Discovery and Evolutionary Analyses of Abiotic Stress-Regulated MicroRNAs in Crops

164 Pages·2014·8.49 MB·English

UC Riverside
UC Riverside Electronic Theses and Dissertations
Title
Discovery and Evolutionary Analyses of Abiotic Stress-Regulated MicroRNAs in Crops
Permalink
https://escholarship.org/uc/item/9718z08s
Author
Wu, Zhigang
Publication Date
2014

Peer reviewed|Thesis/dissertation
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University of California
UNIVERSITY OF CALIFORNIA
RIVERSIDE
Discovery and Evolutionary Analyses of Abiotic Stress-Regulated MicroRNAs in
Crops
A Dissertation submitted in partial satisfaction
of the requirements for the degree of
Doctor of Philosophy
in
Plant Biology
by
Zhigang Wu
June 2014
Dissertation Committee:
Dr. Renyi Liu, Chairperson
Dr. Xuemei Chen
Dr. Jason E. Stajich
Copyright by
Zhigang Wu
2014
The Dissertation of Zhigang Wu is approved:
Committee Chairperson
University of California, Riverside
Acknowledgments
Over the past five years, I was very lucky to have worked with a few knowledgeable
professors and collaborators. I also recieved emotional, financial and intellectual sup-
port from my friends and family. Without them, this dissertation would have not been
possible. I would like to acknowledge them in below.
I am grateful to my advisor Dr. Renyi Liu for introducing me into the field of
bioinformatics. I really enjoyed the way he have trained me over the years. Not only he
had the great patience to teach me things as detailed as debugging a Perl program when
I just joined the lab at the beginning, but also he gave me a lot of flexibility on choosing
what to work on after I gained considerable experience. He taught me how to sharpen
my writing and presentation skills. In addition to the research, he also gave guidances
to me on life.
I am indebted to my committee members: Dr. Xuemei Chen and Dr. Jason E.
Stajich, who have been serving on my committees (guidance committee, qulifying exam
committee and dissertation committee) since I joined UCR in 2009, for being always
available for questions, meetings and recommendation letters. They gave me a variety
of research-related suggestions and improvements as well as career guidances during
different phases of my PhD study.
I also would like to thank Dr. Lei Gao and Dr. Blanca E. Barrera-Figueroa
fortheirhands-onhelpinperformingbioinformaticsanalysesandmolecularexperiments
respectively when I was new to the lab. Thanks to Dr. Lei Gao for lending his hands
when I was blindly searching for a file on a Linux terminal. Thank Dr. Blanca E.
Barrera-Figueroa for teaching me how to treat maize and sorghum with abiotic stresses.
During different phases of my dissertation, I have worked in several labs at
iv
UCR. I would like to thank Dr. Jian-Kang Zhu, Dr. Patricia S. Springer and Dr.
Hailing Jin and their lab members for providing the research space, facilities and help
on performing molecular experiments.
Iwouldliketothankmyfriendsandfellowgraduatestudents: JinYao,Wenhao
Lu and Yizhou Wang. I had many enlightening and pleasant conversations with them
over the years.
I would like to thank my parents, brother and his family for their endless love,
support and encouragement throughout my PhD study. Without them I would not have
been me today. They deserve much credit for what I have acheived so far.
I particularly like to thank my wife Sijia Liu. It is her who always stand by
me for all the joyful and frustrated moments we have been through. Thank her for
bearing with a bad tempered me for all the time. I also owe a great debt to her and
our to-born daughter “Yuanyuan” for indulging me to work on my, what she so-called,
“mystery codes” and dissertation on the nights and weekends, which I should have spent
with them.
It’s impossible to acknowledge everyone here. If I have missed you, I apologize
for that and would like you to know that your help and support is greatly appreciated.
v
Dedicated to my family.
vi
ABSTRACT OF THE DISSERTATION
Discovery and Evolutionary Analyses of Abiotic Stress-Regulated MicroRNAs in
Crops
by
Zhigang Wu
Doctor of Philosophy, Graduate Program in Plant Biology
University of California, Riverside, June 2014
Dr. Renyi Liu, Chairperson
Abioticsstressessuchasdrought,cold,andsaltareverypervasiveinmodernagriculture,
which cause tremendous yield loss each year worldwide. MicroRNAs (miRNAs) play
importantregulatoryrolesinabioticstressresponses. TounderstandtherolesofmiRNAs
in crop responses to abiotic stresses, we used next-generation sequencing techniques to
profile the small RNA population in crops with and without stress treatments. We
identified many novel as well as stress-regulated miRNAs in: cowpea, rice, maize and
sorghum. Specifically, we identified 157 candidate miRNAs in cowpea, 227 in rice, 298
in maize and 407 in sorghum, many of which are novel. We identified 44, 28, 31 and 42
stress-regulatedmiRNAsincowpea, rice, maizeandsorghum, respectively. Interestingly,
majority (81%) of stress-regulated miRNAs in maize were cold stress-regulated, while
the majority (79%) in sorghum are drought stress-regulated. We further show that
the gain of species-specific miRNA families contributes to the different stress tolerances
in maize and sorghum. We provided solid evidence to show that 80 of rice candidate
miRNAs originated from MITEs and transposons. Additionally, homology search and
structural analysis in collinear regions of maize, sorghum and rice recovered another 26,
10 and 3 miRNAs in maize, sorghum and rice, respectively. We provided clear evidence
vii
for orthologous and paralogous miRNA gene expression diversification by northern blot
and/or digital expression analysis. Finally, we estimated that the birth rate of miRNAs
incerealgenomewas23.55genespermillionyearsandthedeathratewas0.63and17.01
genes per million years for orthologous and paralogous miRNA genes, respectively.
viii
Contents
List of Figures xi
List of Tables xii
1 Introduction 1
1.1 Motivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Dissertation overview and organization . . . . . . . . . . . . . . . . . . . . 3
2 Discovery and characterization of abiotic stress-regulated microRNAs
in plants 4
2.1 Biogenesis of small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 Discovery of plant miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 Identification of abiotic stress-regulated miRNAs . . . . . . . . . . . . . . 11
2.4 Identification of miRNA targets . . . . . . . . . . . . . . . . . . . . . . . . 13
2.5 Evolution and expression diversification of stress-regulated miRNAs . . . . 14
3 Identification and comparative analysis of drought-regulated microR-
NAs in two cowpea genotypes 18
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.2 Results and discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.2.1 Identification of miRNAs in cowpea . . . . . . . . . . . . . . . . . 19
3.2.2 Drought-regulated miRNAs . . . . . . . . . . . . . . . . . . . . . . 21
3.3 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.4 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.4.1 Plant materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.4.2 Small RNA library construction and sequencing . . . . . . . . . . . 25
3.4.3 Northern blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4 Abiotic stress-reuglated and repeat-derived microRNAs in the inflo-
rescences of rice 27
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.2 Results and discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.2.1 Identification of miRNAs . . . . . . . . . . . . . . . . . . . . . . . 29
4.2.2 Abiotic stress-regulated miRNAs . . . . . . . . . . . . . . . . . . . 31
4.2.3 Transposon- and Repeat-derived miRNAs . . . . . . . . . . . . . . 32
4.3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.4 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
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