Genetic characterisation of formalin preserved fish tissue

Ignition Grant Round 4 (March 2015)

  • Sharon Appleyard, CSIRO
  • Maxine Piggott, ANU

The Australian National Fish Collection (ANFC) supports research to improve the understanding of biodiversity of marine fishes, sharks and rays from Australia, Antarctica and the Indo-Pacific. Over 150 000 preserved fish specimens representing approximately 3 400 species are held in the collection.

While these specimens provide irreplaceable ecological and evolutionary insights into Australian fish biota, most of these specimens are preserved in formalin. Additionally, many of Australia’s ca. 5 000 fish species are rarely collected (e.g. deepwater or rare) so there are few avenues to obtain fresh tissues.

To access the full genotypic and phenotypic potential of the extensive specimen collection new genomic tools capable of screening degraded DNA need to be trialled and optimised. DNA degradation in archival or formalin preserved specimens often inhibits successful PCRs of fragments larger than 200 base pairs.

This project will give the ANFC the molecular tools to extract DNA from formalin preserved specimens and access to species level genetic information (via mini-barcoding of the mitochondrial cytochrome oxidase COI gene and screening of nuclear variation (e.g. single nucleotide polymorphisms (SNPs)).

This project is also an important step to kick starting ANU:ANFC collaborations and establishing linkages with ANU and CSIRO researchers with interests in fishes and fish communities.

Alongside a current BioPlatforms Australia DNA Reference Barcoding project for fishes (based on full length Sanger sequencing of individual specimens and 650 base pair COI sequence libraries), the outputs from this project will be novel extraction protocols and tools, sequence comparisons between full and mini COI- barcodes for species resolution, genome resources with reduced complexity (RAD tag libraries) and next generation sequencing (NGS) protocols for parallel simultaneous acquisition of DNA barcodes from multiple individuals.

While the degraded DNA and NGS methods outlined in this project are yet to be tested on ANFC specimens, the optimisation of NGS for revealing biodiversity in archival collections is increasingly reported in the literature.

This project will for the first time provide the ANFC with the ability to genetically screen a much larger component of the important preserved material (pre-barcode specimen sampling), including rare material known from few specimens.

Confirmation of species-level identification of preserved specimens against the extensive COI library, especially against old/rare type specimens, will lead to a more ‘research ready’ collection providing valuable sources of new information for Australasian marine fishes, sharks and rays.

The aims of this project are to:

  • develop DNA extraction methods (based on suitable tissue selection (e.g. fin clips v muscle tissue)) for formalin preserved fishes, sharks and rays (utilising a set of time series preserved samples - relatively recently preserved v older preserved samples).
  • develop NGS approaches to the parallel acquisition of COI mini-barcodes (utilizing 10-mer oligonucleotide tags attached to DNA barcoding PCR primers to tag amplicons) through barcoded individuals which are pooled within NGS runs.
  • compare COI mini-barcodes (via NGS) against Sanger gold standard full length COI barcodes for species resolution.
  • develop restriction-associated DNA tags (via RADsequencing libraries run on the Illumina MiSeq platform) for variable genomic region screening (e.g. SNPs) amenable for use with degraded DNA.
  • determine cost effectiveness (considering higher throughput) of NGS v Sanger sequencing for biodiversity and molecular taxonomy assessments in the ANFC.
  • provide tools for Australian fish collections to extend biodiversity investigations and contribute to species information required for conservation and threatened species planning.
  • establish collaboration and direct linkages between ANU and CSIRO researchers interested in fishes and fish communities (including the application of eDNA detection of fish species in fresh and marine water).

This project will assist with conservation planning by discovering and documenting of fish, shark and ray species using alpha and molecular taxonomy (including the detection of cryptic species). It will aslo help with the understanding of genetic diversity and gene flow among threatened fishes including sharks and rays. The project will also lead to improved delineation for cryptic species (particularly east-west species pairs) through access to numerous archived specimens.