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Molecular markers What are molecular markers? n n A readily detectable sequence of DNA or a protein whose inheritance can be monitored Polymorphism in proteins n n Allozymes and isozymes Polymorphism in DNA n n Nuclear Cytoplasmic Desirable properties of molecular markers n Polymorphic Codominat inheritance Easy, fast and inexpensive to detect Reproducible, transferable n No single marker meets all needs! n n n Isozymes and allozymes n n Around 1960 Multiple forms of the same enzyme n n n Isozyme:one enzyme, more than one gene locus Allozyme:one enzyme, one gene locus Methodology n n n n Macerate tissue Separate enzymes by electrophoresis Locate enzymes by histochemical staining Analyze banding patterns Allozymes:methodology Enzyme electrophoresis: Advantages n Robust, cheap reproducible method for: n n n Characterizing/ identifying genotypes Studying population genetics Examining geographical patterns of variation Disadvantages n Limited number of enzymes available n Limited amount of variation RFLP n n n n Restriction Fragment Length Polymorphism Around 1970 RFLP examines differences in size of specific DNA restriction fragments Requires pure, high molecular weight DNA RFLP: methodology n n n n Cut DNA into smaller fragments Separate fragments by gel Transfer DNA fragments to a filter Visualise DNA fragments n radioactive probes n non-radioactive probes RFLP: analysis of results n n Bands scored for presence/absence Differences reflect genetic differences n the choice of restriction enzyme crucial RFLP Advantages n Reproducible n Co-dominant markers n Simple Disadvantages n Time consuming n Expensive n Use of radioactive probes n not needed for mitochondrial/ chloroplast DNA Minisatellites n n n Invented in 1985 (Jeffreys et al.) Like RFLP, but the probe binds in microsatellite area (tandem repeats, 10-50 base pairs) Multi locus minisatellites (DNA-fingerprints) n n paternity analysis, not very useful for population analysis Single locus minisatellites (VNTR = Variable number of tandem repeats) n more useful for population analysis Minisatellites: interpretation RAPD n n n n Random Amplified Polymorphic DNA 1990 (Welsh et al.) PCR-based method Procedure: amplify anonymous stretches of DNA by using arbitrary primers n n n n one primer/reaction generally 10 bases (depends on genome size) Separate fragments by agarose gel Locate fragments by total DNA-staining RAPD: interpreation n Bands scored for presence/absence RAPD Advantages n Fast and simple n Inexpensive n No radioisotopes n n n n Disadvantages Dominant markers Reproducibility problems Problems of interpretation n Same band, same fragment Microsatellites n n 1989 (Tautz, Litt & Luty) (originally CA) Sequence tagged microsatellites (STMS) or Simple Sequence Repeat Polymorphism (SSRP) or Short Tandem Repeats (STR) n n n Motif (1-6 base pairs) repeats e.g. CGCGCGCGCGCGCGCG Procedure: amplify with PCR with specific primers Separate fragments by polyacryalamide sequencing gel (+ stain with silver nitrate) (or agarose + total DNA-stain) or use automated DNA detection with fluorescent based technology Microsatellites Microsatellites Advantages n High amount of variation n Codominant n Highly reproducible n More useful than minisatellites n Known mutation model (?) Disadvantages n Development difficult (separate sheet) and expensive n Interpretation sometimes difficult (stutter bands) Microsatellites: examples Microsatellites: examples AFLP n n n 1995 (Vos et al.) Amplified Fragment Length Polymorphism AFLP is a DNA fingerprinting technique which detects DNA restriction fragments by means of PCR amplification. AFLP: technology n Comprises of the following steps: n n n The restriction of the DNA with two restriction enzymes, preferably a hexa-cutter and a tetracutter The ligation of double-stranded (ds) adapters to the ends of the restriction fragments The amplification of a subset of the restriction fragments using two primers complementary to the adapter and restriction site sequences, and extended at their 3' ends by "selective nucleotides" AFLP: methodology AFLP: methodology n n Gel electrophoresis of the amplified restriction fragments on denaturing polyacrylamide gels ("sequence gels"); The visualization of the DNA fingerprints by means of autoradiography, phosphoimaging, or other methods. AFLP: example AFLP Advantages n No prior gene or sequence information is required n More reliable than RAPD n The technique is very sensitive, and is consequently able to detect low abundance transcripts Disadvantages n Mostly dominat n Technically demanding and expensive Amplifying a specific loci n Single nucleotide polymorphism (SNP) Disadvantages Advantages n Formerly labour-intensive to n It is known what is amplified: known source discover sequences of polymorphism n Formerly rely on relatively few loci: violation of assumptions n Often more reliable than Biallelic anonymous methods (specific primers) n Not known mutation model Kwok P. 2001. METHODS FOR GENOTYPING SINGLE NUCLEOTIDE POLYMORPHISMS. Annual Review of Genomics and Human Genetics 2: 235-258; DOI: 10.1146/annurev.genom.2.1.235 SNP ? SNP discovery by alignment of sequence traces obtained from direct sequencing of genomic PCR products. SNP-revolution n n n SNP arrays available for model species NGS-sequencing allows easy detection of SNP:s even for non-model species Next de facto markers for population genetic CanineHD BeadChip •More than 170,000 evenly studies? spaced and validated SNPs Genomic level markers n n n With the advent of next-generation sequencing (NGS), there are several approaches, which are capable of discovering, sequencing and genotyping not hundreds but thousands of markers across almost any genome of interest in a single step, even in populations in which little or no genetic information is available. Kumar et al. 2012. SNP Discovery through NextGeneration Sequencing. -Int. J. Plant Genom. (http://www.hindawi.com/journals/ijpg/2012/831460/) Davey et al. 2011. Genome-wide genetic marker discovery and genotyping using next-generation sequencing. –Nature Reviews Genetics 12: 503 Platforms n First Generation Sequencing n n (long reads, high quality, low throughput, high cost) Sanger Sequencing Second Generation Sequencing (PCR Needed) decreased cost, short reads, high throughput (massively parallel), clonal template amplification n n n n n Third Generation Sequencing (Single Molecule Sequencing ) n n n n Roche: 454 (pyrosequencing) X Life Science: IONtorrent & IONproton Illumina: MiSeq & HiSeq (Sequencing by Synthesis) ABI: SOLiD Helicos Biosciences: true single molecule sequencing (tSMS) Pacific Biosciences: PacBio RS II (Non--terminal fluorescent sequencing by synthesis, SMRT) Oxford Nanopore: MinION & GridION (single nucleotides are detected as they pass through a nanopore) http://www.molecularecologist.com/next-gen-fieldguide-2014/ Dideoxy sequencing (Sanger) n n n n n 1977 Selective termination of DNA-synthesis Four reactions (lanes) Separate by denaturing PAGE Visualize by autoradiography Automatic Sanger sequencing n Fluorescent dyes: one lane, four bases SGS-methods: Pyrosequencing 1998 Ronaghi M., Uhlen M., Nyren P. (1998b) A sequencing method based on real-time pyrophosphate. Science 281:363–365 Ion Torrent Based on monitoring of base incorporation during DNA synthesis, similar to 454 n 1. Beads placed in wells of a semiconductor chip which contain a pH sensing layer n 2. Single nucleotide types are flowed across the chip one at a time n 3. Incorporation of a nucleotide by DNA polymerase results in the release of a hydrogen ion, which is detected and recorded. – Multiple incorporations lead to a larger signal n Illumina sequencing Metzker 2010 n https://www.youtube.com/watch?v=HMyCqWhwB8E Illumina sequencing n 1. 2. 3. 4. 5. Also based on observation of incorporated nucleotides during DNA synthesis Clonal amplification of template DNA on a glass flow cell Fluorescent, reversibly-terminated nucleotides are added with DNA polymerase. ‘Stalled’ DNA synthesis occurs because of terminating nucleotides System is photographed/scanned in A, G, C and T channel Terminator and fluorophore are removed TGS: Oxford nanopore Oxford nanopore SNP-discovery SNP-calling SNP-validation SNP-genotyping n Kumar et al. 2012. SNP Discovery through Next-Generation Sequencing. -Int. J. Plant Genom. SNP-discovery n SNP is identified when a nucleotide from an accession read differs from the reference genome at the same nucleotide position. Graphical user interface of Tablet, an assembly visualization program, displays the reference genome on top and the mapped reads with color-coded SNPs on the bottom. SNP-discovery Complexity reduction strategies n Often use restriction enzyme digestion of target genomes to reduce the complexity n n n n Reduced-representation sequencing using reducedrepresentation libraries (RRLs) Complexity reduction of polymorphic sequences (CRoPS), restriction-site-associated DNA sequencing (RAD-seq) low coverage genotyping including MSG and genotyping by sequencing (GBS) Davey et al. 2011. Genome-wide genetic marker discovery and genotyping using next-generation sequencing. –Nature Reviews Genetics 12: 503 Genotyping by sequencing (GBS; right panels): • • • barcoded adaptors (yellow) and common adaptors (grey) are ligated to digested fragments, producing fragments with barcode+common, barcode+barcode and common+common adaptor combinations. Samples are pooled and amplified on the Illumina Genome Analyzer flowcell. Only short samples featuring a barcode+common adaptor combination are amplified for sequencing Cost of sequencing