ii BOBLME-2015-Ecology-29 The designations employed and the presentation of material in this publication do not imply the expression of any opinion whatsoever on the part of Food and Agriculture Organization of the United Nations concerning the legal and development status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The BOBLME Project encourages the use of this report for study, research, news reporting, criticism or review. Selected passages, tables or diagrams may be reproduced for such purposes provided acknowledgment of the source is included. Major extracts or the entire document may not be reproduced by any process without the written permission of the BOBLME Project Regional Coordinator. BOBLME contract: CST-RAPARD 165/2014 For bibliographic purposes, please reference this publication as: BOBLME (2015) Report on BOBLME Indian mackerel genetic data analysis including final workshop. 17-19 February 2015, Phuket, Thailand. BOBLME-2015-Ecology-29 ii Report on BOBLME Indian mackerel genetic data analysis including final workshop Executive summary The Indian mackerel genetic analysis workshop was held in Phuket, Thailand, 17-19 February 2015. This follows two previous workshops of the Indian mackerel genetics working group, The Indian mackerel working group meeting 28-29 May 2012 in Colombo, Sri Lanka and the Indian mackerel genetics harmonization training workshop 20-27 August 2013 in Kochi, India. Since the start of the project, initiated at the Colombo meeting, a set of 14 microsatellite markers have been developed specifically for R. kanagurta by cross-species priming and next-generation sequencing. Geneticists from the eight BOBLME countries were trained on the use of these markers. Thirty-one tissue collections representing 2 365 individuals from all eight BOBLME countries were collected and analyzed in seven different genetics laboratories for microsatellite variation at 14 loci representing at least 33 000 PCR reactions that were then sent for genotyping at a facility in India. Microsatellite motifs for the 14 R. kanagurta microsatellites ranged from penta-to di-nucleotide repeats. Data was binned in the corresponding motif sequences determined at the time of locus discovery. Although there appears to be significant inter-lab variability in the data, most of the loci are not conforming to Hardy-Weinberg Equilibrium (HWE), and at least some genotypic data included are from non-target species (R. brachysoma in Bangladesh samples), we can still present a tentative tree structure based on these Indian mackerel markers. The dendrogram shows a pattern of east-west geographic stock structure across the Bay of Bengal where western populations (Indian) are most genetically distant from eastern populations (Malaysia-Indonesia-Thailand). Further work outlined in the recommendations is required to validate the tree structure include: select questionable samples for genetic species identification testing, evaluate the need for additional standardization work between laboratories, support additional regional capacity building in terms of both laboratory equipment and further skills development, evaluate the current set of loci to improve amplification, allele calling and data quality (null alleles, upper allele drop-out, linkage disequilibrium). As well, the working group should remain aware of rapidly changing technologies which allow for the inclusion of both adaptive as well as neutral markers for genetic stock structure work. iii Report on BOBLME Indian mackerel genetic data analysis including final workshop Table of contents 1. Background ........................................................................................................................................ 1 1.1. Indian mackerel working group meeting, 28-29 May 2012, Colombo, Sri Lanka ................... 1 1.2. Indian mackerel genetic harmonization training workshop, 20-27 August 2013, Kochi, India ............................................................................................................................. 2 2. Indian mackerel genetics data analysis workshop, 17-18 February, 2015, Phuket, Thailand ............ 4 2.1. Introduction - Dr Hermes ........................................................................................................ 4 2.2. Country report - India NBFGR - Dr Jena .................................................................................. 4 2.3. Country report - India CMFRI - Dr Sukumaran ........................................................................ 5 2.4. Country report – Indonesia – Mr Suwarso .............................................................................. 6 2.5. Country report – Malaysia-SEAFDEC/MFRDMD - Ms Jamaludin ............................................ 7 2.6. Country report – Malaysia-FRI - Ms Rahim ............................................................................. 9 2.7. Country report – Sri Lanka - Ms Herath .................................................................................. 9 2.8. Country report - Thailand - Dr Kettratad .............................................................................. 10 2.9. Overview of previous R. kanagurta genetic studies - Dr Basheer ........................................ 10 2.10. Allele size calling and Gene mapper software - Dr Divya ..................................................... 11 2.11. Group learning activities overview - Mr J. Candy ................................................................. 11 2.12. Group learning activities - Data conversion program ........................................................... 12 2.13. Group learning activities - Descriptive statistics ................................................................... 12 2.14. Group learning activities – Stock structure programs .......................................................... 13 2.15. Group learning activities – Assignment testing .................................................................... 15 2.16. Workshop conclusions, recommendations and discussion points ....................................... 15 2.16.1. Short-term ..................................................................................................................... 15 2.16.2. Long-term ...................................................................................................................... 15 2.16.3. Recommendation to managers at this point ................................................................ 16 3. Post-workshop genetic data analysis ................................................................................................ 16 3.1. Post-workshop background .................................................................................................. 16 3.2. Base pair calls binning to alleles ........................................................................................... 18 3.3. Descriptive statistics from regional data set......................................................................... 20 3.4. Regional stock structure from multi-lab genetic data .......................................................... 20 3.5. Further work required on regional dataset .......................................................................... 21 3.5.1. DNA amplification from non-target mackerel species .................................................. 21 3.5.2. Upper allele drop-out and null alleles ........................................................................... 24 3.5.3. Inter-lab variability ........................................................................................................ 24 3.6. Recommendations to foster further collaboration on the Indian mackerel genetics project .................................................................................................................... 24 4. References ...................................................................................................................................... 25 iv Report on BOBLME Indian mackerel genetic data analysis including final workshop Appendix I Participant list for Indian mackerel genetic data analysis workshop February 17-19, 2015 Phuket, Thailand ....................................................................................... 28 Appendix II List of 14 microsatellite primers with optimal annealing conditions to be used for genetic analysis of R. kanagurta .................................................................... 30 Appendix III Agenda for the Indian Mackerel Genetic Data Analysis workshop .............................. 31 Appendix IV Pre-workshop preparation ........................................................................................... 33 Appendix V Base pair frequency plots by locus ............................................................................... 34 List of tables Table 1. List of sampling locations, latitude and longitudes, sample sizes, average length and weight of R. kanagurta sampled by the NBFGR lab ........................................................... 5 Table 2. List of sampling locations sample size, maturity stage, average length, and average body weight of R. kanagurta collected by the CMFRI lab .................................................. 6 Table 3. List of sample locations, sample size, sex ratio, and maturity stage for Indonesian R. kanagurta collected by RMIF .............................................................................................