acanthaceae. The distribution of the yellow-fin sea bream penetrates through the Western Pacific of India, from the Bas bay to the Philippines along the coast of India, from Australia to Japan, and in coastal cities such as Guangdong, fujian, guangxi and the like, the yellow-fin sea bream is a rare sea fish and is also an important object of salty and fresh water aquaculture in China. In recent years, the number of the yellow-fin snacks in China is drastically reduced due to factors such as excessive fishing of human beings, water pollution, poor protection consciousness of germplasm resources and the like. In addition, the research on the genetic diversity of the yellow-fin sea bream has not been advanced in recent decades, so the research on the genetic diversity of the yellow-fin sea bream in China is a great trend. The resource status of the main production area of the yellow-fin sea bream in China provides theoretical basis for artificial breeding and variety cultivation, and lays a foundation for molecular marker assisted breeding.
Simple repeat sequences (Simple Sequence Repeats, SSR), i.e. microsatellite markers. Microsatellite markers are composed of a core sequence consisting of 1-6 nucleotide units and a flanking sequence, and simple repeated sequences with the number of unit repetition not less than 5. SSRs are widely distributed in eukaryotic genomes and transcriptomes and therefore become an effective method for eukaryotic genome level genetic diversity assessment. SSR is a co-dominant marker that can directly reflect the genetic information of a species. SSRs have higher polymorphisms and heterozygosity than the bi-allelic markers SNP (Single Nucleotide Polymorphism) and AFLP (Amplified Fragment Length Polymorphism) due to the large probability of mismatches caused by the slide chain during DNA replication. The flanking sequences at the two ends of the microsatellite marker have conservation in the genome of the species with relatively close relativity, so that the microsatellite marker developed by a certain species can be applied to related research of the related species, namely SSR has universality, and the characteristic greatly reduces the workload of developing the microsatellite marker. The microsatellite marker has the advantages of wide distribution, codominance marker, universality and the like, so that the SSR is widely applied to genetic relationship identification, population genetic structure research, genetic linkage map construction and other research.
Disclosure of Invention
The invention aims to provide an SSR marker primer for evaluating genetic diversity of a oplegnathus fasciatus population, which has the advantages of stable amplification, strong polymorphism and high heterozygosity.
The invention also aims at providing a method for evaluating genetic diversity of the group of the oplegnathus fasciatus.
The final object of the invention is to provide the application of the primer or the method in the aspects of the genetic diversity analysis, the variety identification, the genetic map construction or the molecular auxiliary breeding of the yellow-fin sea bream population.
The first object of the present invention is achieved by the following technical solutions: an SSR (simple sequence repeat) marker primer for evaluating genetic diversity of a oplegnathus fasciatus population, wherein the primer comprises 8 pairs, namely a primer 1, a primer 2, a primer3, a primer 4, a primer 5, a primer 6, a primer 7 and a primer 8, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO:1 to 16.
The second object of the present invention is achieved by the following technical solutions: a method of assessing genetic diversity of a group of sparus flavescens, comprising the steps of:
(1) Microsatellite marker development of yellow-fin sea bream
Taking fresh tissues of the brain, kidney, liver, gonad and muscle of the oplegnathus fasciatus, extracting tissue RNA, carrying out transcriptome sequencing, extracting high-quality sequences from the next machine data, searching coding regions of the transcriptome sequences by using software MISA, and searching microsatellite markers;
(2) Polymorphic microsatellite marker screening of yellow-fin sea bream
Using the microsatellite marker predicted in the step (1), detecting the size of an amplified product through PCR amplification and electrophoresis, collecting data, analyzing genetic parameters of SSR through software, and screening to obtain 10 pairs of specific amplified and high polymorphic SSR marker primers;
(3) Genetic diversity analysis of yellow-fin sea bream
Collecting a yellow fin sea bream population sample, extracting individual DNA, respectively carrying out PCR amplification on the yellow fin sea bream population by utilizing 8 pairs of polymorphic SSR marker primers in the step (2) of transcriptome development, carrying out genotyping by capillary electrophoresis, reading capillary electrophoresis data, analyzing the allele factors (A), expected heterozygosity (He), observed heterozygosity (Ho), inbred coefficients (f) and Hartmania temperature balance significance (P) of partial SSR markers in the yellow fin sea bream population by utilizing software FSTAT2.9.3, calculating the allele factors (Na), the observed heterozygosity (Ho), the expected heterozygosity (He) and Polymorphic Information Content (PIC) of each SSR marker in all individuals by utilizing software Cervus, and carrying out cluster analysis on the yellow fin sea bream population by utilizing a UPGMA algorithm of MEGA software.
In the above method for evaluating genetic diversity of the yellow fin sea bream population:
preferably, in step (1), the tissue RNA is extracted by Trizol-chloroform.
Preferably, in step (1) microsatellite markers are developed in the transcriptome coding region.
Preferably, the process of screening 8 pairs of microsatellite primers in the step (2) is as follows: and synthesizing primers according to microsatellite markers predicted by transcriptome sequencing, and respectively carrying out PCR amplification on 6-10 individuals of the oplegnathus fasciatus, and screening 8 pairs of microsatellite primers with stable amplification, strong polymorphism and high heterozygosity.
More preferably, the process of screening 8 pairs of microsatellite primers in the step (2) is as follows: and synthesizing primers according to microsatellite markers predicted by transcriptome sequencing, and respectively carrying out PCR amplification on 8 individuals of the oplegnathus fasciatus, and screening 8 pairs of microsatellite primers with stable amplification, strong polymorphism and high heterozygosity.
Preferably, in the PCR amplification in the step (2), the reaction system is 10. Mu.L: 2 XPCRMIX 5. Mu.L, 10. Mu. Mol/L of each of the forward and reverse primers, 0.4. Mu.L, 100 ng/. Mu.L of DNA template 1. Mu.L, ddH 2 O 3.2μL。
Preferably, the PCR reaction procedure in step (2) is set as follows: pre-denaturation at 94℃for 5min, then at 94℃for 30s, annealing at 48-60℃for 30s, at 72℃for 30s, for a total of 30 cycles, and finally extension at 72℃for 10min.
Preferably, in step (3), FAM fluorophores are labeled at the 5' end of 8 pairs of polymorphic SSR marker primers.
Preferably, step (3) genotyping is performed using ABI 3730XL, individual allele size (bp) is read using Gene mapper v4.0, and 8 pairs of SSR marker primers are analyzed for their allele factor (a), desired heterozygosity (He), observed heterozygosity (Ho), inbreeding coefficient (f) and hastelloy balanced significance (P) in the yellow fin porgy population, respectively, using software FSTAT2.9.3.
The third object of the present invention is achieved by the following technical means: the primer or the method is applied to the aspects of the genetic diversity analysis, variety identification, genetic map construction or molecular assisted breeding of the yellow fin sea bream population.
Compared with the prior art, the invention has the following advantages:
(1) The SSR marker primer developed based on transcriptome data has the advantages of specific amplification, cross-species universality, high polymorphism, co-dominance, easiness in detection and the like, and meanwhile, the SSR markers are all from a transcriptome coding region of the yellow fin porgy and can directly reflect genetic information of a genome, so that the genetic diversity of a yellow fin porgy group is better analyzed, and an SSR marker resource library of the yellow fin porgy is enriched;
(2) The SSR molecular marker developed by the invention can be used in the fields of identification of the germplasm of the yellow-fin sea bream, analysis of genetic diversity and the like, provides a theoretical basis for investigation, development and protection of germplasm resources of the yellow-fin sea bream in the future, provides a theoretical basis for resource status, artificial breeding and variety cultivation of main producing areas of the yellow-fin sea bream in China, and lays a foundation for molecular marker assisted breeding.
Drawings
FIG. 1 is a cluster map of six groups of yellow-fin porgy in example 1.
Detailed Description
The application method of the present invention will be further described with reference to the following specific examples. The following examples and figures are for illustrative purposes only and are not to be construed as limiting the invention. Unless otherwise indicated, the reagent raw materials used in the following examples were conventional commercially available or commercially available biochemical reagent raw materials, and the laboratory instruments used were laboratory conventional instruments, and the methods and apparatuses used in the following examples were methods and apparatuses conventionally used in the art, unless otherwise indicated.
Example 1
The SSR marker primer for evaluating the genetic diversity of the yellow fin sea bream population and the method for evaluating the genetic diversity of the yellow fin sea bream population provided by the embodiment are obtained by the following steps:
(1) Extracting RNA of 5 tissues of brain, gonad, liver, muscle and head kidney of yellow-fin sea bream, mixing the same amount, synthesizing a first cDNA by reverse transcription, amplifying and synthesizing double-chain cDNA by PCR, constructing cDNA libraries with different sizes, purifying and screening, sequencing by using Pacbio sequence platform, extracting high-quality CCS (Circular Consensus Sequence) sequence from the next machine data, removing primer and barcode, poly (A) and interlink structure, and obtaining full-length non-chimeric sequence (FLNC). Clustering similar FLNC ready, and combining the FLNC ready into a complete Isoform;
(2) All isochroms of the transcriptome were searched for SSR sites using MISA software. The set parameters are as follows: the repetition times of the two-base, three-base, four-base, five-base and six-base repeating units are 6, 5, 4 and 4 times respectively;
(3) SSR Primer design is carried out by adopting Primer3 software, wherein the Primer design parameters are that the length of a Primer sequence is 18-27bp, the length of a PCR amplified product is 100-400bp, and the GC content is 57-62%;
(4) Extracting 8 total DNA of the oplegnathus fasciatus, and carrying out PCR amplification to identify the specificity and polymorphism of SSR;
the reaction system was 10. Mu.L: 2 XPCR Mix 5. Mu.L, forward and reverse primers (10. Mu. Mol/L) each 0.4. Mu.L (each primer pair was used alone), DNA template (100 ng/. Mu.L) 1. Mu.L, ddH 2 O 3.2μL。
The PCR amplification procedure was: pre-denatured at 94 ℃ for 5min, then denatured at 94 ℃ for 30 seconds, annealed at 58 ℃ for 30 seconds, extended at 72 ℃ for 30 seconds for 35 cycles, and finally extended at 72 ℃ for 10min. SSRs without bands, without main bands, without single bands were eliminated by 1% agarose gel electrophoresis. Detecting polymorphism of the rest SSR by 8% non-denaturing polyacrylamide gel electrophoresis, and dyeing and developing silver by silver nitrate;
(5) The forward 5' end of the polymorphic SSR marker primer preliminarily screened in the steps is marked with FAM fluorescent groups to synthesize a fluorescent marker primer;
(6) Amplifying the SSR molecular marker primers in the step (5) by using a population, calculating the allele factors (Na), the observed heterozygosity (Ho), the expected heterozygosity (He) and the Polymorphism Information Content (PIC) of SSR in all individuals by using software Cervus3.0, and screening SSR marker primers with multiple allele factors and high expected heterozygosity and polymorphism information content;
(7) Carrying out genetic diversity analysis on the sparus fin group by utilizing SSR marker primers with stable amplification and good polymorphism in the step (6): collecting 6 yellow fin sea bream groups of a plurality of sea areas in China, extracting DNA of 235 individuals as a template for amplification, carrying out capillary electrophoresis genotyping on the obtained fluorescent PCR amplification products, reading capillary electrophoresis data, calculating heterozygosity, genetic distance and the like through allele frequencies of SSR markers appearing in different groups, describing genetic structures of the groups, determining genetic variation of the groups, and clustering the yellow fin sea bream groups.
The method for extracting the yellow fin sea bream tissue RNA in the step (1) specifically comprises the following steps:
1) Taking a proper amount of tissue, fully grinding the tissue in a liquid nitrogen environment, transferring the tissue to a 1.5mL centrifuge tube, adding 1mL Trizol, and immediately and fully mixing the tissue;
2) Placing the uniformly mixed tissue at room temperature for 10min to fully crack;
3) Adding 200 mu L of chloroform, fully shaking and uniformly mixing, centrifuging at 4 ℃, and carrying out 12000g of 10min;
4) Taking the upper aqueous phase and adding an equal volume of phenol: chloroform (25:24), thoroughly mixed, centrifuged at 4 ℃,12000g for 10min;
5) Taking the upper water phase, adding equal volume of chloroform, fully and uniformly mixing, centrifuging at 4 ℃, and carrying out 12000g for 10min;
6) Taking an upper water phase, adding equal volume of isopropanol, standing at-20 ℃ for 1 hour, centrifuging at 4 ℃, and carrying out 12000g for 10min;
7) The supernatant was discarded, 1mL of 75% ethanol was added, the precipitate was washed, centrifuged at 4℃for 8000g of 5min, and the supernatant was discarded;
8) Repeating the previous step;
9) Centrifuging briefly, sucking ethanol by a pipettor, and vacuum drying for 2-4min;
10 Adding 20-50 mu L RNase-FreeWater, dissolving at room temperature for 10min, mixing, and centrifuging instantly;
11 Mixing RNA samples of all tissues of the sparus praecox into a tube, and diluting for use.
Wherein, the step 4 of extracting DNA uses a marine animal DNA extraction kit of Tiangen biochemical technology Co., ltd, and the steps are according to the specification of the kit.
The non-denaturing polyacrylamide gel electrophoresis of the step (4) comprises the following specific steps:
and (4.1) aligning and fixing the square plate and the ear plate on a matched glue making frame, screwing screws on two sides of a base of the glue making frame, and clamping the left side and the right side of the two glass plates by using a long tail clamp to achieve a sealing effect.
(4.2) injecting 40mL of the prepared 8% polyacrylamide gel solution between the two glass plates, inserting a comb when the liquid level reaches the highest position of the ear plates, observing whether leakage exists at any time before solidification, and standing until solidification is completed.
And (4.3) after the gel is fixed, the glass plate is taken off from the gel making frame and fixed on two sides of the electrophoresis tank, the screw is screwed, the screwing degree is not too tight, the gel is easy to deform due to too tight, and the buffer solution above the electrophoresis tank is not easy to loosen due to too loose. A0.5 XTBE buffer was poured over the electrophoresis tank, 2. Mu.L of PCR product was added to the spotted wells, and 50bp DNA Ladder and PBR322DNA Maker were spotted in the middle and leftmost Bian Jiaokong, respectively.
(4.4) covering the upper cover of the electrophoresis tank, running for 10min at 200V and then running for 20min at 600V. And after electrophoresis, switching off the power supply, taking out the gel, dyeing the gel in AgNO3 dyeing liquid for 5min, then placing the gel in clear water for rinsing for 10s, transferring the gel into a color development liquid for color development until clear stripes are observed, and photographing and recording.
Wherein the yellow-fin sea bream material collected in the step (7) is obtained from the tail fin, dorsal fin or pectoral fin of yellow-fin sea bream, and is preserved in 95% ethanol after being removed from living body. The 6 groups of yellow-fin bream were collected from Guangdong Zhuhai (ZH; 44 tails), guangdong Yangjiang (YZ; 41 tails), guangdong Daya Bay (DYW; 36 tails), fujian Zhangzhou (ZZ; 30 tails), fujian Xiamen (XM; 38 tails) and Guangxi Fangdong harbor (FCG; 46 tails), respectively.
The specific results are as follows:
(1) SSR marker development and screening
80 microsatellite markers with 2 bases more than 10 repetition are selected from the SSR data of the transcriptome of the sparus praecox; 3 bases > 7 repeats 83; repeating the steps of 10 times with 4 bases more than 6, wherein the size of the PCR product is between 80bp and 500bp, and 173 microsatellite markers are obtained in total; and 8 individuals of the oplegnathus fasciatus are subjected to PCR amplification, and 8 pairs of primers with stable amplification, strong polymorphism and high heterozygosity are screened out.
The 8 pairs of microsatellite marker primers screened by the invention are as follows: primer 1, primer 2, primer3, primer 4, primer 5, primer 6, primer 7 and primer 8, are shown in Table 1 below.
TABLE 1 SSR marker primer information for 8 pairs of yellow sparus
The 235 individuals were individually subjected to PCR amplification analysis using 8 pairs of primers, and statistical analysis was performed by software, and the results are shown in Table 2.
The allele (A) of each SSR marker is between 7 and 32, and each SSR marker has 17.1 alleles on average, the number of amplified alleles of the primer 8 is maximum, 32, the number of amplified alleles of the primer 1 is minimum, and 7.
The observed heterozygosity is between 0.592 and 0.889, and the average value is 0.740; the expected heterozygosity is between 0.622 and 0.932, with an average value of 0.823, and the observed heterozygosity is significantly lower than the expected heterozygosity, indicating the presence of a loss of heterozygosity. The polymorphic information content of the SSR sites is between 0.576 and 0.933, with an average value of 0.803.
Table 2 8 genetic parameters of SSR in 235 individuals of yellow fin sea bream
(2) Population genetic diversity assessment
The genetic diversity analysis was performed on 6 populations of yellow-fin bream (Guangdong Zhuhai, guangdong Yangjiang, guangdong Daya Bay, fujian Zhangzhou, fujian Xiamen and Guangxi anti-harbor) using the obtained 8 excellent SSR markers with high polymorphism.
As shown in Table 3, the genetic diversity of the urban harbor population was higher than that of the Xiamen population, the allele numbers were 7.625 and 7, respectively, and the expected heterozygosity was 0.765 and 0.741, respectively; yangjiang population has the highest genetic diversity, allele factors and expected heterozygosity of 8.25 and 0.778, respectively, for Daya bay, zhangzhou, and Zhuhai populations of 7, 7.125, and 6.5, respectively, and expected heterozygosity of 0.730, 0.740, and 0.717, respectively.
Table 38 statistical genetic information of SSR in 6 yellow-fin porgy populations
A: number of alleles; he: desired degree of heterozygosity; h0: observing the heterozygosity; f: an inbreeding coefficient; p: the Hardy Winberg equilibrium significance.
The total value of the parilwise Fst obtained through the analysis of the software Arlequin 3.11 shows that high genetic differentiation appears among all the populations, the total differentiation degree of the pearl sea population and other 5 populations is the largest, the average Fst reaches 0.531, and the genetic differentiation degree of the Yangjiang population is the largest (Fst=0.630); the minimal degree of genetic differentiation between Yangjiang and Zhangzhou populations (fst=0.259) and the lesser degree of genetic differentiation between Dayawan and Xiamen populations (fst=0.367).
TABLE 4 genetic differentiation coefficient Fst (lower diagonal) of six populations based on 8 microsatellite marker primers and population genetic distance (upper diagonal) thereof
The MEGA is clustered by a UPGMA method, and a cluster map of six groups of sparus praecox is shown in fig. 1, and the results in fig. 1 show that 6 groups are divided into six clusters: the pearl sea population and the urban port prevention population are independently clustered, the genetic differentiation degree of Zhangzhou and Yangjiang populations is lowest and is clustered into one branch, and the genetic differentiation degree of Dayawan and Xiamen populations is lower and is clustered into one branch.
The results show that the microsatellite marker can accurately evaluate the genetic diversity among a plurality of groups of the yellow fin sea bream, and has important effect on researching the biological diversity and the systematic geography of the yellow fin sea bream.
The foregoing is merely illustrative of the non-limiting embodiments of this invention, and it will be appreciated by those skilled in the art that variations may be made without departing from the principles of the invention, which is defined in the claims.
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Claims (5)
1. An SSR marker primer for evaluating genetic diversity of a group of oplegnathus fasciatus is characterized in that: the primer comprises 10 pairs, namely A.la-110, A.la-139, A.la-97, A.la-164, A.la-79, A.la-175, A.la-169, A.la-113, A.la-86 and A.la-136, and the nucleotide sequences of the primers are respectively shown in SEQ ID NO:1 to 20.
2. A method for evaluating genetic diversity of a group of sparus praecox, comprising the steps of:
collecting a yellow fin sea bream population sample, extracting individual DNA, carrying out PCR amplification on the yellow fin sea bream population by using the 10 pairs of SSR marker primers in claim 1, carrying out genotyping by capillary electrophoresis, reading capillary electrophoresis data, analyzing the allelic factors, expected heterozygosity, observed heterozygosity, inbred coefficients and Hartma temperature-berg balance significance of part of SSR markers in the yellow fin sea bream population by using software FSTAT2.9.3, calculating the allelic factors, observed heterozygosity, expected heterozygosity and polymorphic information content of each SSR marker in all individuals by using software Cervus, and carrying out cluster analysis on the yellow fin sea bream population by using a UPGMA algorithm of MEGA software.
3. The method for evaluating genetic diversity of a group of yellow fin bream according to claim 2, characterized in that: the 5' end of the 10 pairs of SSR marking primers is marked with FAM fluorescent groups.
4. The method for evaluating genetic diversity of a group of yellow fin bream according to claim 2, characterized in that: genotyping was performed using ABI 3730XL, individual allele sizes were read using Gene mapper v4.0, and 10 pairs of SSR markers were analyzed for significance of allele factors, expected heterozygosity, observed heterozygosity, inbreeding coefficients, and hastelligunder balance in the yellow fin porgy population, respectively, using software FSTAT2.9.3.
5. Use of the primer of claim 1 or the method of claim 2 in the analysis of genetic diversity, variety identification or genetic map construction of a group of yellow fin porgy.
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| 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 |
| CN116144788B (en) * | 2022-10-21 | 2024-03-01 | 中山大学 | SSR (simple sequence repeat) marker primer, method and application for evaluating genetic diversity of eleutheronema tetradactylum population |
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