title: Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thaliana seeds.
abstract: BACKGROUND
  Epigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants.
  
  
  RESULTS
  cDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs) displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination). We identified these MEGs by developing a bioinformatics tool (GenFrag) which can directly determine the identities of transcript-derived fragments from (i) their size and (ii) which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1 seeds was confirmed via allele-specific transcript analysis across a range of different accessions. Differentially methylated regions were identified adjacent to ATCDC48 and PDE120, which may represent candidate imprinting control regions. Finally, we demonstrate that expression levels of these three genes in vegetative tissues are MET1-dependent, while their uniparental maternal expression in the seed is not dependent on MET1.
  
  
  CONCLUSIONS
  Using a cDNA-AFLP transcriptome profiling approach, we have identified three genes, ATCDC48, PDE120 and MS5-like which represent novel maternally expressed imprinted genes in the Arabidopsis thaliana seed. The extent of overlap between our cDNA-AFLP screen for maternally expressed imprinted genes, and other screens for imprinted and endosperm-expressed genes is discussed.
authors.affiliation: Genetics and Biotechnology Lab, Botany and Plant Science, National University of Ireland Galway, C306 Aras de Brun, University Road, Galway, Ireland.
authors.forename: Peter C
authors.initials: PC
authors.lastname: McKeown
authors.forename: Sylvia
authors.initials: S
authors.lastname: Laouielle-Duprat
authors.forename: Pjotr
authors.initials: P
authors.lastname: Prins
authors.forename: Philip
authors.initials: P
authors.lastname: Wolff
authors.forename: Marc W
authors.initials: MW
authors.lastname: Schmid
authors.forename: Mark T A
authors.initials: MT
authors.lastname: Donoghue
authors.forename: Antoine
authors.initials: A
authors.lastname: Fort
authors.forename: Dorota
authors.initials: D
authors.lastname: Duszynska
authors.forename: Aurélie
authors.initials: A
authors.lastname: Comte
authors.forename: Nga Thi
authors.initials: NT
authors.lastname: Lao
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Wennblom
authors.forename: Geert
authors.initials: G
authors.lastname: Smant
authors.forename: Claudia
authors.initials: C
authors.lastname: Köhler
authors.forename: Ueli
authors.initials: U
authors.lastname: Grossniklaus
authors.forename: Charles
authors.initials: C
authors.lastname: Spillane
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D017360:Arabidopsis / Q000235:genetics*; D019295:Computational Biology; D019175:DNA Methylation; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D056625:Endosperm / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D018392:Genomic Imprinting; D061346:Laser Capture Microdissection; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics*; D012984:Software
chemical_list: D018076:DNA, Complementary; D018744:DNA, Plant