Transcript The Role of Brassica rapa FLC Genes in Flowering Time

The Role of Brassica rapa FLC Genes in Flowering Time
India Brown, Center for Biotechnology, Fort Valley State University, Fort Valley, GA
Amy Litt, New York Botanical Garden, Fordham University, Bronx, NY
Abstract
Results
Hypotheses
A previous study showed an evolutionary change to earlier
flowering following a drought in the annual plant Brassica rapa.
The goal of the current project was to find out what genes were
responsible for the evolutionary change in flowering time. I
examined the relationship between flowering time and genetic
variation in B. rapa Flowering Locus C (FLC) genes which suppress
flowering. My first question was: is there allelic variation in the
genes, and if so are alleles correlated with flowering time. I
hypothesized that there would be allelic variation correlated with
flowering time. I looked at the 1st intron, because it is responsible
for gene expression. A total of 30 samples, 15 from the 1997
Backbay (BB) population and 15 from the 2004 (BB) population
were used for amplification of the 1st intron using PCR and sent off
for sequencing to look for allelic variation in three of the FLC
genes. Sequence data of the 1st intron revealed no allelic variation,
which did not support my hypothesis. In addition, for future
experiments there needed to be an established method for
studying gene expression through the course of development
within an individual. No one has looked at variation within a single
individual or natural population. My second question was: how
does expression change over the course of development of an
individual. I hypothesized that gene expression would start off
high and then decrease over time. One FLC gene was used to view
gene expression using Quantitative real-time PCR (qRT PCR) to
determine how gene expression changes over the course of
development within a single individual. qRT-PCR showed an
increase in expression from the first to the second sampling time
point, followed by a decrease. The initial increase did not occur in
all of the samples, so more analyses will have to be done.
H1: There are alleles, some correlated with early and
late flowering.
H2: Expression starts high and decreases over the
course of development
Fig.2 Sequence Data revealed almost no allelic
variation
Methods
Sequencing of the 1st Intron:
• 30 DNA samples: (15 from 1997 & 15 from 2004)
st
• Amplified 3 B. rapa FLC genes of the 1 intron using
PCR
• Visualized DNA on a 1% Agarose gel
• Sent samples for sequencing
Fig.3 The Real Time data showed and initial
decrease followed by an increase then a final
decrease in gene expression
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Gene Expression:
• Took hole punched tissue samples of the 1st and 2nd
leaf of Arboretum and Biennials
• Extracted RNA using kit
• cDNA
• Measured concentration levels
• Performed qRT PCR on 1 B.rapa FLC gene
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12
10
8
6
4
RNA Isolation
Background
BB 04
Population
BB H
BB 97
Arb 04
Arb H
Arb 97
40
50
60
2
0
Conclusion
Fig.1 Evolution of earlier flowering
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Comparative RT-PCR
Procedure
70
80
Days to first flowering
Questions
Q1: Do we see different alleles and are they correlated
with flowering time?
Q2: How does the expression on FLC genes change
during the course of development within an
individual?
•There was little to no allelic variation shown with my
sequence data, which resulted in my hypothesis not being
supported.
•This could be because random samples were used with
little difference in flowering time.
•I hypothesized that gene expression would first increase
and then decrease, but my results showed an initial
decrease then increase.
•Based on these results, there will need to be more tests
and analyses done.
Acknowledgements
This research was supported by the NSF HBCU-UP grant
awarded to Sarwan K. Dhir at the Fort Valley State
University, Fort Valley, GA.