DNA Extraction and RAPD Profiling

Whole genomic DNA was extracted by grinding the whole leg in liquid nitrogen. The obtained fine powder was homogenized in 500 jmL buffer (100 mM Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0), 0.5% SDS, and 50 mM DTT). The homogenate was incubated at 40°C for four hours in the presence of RNAse (1 unit), and proteinase K (0.25 mg). Proteins were removed by extraction, first with phenol-chloroform and then with chloroform-isoamyl alcohol (2%). DNA was concentrated by ethanol precipitation (Sambrook et al. 1989). DNA samples were stored in TE 1X at a standardized concentration of 50 ng/^L.

RAPD-PCR markers have been successfully used to detect genetic polymorphism in other Odonata species (Andrés et al. 2000). We tested a total of 60 deca-meric random oligonucleotides (0PA01-0PA20, 0PD01-0PD20, 0PE01-0PE20, Operon Technologies, Inc.) in order to find polymorphism in a subsample of three individuals per species and from different localities. Eight of the primers (four per species, Table 2) proved to be highly polymorphic and were used to screen the entire collection.

Amplification reactions were performed in a final volume of 25 jmL containing 1 jmL 10X reaction buffer (Sigma), 1.5 mM MgCl2, 10 mM dNTPs, 2 pmol primer, 1 U Taq (DNA polymerase (Sigma)), and 50 ng genomic DNA. The thermocycler program used for amplification was: initial denaturation step (94°C for 5 min), 45 cycles (94°C for 1 min, 35°C for 1 min, 72°C for 2 min), final extension (72°C for 5 min), and cooling at 4°C. A negative control was always included to monitor any

Table 2 RAPD primers (Operon Technologies Inc.) and summary of RAPD markers obtained for each studied species

Primer name

Primer sequence (5 ' -3 ' )

Macromia splendens

Oxygastra curtisii

OPA-01

CAGGCCCTTC

12, 50-1,000, 6*

-

OPA-02

TGCCGAGCTG

15, 50-750, 7

-

OPA-04

AATCGGGCTG

11, 50-750, 8

-

OPA-08

GTGACGTAGG

-

13, 50-750, 5

OPC-09

CTCACCGTCC

-

17, 50-750, 11

OPA-09

GGGTAACGCC

10, 50-1,000, 4

-

OPE-11

GAGTCTCAGG

-

9, 50-1,000, 6

OPD-01

ACCGCGAAGG

-

10, 150-500, 11

*The three values provided account for (1) number of scored bands, (2) size range of the marker (in base pairs), and (3) number of polymorphic bands (shared by < 99% individuals)

*The three values provided account for (1) number of scored bands, (2) size range of the marker (in base pairs), and (3) number of polymorphic bands (shared by < 99% individuals)

possible DNA contamination. Fragments were resolved on 1.5% agarose gels and sized against a one Kilobase ladder (Sigma). Gels were run for 1.75 h at 120 V/400 A. DNA bands, stained with ethidium bromide, were visualized under UV light and photographed. All amplifications were repeated twice to prove the reproducibility of the RAPD-PCR bands. Only well-amplified and reproducible bands were considered for further analyses. RAPD bands were scored as present (1) or absent (0), and a binary matrix of different RAPD phenotypes was so assembled. RAPD bands shared by 99% or more individuals of the same species across the whole dataset were regarded as monomorphic and excluded from genetic analyses.

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