Types of genetic markers • multilocus markers (RAPD, AFLP, minisatelitte DNA fingerprinting) • single-locus markers (allozymes, microsatelittes, SNPs) • dominant markers – scored as present or absent (RAPD, AFLP, ...) • codominant markers – identification of homologous alleles, i.e. scoring of homozygote and heterozygote states (allow estimation of allele frequencies – SNPs, microsatelittes, ...) Main markers used in molecular ecology Multi-locus genetic markers • screening of many loci distributed randomly throughout the genome Ø minisatellite DNA fingerprinting Ø RAPD (randomly amplified polymorphic DNA) Ø AFLP (arbitrary or amplified fragment length polymorphism) • presence vs. absence - codominant Minisatellite DNA fingerprinting RAPD (randomly amplified polymorphic DNA) AFLP (amplified fragments length polymorphism) • cheap, easy, fast and reliable method to generate hunderds of informative genetic markers • simultaneous screening of many different DNA regions distributed randomly throughout the genome • more reproducible banding pattern than RAPD Generating AFLP markers Generating AFLP markers Single-locus genetic markers • allozymes and other transcribed genes • SNPs (single nucleotide polymorphisms) • microsatelittes (length polymorphism) Single nucleotide polymorphisms (SNPs) Example of SNP marker Use of SNPs markers • species (or genetical group) identification and analysis of hybridization • phylogeography • population genetics (genetic variation, individual identification – parentage, relatedness, population structure, population size, changes in population size) Advantages • abundant and widespread in many genomes (in both coding and non-coding regions) – milions of loci • spaced every 300-1000 bp • biparentaly inherited (vs. mtDNA) • evolution is well described by simple mutation models (vs. microsatellites) • shorter fragments are needed – using in non-invasive methods Disadvantages • ascertainment bias – selection of loci from an unrepresentative sample of individuals • low variability per locus (usually bi-allelic) • higher number of loci is needed in population genetic applications (4-10 times more loci) Methods • Locus discovery (ascertainment) • Genotyping SNPs discovery Sequencing Sangrova dideoxy metoda • Sekvence délky 500 – 1000 bp • 4 kapiláry - destička s 96 vzorky za noc • Jsou i sekvenátory s 96 kapilárami Použití nových přístupů Metagenomické knihovny Ursus spelaeus > 28 000 bp (jaderná i mitochondriální DNA) (Noonan et al. 2005) „Next generation“ sequencing (Hudson 2008) 454 pyrosequencing • emulzní techniky amplifikace pikolitrové objemy • simultánní sekvenování na destičce z optických vláken detekce pyrofosfátů uvolňovaných při inkorporaci bazí • První generace GS20 → 200 000 reakcí najednou (zhruba 20 milionů bp) dnes FLX → 400 000 reakcí najednou • Problémy s homopolymery • Délka jednotlivých sekvencí 100 – 400 Solexa/Illumina 1G SBS technology (SBS = sequencing by synthesis) • 1 Gb (šestinásobek genomu Drosophily) • Výrazně levnější • Sekvence délky 35 bp • Flourescence, reversibilní terminátory • Spíš pro resequencing SOLiD (sequencing by Oligonucleotide Ligation and Detection) SNP genotyping - old standards SNP genotyping – new methods SNP genotyping – new methods Detection or SBE products