1. Microsatellite identification via ISSR Protocol:
Identifying genus and species specific microsatellite loci for Culex modestus
Waymouth,T., Strode,C., Dallimore, T.,
Introduction:
There has been a recent emergence of Cx.modestus across southern parts of England1. Populations have never been documented this far north, a possible result of climate change, altering the distance migratory species such as this inhabit.
Cx.modestus has been confirmed to be a susceptible vector for multiple diseases2. This is makes it important to identify molecular tools capable of locating origins of newly documented populations.
Methodology:
ISSR Primer Cloning Protocol
Inter Simple Sequence Repeats (ISSR) primers have been used in previous microsatellite identification projects3. These target regions of DNA suspected to have a high concentration of microsatellites, to locate species specific microsatellite loci for Cx.modestus.
Data-mining
To locate genus specific microsatellites, papers publishing microsatellite primers of closely related species, such as Cx.pipiens4 have been identified. These will be tested on Cx.modestus samples to determine efficacy.
Microsatellite identification via ISSR Protocol:
Fig.1 : Chromatograph showing a microsatellite region (CA)9A(AC)7T(CA)3
Sampling
DNA extraction and PCR
ISSR Cloning Protocol
PCR and Purification
DNA Sequencing
Samples collected by Public Health
England from Thames Estuary
Qiagen DNeasy protocol followed.
DNA amplified using a variety of ISSR primers
DNA fragments spliced into E.coli
competent cells and grown on nutrient agar.
192 white colonies picked and
amplified with M13 primers.
ExoSAP performed to clean
PCR with only forward M13, and Big Dye Terminator ddNTP’s.
Cleaned through Ethanol/EDTA precipitation.
Ready for sequencing using Genetic Analyser
Fig.2: Culex pipiens
Fig.3: Culex modestus
Results:
DNA fragments were analysed with the software MEGA. This was used to form chromatographs to display DNA base sequence.
Searching through these chromatographs have currently yielded 6 potential candidates for microsatellite loci of various sizes.
Primers were then designed flanking these microsatellite regions as according to primer design rules, such as melting temperature, GC content, and fragment size.
Table.1: Table of primers designed for all 6 potential microsatellite loci
References:
Golding, N., Nunn, M., Medlock, J., Purse, B., Vaux, A. and Schäfer, S. (2012). West Nile virus vector Culex modestus established in southern England. Parasites & Vectors, 5(1), p.32.
BICOUT, D., ZELLER, H., SCHAFFNER, F., REITER, P., VAZEILLE, M. and BALENGHIEN, T. (2009). Evidence of Laboratory Vector Competence of Culex Modestus for West Nile Virus.
WILSON, P. and PROVAN, J. (2006). Development of microsatellites for the peat mossSphagnum capillifoliumusing ISSR cloning. Molecular Ecology Notes, 7(2), pp
Keyghobadi, N., Matrone, M., Ebel, G., Kramer, L. and Fonseca, D. (2003). Microsatellite loci from the northern house mosquito (Culex pipiens), a principal vector of west Nile virus in north America. Molecular Ecology Notes, 4(1), pp
Further Study:
Follow up DNA fragment analysis on potential microsatellites that were located.
Testing Cx.pipiens microsatellite primers on Cx.modestus.