The first task of the project was concerned with obtaining the biological material from Greenland. An expedition to West Greenland provided replicate sediment samples from 4 lakes. Upon returning the material to the UK, sediment was processed to quantify ephippia with dormant eggs for DNA extraction or whole genome amplification. All lakes contained Daphnia pulex ephippia at least throughout the past several hundred years, however, only 2 lakes contained sufficient egg numbers for further analysis. Eggs were used for initial genotyping (microsatellite marker) from 3 lakes covering a period of several hundred years. Loss-On-Ignition (LOI) profiles were constructed of selected sediment cores using sediment subsamples along the entire core for correlation with previous core collections, providing a comprehensive archive of historical environmental data from previous work.
Ephippia were collected and quantified from Lake SS4 which contained the best-preserved eggs for DNA analysis. Eggs (for observation of genomic changes over time) were collected at a high temporal resolution of 30-year intervals, covering a total of ~300 years. From 10 time periods, 30 eggs were collected and frozen at -80 C until further analysis, yielding a total of 300 isolates.
The success of the project depended heavily upon successful methodological optimisation for processing of isolated Daphnia eggs. This required rigorous optimisation through a series of steps: (1) pretreatment (cleaning) of eggs to avoid external contamination, (2) egg homogenization and subsequent whole genome amplification, (3) quantification and quality control of amplified DNA, (4) genomic library construction. Further, (5) a sequencing test was run to provide information for the optimal strategy of the final sequencing of 300 egg isolates (whole genome amplified). Finally (6) bioinformatic analysis of test samples was carried out to identify the fraction of foreign DNA (a common problem in ancient DNA samples) and the resulting calculation of the targeted depth-of-coverage for the final sequencing of 300 samples.
A preliminary study was performed, using microsatellite analysis of individual eggs isolated from the sediment. This included egg from three lakes, across 50 - 300 years. Ten microsatellite markers were used to genotype individual eggs, revealing low clonal diversity in the lakes at all time periods tested, detecting distinct genotypes unique to each lakes despite their geographic proximity. Further, the Comet Assay, a method by which DNA damage can be quantified, was optimised for its use on individuals eggs and will be a useful tool for future studies.
Results of this study were partly included in the reports of 3 student projects supervised by Dr Frisch: (1) O'Grady, C. The development and optimization of whole genome and whole transcriptome amplification in historic Daphnia magna and Daphnia pulex diapause eggs. University of Birmingham MIBTP Rotation project, 2016; (2) Dane, M. Stability of spatio-temporal genetic variation across the centuries: a case study of arctic asexual Daphnia pulex populations from three West Greenland lakes. University of Birmingham. Third Year Practical Project Report, 2016; (3) Salimraj, R. Optimization of the Comet Assay to assess environmental impact on DNA damage in diapause eggs of Daphnia. University of Birmingham. Third Year Practical Project Report, 2017. Based on these project outcomes, three manuscripts are currently in advanced preparation, to be submitted to journals included in the science citation index. Additional publications are expected as soon as the current sequencing experiment is finalised.
