Procedures in RFLP. RFLP analysis can detect Point mutations Length mutations Inversions.

Procedures in RFLPProcedures in RFLP

RFLP analysis can detectRFLP analysis can detect

• Point mutations• Length mutations• Inversions

Effect of base changes on RFLP DNA profilesEffect 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 b c

a c

a b’ b c

h g c

e

d

c── ── ── ── ──

── ── ── ──── ── ── ── ──── ── ── ── ──

── ── ── ── ──

── ── ── ──

── 10── 9

── 8

── 7

── 6

── 5

── 4

── 3

── 2

── 1

── ── ── ── ──

gd

be

a

f

h

-

+

P = DNA marker Restriction site

── ── ── ──

f

Limitations in RFLPLimitations 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, Ae

• Ae = 1/ i i2 = 1/(1- He)

i is i-th allele frequency

Genetic diversity parametersGenetic diversity parameters

Genetic diversity parametersGenetic 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

Genetic diversity parametersGenetic 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

Genetic diversity parametersGenetic 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

Genetic diversity parametersGenetic 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