Formalin can cross-link with the biological macromolecules such as proteins and DNA to produce a stable, closed cross-networking, so that it can maintain a good shape for organism and is widely used for long-term preservation of specimens. Formalin-fixed samples contain DNA, but the classical method was very difficult to extract genomic DNA and DNA fragment degraded seriously [1] [2] . With the birth and development of PCR, it is possible to extract and amplify DNA directly from DNA from formalin fish fin tissues. In this study, the developed DNA extraction method from formalin fish (Coilia macrognathos) fin tissues by pretreatment with a gradient of ethanol system is used to study the genetic structure and phylogenesis of endangered fish, using the specimens in museum and herbarium.

Six specimens of Coilia macrognathos were conserved in Fishery Research Institute of Hunan Province for 35 years. Approximately 200 mg of fin tissue from each specimen was cut into pieces and treated with a gradient of ethanol (65%, 70%, 75%, 80%, 85%, 90%, 95% and 100%), each for 20 minutes, centrifuged 3000 g × 15 minutes to gradually remove the formalin. Then they were put into the dryer for overnight. The fin tissue was subsequently digested with 500 μL cell lysate ( 10 M NaCl, 500 mM Trisbase, 20 mM EDTA and 0.5% SDS, pH 8.0) and 20 μl protenase K (20 mg/ml). The samples were incubated at 55˚C overnight, after which 500 μL of 4.5 M NaCl and 300 μL chloroform were added to each sample. Samples were vortexed for 1 min at maximum speed, and tubes were centrifuged for 10 min at 10, 000 g . The supernatant was transferred to fresh tubes. An equal volume of isopropanol was added to each sample, mixed well, and samples were incubated at ?20˚C for 30 min. Samples were then centrifuged for 20 min, 4˚C, at 10, 000 g . The deposit was washed with 70% ethanol, dried and finally were dissolved in 500 μL dd H₂O.

The quantity and quality of DNA was measured using a UV-2450 Spectrophotometer (Shimaseiki, JAPAN). Additionally, DNA quality was visually checked on 1.0% agarose gel after staining with ethidium bromide. Furthermore, PCR amplification for mtDNA cyt b gene was carried out with a 50 μL reaction volume with a final concentration of 1× Taq polymerase buffer and 0.6 U of Taq polymerase, 1.5 mM MgCl₂, 50 mM of each dNTP and 2.5 pM of each primer. Primers used in the experiment were as follows: L14322 (forward) 5'-CGC- CTGTTTATCAAAAACAT-3'; H15576 (reverse): 5'-GCGCTAGGGAGGAATTTAACCTCC-3' for the cyt b gene [3] . The thermal profile for hot-start polymerase chain reaction (PCR) included an initial denaturation 94˚ for 5 min, followed by 35 cycles of 30 s at 94˚C, 30 s at 54˚C, 60 s at 72˚C and a final cycle of 10 min at 72˚C. Amplicons were purified with QIAquick kit (QIAGEN) following the manufacturer’s instructions. Both strands were sequenced using an ABI 3730XL Genetic analyser (Applied Biosystems, Inc.).

The results show that DNA can be extracted from formalin fish fin tissues by pretreatment with a gradient of ethanol. Agarose gel visualization of the DNA confirmed this. Genomic dna molecular weight, extracted from Tenualosa reevesii formalin-fixed specimens, ranged from 500 to 1800 bp, with that of Psephuyrus gladius ranging from 600 to 2000 bp; that of Lipotes vexillifer ranging from 200 to 2000 bp [4] [5] . The results suggested there were more consistent with this experimental result. MtDNA cyt b gene banding pattern of samples with agarose gel were clearly visible (Figure 1) and could be used to be sequenced. In terms of DNA purity, the 260/280 ratio of 87.5% samples ranged between 1.8 and 2.0, indicating that DNA of the majority of the samples was pure [6] .

In summary, the developed DNA extraction from formalin fish fin tissues by the pretreatment with a gradient of ethanol system can be a useful tool to study the genetic structure and phylogenesis of endangered fish, using formalin-fixed preserved specimens from museums and herbarium.

This study was supported by the project of Fishery Resources and Environment Key Field Station of Upper-

middle Reaches of Yangtze River (YWTZ1003), of Key Laboratory of Freshwater Ecology and Aquaculture of Ministry of Agriculture (2010FEA03017) and of Key Laboratory of Freshwater Biodiversity Conservation and Utilization, Ministry of Agriculture (LFBCU0715).