PCB Fa Falll l2012

Transcription

1 PCB 5065 Fall 2012 Molecular Markers

2 Bassi and Monet (2008) Morphological Markers

3 Cai et al. (2010) JoVE Cytogenetic Markers

4 Boskovic and Tobutt, 1998 Isozyme Markers

5

6 What Makes a Good DNA Marker? High level of polymorphism multi allelic Exhibit codominance (heterozygote can be distinguished)

7 What Makes a Good DNA Marker? Locus specific Even and frequent distribution over the genome Easy detection/visualization methods High repeatability Scalable for high throughput genotyping Low development and operational costs

8 Xu, 2010 Molecular Plant Breeding How Do Markers Arise?

9 Xu, 2010 Molecular Plant Breeding How Do Markers Arise?

10 Xu, 2010 Molecular Plant Breeding How Do Markers Arise?

11 Markers Where would you find a perfect marker? Functional i l Marker k Buchanan et al, 2000, Biochemistry & Molecular Biology of Plants

12 Hybridization Based Markers Require prior sequence knowledge for probe development PCR not necessary

13 RFLP (Restriction Fragment Length Polymorphism) Botsteinet et al. (1980) Construction of a genetic linkage map in man using restriction fragment lengthpolymorphisms. Amer. J. Hum. Genet. 32: Watson et al., 2008 Recombinant DNA

14 Restriction Enzymes EcoRI 5..ACGTGAATTCACTG TGCACTTAAGTGAC base recognition site 4 6 (4,096) average cut site frequency A 100 Mb genome (100,000,000 base pairs) would have over 24,000 fragments

15 RFLP Genomic DNA digested with one or more restriction enzymes Probes (the actual markers in this case) are developed fromest libraries Probes tend to be conserved = repeatability Need lots of high quality DNA

16 Hosaka and Spooner, 1992 RFLP

17 PCR Based Markers 1. Require prior sequence knowledge for primer development 2. Use arbitrary sequences to prime multiple loci in the genome

18 SSR (Simple Sequence Repeat) Also known as: Microsatellites Short Tandem Repeats Agarwal et al., 2008

19 SSR Repeated units of nucleotide motifs < 10bp Examples: (CA)n, (AAT)n, (GATA)n Widely distributed in eukaryotic genomes High level of allelic variation Hypervariable DNA strand mis pairing ii during replication Primers designed from unique sequence in flanking regions

20 Ellegren, 2004 SSR

21 SSR Marker CMTm120 (C. moschata) ct repeat motif Marker CMTm009 (C. moschata) aag + at repeat motif

22 SSR SSR sites are typically identified from genomic or cdna libraries: Hybridize clones with labeled oligonucleotide containing the SSR motif Sequence positive clones Design primers from flanking regions Often work in related ltdspecies

23 SSR Silver stained Polyacrylamide gel Parental survey for allele differences

24 SNP (Single Nucleotide Polymorphism)

25 SNP Most common type of sequence variation available for markers Estimate of 1 SNP every bp in eukaryotes Most SNPs are identified from genomic sequence or EST libraries Number of SNP markers possible makes genotyping challenging

26 SNP Genotyping Examples Low throughput Allele specific PCR amplification High throughput Many methods, constantly being improved Differential hybridization Enzymatic reactions (primer extension or y (p DNA ligation)

27 Chagne et al., 2007 SNP Genotyping Examples

28 SNP Genotyping Examples 3 unique oligos are designed for each SNP site 3 rd oligo has a unique identifier ifi that allows multiplexing Shen et al., 2005

29 Ding and Jin, 2009 SNP Genotyping Examples

30 PCR Based Methods 1. Require prior sequence knowledge for primer development 2 U bit t i 2. Use arbitrary sequences to prime multiple loci in the genome

31 RAPD (Random Amplified Polymorphic DNA) Utilizes small primers of arbitrary sequence 10 base pair (10 mer) is common Primers vary in GC content Primers should bind to many different sites in the genome When two primers bind within ~3000 bp in the correct orientation for PCR, amplification will occur Williams et al., 1990

32 AFLP (Amplified Fragment Length Polymorphism) Proprietary technology (Keygene) AFLP combines restriction digestion with arbitrary primer amplification Best of RFLP and RAPD techniques No prior sequencing needed dd High level of polymorphism p for each selective primer combination Vos et al., 1995

33 RAPD and AFLP RAPD markers often have reproducibility issues AFLP markers are combine the best of RAPD and RFLP methods Both RAPD and AFLP markers opened genomics to all species

34 AFLP EcoRI+AC and MseI +CTA

35 Schlotterer, 2004

36 Single nucleotide polymorphism (SNP) discovery and applications of SNP genotyping in nonmodel organisms Molecular Ecology Resources pages 1 8, 17 FEB 2011 DOI: /j x x/full#f1

37 Conclusions Many types of molecular markers available, non are ideal Type(s) chosen for use will depend on many factors As sequencing costs become lower, random markers are less desirable Polymorphism is necessary for genetic mapping and screening, but not for physical mapping