Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

BackgroundMicro RNAs are a class of small non coding RNAs of 20–24 nucleotides transcribed as single stranded precursors from MIR gene loci. Initially described as post-transcriptional regulators involved in development, two decades ago, miRNAs have been proven to regulate a wide range of processes in plants such as germination, morphology and responses to biotic and abiotic stress. Despite wide conservation in plants, a number of miRNAs are lineage specific. We describe the first genome wide survey of Eucalyptus miRNAs based on high throughput sequencing.ResultsIn addition to discovering small RNA sequences, MIR loci were mapped onto the reference genome and interspecific variability investigated. Sequencing was carried out for the two most world widely planted species, E. grandis and E. globulus. To maximize discovery, E. grandis samples were from BRASUZ1, the same tree whose genome provided the reference sequence. Interspecific analysis reinforces the variability in small RNA repertoire even between closely related species. Characterization of Eucalyptus small RNA sequences showed 95 orthologous to conserved miRNAs and 193 novel miRNAs. In silico target prediction confirmed 163 novel miRNAs and degradome sequencing experimentally confirmed several hundred targets. Experimental evidence based on the exclusive expression of a set of small RNAs across 16 species within Myrtaceae further highlighted variable patterns of conservation and diversity of these regulatory elements.ConclusionsThe description of miRNAs in Eucalyptus contributes to scientific knowledge of this vast genre, which is the most widely planted hardwood crop in the tropical and subtropical world, adding another important element to the annotation of Eucalyptus grandis reference genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2322-6) contains supplementary material, which is available to authorized users.

Several studies suggest the relation of DNA methylation to diseases in humans and important phenotypes in plants drawing attention to this epigenetic mark as an important source of variability. In the last decades, several methodologies were developed to assess the methylation state of a genome. However, there is still a lack of affordable and precise methods for genome wide analysis in large sample size studies. Methyl sensitive double digestion MS-DArT sequencing method emerges as a promising alternative for methylation profiling. We developed a computational pipeline for the identification of DNA methylation using MS-DArT-seq data and carried out a pilot study using the Eucalyptus grandis tree sequenced for the species reference genome. Using a statistic framework as in differential expression analysis, 72,515 genomic sites were investigated and 5,846 methylated sites identified, several tissue specific, distributed along the species 11 chromosomes. We highlight a bias towards identification of DNA methylation in genic regions and the identification of 2,783 genes and 842 transposons containing methylated sites. Comparison with WGBS, DNA sequencing after treatment with bisulfite, data demonstrated a precision rate higher than 95% for our approach. The availability of a reference genome is useful for determining the genomic context of methylated sites but not imperative, making this approach suitable for any species. Our approach provides a cost effective, broad and reliable examination of DNA methylation profile on MspI/HpaII restriction sites, is fully reproducible and the source code is available on GitHub (https://github.com/wendelljpereira/ms-dart-seq).

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