Open Access Highly Accessed Research article

Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thaliana seeds

Peter C McKeown1, Sylvia Laouielle-Duprat1, Pjotr Prins2, Philip Wolff34, Marc W Schmid5, Mark TA Donoghue1, Antoine Fort1, Dorota Duszynska1, Aurélie Comte1, Nga Thi Lao1, Trevor J Wennblom6, Geert Smant2, Claudia Köhler34, Ueli Grossniklaus5 and Charles Spillane1*

Author Affiliations

1 Genetics and Biotechnology Lab, Botany and Plant Science, National University of Ireland Galway (NUIG), C306 Aras de Brun, University Road, Galway, Ireland

2 Laboratory of Nematology, Wageningen University, Droevendaalsesteeg 1, Wageningen, The Netherlands

3 Department of Biology and Zürich-Basel Plant Science Center, Swiss Federal Institute of Technology, ETH Centre, CH-8092 Zürich, Switzerland

4 Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden

5 Institute of Plant Biology and Zürich-Basel Plant Science Center, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland

6 Silicon Life Sciences, Minneapolis, MN, USA

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BMC Plant Biology 2011, 11:113  doi:10.1186/1471-2229-11-113

Published: 12 August 2011

Additional files

Additional file 1:

Table S1 - Known imprinted genes in flowering plants. In the angiosperms, eleven imprinted genes had been reported from Arabidopsis thaliana and related species, six from maize and one from rice. All but three are expressed solely from the maternally inherited allele. With the exception of MEDEA, for which conflicting reports have been published (for discussion see [72]), all imprinted Arabidopsis thaliana genes show mono-allelic expression only in the terminally differentiating endosperm, whereas maize Mee1 clearly shows imprinted expression in the maize embryo.

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Additional file 2:

Figure S1 - Flow chart of the GenFrag program. Generation of the Transcript Derived Fragment (TDF) by poly-A capture of mRNA from silique tissue, followed by two digestions using first BstYI then MseI restriction enzymes. The fragments were ligated to adapters and selectively amplified using the primer combinations described (Additional file 4, Table S3). Transcript sizes were then determined on the ABI3130xl Genetic Analyzer. TDFs which were called as present in the maternal hybrid cross direction but absence from the reciprocal cross were considered as being derived from candidate MEGs (Additional file 3, Table S2). Subsequently, transcript sequences were assigned to progenitor genes by GenFrag using publicly available Arabidopsis transcript sequences. The transcript goes through two in silico digestions using first BstYI then MseI restriction enzymes to produce one fragment per transcript mimicking the in vitro protocol from which the gene identify is determined (Table 1).

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Additional file 3:

Table S2 - Relative proportions of uniparental TDFs expressed in siliques as determined by cDNA-AFLP of hybrid Col-0 × Ler-0 reciprocal crosses across three timepoints.

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Additional file 4:

Table S3 - Identification of 93 TDFs showing maternal-specific expression using the GenFrag software. 93 TDFs showing maternal-specific expression were analysed by GenFrag. Gene identitites were predicted using combinations of primers designed against BstYI and MseI cutting sites (3rd and 4th columns) and the size of the TDF as determined by capillary electrophoresis (5th column) as unique identifiers. Single unique genes were predicted for 52 TDFs out of the 93 maternal-specific TDFs (7th column, TDFs 1-52). A further 21 TDFs (TDFs 53-73) rendered non-unique predictions and 20 others (TDFs 74-93) could not be matched by GenFrag to any known Arabidopsis thaliana sequence. For 8 TDFs, the predicted size (marked with *) differed from that determined by capillary electrophoresis, by amounts indicated in parentheses.

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Additional file 5:

Table S4 - Lack of detection of known imprinted genes is due to lack of SNPs in restriction sites. MseI cuts in the T/TAA context, Bst YI cuts in the R/GATCY context (source: SALK SNP viewer, TAIR).

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Additional file 6:

Table S5 - Relative expression levels of maternally-expressed seed genes in the endosperm and seed coat. Genes detected as maternal by cDNA-AFLP with a log2-ratio of higher than 1 (indicating expression twice as high in endosperm vs. seed coat) are listed.

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Additional file 7:

Figure S2 - Analysis of expression profiles of ATCDC48, PDE120 and MS5-like in LCM tissues of Arabidopsis thaliana seeds (4 dap). Results of RT-PCR performed on cDNA derived from LCM endosperm (ES), seed coat (SC) and embryo (EM) tissues, shown for one representative replicate of two. ACT11 and UBC9 were used as loading controls.

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Additional file 8:

Figure S3 - Confirmation of maternal expression of cDNA-AFLP candidate genes in crosses to Ler-0. cDNA from 4 dap tissue was amplified and sequenced from F1 Ler-0 × Col-0 hybrid seeds and MS5-like and PDE120 found to be maternally expressed.

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Additional file 9:

Figure S4 - Confirmation of six further cDNA-AFLP genes as maternal in seed tissue. cDNA from F1 4 dap seed tissue was amplified and sequenced. In each case, cDNA from Col-0 × C24 is shown on the left, C24 × Col-0 is shown on the right. SNPs are marked with asterisks.

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Additional file 10:

Figure S5 - Divergent candidate imprinted genes identified by different screens in Arabidopsis. The overlap between maternally-expressed genes identified from this cDNA-AFLP screen performed in 3-5 dap Col-0 × Ler-0 crosses (red); those predicted from analysis of DMRs identified from Col-0 × Ler-0 endosperm (blue, [25]) and those identified by next generation screening approaches ([24] yellow; [23]; green) (see descriptions in Introduction).

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Additional file 11:

Figure S5 - Identification of DMRs located in the vicinity of candidate imprinted genes (Table 2) and other maternally inherited genes. Difference in methylation percentage between dme and wild-type endosperm for cytosine bases in the vicinity of three candidate imprinted genes (Table 2). Grey boxes represent the gene body in a 5'-3' orientation, red bars highlight DMRs.

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