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The present study reports the genetic variation observed among five anthropologically distinct population groups of India, using four highly polymorphic minisatellite loci (D1S80, D17S5, D19S20, and APOB 3′ VNTR) in order to examine the effect of geographical and linguistic affiliations on the genetic affinities among these groups. Random individuals from five ethnic groups were studied; the sample size ranged from 235 to 364. The population groups belong to two geographically separated regions of India, the state of Maharashtra (western India) and the state of Kerala (southern India). The two Maharashtrian groups (Konkanastha Brahmins and Marathas) speak “Marathi,” an Indo-European language, whereas the three Kerala population groups (Nairs, Ezhavas, and Muslims) speak “Malayalam,” an Indo- Dravidian language. Genomic DNA was extracted from peripheral blood samples and analyzed using amplified fragment length polymorphism (Amp- FLP) technique. All four loci displayed high heterozygosity with average heterozygosity in the range of 0.82 to 0.84. The Polymorphic Information Content and Power of Discrimination were ≥0.75 and ≥0.80, respectively. The coefficient of gene differentiation was found to be low (average GST = 1.2%; range between 0.6% at D1S80 locus to 1.6% at APOB 3′ VNTR locus) across the loci, indicating close affinity among the population groups. The neighborjoining tree revealed two clear clusters, one for the two Maharashtrian population groups and the other for the three Kerala population groups. The results obtained are in conformity with the geographical and linguistic backgrounds of the studied populations.