CN113667760A - SSR marker primer and method for evaluating genetic diversity of sparus latus population - Google Patents
SSR marker primer and method for evaluating genetic diversity of sparus latus population Download PDFInfo
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Abstract
The invention discloses SSR labeled primers for evaluating genetic diversity of sparus latus population, which comprise 8 pairs of primers 1-8, wherein the nucleotide sequences of the primers are respectively shown as SEQ ID NO: 1 to 16. The invention also discloses a method for evaluating the genetic diversity of the sparus latus group, which comprises the steps of carrying out RNA extraction on sparus latus tissues, carrying out sequencing and SSR excavation, taking sparus latus genome DNA in a plurality of regions as a material, and verifying the developed SSR marker primers to obtain 8 pairs of excellent SSR molecular marker primers. The SSR molecular marker can be used in the fields of yellow-fin sparus germplasm identification, genetic diversity analysis and the like, and simultaneously provides a theoretical basis for investigation, development and protection of yellow-fin sparus germplasm resources in the future.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an SSR marker primer and an SSR marker method for evaluating genetic diversity of a sparus latus population, which are obtained based on sequencing of a sparus latus transcriptome, and application of the SSR marker primer and the method in population genetic diversity analysis.
Background
Yellow fin sea bream (Acanthopaggrus latus) belongs to the order Perciformes, family Paciformes, genus Acanthopagrus. The yellow-fin sea bream is distributed throughout the western Pacific India, from the Baus along the Indian coast to the Philippines, from Australia to Japan, and mainly distributed in coastal cities such as Guangdong, Fujian, Guangxi, and the like in China, is a rare sea fish and is also an important object for the salt water culture in China. In recent years, the number of yellow-fin sea breams in China is sharply reduced due to factors such as over-fishing of human beings, water pollution, and low protection consciousness of germ plasm resources. In addition, the research on the genetic diversity of the yellow fin sea bream has not been developed in the last decade, so the research on the genetic diversity of the yellow fin sea bream in China is in the greater trend. The resource status of the main producing area of the yellow fin porgy in China provides a theoretical basis for artificial breeding and variety cultivation of the yellow fin porgy, and lays a foundation for molecular marker-assisted breeding.
Simple Sequence Repeats (SSR), i.e. microsatellite markers. The microsatellite marker is a simple repetitive sequence which consists of a core sequence and a flanking sequence, wherein the core sequence consists of 1-6 nucleotide composition units, and the repetition frequency of the units is not less than 5 times. SSRs are widely distributed in eukaryotic genomes and transcriptomes and thus become an effective method for genetic diversity assessment at the eukaryotic genome level. SSRs are co-dominant markers that can directly reflect the genetic information of a species. Due to the high probability of mismatch caused by the slide strand during DNA replication, SSR has higher polymorphism and heterozygosity compared with double allele markers SNP (Single Nucleotide polymorphism) and AFLP (amplified Fragment Length polymorphism). The flanking sequences at the two ends of the microsatellite marker have conservation in the genomes of species with relatively close relativity, so that the microsatellite marker developed by a certain species can be applied to the related research of the closely related species, namely the SSR has universality, and the characteristic greatly reduces the workload of developing the microsatellite marker. The microsatellite markers have the advantages of wide distribution, co-dominant markers, universality and the like, so the SSR is widely applied to the researches in the aspects of genetic relationship identification, population genetic structure research, genetic linkage map construction and the like.
Disclosure of Invention
The invention aims to provide an SSR marker primer for evaluating the genetic diversity of a sparus latus population, which has the advantages of stable amplification, strong polymorphism and high heterozygosity.
The invention also aims to provide a method for evaluating the genetic diversity of the sparus latus population.
The final purpose of the invention is to provide the application of the primer or the method in the aspects of analysis of genetic diversity of the sparus latus population, variety identification, construction of genetic maps or molecular assisted breeding.
The first object of the present invention is achieved by the following technical solutions: an SSR labeled primer for evaluating genetic diversity of sparus latus population comprises 8 pairs of primers 1, 2, 3, 4, 5, 6, 7 and 8, wherein 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 for evaluating genetic diversity of a sparus latus population, comprising the steps of:
(1) microsatellite marker development of sparus latus
Taking fresh tissues of brain, kidney, liver, gonad and muscle of the sparus latus, extracting tissue RNA, performing transcriptome sequencing, extracting a high-quality sequence from off-line data, searching a coding region of the transcriptome sequence by using software MISA, and searching for a microsatellite marker;
(2) polymorphic microsatellite marker screening of sparus latus
Using the microsatellite markers predicted in the step (1), carrying out PCR amplification and electrophoresis detection on the sizes of amplification products, collecting data, analyzing genetic parameters of SSR by using software, and screening to obtain the 10 pairs of SSR marker primers with specific amplification and high polymorphism;
(3) genetic diversity analysis of sparus latus
Collecting a yellow fin sea bream population sample, extracting individual DNA, respectively carrying out PCR amplification on the yellow fin sea bream population by using 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 allele factors (A), expected heterozygosity (He), observed heterozygosity (Ho), inbreeding coefficients (f) and Hawthorn Weinberg balance significance (P) of partial SSR markers in the yellow fin sea bream population by using software FSTAT2.9.3, calculating the allele factors (Na), observed heterozygosity (Ho), expected heterozygosity (He) and Polymorphic Information Content (PIC) 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.
In the above method for assessing genetic diversity of a sparus latus population:
preferably, the tissue RNA is extracted by Trizol-chloroform in step (1).
Preferably, in step (1) microsatellite markers are developed in the coding region of the transcriptome.
Preferably, the process of screening 8 pairs of microsatellite primers in step (2) is as follows: synthesizing primers according to microsatellite markers predicted by transcriptome sequencing, performing PCR amplification on 6-10 sparus latus individuals, and screening 8 pairs of microsatellite primers with stable amplification, strong polymorphism and high heterozygosity.
Preferably, the process of screening 8 pairs of microsatellite primers in the step (2) is as follows: synthesizing primers according to the microsatellite markers predicted by transcriptome sequencing, performing PCR amplification on 8 sparus latus individuals, and screening 8 pairs of microsatellite primers with stable amplification, strong polymorphism and high heterozygosity.
Preferably, in the PCR amplification in step (2), the reaction system is 10 μ L: 2 XPCRMix 5. mu.L, forward and reverse primers at a concentration of 10. mu. mol/L each at 0.4. mu.L, DNA template at a concentration of 100 ng/. mu.L at 1. mu.L, ddH2O 3.2μL。
Preferably, the PCR reaction program in step (2) is set as follows: pre-denaturation at 94 deg.C for 5min, then annealing at 94 deg.C for 30s, annealing at 48-60 deg.C for 30s, and annealing at 72 deg.C for 30s for 30 cycles, and finally extension at 72 deg.C for 10 min.
Preferably, FAM fluorescent group is marked at 5' end of 8 pairs of polymorphic SSR marker primers in step (3).
Preferably, step (3) is performed by genotyping using ABI 3730XL, reading the size (bp) of the allele of an individual by using Gene mapper v4.0, and analyzing the allele factors (a), the expected heterozygosity (He), the observed heterozygosity (Ho), the inbreeding coefficient (f) and the significance of hao wenberg balance (P) of 8 pairs of SSR-labeled primers in the sparus latus population by using software FSTAT2.9.3.
The third object of the present invention is achieved by the following technical solutions: the primer or the method is applied to the aspects of analysis of genetic diversity of sparus latus population, variety identification, genetic map construction or molecular assisted breeding.
Compared with the prior art, the invention has the following advantages:
(1) the developed SSR marker primer has the advantages of specific amplification, cross-species universality, high polymorphism, codominance, easiness in detection and the like, and the SSR markers are from the coding region of the Sparus latus transcriptome and can directly reflect the genetic information of a genome, so that the genetic diversity of the Sparus latus colony is better analyzed, and the SSR marker resource library of the Sparus latus is enriched;
(2) the SSR molecular marker developed by the invention can be used in the fields of yellow-fin sparus germplasm identification, genetic diversity analysis and the like, provides a theoretical basis for the survey, development and protection of sparus latus germplasm resources in the future, provides a theoretical basis for the resource condition, artificial culture and variety cultivation of the major producing areas of sparus latus in China, and lays a foundation for molecular marker-assisted breeding.
Drawings
Fig. 1 is a graph of the clusters of six yellow fin breams in example 1.
Detailed Description
The method of the present invention is further illustrated by the following examples. The following examples and drawings are illustrative only and are not to be construed as limiting the invention. Unless otherwise specified, the reagent raw materials used in the following examples are biochemical reagent raw materials which are conventionally commercially available or commercially available, and the laboratory instruments used are laboratory conventional instruments, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.
Example 1
The SSR marker primer for evaluating genetic diversity of sparus latus population and the method for evaluating genetic diversity of sparus latus population provided by the embodiment are obtained by the following methods:
(1) extracting RNA of 5 tissues of brain, gonad, liver, muscle and head kidney of the yellow fin sea bream, mixing the RNA in equal quantity, synthesizing a first cDNA by reverse transcription, synthesizing a double-stranded cDNA by PCR amplification, constructing cDNA libraries with different sizes, performing sequencing by using a Pacbio sequence platform after purification and screening, extracting a high-quality CCS (circular Consensus sequence) sequence from the next-generation data, and removing a primer, a barcode, a poly (A) and a connecting ring structure to obtain a full-length non-chimeric sequence (FLNC). Clustering similar FLNC reads, and combining into a complete Isoform;
(2) all isofoms of the transcriptome were searched using MISA software to find SSR loci. 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 respectively 6, 5, 4 and 4;
(3) SSR Primer design is carried out by adopting Primer3 software, the Primer design parameters are that the length of a Primer sequence is 18-27bp, the length of a PCR amplification product is 100-400bp, and the GC content is 57-62%;
(4) extracting 8 total DNA of sparus latus, and performing PCR amplification to identify the specificity and polymorphism of SSR;
the reaction system is 10 μ L: 2 XPCR Mix 5. mu.L, forward and reverse primers (10. mu. mol/L) 0.4. mu.L each (used alone for each pair of primers), DNA template (100 ng/. mu.L) 1. mu.L, ddH2O 3.2μL。
The PCR amplification procedure was: pre-denaturation at 94 ℃ for 5min, followed by denaturation at 94 ℃ for 30 sec, annealing at 58 ℃ for 30 sec, extension at 72 ℃ for 30 sec for 35 cycles, and final extension at 72 ℃ for 10 min. SSRs without bands, main bands and single bands are eliminated by 1% agarose gel electrophoresis. Detecting polymorphism of the rest SSR by 8% non-denaturing polyacrylamide gel electrophoresis, dyeing with silver nitrate, and developing with silver dye;
(5) marking FAM fluorescent group at the positive 5' end of the polymorphic SSR marking primer preliminarily screened in the step and synthesizing a fluorescent marking primer;
(6) using the SSR molecular marker primer in the group de-amplification step (5), calculating the allelic base factors (Na), the observed heterozygosity (Ho), the expected heterozygosity (He) and the Polymorphic Information Content (PIC) of the SSR in all individuals by using software Cervus3.0, and screening the SSR marker primer with multiple allelic base factors, the expected heterozygosity and high polymorphic information content;
(7) and (5) performing genetic diversity analysis on the sparus latus population by using the SSR marker primer which is amplified stably and has good polymorphism in the step (6): collecting 6 sparus latus populations in multiple sea areas of China, counting 235 individuals, extracting DNA of the individuals as a template for amplification, carrying out capillary electrophoresis genotyping on the obtained fluorescence PCR amplification product, reading capillary electrophoresis data, calculating heterozygosity, genetic distance and the like according to allele frequency of SSR marks appearing in different populations, thereby describing the genetic structure of the populations, determining the genetic variation of the populations, and clustering the sparus latus populations.
The method for extracting the RNA of the sparus latus tissue in the step (1) comprises the following specific 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 and the Trizol;
2) standing the mixed tissue at room temperature for 10min for full lysis;
3) adding 200 μ L chloroform, shaking thoroughly, mixing, centrifuging at 4 deg.C, 12000g for 10 min;
4) taking the upper aqueous phase, adding equal volume of phenol: chloroform (25: 24), mixing well, centrifuging at 4 deg.C, 12000g for 10 min;
5) adding equal volume of chloroform into the upper water phase, mixing, centrifuging at 4 deg.C, and 12000g for 10 min;
6) taking the upper water phase, adding isopropanol with the same volume, standing for 1 hour at the temperature of minus 20 ℃, centrifuging at the temperature of 4 ℃, and performing 12000g for 10 min;
7) discarding the supernatant, adding 1mL of 75% ethanol, washing the precipitate, centrifuging at 4 ℃, 8000g for 5min, and discarding the supernatant;
8) repeating the previous step;
9) centrifuging for a short time, sucking dry ethanol by a pipettor, and vacuum drying for 2-4 min;
10) adding 20-50 μ L RNase-FreeWater, dissolving at room temperature for 10min, mixing, and centrifuging instantly;
11) the RNA samples of all the tissues of the yellow-fin sea bream were mixed into a tube and diluted for use.
Wherein, the DNA extracted in the step 4 is extracted by using a marine animal DNA extraction kit of Tiangen Biochemical technology Co.
Wherein, the non-denaturing polyacrylamide gel electrophoresis of the step (4) comprises the following specific steps:
(4.1) aligning and fixing the square plate and the ear plate on a matched glue making frame, screwing the screws on the two sides of the 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 the prepared 40mL of 8% polyacrylamide gel solution between the two glass plates, inserting a comb when the liquid level reaches the highest position of the ear plate, observing whether leakage occurs at any time before coagulation, and standing until complete coagulation.
(4.3) after the gelatinization is fixed, taking down the glass plate from the gel making frame, fixing the glass plate to the two sides of the electrophoresis tank, screwing the glass plate, wherein the screwing degree is not too tight, the too tight is easy to cause gel deformation, and the too loose is not easy to cause the buffer solution above the electrophoresis tank to leak to the lower side. 0.5 XTBE buffer was poured over the electrophoresis chamber, 2. mu.L of PCR product was added to the wells, and 50bp DNA Ladder and PBR322DNA Maker were spotted in the middle and left-most wells, respectively.
(4.4) covering the upper cover of the electrophoresis tank, firstly using the voltage of 200V to run for 10min, and then using the voltage of 600V to run for lh20 min. And (3) cutting off the power supply after the electrophoresis is finished, taking out the gel, dyeing the gel in AgNO3 dyeing solution for 5min, rinsing the gel with clear water for 10s, transferring the gel into color development solution for color development until a clear strip appears, and taking a picture for recording.
Wherein, the materials of the yellow fin porgy collected in the step (7) are all taken from the tail fin, dorsal fin or pectoral fin of the yellow fin porgy, and the living body is stored in 95 percent ethanol after being taken down. 6 Pagrus latus groups were collected from Guangdong Pearl sea (ZH; 44 tails), Guangdong Yangjiang (YZ; 41 tails), Guangdong great Yawan (DYW; 36 tails), Fujian Zhang (ZZ; 30 tails), Fujian Xiamen (XM; 38 tails) and Guangxi defense harbor (FCG; 46 tails), respectively.
The specific results are as follows:
(1) development and screening of SSR markers
Selecting 80 repeated microsatellite markers with 2 bases more than 10 from SSR data of the Sparus latus transcriptome; 3 bases > 7 repeats 83; the number of the 4 bases is more than 6, the number of the repeats is 10, the size of a PCR product is between 80bp and 500bp, and 173 microsatellite markers are obtained in total; and 8 sparus latus individuals 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, as shown in Table 1 below.
TABLE 1 Sparus latus 8 information on SSR-labeled primers
235 individuals were analyzed by PCR amplification using 8 pairs of primers and statistical analysis was performed by software, and the results are shown in Table 2.
The allele factors (A) of each SSR marker are between 7 and 32, the average number of alleles of each SSR marker is 17.1, the number of amplified alleles of the primer 8 is the largest and is 32, and the number of alleles of the primer 1 is the smallest and is 7.
Observing heterozygosity of 0.592-0.889, 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 locus is 0.576 to 0.933, and the average value is 0.803.
TABLE 28 genetic parameters of SSR in 235 tailed yellow fin Pagrus individual
(2) Population genetic diversity assessment
By using the obtained 8 excellent SSR markers with high polymorphism, genetic diversity analysis is carried out on 6 yellow fin sea bream populations (Guangdong Zhuhai, Guangdong Yangjiang river, Guangdong great gulf of Asia, Fujian Zhangzhou, Fujian Xiamen and Guangxi defense harbor).
As a result of the analysis, as shown in Table 3, the genetic diversity of the anti-harbor population was higher than that of the mansion population, the numbers of alleles were 7.625 and 7, respectively, and the desired heterozygosity was 0.765 and 0.741, respectively; yangjiang populations have the highest genetic diversity with allele factors and desired heterozygosity of 8.25 and 0.778, respectively, for gulf, Zhangzhou, and Pearl sea populations of 7, 7.125, and 6.5, respectively, and desired heterozygosity of 0.730, 0.740, and 0.717, respectively.
TABLE 38 statistical genetic information of SSR in 6 Sparus latus populations
A: the number of alleles; he: a desired heterozygosity; h0: observing the heterozygosity; f: the coefficient of inbreeding; p: significance was balanced by the Hazberg equilibrium.
The values of pairwise Fst obtained by the analysis of software Arlequin 3.11 show that high genetic differentiation occurs among the populations, the population of the Zhuhai and the other 5 populations are differentiated to the maximum extent, the average Fst reaches 0.531, and the genetic differentiation with Yangjiang population is the maximum (Fst is 0.630); the genetic differentiation degree between Yangjiang and Zhangzhou populations is minimal (Fst is 0.259), and the genetic differentiation degree between the gulf and Xiamen populations is small (Fst is 0.367).
TABLE 4 genetic differentiation coefficients Fst (lower diagonal) of the six populations based on 8 microsatellite marker primers and their population genetic distance (upper diagonal)
The MEGA was subjected to clustering analysis by the UPGMA method, and the clustering chart of six sparus latus populations is shown in fig. 1, and the results in fig. 1 show that 6 populations were divided into six clusters: the Zhangzhou and Yangjiang river populations independently form a cluster, the genetic differentiation degrees of the Zhangzhou and Yangjiang river populations are the lowest to form a branch, and the genetic differentiation degrees of the great gulf and mansion populations are lower to form a branch.
The results show that the microsatellite marker can accurately evaluate the genetic diversity among multiple groups of the yellow fin sea bream, and has an important role in researching the biological diversity and the systematic geography of the yellow fin sea bream.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof, since modifications may be made by those skilled in the art without departing from the spirit of the invention and are to be considered within the scope of the invention.
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Claims (10)
1. An SSR marker primer for evaluating genetic diversity of sparus latus population is characterized in that: the primers comprise 8 pairs of primers 1, 2, 3, 4, 5, 6, 7 and 8, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO: 1 to 16.
2. A method for evaluating the genetic diversity of sparus latus population is characterized by comprising the following steps:
(1) microsatellite marker development of sparus latus
Taking fresh tissues of brain, kidney, liver, gonad and muscle of the sparus latus, extracting tissue RNA, performing transcriptome sequencing, extracting a high-quality sequence from off-line data, searching a coding region of the transcriptome sequence by using software MISA, and searching for a microsatellite marker;
(2) polymorphic microsatellite marker screening of sparus latus
Using the microsatellite markers predicted in the step (1), carrying out PCR amplification and electrophoresis detection on the sizes of amplification products, collecting data, using software to analyze genetic parameters of SSR, and screening to obtain 8 pairs of SSR marker primers with specific amplification and high polymorphism in claim 1;
(3) genetic diversity analysis of sparus latus
Collecting a yellow fin sea bream population sample, extracting individual DNA, respectively carrying out PCR amplification on the yellow fin sea bream population by using 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 allele factors (A), expected heterozygosity (He), observed heterozygosity (Ho), inbreeding coefficients (f) and Hawthorn Weinberg balance significance (P) of partial SSR markers in the yellow fin sea bream population by using software FSTAT2.9.3, calculating the allele factors (Na), observed heterozygosity (Ho), expected heterozygosity (He) and Polymorphic Information Content (PIC) 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 of assessing the genetic diversity of a yellow fin sea bream population according to claim 2, wherein: in the step (1), the Trizo 1-chloroform method is adopted to extract the tissue RNA.
4. The method of assessing the genetic diversity of a yellow fin sea bream population according to claim 2, wherein: in step (1), microsatellite markers are developed in the coding region of the transcriptome.
5. The method of assessing the genetic diversity of a yellow fin sea bream population according to claim 2, wherein: the process of screening 8 pairs of microsatellite primers in the step (2) comprises the following steps: synthesizing primers according to microsatellite markers predicted by transcriptome sequencing, performing PCR amplification on 6-10 sparus latus individuals, and screening 8 pairs of microsatellite primers with stable amplification, strong polymorphism and high heterozygosity.
6. The method of assessing the genetic diversity of a yellow fin sea bream population according to claim 2, wherein: during PCR amplification in the step (2), the reaction system is 10 mu L: 2 XPCR Mix 5. mu.L, forward and reverse primers at a concentration of 10. mu. mol/L each 0.3. mu.L, DNA template at a concentration of 100 ng/. mu.L 1. mu.L, ddH2O 3.4μL。
7. The method of assessing the genetic diversity of a yellow fin sea bream population according to claim 2, wherein: the PCR reaction program in step (2) is set as follows: pre-denaturation at 94 deg.C for 5min, then annealing at 94 deg.C for 30s, annealing at 48-60 deg.C for 30s, and annealing at 72 deg.C for 30s for 30 cycles, and finally extension at 72 deg.C for 10 min.
8. The method of assessing the genetic diversity of a yellow fin sea bream population according to claim 2, wherein: and (3) marking FAM fluorescent groups at the 5' ends of the 8 pairs of polymorphic SSR marking primers.
9. The method of assessing the genetic diversity of a yellow fin sea bream population according to claim 2, wherein: and (3) adopting ABI 3730XL to perform genotyping, reading the size (bp) of an individual allele by utilizing Gene mapper v4.0, and analyzing the allele factors (A), the expected heterozygosity (He), the observed heterozygosity (Ho), the inbreeding coefficients (f) and the Hawthorn-Wenberg balance significance (P) of 8 pairs of SSR labeled primers in the yellow fin sea bream population by utilizing software FSTAT2.9.3.
10. The primer according to claim 1 or the method according to claim 2, wherein the primer is used for genetic diversity analysis, variety identification, genetic map construction or molecular assisted breeding of sparus latus populations.
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