DNA polymorphisms • Insertion-deletion length polymorphism – INDEL • Single nucleotide polymorphism – SNP • Simple sequence repeat length polymorphism – mini- and micro-satellites
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 Labeled 3’ TGGCTAGCT 5’ Probe 3’ TGGCTAGCT 5’ ||||||||| Target 1 5’-CCTAACCGATCGACTGAC-3’ 2 5’-GGATTGGCTAGCTGACTG-3’
RFLP
RFLPs Allele A Allele a a a A a A a a A Ind 5 Ind 1 Ind 3 Ind 4 Ind 2 Ind 8 Ind 6 Ind 7
RFLPs Allele A Allele a a a A a A a a A Ind 5 Ind 1 Ind 3 Ind 4 Ind 2 Ind 8 Ind 6 Ind 7
RFLPs Allele B Insertion Allele b b b B b B b b B Ind 5 Ind 1 Ind 3 Ind 4 Ind 2 Ind 8 Ind 6 Ind 7
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.
EcoRI MseI Digestion with 2 restriction enzymes Restriction site adapter ligation Selective preamplification 3’ T T 5’ 5’ 3’ A A Amplification 3’ C T T 5’ 5’ A T G 3’
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 109 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
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
SSR Individual 1 (AC)x9 Individual 2 (AC)x11 51 bp 55 bp Chloroplast SSRs of pine Powell et al. 1995. ProcNatlAcadSci U S A. 92(17): 7759–7763.
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
CAPS Forward Primer Indiv. 1 Reverse Primer Forward Primer Indiv. 2 Reverse Primer EcoRI EcoRI Indiv. 1 Indiv. 2 EcoRI
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)
RAPDs 5’ 3’ Indiv. 1 3’ 5’ 5’ Indiv. 2 3’ 3’ 5’ (Brahm et al. 2000)
Features of RAPDs • Simple assay • Dominant • Unrestricted access to primers • Requires little initial investment • Not very reproducible
