Procedures in RFLP

RFLP analysis can detect Point mutations Length mutations Inversions

Effect of base changes on RFLP DNA profiles Reference ── GATC ─── GATC ───── A Base substitution ── GACC ─── GATC ───── B Deletion ─────────────── C Addition ───────────────── D Inversion ────┬──────────── E a b c a c a b’ b c h g c e d  c ── ── ── ── ── ── ── ── ── ── ── ── ── ── ── 10 ── 9 ── 8 ── 7 ── 6 ── 5 ── 4 ── 3 ── 2 ── 1 ── ── ── ── ── g d b e a f h - + P = DNA marker Restriction site ── ── f

Limitations in RFLP Intensive work High cost High amount of DNA Use of radioactive

Co-dominant data (isozymes, RFLPs, SSRs) –Percentage of polymorphic loci, P –Mean no. of alleles per locus, A –Effective no. of alleles per locus, A e A e = 1/  i  i 2 = 1/(1- H e )  i is i-th allele frequency Genetic diversity parameters

Co-dominant data (isozymes, RFLPs, SSRs) –Observed heterozygosity per locus, H o –Expected heterozygosity per locus, H e H e = (1-  i  i 2 )  i is i-th allele frequency

Genetic diversity parameters Co-dominant data (isozymes, RFLPs, SSRs) –Fixation index, F is F is = 1- Ho/He –Genetic differentiation, G ST G ST = D ST /H T where H T = H s + D ST H T is total gene diversity; H S is gene diversity within population; D ST is gene diversity between population

Genetic diversity parameters Co-dominant data (isozymes, RFLPs, SSRs) –Genetic similarity, I J xy /  J x J y where J = 1- H e, X = population X; Y = population Y –Genetic distance, D D = -ln I

Genetic diversity parameters VNTR used as probes in RFLP analysis –Percentage of polymorphic loci –Shannon diversity index, H H =  n i=1 -  i ln  i –Genetic similarity, F F = 2m xy / (m x +m y ) m xy is number of shared fragments by X and Y m x is number of fragments present in X m y is number fragments present in Y –Genetic distance, 1- F