Identification of Small RNAs In Common Bean (Phaseolus vulgaris) from 454
transcriptome sequencing data
Yaqoob Thurston1 , Zhanji Liu2, Venu Kalavacharla1,3
1Department
of Agriculture & Natural Resources, Delaware State University, Dover, DE 19901 2College of Agriculture & Related Sciences, Delaware State University, Dover, DE 19901, 3Center for Integrated Biological and Environmental Research
(CIBER), Delaware State University, Dover, De, 19901
Abstract
Common bean (Phaseolus vulgaris L.) is a low cost source of protein that is nutritionally and
economically important to U.S. agriculture. Compared to most plant genomes, common bean is
relatively small between 570-650 mega base pairs (Mbp) making it a good model species for
studying sequence organization and evolution of the legume family. MicroRNAs, conserved in
eukaryotic organisms, are involved in gene regulation and are known to play a major role in
development,
nutrient homeostasis, abiotic stress and pathogen responses via interactions with
.
specific target mRNAs in plants. The goals of our research are to predict secondary structures,
predict potential miRNA candidates and validate predicted miRNA candidates in order to better
understand miRNA involvement in common bean gene regulation. In the future, our ability to
understand plant genetics may aid in the production of more sustainable crops. In this study, we
analyzed 454 sequences from common bean derived from transcriptome sequencing to identify
candidate miRNAs. We report on progress towards the identification of potential miRNAs using a
combination of computational methods: (mfold software, and mirBase database), evaluation
against published miRNAs criteria, and current genetic experimental approaches (RNA
extraction, cDNA synthesis, and RT-PCR). We have found that our 454 transcriptome
sequences (861 which were classified into 64 putative families) have secondary structures. Our
analyses revealed that four sequences carry signature hairpin stem loops that have potential
miRNA locations on the hairpin on the secondary structure. Through our work, we were unable
to amplify cDNA using primers constructed from the four potential miRNA. However, we have
found that predicting miRNA candidates requires a thorough reenactment of the developmental
progress from which potential candidates are derived. Our findings will serve as a foundation for
future work involving the validation of the 454 transcriptome sequence data.
Introduction
• Common beans have proven to be an economically important crop (worth approximately $11
billion) that provides a low-cost source of protein and fiber.
Results
6) predicted mature miRNAs with no more than 3 nt substitutions as compared with A.
thaliana, rice, Populus and Physcometrella mature miRNAs
64 sequences
had homology
to miRNA
mirBase
Checked primers
mFold
Designed
primers
Check
criteria
Flow Chart of Analysis
• DNA extraction was performed on five genotypes according to Kalavacharla et. al. (2000).
•Primer design was performed using the Integrated DNA Technology website
(www.idtdna.com)
(2) a mature miRNA sequence site in one arm of the hairpin structure
Δ3
N
Discussion
• Results show that we should continue our research on identification of small in
RNAs common bean .
• After secondary structures were predicted using the mfold software, mirBase was
then used to observe the location on possible miRNA candidates. Subsequently, the
miRNA criteria was used to validate candidates.
Figure 3: Location of miRNA on secondary structure
(3) miRNAs having less than six mismatches with the opposite miRNA* sequence in
the other arm
Objectives
• To predict secondary structures for possible miRNA candidates identified by 454
transcriptome sequencing
oIdentify where miRNA is located on secondary structure
oDetermine if candidate miRNA fits known miRNA criteria
• To validate miRNA candidates
Figure 4: miRNA sequence on secondary structure
(5) predicted secondary structures with higher MFELs and negative MFEs
• 961 candidate miRNA sequences were identified from a BLAST analysis of plant known
miRNA sequence and 454 sequences
• Results show that the genomic DNA of the five genotypes was amplified with
miRNA primers. Further studies need to be carried out to identify large scale
secondary structures and small RNAs.
• Future work should also be done to identify other small RNAs such as siRNAs.
References
1. Kalavacharla, V., Stavely, JR., Myers, JR., McCleam, PE. Crg, a Gene Required
for Ur-3 Mediated Rust Resistance in Common Bean, Maps to a Resistance Gene
Analog Cluster. Molecular Plant- Microbe Interactions 13(11), 1237-1242 (2000).
2. Ambros, V., Bartel, B., Bartel, D., Burge, C., Carrington, J., Chen, X., Dreyfuss, G.,
Eddy, S., Griffiths-Jones, S., Marshall, M., Matzke, M., Ruvkun, G. and Tuschl, T.
(2003b). A uniform system for microRNA annotation. RNA 9, 277-279
3. Elbashir, Sayda M., Javier Martinez, Agnieszka Patkaniowska, Winfried Lendeckel
and Thomas Tuschl. Functional anatomy of siRNAs for mediating efficient RNAi in
Drosophila melanogaster embryo lysate. The EMBO Journal, 20 (23), 6877-6888,
2001
4. Integrated DNA Technologies (www.idtdna.com)
Acknowledgement
s would like to thank Dr. Jyothi Thimmapuram for assistance with the
We
• mFold software was used to predict secondary structures on RNA
•The miRBase database was used to identify where the miRNA candidates are located on
the secondary structure
• miRNA criteria was used to validate if RNA sequences have homology to miRNAs
Δ2
• Because we were able to predict a secondary structure from 454 transcriptome
sequences, our research shows promise that there are other small RNAs to be
validated.
(4) no loop or break in miRNA* sequences
• Five common bean genotypes (listed in Figure 1)
C
Figures 7 : Gel electrophoresis of PCR product with miRNA primers
• Small RNAs are (21-26 nt, flexible repressors of gene expression in plants, animals and many
fungi
Materials and Methods
O
*
• Secondary structures can show the location of small RNAs
• MicroRNAs are a class of non–protein-coding RNAs normally 19-24 nucleotides in length that
mediate the down regulation of gene expression. (Elbashir et. at., 2001).
S
miRNA criteria (Ambros et. at.,2003)
(1) a RNA sequence folding into an appropriate stem-loop hairpin secondary structure
Figure 2: Secondary structure from candidate RNA sequence 21
• Small RNAs encompass many different classes of non-coding RNAs each with their own
properties and functions.
1 kb
•PCR with primers constructed from miRNA was carried out to amplify DNA of the five
genotypes followed by gel electrophoresis to measure and validate amplification of PCR
product
• Similar to lima beans and soybeans, common bean is high in starch, protein, and dietary fiber
and is an excellent source of iron, potassium, selenium, molybdenum, thiamine, vitamin B6
and folic acid.
Figure 1: Image of five common bean genotypes (Sierra, Olathe, crg, Δ2 and Δ3 used in
experiments)
• Secondary structures are RNA structures characterized by folding of the peptide chain into an
alpha helix, beta sheet, or random coil
Figure 6 : miRBase output displaying mature miRNAs that were compared to other plant
species
Figure 5 : mFold output of MFELs
assembling of the 454 sequences, Dr. Kalpalatha Melmaiee and Ms.
Antonette Todd for advice and suggestions. YT acknowledges HBCU –
STEM fellowship and VK acknowledges funding by NSF grant EPS0814251 and support for YT.