The Role of Brassica rapa FLC Genes in Flowering Time

1. NSF HBCU-UP Program Fort Valley State University Comparative RT-PCR Procedure RNA Isolation 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 H1: There are alleles, some correlated with early and late flowering. H2: Expression starts high and decreases over the course of development Fig.2 SequenceData revealed almost no allelic variation 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. rapaFlowering 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. Methods • Sequencing of the 1st Intron: • 30 DNA samples: (15 from 1997 & 15 from 2004) • Amplified 3 B. rapa FLC genes of the 1st intron using PCR • Visualized DNA on a 1% Agarose gel • Sent samples for sequencing • 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 Fig.3 The Real Time data showed and initial decrease followed by an increase then a final decrease in gene expression Background Conclusion Fig.1 Evolution of earlier flowering • 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. Questions Acknowledgements 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? This research was supported by the NSF HBCU-UP grant awarded to Sarwan K. Dhir at the Fort Valley State University, Fort Valley, GA.