Ancient cattle DNA from Bhirrana: A Hakra culture/preharappan settlement of the Indus valley civilization, India

Bhirrana a settlement of the Indus valley civilization in northern India is important for revealing a cultural deposit dating from the preharappan (8-9 ka Before Present (BP)) to the mature Harappan period and also inferred to be the oldest (>9 ka BP) in the Indian subcontinent. Among the numerous animal remains excavated from Bhirrana noteworthy is the large-scale presence of bovids besides caprines and wild fauna, however, identification of cattle among the fragmented bovid bones is a difficult task. As cattle have played an important role since the Neolithic times in the origin and evolution of agro-pastoral societies, their identification is critical for studying the early development of animal husbandry practices in South Asia. To address this, using the ancient DNA method, we successfully identified 2 cattle bones out of 16 bovid samples. Our newly designed cattle-specific primers were able to amplify 70 bp of the maternally inherited cytochrome b gene found in the mitochondria. Homology searching of this ancient gene with modern cattle in the database confirms it to be of cattle. Of note, we found a unique haplotype in these two ancient cattle samples marked by a single mutation at 10th bp. Our phylogenetic tree construction also supports ancestral position of sequenced ancient Indus cattle with respect to their extant bovid relatives.