Procedures in RFLP

1. Procedures in RFLP

4. RFLP analysis can detect • Point mutations • Length mutations • Inversions

5. Effect of base changes on RFLP DNA profiles - c a b c ── ── ── ── ── ── 10 ── 9 ── 8 ── 7 ── 6 ── 5 ── 4 ── 3 ── 2 ── 1 d ── ── ── ── g f d ──── ── ── ── ── ── ── ── b ── ── ──── e a b c ── ── ── ── ── e a a c  ── ── ── ── ── f a b’ b c h ── ── ── ── ── h g c + P = DNA marker Restriction site

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

8. Genetic diversity parameters • Co-dominant data (isozymes, RFLPs, SSRs) • Percentage of polymorphic loci, P • Mean no. of alleles per locus, A • Effective no. of alleles per locus, Ae • Ae = 1/ ii2 = 1/(1- He) • i is i-th allele frequency

9. Genetic diversity parameters • Co-dominant data (isozymes, RFLPs, SSRs) • Observed heterozygosity per locus, Ho • Expected heterozygosity per locus, He • He = (1- ii2 ) • i is i-th allele frequency

10. Genetic diversity parameters • Co-dominant data (isozymes, RFLPs, SSRs) • Fixation index, Fis • Fis = 1- Ho/He • Genetic differentiation, GST • GST = DST/HT where HT = Hs + DST • HT is total gene diversity; HS is gene diversity within population; DST is gene diversity between population

11. Genetic diversity parameters • Co-dominant data (isozymes, RFLPs, SSRs) • Genetic similarity, I • Jxy / JxJy • where J = 1- He, • X = population X; Y = population Y • Genetic distance, D • D = -ln I

12. Genetic diversity parameters • VNTR used as probes in RFLP analysis • Percentage of polymorphic loci • Shannon diversity index, H • H = ni=1 -i ln i • Genetic similarity, F • F = 2mxy / (mx+my) • mxy is number of shared fragments by X and Y • mx is number of fragments present in X • my is number fragments present in Y • Genetic distance, 1- F