The present study was set to characterise the DNMT
The present study was set to characterise the DNMT gene orthologs and describe the expression patterns of these genes during seed development in natural diploid apomict B. divaricarpa and diploid sexual B. stricta.
Material and methods
Results The B. stricta genome v1.2, was sequenced and assembled by the Department of Energy's Joint Genome Institute (JGI) and can be reached through the Phytozome 10.2 web page (http://phytozome.jgi.doe.gov/). We performed a BLASTp search against the diploid sexual B. stricta genome using the DNMT protein sequences from A. thaliana as query. The only protein that showed high sequence similarity (86.8% identical) to AT5G49160 (MET1) was Bostr.15774s0046. This protein contains 1546 amino acids, encoded from a locus comprising 11exons that spans 6553bp in total and annotated as DNA (CYTOSINE-5) –METHYLTRANSFERASE 1 (MET1). The database also reveals that Bostr.15774s0046 contains a Bromo-Adjacent Homology (BAH) (Callebaut et al., 1999) and cytosine specific DNA methyltransferase replication foci domains (Rountree et al., 2000) that are common with A. thaliana MET1. The BLASTp search also indicated that two proteins (Bostr.2902s0198.1 and Bostr.2902s0198.2) show high sequence similarity (86.9% identical) to AT5G14620 (DRM2). These proteins are encoded by same locus as alternative transcripts, although the amino AZD 3965 sequences are the same. This locus comprises 10exons that spans 4228bp in total, and encodes a protein contains 631 amino acids annotated as DNA (CYTOSINE-5-) –METHYLTRANSFERASE 3-RELATED. The database shows that Bostr.2902s0198.1 and Bostr.2902s0198.2 are both contain C-5 cytosine-specific DNA methylase domain common with A. thaliana DRM2. Bostr.10273s0387.1 had the highest sequence similarity (76.5%) with AT1G69770. This protein is encoded by a locus comprised 21exons that spans 5739bp in total and contains 827 amino acids is annotated as DNA (cytosine-5-) –methyltransferase (CMT3). The database also shows that Bostr.10273s0387.1 contains a BAH and Chromo domains in common with AT1G69770 (Callebaut et al., 1999, Min et al., 2003). Then, we designated these genes as Bostr.MET1, Bostr.DRM2 and Bostr.CMT3 respectively, based on homology and conserved domains. We also downloaded the raw sequencing data of diploid apomict B. holboellii cv. Panther genome from the SRA database and trimmed the low quality (Q<30) reads. This library contains 1.427.651.762 short reads. We obtained the apomict B. holboellii DNMTs genomic and putative amino acid sequences using the Geneious R8 software “map to references” option. We used the B. stricta DNMT sequences as reference sequences to map the B. holboellii genome library. The mappings enabled us to assemble 45.736 reads for Bostr.MET1, 17.464 reads for Bostr.DRM2 and 20.506 reads in total for Bostr.CMT3. The consensus sequences were saved and we annotated the B. holboellii DNMT gene sequences using the “find annotations” feature in Geneious, transferring annotations with a 50% or greater similarity of reference sequences on Phytozome V10.3. Our results showed that the genomic sequences of Boholb.MET1 are comprised of 11exons which span 6532bp in total, encodes a protein contains 1546 amino acids that is highly identical to Bostr.MET1 (99.1%) and to AT5G49160 (87%). Similarly, the genomic sequence of Boholb.DRM2 was comprised of 10exons which span 4233bp in total, encodes a protein contains 631 amino acids that is highly identical to Bostr.DRM2 (99.2%) and to AT5G14620 (98%). The genomic sequences of Boholb.CMT3 are composed of 21exons which span 5732bp in total, encodes a protein contains 827 amino acids that are highly identical to Bostr.CMT3 (99.4%) and to AT1G69770 (77%). The diploid apomict B. divaricarpa transcriptome data contains 203.736.114 reads in total. The mapping studies reveal that the transcriptome data provides 6750 reads for CMT3, 8477 reads for DRM2 and 8622 reads for MET1. We saved the consensus sequences and transferred the annotations with a 50% or greater similarity of reference sequences using Geneious R8 software. The lengths of B. divaricarpa DNMT coding sequences (CDS) were 4641bp for MET1, 1896bp for DRM2 and 2484bp for CMT3. These CDS encoded 1546, 631 and 827 amino acids in total respectively. The Blast analyses showed that the B. divaricarpa DNMTs are all highly identical to B. stricta and Arabidopsis DNMTs. Bodiv.MET1 is 86.8% identical to MET1 and 99.3% to Bostr.MET1. Bodiv.DRM2 is 86.9% identical to MET1 and 99.2% to Bostr.MET1. Bodiv.CMT3 is only 76.8% identical to MET1, and 99.9% to Bostr.CMT3.