Document Type

Thesis

Date of Award

5-2011

School/College

College of Science, Engineering, and Technology (COSET)

Degree Name

MS in Biology

First Advisor

Professor Hector C. Miranda

Abstract

Molecular markers, also called genetic barcodes, are those segments of DNA that have unique characteristics which can be used in the identification and delineation of species, or phylotypes, in organisms. This is important in the study of phylogenetic diversity of microorganisms, 95 percent of which are unculturable. There have been ongoing research and success in recognizing markers for many organisms, such as 16S rRNA for bacteria and cytochrome oxidase I for animals. The Internal Transcribed Spacer (ITS) region has been gaining popularity as a marker in fungal species identification. However, the ability of the ITS region to discriminate among geographic populations within the same phylotype has not been explored. Because of the importance of phylogenetic systematics and the use of appropriate molecular markers in fungal biology, medicine, and agriculture, a refined protocol for amplifying ITS is used for the Aspergillus species/phylotypes. Additional Aspergillus ITS sequences were collected from GenBank to compare with those sequenced in the lab. The problem this research is addressing is can ITS be used to identify and differentiate phylogenetic relationships among and within the Aspergillus species. One criterion for an effective marker is the ability to discriminate among different 1 2 species and geographic isolates within the same species, or phylotype. The methodology includes obtaining multiple samples of different Aspergillus species (along with a few Penicillium species and non-related species of fungi), DNA extraction, peR amplification, DNA purification, and phylogenetic analysis of DNA sequences. Maximum parsimony, distance, and maximum likelihood methods, 􁪽p.__ coupled with bootstrap analysis will be employed to assess the rate evolution and the 􁪽-:􁪽:,􁪽. 􁪽 ",:􁪽,􁪽' level' of variability within and between Aspergillus phylotypes. The different optimality criteria used corroborated each other, suggesting that Aspergillus is not monophyletic. At least two Penicillium clades are embedded within the phylogeny. One to two groups of toxic/allergenic fungi sampled from the environment were also embedded within the Aspergillus clade and were found to more closely related to Cladosporium, Toxicocladosporium, Alternaria and Penicillium. The low level of sequence similarity between the cultured samples and their corresponding most closely related taxa suggest that the cultures may represent novel and undescribed species. Further morphological and genetic evaluations are suggested

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