Analysis of genetic variation in Drosophila pools by AFLP markers.

Freschi, Ana Paula P., Marlene K.H. Kobayashi, and Wlademir J. Tadei.  Departamento de Biologia, Universidade Estadual Paulista-UNESP, Rua Cristóvão Colombo 2265. CEP 15054-000, São José do Rio Preto-SP, Brazil.

      The analysis of genetic variation using DNA fingerprinting techniques has become an important approach in the taxonomy, population genetic and evolutionary studies of a variety of animal species. The most frequently used DNA markers include restriction fragment length polymorphism (RFLP) of nuclear or mitochondrial DNA (Gibson, 1989;  Gibson and Whittington, 1993), DNA fingerprinting of microsatellite sequences, standard polymerase chain reaction – PCR (Beckmann and Soller, 1990; Su and Wellems, 1996), and random amplified polymorphic DNA analysis (RAPD) of nuclear DNA (Williams et al., 1990;  Welsh and McCleland, 1990). Recently, a novel DNA fingerprinting technique called AFLP (Amplified Fragment Length Polymorphism) was presented by Vos et al. (1995). The AFLP technique is based on selective amplifications of a DNA fragment subset generated by restriction enzymes and ligated to adapters of known sequences. Although AFLP markers have been ordinarily studied in plants (for example, Maughan et al., 1996;  Piepho and Kock, 2000;  Jansen et al., 2001), they have been hardly reported in the DNA of insects. The purpose of this study was to detect genetic variation in two Drosophila sibling species by the use of AFLP markers in samples formed by a variable number of individuals.

Figure 1.  Electrophoresis on 8% denaturing polyacrylamide gel of the amplified products with the primer 7 to pools of 5, 7, 10, 15 and 20 individuals of Drosophila saltans (S6 strain) and of D. prosaltans (P76 strain). M = molecular size standard (100 bp Ladder); B = negative control.

      In this study, two sibling species were used, Drosophila prosaltans and D. saltans, which belong to the saltans subgroup of the saltans group (Magalhães, 1962). The strain of D. prosaltans (P76) used is a mixture of two Brazilian strains, one from Eldorado (State of Rio Grande do Sul) and the other from Belém (State of Pará), and the strain of D. saltans (S6) comes from Huychiauyan, Mexico (Tadei and Bicudo, 1981).  Males of these two strains were etherized, individually separated in tubes of 1.5 ml and frozen –20^oC until later extraction of the DNA.

      The genomic DNA was individually extracted and then reunited in pools of 5, 7, 10, 15 and 20 flies for Drosophila prosaltans and D. saltans, respectively. The use of pools is recommended because the analysis of a single individual can led to erroneous conclusions if this individual is not representative of the genetic variability of the population or species studied. Each pool was formed by a mixture of 3 ml of the extracted DNA, whose individual spectrophotometric measurement was around 80 ng/ml of solution.  All pools of DNA have been analyzed using the "AFLP^Ò Analysis System I", Gibco-BRL Products, in a thermocycler (MJ Research Minicycler), according to description of Vos et al. (1995) and to Vandermark (1999) modification.  The technique consists basically of four stages: restriction of the DNA with EcoRI and MseI, ligation of oligonucleotide adapters, preampli-fication and selective amplification.  For the selective amplification, 15 combi-nations of primers of three nucleotides have were tested:

Primer 1 ®  E-ACA/M-CAC

Primer 2 ®  E-AAC/M-CTT

Primer 3 ®  E-AAG/M-CTC

Primer 4 ®  E-ACC/M-CTA

Primer 5 ®  E-AGC/M-CTG

Primer 6 ®  E-AGG/M-CAG

Primer 7 ®  E-ACT/M-CAA

Primer 8 ®  E-ACG/M-CAT

Primer  9  ®  E-AAC/M-CTC

Primer 10 ®  E-AAG/M-CAG

Primer 11 ®  E-ACA/M-CAC

Primer 12 ®  E-ACC/M-CTG

Primer 13 ®  E-AGG/M-CTT

Primer 14 ®  E-AGC/M-CAA

Primer 15 ®  E-ACA/M-CAC

Figure 2.  Electrophoresis on 8% denaturing polyacrylamide gel of the amplified products with the primer 6 to pools of 5, 7, 10, 15 and 20 individuals of Drosophila saltans (S6 strain) and of D. prosaltans (P76 strain). White arrows indicate the absent bands and black arrows indicate the exclusive bands in pool of 7 individuals of the strain P76. M = molecular size standard (100 bp Ladder); B = negative control.

     The separation of the fragments for electrophoresis was made on 8% denaturing polyacrylamide gel applying 7 ml of DNA amplified of each pool, including the negative control and the marker of molecular size DNA Ladder 100 bp (Gibco-BRL).  The gel was stained with silver nitrate and later dried between two sheets of cellophane paper and 5% gelatin. The gels, after being dried, have been analyzed with fluorescent light for counting and classification, regarding  presence or absence of bands.

      From the 15 tested combinations of primers in the reactions of selective amplification of the DNA, five of them presented good amplification quality, with well-defined bands and in adequate number for the analysis. Only the DNA fragments with size lower than 1500 bp were considered. The pattern of bands of the amplified fragments of D. saltans is distinct from the pattern of D. prosaltans, except for the occurrence of common bands. The number of bands produced by the five primers varied from 19 to 37 in D. saltans, a total of 132 bands, 6 of which are exclusive for this species.  In D. prosaltans,  the number varied from 22 to 42, resulting in a total of 164 bands, being 12 exclusive.

      Amongst the five primers selected, primer 5 (E-AGC/M-CTG) was the one which presented the most diverse pattern of bands among the pools studied. The biggest difference occurred in D. prosaltans, where each pool presented a unique banding pattern, even having some shared bands.  In D. saltans the pattern of bands was similar for three pools (5, 7 and 10); however, it differed from the other two (15 and 20 flies).

      Regarding the other primers, there was roughly no variation in the pattern among different pools of flies of the same species, when the same primer is used.  Figure 1 presents the products amplified by primer 7 (E-ACT/M-CAA), which is an example of the pattern of bands shown by the two species.  For primer 6 (E-AGG/M-CAG), the characteristic pattern of bands of D. saltans was the same for all pools;  however, in D. prosaltans the band pattern of the pool of 7 flies differed from the characteristic shown by the other pools, due to the absence of two bands and presence of two others (Figure 2).

      The AFLP technique was shown to be efficient for studies with insects emphasizing genetic variability of the DNA of Drosophila sibling species.  In pools formed by DNA samples of 10, 15 or 20 individuals, in four of five tested combinations of primers, invariability in the banding pattern was observed. Therefore, the use of DNA samples of 10 individuals is recommended, because besides being operationally viable in the routine of the laboratory, it assures a good probability of the samples contain the genetic variability of the studied species.  Besides the size of the sample, the choice of the combinations of primers to be used is an important factor in the studies with AFLP markers to provide evidence of the genetic variability in populations.

      Acknowledgments:  The authors would like to thank Dr. Luiz R. Goulart Filho for suggestions and Solange Aranha for English language review. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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