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CRESH Task 6: Genetic differentiation, and genetic barcoding of squid paralarvae

CRESH Task 6: Genetic differentiation, and genetic barcoding of squid paralarvae. Paul Shaw & Niall McKeown. Objective Analysis of genetic differentiation between cuttlefish spawning populations.

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CRESH Task 6: Genetic differentiation, and genetic barcoding of squid paralarvae

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  1. CRESH Task 6: Genetic differentiation, and genetic barcoding of squid paralarvae Paul Shaw & Niall McKeown Objective Analysis of genetic differentiation between cuttlefish spawning populations. Development of molecular markers for genetic barcoding of squid paralarvae, and species determination in early stages of Loligo forbesi, Loligo vulgaris, and Alloteuthis subulata.

  2. Task 6.1 Development of DNA barcoding methods for identification of early life stages of squid to species. DNA sequence variation within two regions (COI and cytb) of the mitochondrial genome of L.forbesi, L.vulgaris, and A.subulata will be assayed to identify species-specific marker sequences that can be used to develop species-specific genetic ID assays (RFLP and/or Taqman-based RT PCR). Other cephalopod species co-occurring in the study region will also be screened for sequence variation to check species-specificity. • Progress (3 months) • perfection of PCR-based method for ID of L. forbesi and L. vulgaris • (No discriminating RFLP sites in COI or 12s genes) • (no Alloteuthis available at present, but DNA sequence more divergent so extension of PCR method should be routine) Next tasks (in order of priority) 1. Extension of method to Alloteuthis 2. Provision of DNA barcode sequences for described specimens

  3. L. vulgaris primer L. forbesi primer 1: Species-specific DNA primers designed 3: PCR produces distinct patterns for each species 2: Primers exhibit species-specific PCR success or failure PCR product L. vulgaris DNA L. vulgaris L. forbesi X NO PCR product X NO PCR product L. forbesi DNA PCR product Objective: To design a genetic assay permitting identification/ discrimination of Loligo vulgaris & L. forbesi

  4. Primer set 2 Primer set 1 L.forbesi - L. vulgaris + L. vulgaris + L.forbesi - Primer set 3 Primer set 4 L. vulgaris + L.forbesi - L. vulgaris - L.forbesi + Combining primer sets 1,3 & 4 into a single reaction Lf Lf Lf Lf Lf Lf Lf Lf Lf Lf Lf Lf Lv Lv Lv Lv Lv Lv Lv Lv Lv Lv Lv Lv Species identification using a single PCR-based assay and agarose gel electrophoresis - Reliable, Easily Interpreted, Fast & Cheap

  5. Task 6: Genetic differentiation, and genetic barcoding of squid paralarvae Task 6.2 Identification of field samples of squid eggs/larvae to species using genetic methods. Application of the species-specific genetic ID assay to samples of eggs and larvae collected under Tasks 3, 4, 5 and 7, to identify to species (and so aid determination of relative species usage of different habitats in crucial spawning and early life history stages). • Progress • no results expected at this stage – technique development period Next tasks (in order of priority) 1. Testing of species-specific PCR on known egg and larval material

  6. Task 6: Genetic differentiation, and genetic barcoding of squid paralarvae Task 6.3 Analysis of genetic differentiation among spawning populations of cuttlefish. Application of existing genetic markers (microsatellites – Shaw & Perez Losada 2000) and methods (Perez Losada et al. 2002) to samples of Sepia officinalis spawning adults collected from target spawning areas, to assess genetic diversity within and differentiation between local spawning populations. Results will be analysed to assess relative genetic (i.e. reproductive) isolation of spawning populations using different habitats, and rates of genetic exchange (i.e. migration) between local populations, in order to determine the extent to which local stocks are self-recruiting and so sensitive to localised depletion or habitat loss. Samples of paralarvae will be screened to assess the potential for genetic assignment back to population of origin (for assessment of dispersal processes).

  7. Task 6.3 Analysis of genetic differentiation among spawning populations of cuttlefish. Genetic connectivity between populations – amount of interbreeding and gene flow = gene flow = widespread interbreeding = genetic homogeneity Breaks in homogeneity = breaks in gene flow / interbreeding

  8. Allozymes + microsatellites: • - clinal gene frequencies • Isolation-By-Distance (IBD)? Perez Losada et al. (2002) Heredity 89 Sepia officinalis

  9. Frequencies of 4 major mtDNA clades across Europe

  10. EZ SE M AL ML All samples significantly different – v. limited gene flow Cuttlefish: microsatellite DNA – inter-sample differences (FST)

  11. K2 E MED AEGEAN K3 ML AL M SE EZ K4 K5 Task 6.3 - genetic ID of paralarvae? Microsatellites – STRUCTURE analysis - most likely K = 5 (populations) - E Med v Aegean - E Med v Aegean v N Aegean - ML v AL v Aegean v N Aegean - All semi-isolated = most likely Potential for ID if individuals back to population-of-origin – only main areas?

  12. Other genetic studies of Sepia officinalis FAIR 2 project - no significant differences between East and West Channel Garoia et al 2004 - no significant differences within Adriatic (small samples sizes?) Wolfram et al 2006 - significant differences between Biscay, Channel and North Sea samples (small samples sizes within Channel?) Inconclusive for structuring within Channel and associated areas – the potential effect of localised spawning populations?

  13. Task 6: Genetic differentiation, and genetic barcoding of squid paralarvae Task 6.3 Analysis of genetic differentiation among spawning populations of cuttlefish. • Progress • Testing of existing marker loci completed (verification of methods) • Identification of further potential marker loci • Collation of existing dataset for Channel – Biscay samples • (no new samples available yet?) Next tasks (in order of priority) 1. Development of further loci – improved testing / discrimination 2. Screening of samples from key spawning sites (Adults? Eggs/hatchlings?) 3. Screening of repeat samples (temporal stability?)

  14. Task 6.3 Analysis of genetic differentiation among spawning populations of cuttlefish. • Thoughts? • Sampling reproductive populations – adults or offspring? • adults better, but are they spawning? • juveniles definitely a reproductive population, but relatedness?? • can test relatedness with genetic markers – more markers needed • combine pop sampling with viability material??

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