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Published on November 16, 2007; 10.1104/pp.107.108597

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Received September 3, 2007
Accepted October 26, 2007

Transcript Profiling by 3'UTR Sequencing Resolves Expression of Gene Families

Andrea L. Eveland , Donald R. McCarty , and Karen E. Koch *

Department of Horticultural Sciences, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, Gainesville, 32611, USA

* Corresponding author; email: kekoch{at}ufl.edu.

Differences in gene expression underlie central questions in plant biology extending from gene function to evolutionary mechanisms and quantitative traits. However, resolving expression of closely-related genes (e.g. alleles and gene family members) is challenging on a genome-wide scale due to extensive sequence similarity and frequently incomplete genome sequence data. We present a new expression-profiling strategy that utilizes long-read, high-throughput sequencing to capture the information-rich 3'-untranslated region (UTR) of mRNAs. Resulting sequences resolve gene-specific transcripts independent of a sequenced genome. Analysis of ~229,000 3'-anchored sequences from maize (Zea mays L.) ovaries identified 14,822 unique transcripts represented by ≥ 2 sequence reads. Total RNA from ovaries of drought-stressed wild-type and viviparous-1 mutant plants was used to construct a multiplex cDNA library. Each sample was labeled by incorporating one of 16 unique three-base key codes into the 3'-cDNA fragments, and combined samples were sequenced using a GS 20 454 instrument. Transcript abundance was quantified by frequency of sequences identifying each unique mRNA. At least 202 unique transcripts showed highly significant differences in abundance between wild-type and mutant samples. For a subset of mRNAs, quantitative differences were validated by Q-PCR. The 3'UTR profile resolved 12 unique Cellulose Synthase (CesA) transcripts in maize ovaries and identified previously-uncharacterized members of a Histone-1 gene family. In addition, this method resolved nearly-identical paralogs, as illustrated by two Auxin Repressed Dormancy Associated transcripts, which showed reciprocal mRNA abundance in wild-type and mutant samples. Our results demonstrate the potential of 3'UTR profiling for resolving gene- and allele-specific transcripts.




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