Background

Genetically improved farmed tilapia, GIFT, is a Nile tilapia strain directly introduced by the International Centre for the Management of Aquatic Biological Resources (ICHRM) through four African origin (Egypt, Ghana, Kenya, Senegal) and four widely cultivated Nile tilapia strains (Israel, Singapore, Thailand, Taiwan of China) in Asia. China existing GIFT tilapia is mainly the offspring of third generation of tilapia imported from Philippines in two batches in June and September 1994 by College of Fisheries and Life Science of Shanghai Ocean University, which was confirmed as a new tilapia variety in 2006 by the national aquaculture and Seed Certification Committee after many generations of breeding (Li, 2001; Li and Cai, 2008). GIFT tilapia has the advantages of fast growth, high yield, wide food, strong ability to adapt to the environment and delicious meat. It has been widely cultured in China and has become one of the important freshwater breeding varieties in China. Therefore, the detection and genetic analysis of the GIFT tilapia populations to reveal the genetic diversity and inter-species genetic difference of the population were significant for the breeding of the fine species of GIFT tilapia and the molecular marker assisted breeding.

Holland scientists Zabeau and Vos (1993) developed a new molecular marker technique for the detection of DNA which is polymorphism amplified fragment length polymorphism, AFLP. This technique has the characteristics of large amount of information, high sensitivity, conforming to Mendelian heredity, and does not need to know the genetic background of the research object in advance, so it has been f it has been widely used in various fields of biology by the vast majority of researchers. At present, AFLP markers have been widely used in genetic diversity analysis, genetic map construction, gene location and analysis, germplasm resources identification and so on. Yang et al. (2006) have used AFLP technique to analyze the genetic diversity of Oreochromis Mossambicus and Oreochromis Hornorum population. The results showed that the genetic diversity of these two populations was abundant. And the genetic diversity of Oreochromis Mossambicus population was more abundant. Li et al. (2007) have used AFLP to analyze the genetic diversity of different breeding populations of GIFT tilapia. The results showed that GIFT tilapia had potential for further breeding. Kocher et al. (1998) and Jeremy et al. (2000) constructed Genetic maps of tilapia from different sources by AFLP markers. AFLP technique was used to analyze the genetic characteristics of GIFT tilapia population (F16), which might provide a theoretical basis for the subsequent breeding of GIFT tilapia.

1 Results and Analysis

1.1 DNA extraction, enzyme digestion, pre-amplification results

Partial genomic DNA of GIFT tilapia (genetically improved farmed tilapia) and AFLP pre-amplified products which were double enzyme digested by Ecor I and MSE I were analyzed by 1% agarose gel electrophoresis. The results are shown in from Figure 1, Figure 2, and Figure 3.

1.2 Results of selective amplification of AFLP

The selected 5 pairs of primer combinations were used to amplify the genomic DNA of GIFT tilapia F16, and some electrophoresis results were shown in Figure 4.

1.3 Data analysis results

AFLP amplification was carried out on individuals from Gifel tilapia F16 by using the primer combination E-AGG/M-CTT, E-AGG/M-CTG, E-AAC/M-CAG, E-ACA/M-CTG, E-ACA/M-CAG. A total of 187 loci were amplified from 27 individuals. The number of loci amplified by each pair of primer combinations ranged from 32 to 55, with an average of 37.4 loci per primer pair, including 163 polymorphic loci and 87.17% polymorphic loci. Among them, the maximum number of E-AGG/M-CTT was 55, and the minimum number of bands detected by E-AAC/M-CAG was 32. The primer combination with the highest proportion of polymorphic loci was E-AGG/M-CTT, which was 100.00%, and the lowest was E-AGG/M-CTG, which was 73.53%. As shown in Table 1. AFLP genetic marker obeyed the Mendelian law (Lerceteau and Szmidt, 1999). Therefore, one amplified fragment could be regarded as a gene, and the combination of amplified fragments of one individual could be regarded as the genotype of this individual. Table 1 showed that the genotypes detected by 5 pairs of primer combinations were consistent with the number of experimental specimens, which was, the amplification patterns of each individual were different from those of other individuals, and the primer combinations could effectively detect this difference.

The average Nei gene diversity (H) of GIFT tilapia (F16) was 0.288,6 by Pop-Gene analysis. The highest value of E-AAC/M-CAG was 0.306,2, and the lowest value was 0.263,8. The average Shannon’s information index (I) was 0.433,9, in which the highest value of E-AGG/M-CTT was 0.458,0, and the lowest value of E-AGGM-CTG was 0.388,3, as shown in Table 2.

2 Discussion

Amplified fragment length Polymorphism (AFLP) is based on PCR. First, the genomic DNA was cut by two kinds of restriction endonuclease, and then the double strand joint was connected to the end of the DNA fragment. The primer binding sites were the joint sequence and the sequence of the endonuclease site adjacent to the joint sequence, respectively. Two endonuclease used were six base common cleavage enzyme and four base rare cleavage enzyme (Chen, 2005). AFLP has the stability of RFLP and the high efficiency of PCR technology, and can obtain abundant and stable genetic markers. AFLP is considered to be one of the ideal molecular markers, which has been widely used in genetic map construction, genetic diversity analysis, phylogeny and taxonomy, genetic breeding and germplasm identification, etc.

The proportion of polymorphic loci is an important index to measure population diversity. In this experiment, 187 loci were obtained by using 5 pairs of primer combinations, including 163 polymorphic loci and the proportion of polymorphic loci was 87.17%, which indicated that the breeding population had abundant genetic diversity. Wang et al. (2002) used 5 pairs of primer combinations to study wild populations and two breeding populations of Guanjingyang Pseudosciaena Crocea of Fujian Province, the percentage of polymorphic loci was 76.6%, 70.6% and 69.2%, respectively. Kuang et al. (2007) used 12 pairs of primer combinations to study the Huma River taimen of the Huma River in Heilongjiang River. The proportion of polymorphic loci was 84.43%. The genetic polymorphism obtained in this experiment was slightly higher than that of the above fish, which might be related to the strict control of inbreeding in the breeding of GIFT tilapia population used in this study. However, Jie et al. (2011) used AFLP markers to study the genetic variation of GIFT tilapia. The results showed that the percentage of polymorphic loci in F0 generation was 51.13, in F6 generation was 48.17%, in F7 generation was 47.11%, in F8 generation was 46.11%, and in F9 generation was 43.18%, which showed a downward trend, which was different from the results obtained in this experiment. This might be related to the selection of the breeding population and the selection of primers and image processing methods.

Average Nei’s gene diversity (H), also called genetic heterozygosity, is the average heterozygosity of each locus, which can reflect the genetic variation of each population at several loci. It is generally considered to be a more suitable parameter to measure population genetic variation (Song et al., 2008). Average Nei’s gene diversity of F16 population of GIFT tilapia was 0.288,6, average Shannon’s information index (I) was 0.433,9. These results were obviously higher than that of Li (2007) on genetic diversity of different breeding populations of Tilapia japonicas, and also showed that the experiment group deposit had rich genetic diversity and maintained a high genetic heterozygosity, had value of further breeding and development.

3 Materials and Methods

3.1 Sample source

The experiment used 27 tilapia tails, the 16th generation group (F16) collected by Guangdong Huazhou Guanghui farm co., LTD in January 2011. Back muscles were taken to put in absolute alcohol, reserved at -20°C.

3.2 Extraction of genomic DNA

Genomic DNA extraction was carried out by phenol/chloroform method (Lu, 1999) and stored at 4°C by 1% agarose gel electrophoresis.

3.3 AFLP analysis

3.3.1 Genomic DNA digestion

Restriction endonuclease Ecor I and MSE I were used for double enzyme digestion. First, the MSE I endonuclease was used for enzyme digestion, and the reaction system was as follows: 10×Tango Buffer 2.5 μL, Mse I 0.5 μL (10 U/μL), DNA template 3 μL, the double distilled water 14.0 μL. After 3 h of warm bath at 65°C, EcoR I endonuclease 0.5 μL (10 U/μL) and double distilled water 1.5 μL (37°C) were added, enzyme digestion at 37°C for 3 h, then inactivated at 85°C for 15 min, waiting for connection.

3.3.2 Coupled reaction

The ligation reaction was carried out immediately after the enzyme digestion. Each reaction system consisted of 10 × Buffer 2 μL, T4 ligase 0.5 μL (5 U/μL), the double digested product 10 μL, Mse I connected with 0.5 μL (50 μmol/L), EcoR I connected with 0.5 μL (5 μmol/L), and the double distilled water was added to 20 μL for overnight at 20°C.

3.3.3 AFLP pre-amplification reaction

The product was diluted 10 times and used for AFLP pre-amplification. The pre-amplified primers were as follows: EcoR I pre-amplification primer (E-p): 5’-GACTGCGTACCAATTC-3’; Mse I pre-amplification primer (M-p): 5’-GATGAGTCCTGAGTAA-3’. The reaction system was as follows: 10×PCRBuffer (containing Mg²⁺) 2.5 μL, E-p 1 μL (5 μmol/L), M-p 1 μL (5 μmol/L), dNTPs 2 μL (2.5 mmol/L), bonding product (dilution 10 times) 4 μL, Taq enzyme 0.2 μL (5 U/μL), add double steamed water 14.3 μL. Reaction procedure: first, 2 min of pre-denaturation at 94°C, then, 30 s of denaturation at 94°C, 45 s of annealing at 56°C, 60 s of extention at 72°C, carried out 20 cycles; at the final, 10 min of extention at 72°C. AFLP pre-amplification reaction was detected by 1% agarose gel and the products were preserved at 4°C.

3.3.4 AFLP selective amplification reaction

The AFLP primer combinations were as follows: E-AGG/M-CTT; E-AGG/M-CTG; E-AAC/M-CAG; E-ACA/M-CTG; and EACA/M-CAG. The reaction system was as follows: 10×PCRBuffer (containing Mg2+) 2.0 μL, EcoR I primer1.0 μL (5 μmol/L), Mse I primer1.0μL (5 μmol/L), dNTPs 1.5 μL (2.5 mmol/L), pre-amplification products (dilution 20 times) 3.0 μL, Taq enzyme 0.2 μL (5 U/μL), add double steamed water supplemented to 25 μL.

AFLP selective amplification reaction procedure adopts “touch-down” strategy, reaction procedure: 4 min of pre-denaturation at 94°C, 30 s of denaturation at 94°C, 30 s of annealing at 65°C, 60 s of extention at 72°C, carried out 12 cycles, the annealing temperature of each cycle was reduced 0.7°C, and other conditions remain unchanged. Then 24 cycles were carried out, and the reaction conditions were as follows: 30 s of denaturation at 94°C, 30 s of annealing at 56°C, 60 s of extention at 72°C; at the final, 10 min of extention at 72°C, and the amplified product was stored at 4°C.

3.4 Detection of AFLP selective amplification products by polyacrylamide gel electrophoresis

AFLP products were amplified by 6% polyacrylamide gel electrophoresis, silver staining (Wang et al., 2000) and took pictures by a digital camera.

3.5 Data statistics

Using Gel-Pro analyzer 4.0 software to read electrophoresis map and established 1/0 matrix. The band (dominant phenotype) was 1, and the no band (invisible phenotype) was 0. PopGene32 was used to analyze the number of alleles (Na), effective allele number (Ne), polymorphism site ratio, Nei’s gene diversity index (H) (Nei, 1973) and Shannon’s index (I) (Lewontin, 1972).

Authors’ contributions

YT is responsible for the operation of the experiment and the writing of the article. WZD provided guidance and assistance for the operation, data processing and the writing of the experiment. LCW is in charge of the project that is responsible for the selection, design and revision of the experiment. GYS and LL provide guidance and help for this experiment operation and article writing. All the authors read and approved the final text.

Acknowledgments

Thanks for the financial support of Guangdong Marine Fisheries Science and Technology Research and Development Project (A201101B02; A201008C03). Thanks for the experimental materials provided by Guanghui Farm Limited Company, Huazhou (Guangdong, China). Thank Professor Luo Jie for his support and assistance in sample collection.

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