Optimization of Degenerate Oligo nucleotide Primed Polymerase Chain Reaction (DOP-PCR) and preliminary characterization of genetic diversity in Asian corn borer Ostrinia furnacalis Guenee (Lepidoptera), from selected provinces of Luzon and Mindanao

Date

4-2013

Degree

Bachelor of Science in Biology

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Dela Viña, Celia B.

Abstract

Optimization of Degenerate Oligonucleotide Primed Polymerase Chain Reaction (DOP-PCR) was done using Asian corn borer (ACB), Ostrinia furnacalis Guenee, collected from selected provinces in Luzon (Isabela, Camarines Sur, and Pangasinan) and Mindanao (Bukidnon). DNA was extracted from third and fourth instar larvae from each population and was amplified by DOP-PCR using four different primers (6N, 8A, 10A and 10B) and different protocols like varying PCR profiles, reaction volumes, primer concentrations, and DNA concentrations, 10mM DOP-PCR primers 6N and 8A produced smears with no distinc bands. The results were indicative of whole genome amplifications. Using 0.75 mM DOP-PCR primer 10A in 15 uL reaction volumes and 1:30 genomic DNA concentrations yielded PCR products ranging from 300 to 2000 bp. With similar conditions except using 15 uL reaction volume, DOP-PCR primer 10B produced distinc bands that ranged from 100bp to 500bp. With the protocol tested, use of DOP-PCR primers 10A and 10B revealed differences in the PCR products of ACB collected from different provinces in the PCR products of ACB collected from different provinces. When DOP-PCR primer 10A was used, a 2kb product was observed only in the induced population from Isabela previously exposed to Cryl Ab protein for at least 10 generations and Pangasinan while a 500 bp PCR product was observed only in ACB sampled from Pangasinan and the induced population from Isabela when DOP-PCR primer 10B was used. This may suggest genetic variability among the populations of ACB sampled from the selected provinces. Based on Nei's original measures of genetic distance it was found that the population from Pangasinan and the induced population from Isabela were most closely related as compared to the other ACB population used in the study. The relatedness of the two populations may suggest that the variability may be linked to Cry1Ab susceptibility but additional studies have to be done. Sequencing of the PCR products and analysis of the putative roles of gene products are recommended.

Language

English

Location

UPLB Main Library Special Collections Section

Call Number

Thesis

Document Type

Thesis

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