Optimization of mtCOI based Barcoding and Sequence Analysis for Species Demarcation

Abstract

Aphids are small insects that feed on plants and cultivated crops by sucking their sap. They belong to Aphidoidea, a superfamily of insects and have immense genetic and morphological variation among species. Usually animal species are recognized based on morphological characteristics but morphological documentation of aphids is complicated and not so reliable due to the unique biological properties of aphids such as complex life cycle that alternates between sexual and asexual phase of reproduction, polyphenism that refers to the expression of distinct phenotypes from a single genotype, and host plant alteration. Thus, this study was conducted to identify aphid species on molecular basis using mitochondrial cytochrome c oxidase subunit 1 gene as a DNA barcode. Samples were gathered from six different host plants at different locations across Pakistan. After PCR amplification using universal primers, the sequences were then commercially sequenced. BLASTn using NCBI and phylogenetic analysis of the obtained sequences suggested that 8 sequences represent aphid species while 2 sequences relate to parasites of aphids. Aphid species identified through DNA barcoding in this study exclusively belong to the sub-family Aphidinae which is also the biggest sub-family of aphids. While the aphid’s parasites identified in this study were related to Coccinellidae, Eupelmidae and Aphelinidae. Pakistan as an agrarian country requires a detailed and comprehensive study for profiling of all the aphid species infesting different agricultural crops across Pakistan through DNA barcoding to generate a completer DNA barcode reference library. This DNA barcode library of aphid species will enhance our knowledge about aphid diversity, will add to the global data set of DNA barcodes, will help us in early detection of alien species, will improve our understanding regarding the transmitted viruses and disease development and most importantly will help us in formulating productive integrated pest management strategies.