VALIDATING APPROACHES FOR STUDYING MICROBIAL DIVERSITY TO CHARACTERIZE ...

166 Pages·2016·5.38 MB·English

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Doctoral Dissertations Graduate School
8-2013
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Migun Shakya
[email protected]
Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss
Part of the Environmental Microbiology and Microbial Ecology Commons
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Shakya, Migun, "VALIDATING APPROACHES FOR STUDYING MICROBIAL DIVERSITY TO CHARACTERIZE
COMMUNITIES FROM ROOTS OF Populus deltoides. " PhD diss., University of Tennessee, 2013.
https://trace.tennessee.edu/utk_graddiss/2482
This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee
Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized
administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact
[email protected].
To the Graduate Council:
I am submitting herewith a dissertation written by Migun Shakya entitled "VALIDATING
APPROACHES FOR STUDYING MICROBIAL DIVERSITY TO CHARACTERIZE COMMUNITIES
FROM ROOTS OF Populus deltoides." I have examined the final electronic copy of this
dissertation for form and content and recommend that it be accepted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy, with a major in Life Sciences.
Mircea Podar, Major Professor
We have read this dissertation and recommend its acceptance:
Christopher W. Schadt, Mitch Doktycz, Alison Buchan, Igor Jouline
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
University of Tennessee, Knoxville
Trace: Tennessee Research and Creative
Exchange
Doctoral Dissertations Graduate School
8-2013
VALIDATING APPROACHES FOR
STUDYING MICROBIAL DIVERSITY TO
CHARACTERIZE COMMUNITIES FROM
ROOTS OF Populus deltoides
Migun Shakya
[email protected]
This Dissertation is brought to you for free and open access by the Graduate School at Trace: Tennessee Research and Creative Exchange. It has been
accepted for inclusion in Doctoral Dissertations by an authorized administrator of Trace: Tennessee Research and Creative Exchange. For more
information, please [email protected].
To the Graduate Council:
I am submitting herewith a dissertation written by Migun Shakya entitled "VALIDATING
APPROACHES FOR STUDYING MICROBIAL DIVERSITY TO CHARACTERIZE
COMMUNITIES FROM ROOTS OF Populus deltoides." I have examined the final electronic copy of
this dissertation for form and content and recommend that it be accepted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy, with a major in Life Sciences.
Mircea Podar, Major Professor
We have read this dissertation and recommend its acceptance:
Christopher W. Schadt, Mitch Doktycz, Alison Buchan, Igor Jouline
Accepted for the Council:
Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official student records.)
VALIDATING APPROACHES FOR STUDYING
MICROBIAL DIVERSITY TO CHARACTERIZE
COMMUNITIES FROM ROOTS OF Populus deltoides
A Dissertation
Presented for the
Doctor of Philosophy
Degree
The University of Tennessee, Knoxville
Migun Shakya
August 2013
(cid:13)c by Migun Shakya, 2013
All Rights Reserved.
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For my parents,
Buddha Ratna Shakya
Mina Shakya
loving wife,
Urshula Shakya
and my beloved aunt,
Late Anjali Shakya
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Acknowledgements
This dissertation research would not have been possible without the help and support of
my mentors, friends, and family. First and foremost, I would like to express my sincere
gratitudetomyadvisors,Drs. MirceaPodarandChristopherW.Schadt. Theyhaveworked
tirelessly to improve my scientific execution, writing, publication, and overall scientific way
of thinking. Thank you for your support, mentorship, and dedication.
I would not have been able to be a scientist that I am today without my committee
members in Drs. Mitch Doktycz, Alison Buchan, and Igor B. Jouline. Thank you for your
suggestions, constructive criticisms, and insightful comments.
During my graduate studies, I met some of the most amazing colleagues and friends
in Alisha Campbell, James Campbell, Scott Hamilton-Brehm, Mike Robeson, Anil
Somenahally, Richard Hurt, Neil Gottel, Marilyn Kerley, Santosh Bhatt, Zamin K. Yang,
Hector Castro, Jerreme Jackson, and Ritin Sharma. I would like to acknowledge all of
them for their friendship, support, and comments on matters both inside and outside of
the lab. Throughout the graduate school, I have also had the pleasure of collaborating
with a number of fellow scientist including Anna Louise Reysenbach and Gilberto Flores
at Portland State University, Dwayne Elias at Oak Ridge National Lab, and Christopher
Quince at University of Glasglow. I am grateful for the opportunity.
None of this would have been possible without constant support and sacrifice from my
family. My parents, grand parents, uncles, and aunts have all contributed in one way or
another towards my success. I will be forever indebted to them. Lastly, a special thanks to
my wife Urshula for keeping me happy during arduous parts of my dissertation.
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”Science is a way of thinking
much more than it is a body of knowledge.”
-Carl Sagan, The fine art of baloney detection
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Abstract
Microbial (archaeal, bacterial, and fungal) communities associated with plant roots
are central to its health, survival, and growth. However, a robust understanding of
root microbiota and the factors that govern their community structure and dynamics
have remained elusive, especially in mature perennial plants from natural settings.
Although the advent of Next Generation Sequencing (NGS) technologies have changed the
scale of microbial ecological studies by enabling exhaustive characterization of microbial
communities,theaccuracyoftaxonomicandquantitativeinferencesareaffectedbymultiple
experimentalandcomputationalstepsandlackofknowledgeofthetrueecologicaldiversity.
To test for inaccuracies and biases, I assembled diverse bacterial and archaeal ‘synthetic
communities’ from genomic DNAs of sequenced organisms. I tested and compared different
approaches that included metagenomic and small subunit rRNA (SSU rRNA) amplicon
sequencing. The outcome was dependent on primer pairs, analysis parameters, and
sequencingplatforms. Nevertheless,newapproachesinprocessingandclassifyingamplicons
were able to recapitulate microbial diversity with high reproducibility within primer sets,
even though all tested primers sets showed taxon-specific biases. Consequently, inferences
from‘syntheticcommunities’studywereimplementedinexperimentaldesignandanalysisof
microbial communities from roots of naturally occurring mature riparian plants of Populus
deltoides. Thaumarchaeota, Proteobacteria and Ascomycota dominated the overall archaeal,
bacterial, and fungal communities respectively. Further, I investigated relationships of
bacterialandfungalcommunitiesinrhizosphereandendospherewithsoilandenvironmental
properties, host genotype, season, and geographic setting. The variation of bacterial and
fungal communities between each sampled roots were explained on the basis of seasonal,
soil properties, and geographical settings (4% to 23%), however, most variations remain
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