Research on control region or mitochondrial d-loop is of special interest in all mammalian species because they the displacement loop hyper-variable region of mtDNA (D-loop), which is of 1200 bp, is very important for genetic variations. It is also important for amtDNA in Nuclear Mitochondrial sequences (NUMTs) in vertebrates species so that it rarely occurring NUMTs. The control region in equine consist of two highly variable regions (HVR1 and HVR2), 4 conserved blocks (CSB), and variable repeats of 8 bp motifs (Cothran, et al. 2013).
The displacement loop hyper-variable region of mtDNA (D-loop) of Pak- local donkeys are very similar to genome of other species; however there are considerable differences in the mtDNAevolutionary rate for different taxonomic groups.
The phylogenetic tree based on consensus sequences of 12 Asian Donkeys breeds available on NCBI and sequences of local Pakistani donkey breeds showed their genetic relationship among each other. The clade was consisting of Pakistani local donkey breeds i.e. 3-SF, 7-SF,10-SF,11-SF,12-SF,13-SF, K-3,K-6,K-11,K-13,K-16,K18,K-19 and K-20 showing their high relatedness.K-8 and 8-SF have more mutation rate in the sequence and have more diversity from other individuals.

The second major branch was furher divided in two sub branches i.e. Donkey family representing Equusburchellichapmani (JX312729), Equusburchelliquagga (JX312733), Equusgreyvi(NC020432) and Equus zebra (JX312718) clustered together. The second sub branch was consisting of other donkey breeds i.e. Equushemionusonager (JX312730), Equushemionus(NC016061) and Equushemionuskulan (NC018782) grouped together while two Pakistani local donkey breeds i.e. SF-8 and K-8 were clusterd with Equusasinussomalicus (AP012271) and Equusasinus (X97337).
The d-loop sequence of Human (Homo sapiens) was taken as out group and it clearly differed and separated from rest of phylogenetic tree i.e. camel, bovine, ovine, caprine and other mammals.
The phylogenetic tree constructed under this study not only confirmed the status of Pak- local donkey breeds but also confirmed the genetic relationship among other mammalian species, thus reconfirming the already established biological classification.