CN111286547A - Tilapia and microsatellite identification primer and method for genetic diversity of Tilapia - Google Patents
Tilapia and microsatellite identification primer and method for genetic diversity of Tilapia Download PDFInfo
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Abstract
The invention provides a microsatellite identification primer and a microsatellite identification method for tilapia and genetic diversity thereof, comprising the microsatellite identification primer and the microsatellite identification method, wherein the method comprises the following steps: selecting materials, extracting genome DNA, screening a microsatellite primer, carrying out a microsatellite primer PCR reaction, carrying out electrophoresis detection and dyeing, and analyzing data; the method takes the later generation (F13, F14 and F15) population of the new lucky tilapia as a research object, utilizes the microsatellite marking technology to track and monitor the genetic variation of the later generation population of the breeding, on one hand, the genetic diversity level of different breeding generation populations is compared, the influence of the breeding process on the genetic structure of the breeding population is analyzed, on the other hand, the microsatellite identification marker capable of distinguishing different breeding generation populations is searched, and the scientific basis is provided for the continuous breeding and the breed conservation work of the later generation of the new lucky tilapia through the comprehensive analysis of the two aspects.
Description
Technical Field
The invention relates to the technical field of fish breeding, in particular to a microsatellite identification primer and a microsatellite identification method for tilapia and genetic diversity thereof.
Background
"New Jifu" tilapia is a new variety which is systematically bred from 1996 to 10 years by the third generation of nile tilapia "GIFT" strain introduced in 1994 (F0) (Li Si Fa, 2001) at the university of high-salt water production, wherein F8 is examined and named by the national aquatic breeder and improved breed examination committee in 2006 1 month (registration number GS 01-001-. In view of the fact that no ever improved breed concept exists and new era requirements are developed, on the basis of the breeding line F8, the subject group insists on the continuous breeding of the new Jifu tilapia from the 15 th generation (F15) in 2011. However, along with the artificial breeding process, the closed breeding population is easily affected by factors such as non-random mating, genetic drift, artificial directional selection pressure and the like, so that the number of effective populations is reduced, and the inbreeding probability is increased. Therefore, in the breeding process, it is necessary to track and monitor the genetic variation of the breeding population and know the change of the genetic structure in time, so as to make corresponding scientific measures to ensure the smooth breeding. Meanwhile, artificial breeding is a long-term and complex process, in the process, the preservation of the germplasm resource library of each generation of breeding population is a difficult task, and because the breeding populations of each generation are very similar in body type characteristics, the phenomenon of germplasm mixing among the populations is easy to occur due to factors such as insufficient isolation measures and the like, scientific means (such as a molecular genetic marker technology) is necessary to perform germplasm identification on continuous breeding generation populations from the aspect of genotypes, so that the germplasm mixing among the populations is fundamentally avoided.
Disclosure of Invention
Aiming at the problems, the invention provides a microsatellite identification primer and a microsatellite identification method for tilapia and genetic diversity thereof.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
the first purpose of the invention is to provide a microsatellite identification primer for tilapia and genetic diversity thereof, wherein the microsatellite identification primer comprises 15 pairs which are respectively: UNH904, GM532, UNH846, UNH1007, HM370073, HM370078, HM370085, HM370111, UNH878, UNH896, UNH901, GM047, GM294, GM323 and GM578, wherein the microsatellite identification primers are selected by searching 50 published Nile tilapia microsatellite loci from a GenBank database and designing primers by using Primer5.0 software.
The second purpose of the invention is to provide a microsatellite identification method of tilapia and genetic diversity thereof, which comprises the following steps:
(1) selecting materials: respectively selecting new Jifu tilapia breeding lines F13, F14 and F15, wherein the number of samples F13 is 29, the number of samples F14 and F15 is 30, cutting each tail of the tail fin, numbering and storing in 95% ethanol for later use;
(2) extraction of genomic DNA: extracting the genome DNA of the tail fin of each sample in the step (1) by using a tissue/cell genome DNA rapid extraction kit, detecting the concentration and purity of the DNA by agarose gel electrophoresis, numbering the detected DNA extracts, and storing the DNA extracts in a refrigerator at the temperature of minus 40 ℃ for later use;
(3) screening microsatellite primers: searching 50 published nile tilapia microsatellite loci from a GenBank database, designing primers by using Primer5.0 software, and screening 15 pairs of effectively amplified microsatellite primers which are respectively: UNH904, GM532, UNH846, UNH1007, HM370073, HM370078, HM370085, HM370111, UNH878, UNH896, UNH901, GM047, GM294, GM323, GM 578;
(4) performing microsatellite primer PCR reaction, electrophoretic detection and dyeing: the PCR amplification reaction was 10. mu.L in total volume and contained 1L of genomic DNA template (20 ng/. mu.L), 0.5L (0.5. mu.M) of each of the forward and reverse primers, 5L of buffer (0.2mM dNTPs, 1.5mM MgCl2, 0.5M Taq DNA polymerase), 3L of sterile deionized water; detecting the PCR product by using non-denaturing polyacrylamide gel electrophoresis, wherein the gel concentration is 8 percent, and the electrophoresis buffer solution is 1 xTBE; after electrophoresis is finished, dyeing is carried out by using a rapid silver staining method, and the amplification condition of the PCR product is observed;
(5) and (3) data analysis: judging according to the DNA migration distance on the gel, and if the electrophoresis strip shows one strip and the migration distances are consistent, considering that the allele locus is homozygous; if two bands appear, the seat is considered to be heterozygous; estimating the molecular weight size of each band in the electrophoretic bands by using AlphaEaseFC 4.0 software, converting the size into letters, and calculating the allele factor (Na), the effective allele factor (NE), the observed heterozygosity (Ho) and the expected Heterozygosity (HE) of each population by using POPGENE software (Yeh et al, 1997); calculating an inter-population genetic similarity coefficient and an inter-population genetic distance according to the method of Nei (1972,1978); calculating a population genetic differentiation index (FST) by using ARLEQUIN 3.5 software (Excoffeer et al,2010), estimating genetic differentiation between populations, analyzing molecular Variance (AMOVA) in and between populations, and detecting the significance of the FST (the number of repetitions is 1000) by using a permutation test (persistence test); and the Polymorphic Information Content (PIC) was calculated according to the following formula:
in the formula: pi and pj are the frequency of the ith and jth alleles, respectively, and n is the number of alleles.
The PCR amplification program in the step (4): pre-denaturation at 94 ℃ for 4min followed by 35 cycles, each cycle comprising denaturation at 94 ℃ for 30s, annealing for 45s (see table 1 for specific annealing temperatures for each primer pair), and extension at 72 ℃ for 1 min; after 35 cycles, extension was carried out at 72 ℃ for 10min and the PCR product was stored at 4 ℃ for detection.
The reagent preparation formula of the non-denatured polyacrylamide gel in the step (4) is as follows:
(1)5 × TBE: 54g of Tris alkali, 27.5g of boric acid and 2.92g of EDTA, adding double distilled water to a constant volume of 1L, and storing at room temperature;
(2) 30% acrylamide (29% acrylamide + 1% N, N' -methylenebisacrylamide): adding 29g of acrylamide and 1g of N, N' -methylene bisacrylamide into 60mL of double distilled water, heating to 37 ℃ to dissolve the reagent, and fixing the volume to 100 mL; the solution can be prepared 1 month in advance, and stored in brown bottle at 4 deg.C;
(3) 10% Ammonium Persulfate (APS): 1g of ammonium persulfate is dissolved to 10mL by adding double distilled water, and the ammonium persulfate is preserved at 4 ℃ in a dark place and is prepared fresh every week;
(4) 8% polyacrylamide gel buffer (50 mL): 13.3mL of 30% acrylamide, 10mL of 5 XTBE, 350 mu L of 10% ammonium persulfate, 17.5 mu L of TEMED and 26.35mL of double-distilled deionized water;
(5) staining solution (0.1% silver nitrate): 0.5g of silver nitrate, adding double distilled water to a constant volume of 500mL, and preparing the silver nitrate fresh before each use;
(6) color development liquid: 10g of sodium hydroxide, 0.2g of sodium carbonate and 750 mu L of 37 percent formaldehyde, adding double distilled water to a constant volume of 500mL, and preparing the mixture fresh before each use;
(7) stopping liquid: double distilled water.
The dyeing procedure in step (4) is as follows:
(1) rinsing: rinsing with double-distilled deionized water twice, each for 5 min;
(2) dyeing: soaking the gel in 0.1% silver nitrate, and gently mixing in shaking table for 8 min;
(3) rinsing: rapidly rinsing twice (each time not exceeding 5s) with double distilled deionized water;
(4) color development: adding color development solution (prepared temporarily), adding 750L of 37% formaldehyde when in use, and gently mixing until the DNA bands are clear (about 10 min);
(5) fixing: soaking in double distilled water;
(6) and (3) storage: and after color development, taking a picture by using a digital camera and storing the picture.
The invention has the beneficial effects that: the low-voltage electrophoresis of 8% non-denatured polyacrylamide gel is adopted, and the DNA strip separation effect is good; the method takes the later generation (F13, F14 and F15) population of the new lucky tilapia as a research object, utilizes the microsatellite marking technology to track and monitor the genetic variation of the later generation population of the breeding, on one hand, the genetic diversity level of different breeding generation populations is compared, the influence of the breeding process on the genetic structure of the breeding population is analyzed, on the other hand, the microsatellite identification marker capable of distinguishing different breeding generation populations is searched, and the scientific basis is provided for the continuous breeding and the breed conservation work of the later generation of the new lucky tilapia through the comprehensive analysis of the two aspects.
Drawings
FIG. 1 is a polyacrylamide gel electrophoresis diagram of the microsatellite locus GM047 of the present invention, wherein 01-07 is F13,08-15 is F14,16-23 is F15, and the unlabeled lane is Marker (pBR322DNA/Msp I);
FIG. 2 is a polyacrylamide gel electrophoresis diagram of the microsatellite locus GM294 of the present invention, wherein 01-07 is F13,08-14 is F14,15-21 is F15, and the unlabeled lane is Marker (pBR322DNA/Msp I);
FIG. 3 is a graph of allele frequency distribution of GM294 in 3 breeding generation populations according to the present invention;
FIG. 4 is a graph of allele frequency distribution of GM532 in 3 breeding generation populations according to the present invention;
FIG. 5 is a graph showing the allele frequency distribution of GM323 in a population of 3 breeding generations according to the present invention;
FIG. 6 is a graph of the allele frequency distribution of UNH846 in a population of 3 breeding generations in accordance with the present invention;
FIG. 7 is a variation curve of 4 genetic parameters in the new Jifu tilapia breeding line F13-F15 according to the invention;
Detailed Description
The present invention will be described in detail with reference to the following examples:
example 1:
a microsatellite identification method for tilapia and genetic diversity thereof comprises the following steps:
(1) selecting materials: respectively selecting new Jifu tilapia breeding lines F13, F14 and F15, wherein the number of samples F13 is 29, the number of samples F14 and F15 is 30, cutting each tail of the tail fin, numbering and storing in 95% ethanol for later use;
(2) extraction of genomic DNA: extracting the genome DNA of the tail fin of each sample in the step (1) by using a tissue/cell genome DNA rapid extraction kit, detecting the concentration and purity of the DNA by agarose gel electrophoresis, numbering the detected DNA extracts, and storing the DNA extracts in a refrigerator at the temperature of minus 40 ℃ for later use;
(3) screening microsatellite primers: searching 50 published nile tilapia microsatellite loci from a GenBank database, designing primers by using Primer5.0 software, and screening 15 pairs of effectively amplified microsatellite primers which are respectively: UNH904, GM532, UNH846, UNH1007, HM370073, HM370078, HM370085, HM370111, UNH878, UNH896, UNH901, GM047, GM294, GM323, GM578, and the primer properties and PCR reaction conditions are shown in Table 1;
TABLE 115 pairs of efficiently amplified microsatellite primers
Note: f, a forward primer; r, a reverse primer.
(4) Performing microsatellite primer PCR reaction, electrophoretic detection and dyeing:
the total volume of the PCR amplification reaction was 10. mu.L, containing 1L of genomic DNA template (20 ng/. mu.L), 0.5L (0.5. mu.M) of each of the forward and reverse primers, 5L of buffer (0.2mM dNTPs, 1.5M MgCl2, 0.5M Taq DNA polymerase), 3L of sterile deionized water;
amplification procedure of PCR: pre-denaturation at 94 ℃ for 4min followed by 35 cycles, each cycle comprising denaturation at 94 ℃ for 30s, annealing for 45s (see table 1 for specific annealing temperatures for each primer pair), and extension at 72 ℃ for 1 min; after 35 cycles, extending for 10min at 72 ℃, and storing the PCR product to be detected at 4 ℃;
detecting the PCR product by using non-denaturing polyacrylamide gel electrophoresis, wherein the gel concentration is 8 percent, and the electrophoresis buffer solution is 1 xTBE; after electrophoresis is finished, dyeing is carried out by using a rapid silver staining method, and the amplification condition of the PCR product is observed;
(5) and (3) data analysis: judging according to the DNA migration distance on the gel, and if the electrophoresis strip shows one strip and the migration distances are consistent, considering that the allele locus is homozygous; if two bands appear, the seat is considered to be heterozygous; estimating the molecular weight size of each band in the electrophoretic bands by using AlphaEaseFC 4.0 software, converting the size into letters, and calculating the allele factor (Na), the effective allele factor (NE), the observed heterozygosity (Ho) and the expected Heterozygosity (HE) of each population by using POPGENE software (Yeh et al, 1997); calculating an inter-population genetic similarity coefficient and an inter-population genetic distance according to the method of Nei (1972,1978); calculating a population genetic differentiation index (FST) by using ARLEQUIN 3.5 software (Excoffeer et al,2010), estimating genetic differentiation between populations, analyzing molecular Variance (AMOVA) in and between populations, and detecting the significance of the FST (the number of repetitions is 1000) by using a permutation test (persistence test); and the Polymorphic Information Content (PIC) was calculated according to the following formula:
in the formula: pi and pj are the frequency of the ith and jth alleles, respectively, and n is the number of alleles.
Results and analysis:
1. and (3) PCR amplification result:
comparing and analyzing 89 samples of 3 breeding generations of the selected 15 pairs of microsatellite primers; because of different primer sequences, the number of alleles amplified by each primer is 3-9 respectively, and the number of the alleles is 101 in total, and the size range is 130-300 bp (table 1 and table 3); FIGS. 1 and 2 show the amplification maps of primers GM047 and GM294, respectively, over 3 breeding generations.
2. Microsatellite genotyping results:
a total of 101 alleles were detected at 15 microsatellite loci in this study, with 91 detected at F13, 77 detected at F14, and 76 detected at F15 (table 3).
TABLE 3 number of alleles of F13/F14/F15
Details of the alleles amplified from the 15 microsatellite loci over 3 generations of breeding are shown in Table 4.
Among these 15 sites, UNH846 amplified the highest number of allele factors (10) and the lowest GM578 (4). F15 presents two unique alleles at the site of UNH896, possibly due to mutations. The allele frequencies ranged from 0.0167 to 0.7667.
TABLE 4 allele frequencies of the "New Jifu" Tilapia F13-F15 population at 15 microsatellite loci
3. Microsatellite identification marker between breeding lines
Further comparing the frequency of all the alleles amplified by each microsatellite marker in three breeding generations (FIG. 3-FIG. 6), it was found that the frequency of 3 alleles (GM294-195bp, GM532-241bp and GM532-261bp) amplified by 2 microsatellite markers (GM294 and GM532) decreased with the progression of the breeding generations (i.e., F13> F14> F15), wherein, the frequencies of the 195bp allele amplified by GM294 in 3 breeding generations are respectively F13(0.5172), F14(0.2500) and F15(0.1833) (FIG. 3), the frequencies of the 241bp allele amplified by GM532 in 3 breeding generations are respectively F13(0.2414), F14(0.1500) and F15(0.1000) (FIG. 4), and the frequencies of the 261bp allele amplified by GM532 in 3 generations are respectively F13(0.5517), F14(0.5000) and F15(0.3500) (FIG. 4).
And the occurrence frequency of 4 alleles (GM294-175bp, GM294-183bp, GM532-271bp and GM323-150bp) amplified by other 3 microsatellite markers (GM294, GM532 and GM323) is increased along with the progressive breeding generations (namely F13< F14< F15), wherein the occurrence frequency of the 175bp alleles amplified by GM294 in the 3 breeding generations is F13(0.1379), F14(0.2333) and F15(0.4000) respectively (FIG. 3); the frequency of occurrence of the 183bp allele amplified by GM294 in 3 breeding generations was F13(0.0862), F14(0.1000) and F15(0.1500), respectively (FIG. 3); the occurrence frequency of the 271bp allele amplified by GM532 in 3 breeding generations was F13(0.1207), F14(0.2167) and F15(0.3000), respectively (FIG. 4); the 150bp allele amplified by GM323 appeared in 3 generations of breeding at F13(0.3103), F14(0.6667) and F15(0.7667), respectively (FIG. 5).
Notably, the number of alleles amplified by 1 microsatellite marker (UNH846) decreased with the progression of breeding generations (i.e., F13> F14> F15), wherein the number of alleles amplified by UNH846 over 3 breeding generations were F13 (9), F14 (7) and F15 (3), respectively (FIG. 6).
In conclusion, the inventor believes that GM294, GM532, GM323 and UNH846 can be used as identification markers among 3 breeding generations according to the identification frequency that ① GM294-195bp, GM532-241bp and GM532-261bp reduce generation by generation among 3 breeding generations, or the occurrence frequency that ② GM294-175bp, GM294-183bp, GM532-271bp and GM323-150bp increase generation by generation among 3 breeding generations, or the trend that the number of alleles amplified by ③ UNH846 reduces generation by generation among 3 breeding generations.
4. Genetic differences among breeding groups:
the similarity coefficient and genetic distance between the breeding populations are shown in Table 5. The similarity coefficient among different groups is 0.8401-0.9360, and the similarity coefficient after deviation correction is 0.8536-0.9492. The similarity coefficient and genetic distance between the populations obtained by the two calculation methods are slightly different, but both indicate that 3 populations have certain genetic differentiation. Wherein, the similarity coefficient between F13 and F14 and F15 is 0.8706 and 0.8536 respectively, which are relatively low; whereas the similarity coefficient between F14 and F15 is 0.9492, which is relatively high. The genetic distances between F13 and F14, F15 were 0.1386 and 0.1583, respectively, and were relatively large; whereas the genetic distance between F14 and F15 is 0.0522, which is relatively small. This result indicates that the genetic difference between the breeding generations is gradually reduced.
TABLE 53 Nei similarity coefficient (over diagonal) and genetic distance (under diagonal) for breeding generations
5. And (3) breeding genetic diversity among generations:
the allelic factor can reflect the genetic variation of a population at multiple sites, and is one of important parameters for measuring the genetic diversity of the population. A total of 101 alleles were detected at 15 microsatellite loci in this study, with 91 detected at F13, 77 detected at F14, and 76 detected at F15. As shown in table 6, the average allelic factors of the 3 breeding generations were F13(6.0667) > F14(5.1333) > F15(5.0667) in sequence, and the average effective allelic factors were F13(3.6543) > F14(3.1536) > F15(2.8139) in sequence, and as a whole, there was a tendency of decreasing with the progression of the breeding generations, but the decrease was smaller in magnitude, and the t-test showed no significant difference between the breeding generations (P >0.05) (table 8).
TABLE 6 genetic diversity of the "New Jifu" Tilapia F13-F15 population
Note: na: an average allelic factor; NE: an average effective allelic factor; ho: averagely observing the heterozygosity; he:
average expected heterozygosity; i: shannon diversity index; PIC: average polymorphic information content.
Polymorphic information content, genetic heterozygosity and Shannon diversity index are common parameters for measuring population genetic diversity. In the research, the variation ranges of 4 genetic parameters of the average observed heterozygosity (Ho), the average expected Heterozygosity (HE), the Polymorphic Information Content (PIC) and the Shannon diversity index (I) of 15 microsatellite loci in 3 breeding generations are respectively as follows: 0.37111-0.46667, 0.61680-0.69470, 0.56245-0.63977 and 1.1835-1.4114 (Table 6), the 4 parameters show a trend of decreasing with the increase of the number of breeding generations in 3 breeding generations (FIG. 7). We note that the average observed heterozygosity for each of the 3 breeding generations was slightly lower than the average expected heterozygosity, indicating that there was some loss of heterozygosity.
The results of molecular analysis of variance (AMOVA) showed that the variance among the populations accounted for 4.81% of the total variance, while the variance within the populations accounted for 95.19% of the total variance, as shown in Table 7, indicating that the majority of the genetic variation of the breeding populations originated from the genetic differences among individuals within each generation.
TABLE 7 molecular ANOVA of New Guifu Tilapia F13-F15
The paired comparison FST values and t-test P values for the genetic diversity parameters are shown in table 8. The FST value range between generations is 0.02156-0.06752, and the genetic differentiation between two generations is not significant (P is more than 0.05) through inspection.
TABLE 8 pairwise comparison of FST values between New Jifu Tilapia F13-F15 populations (bottom left corner) and t-test P values for genetic diversity parameters (top right corner)
The results show that:
(1) on 15 microsatellite loci, 101 alleles are detected in total, and the length of an amplified fragment is 130-300 bp.
(2) The average effective allele Number (NE), average expected Heterozygosity (HE) and average Polymorphic Information Content (PIC) ranges of 3 breeding generations are respectively as follows: 3.6543-2.8139, 0.69470-0.61680 and 0.63977-0.56245 all show a trend of stable decline along with the progressive breeding generations; no significant difference exists among generations through t test (P > 0.05).
(3) The FST value between every two of 3 breeding generations is 0.02156-0.06752, and no significant genetic differentiation exists between generations (P is more than 0.05). (4)4 microsatellite markers (GM294, GM532, GM323 and UNH846) can be used as identification markers in 3 breeding generations.
Therefore, the genetic homogeneity of the new Jifu tilapia in the population shows the trend of generation-by-generation improvement in the long-term artificial directed breeding process, and the genetic differentiation is generated to a certain degree among the populations.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the present specification and the drawings, which are directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (5)
1. A microsatellite identification primer for tilapia and genetic diversity of tilapia is characterized in that the microsatellite identification primer comprises 15 pairs which are respectively: UNH904, GM532, UNH846, UNH1007, HM370073, HM370078, HM370085, HM370111, UNH878, UNH896, UNH901, GM047, GM294, GM323 and GM578, wherein the microsatellite identification primers are selected by searching 50 published Nile tilapia microsatellite loci from a GenBank database and designing primers by using Primer5.0 software.
2. A microsatellite identification method for tilapia and genetic diversity thereof is characterized by comprising the following steps:
(1) selecting materials: respectively selecting new Jifu tilapia breeding lines F13, F14 and F15, wherein the number of samples F13 is 29, the number of samples F14 and F15 is 30, cutting each tail of the tail fin, numbering and storing in 95% ethanol for later use;
(2) extraction of genomic DNA: extracting the genome DNA of the tail fin of each sample in the step (1) by using a tissue/cell genome DNA rapid extraction kit, detecting the concentration and purity of the DNA by agarose gel electrophoresis, numbering the detected DNA extracts, and storing the DNA extracts in a refrigerator at the temperature of minus 40 ℃ for later use;
(3) screening microsatellite primers: searching 50 published nile tilapia microsatellite loci from a GenBank database, designing primers by using Primer5.0 software, and screening 15 pairs of effectively amplified microsatellite primers which are respectively: UNH904, GM532, UNH846, UNH1007, HM370073, HM370078, HM370085, HM370111, UNH878, UNH896, UNH901, GM047, GM294, GM323, GM 578;
(4) performing microsatellite primer PCR reaction, electrophoretic detection and dyeing: the total volume of the PCR amplification reaction was 10. mu.L, containing 1L of genomic DNA template (20 ng/. mu.L), 0.5L (0.5. mu.M) of each of the forward and reverse primers, 5L of buffer (0.2mM dNTPs, 1.5M MgCl2, 0.5M Taq DNA polymerase), 3L of sterile deionized water; detecting the PCR product by using non-denaturing polyacrylamide gel electrophoresis, wherein the gel concentration is 8 percent, and the electrophoresis buffer solution is 1 xTBE; after electrophoresis is finished, dyeing is carried out by using a rapid silver staining method, and the amplification condition of the PCR product is observed;
(5) and (3) data analysis: judging according to the DNA migration distance on the gel, and if the electrophoresis strip shows one strip and the migration distances are consistent, considering that the allele locus is homozygous; if two bands appear, the seat is considered to be heterozygous; estimating the molecular weight size of each band in the electrophoretic bands by using AlphaEaseFC 4.0 software, converting the size into letters, and calculating the allele factor (Na), the effective allele factor (NE), the observed heterozygosity (Ho) and the expected Heterozygosity (HE) of each population by using POPGENE software (Yeh et al, 1997); calculating an inter-population genetic similarity coefficient and an inter-population genetic distance according to the method of Nei (1972,1978); calculating a population genetic differentiation index FST by using ARLEQUIN 3.5 software (Excofnier et al,2010), estimating genetic differentiation among populations, analyzing molecular variance (AMOVA) in and among populations, and detecting the significance of the FST (the number of repetitions is 1000) by using a permutation test (persistence test); and the Polymorphic Information Content (PIC) was calculated according to the following formula:
in the formula: pi and pj are the frequency of the ith and jth alleles, respectively, and n is the number of alleles.
3. The method for microsatellite identification of tilapia mossambica and genetic diversity thereof according to claim 2, wherein the amplification process of PCR in step (4): pre-denaturation at 94 ℃ for 4min followed by 35 cycles, each cycle comprising denaturation at 94 ℃ for 30s, annealing for 45s (see table 1 for specific annealing temperatures for each primer pair), and extension at 72 ℃ for 1 min; after 35 cycles, extension was carried out at 72 ℃ for 10min and the PCR product was stored at 4 ℃ for detection.
4. The method for identifying tilapia mossambica and microsatellite of genetic diversity thereof according to claim 2, wherein the reagent formulation of said non-denatured polyacrylamide gel in step (4) is as follows:
(1)5 × TBE: 54g of Tris alkali, 27.5g of boric acid and 2.92g of EDTA, adding double distilled water to a constant volume of 1L, and storing at room temperature;
(2) 30% acrylamide (29% acrylamide + 1% N, N' -methylenebisacrylamide): adding 29g of acrylamide and 1g of N, N' -methylene bisacrylamide into 60mL of double distilled water, heating to 37 ℃ to dissolve the reagent, and fixing the volume to 100 mL; the solution can be prepared 1 month in advance, and stored in brown bottle at 4 deg.C;
(3) 10% Ammonium Persulfate (APS): 1g of ammonium persulfate is dissolved to 10mL by adding double distilled water, and the ammonium persulfate is preserved at 4 ℃ in a dark place and is prepared fresh every week;
(4) 8% polyacrylamide gel buffer (50 mL): 13.3mL of 30% acrylamide, 10mL of 5 XTBE, 350 mu L of 10% ammonium persulfate, 17.5 mu L of TEMED and 26.35mL of double-distilled deionized water;
(5) staining solution (0.1% silver nitrate): 0.5g of silver nitrate, adding double distilled water to a constant volume of 500mL, and preparing the silver nitrate fresh before each use;
(6) color development liquid: 10g of sodium hydroxide, 0.2g of sodium carbonate and 750 mu L of 37 percent formaldehyde, adding double distilled water to a constant volume of 500mL, and preparing the mixture fresh before each use;
(7) stopping liquid: double distilled water.
5. The method for microsatellite identification of tilapia mossambica and genetic diversity thereof according to claim 2, wherein the staining procedure in step (4) is as follows:
(1) rinsing: rinsing with double-distilled deionized water twice, each for 5 min;
(2) dyeing: soaking the gel in 0.1% silver nitrate, and gently mixing in shaking table for 8 min;
(3) rinsing: rapidly rinsing twice (each time not exceeding 5s) with double distilled deionized water;
(4) color development: adding color development solution (prepared temporarily), adding 750L of 37% formaldehyde when in use, and gently mixing until the DNA bands are clear (about 10 min);
(5) fixing: soaking in double distilled water;
(6) and (3) storage: and after color development, taking a picture by using a digital camera and storing the picture.
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