DNA polymorphisms Insertion-deletion length polymorphism – INDEL Single nucleotide polymorphism – SNP Simple sequence repeat length polymorphism – mini- and micro-satellites

Labeled 3’ TGGCTAGCT 5’ Probe 3’ TGGCTAGCT 5’ ||||||||| Target 1 5’-CCTAACCGATCGACTGAC-3’ 2 5’-GGATTGGCTAGCTGACTG-3’ Restriction Fragment Length Polymorphism (RFLP) RFLPs (Botstein et al. 1980) are differences in restriction fragment lengths caused by a SNP or INDEL that create or abolish restriction endonuclease recognition sites. RFLP assays are based on hybridization of a labeled DNA probe to a Southern blot (Southern 1975) of DNA digested with a restriction endonuclease

RFLP

Allele A Allele a A a a a A a a A Ind 1 Ind 2 Ind 5Ind 3 Ind 4 Ind 8Ind 6 Ind 7 RFLPs

Allele A Allele a A a a a A a a A Ind 1 Ind 2 Ind 5Ind 3 Ind 4 Ind 8Ind 6 Ind 7 RFLPs

Allele B Allele b B b b b B b b B Ind 1 Ind 2 Ind 5 Ind 3 Ind 4 Ind 8Ind 6 Ind 7 Insertion RFLPs

Features of RFLPs Co-dominant Locus-specific Genes can be mapped directly Supply of probes and markers is unlimited Highly reproducible Requires no special instrumentation

Amplified Fragment Length Polymorphism (AFLP) AFLPs (Vos et al. 1995) are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites. AFLP assays are performed by selectively amplifying a pool of restriction fragments using PCR.

Digestion with 2 restriction enzymes Restriction site adapter ligation EcoRI MseI T A 5’5’ 3’ 5’5’ 3’3’ T A Selective preamplification C T T A T G 5’5’ 3’ 5’5’ 3’3’ Amplification

Amplified Fragment Length Polymorphism (AFLP) Polymorphisms betwwen genotypes may arise from: –Sequence variation in one or both restriction sites –Sequence variation in the region immediately adjacent to the restriction sites –Insertions or deletions within an amplified fragment

AFLP The predicted number of DNA fragments amplified by AFLP primers with n selective nucleotides is N is genome size in base-pairs b is the number of nucleotides in the recognition site of a restriction endonuclease

AFLP DNA restriction fragments produced by a six-base cutter (n = 6) in soybean (N = 1 X 10 9 bp, ~1,000 Mb/1C) N = genome size in base-pairs b = the No. of nucleotides in in the recognition site n = No. of selective nucleotides nCalculationNo. of restriction fragments 01/4 6 (1 X 10 9 )244,141 11/4 6 (1 X 10 9 ) 1/4 2 15,259 21/4 6 (1 X 10 9 ) 1/ /4 6 (1 X 10 9 ) 1/4 6 60

Features of AFLPs Very high multiplex ratio Very high throughput Off-the-shelf technology Fairly reproducible Dominant and co-dominant

Simple Sequence Repeats (SSR) SSRs or microsatellites (Nakamura et al. 1987) are tandemly repeated mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs SSR length polymorphisms are caused by differences in the number of repeats Assayed by PCR amplification using pairs of oligonucleotide primers specific to unique sequences flanking the SSR

Individual 1 (AC)x9 Individual 2 (AC)x11 51 bp 55 bp Powell et al Proc Natl Acad Sci U S A. 92(17): 7759–7763. Chloroplast SSRs of pine SSR

Features of SSRs Highly polymorphic Highly abundant and randomly dispersed Co-dominant Locus-specific High throughput Can be automated

SSR Sources SSRs are often found in cDNA and genomic DNA sequences SSRs are developed by screening genomic DNA libraries enriched for one or more repeat motifs. SSR-enriched libaries can be commercially purchased

SSR Repeat Motifs AC repeats tend to be more abundant than other di-nucleotide repeat motifs in animals (Beckmann and Weber 1992) The most abundant di-nucleotide repeat motifs in plants, in descending order, are AT, AG, and AC (Lagercrantz et al. 1993; Morgante and Oliveri 1993) Typically, SSRs are developed for di-, tri-, and tetra-nucleotide repeat motifs CA and GA have been widely used in plants Tetra-nucleotide repeats have the potential to be very highly polymorphic; however, many are difficult to amplify

Cleaved Amplified Polymorphic Sequences (CAPS) CAPS polymorphisms are differences in restriction fragment length caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites in PCR amplicons produced by locus-specific oligonucleotide primers (Tragoonrung et al. 1992; Konieczny and Ausubel 1993) Assays are performed by digesting locus-specific PCR amplicons with one or more restriction enzymes and separating the digested DNA on gels

Indiv. 1 Indiv. 2 Forward Primer Reverse Primer Forward Primer Reverse Primer Indiv. 1 Indiv. 2 EcoRI CAPS

Features of CAPS Locus-specific - PCR-based assay for a marker with known DNA sequence Method to map markers without Southern blotting Co-dominant and dominant

Random Amplified Polymorphic DNA (RAPD) DNA polymorphism produced by rearrangements at or between oligonucleotide primer binding sites in the genome (Williams et al. 1990) Assays are performed using single short oligonucleotide primers of arbitrary sequence (typically 10-mers)

Indiv. 1 Indiv. 2 RAPDs (Brahm et al. 2000) 3’ 5’ 3’ 5’ 3’

Features of RAPDs Simple assay Dominant Unrestricted access to primers Requires little initial investment Not very reproducible