IICAR   25568
INSTITUTO DE INVESTIGACIONES EN CIENCIAS AGRARIAS DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Characterization of the floral transcriptome small RNA component from Paspalum notatum sexual and apomictic genotypes
Autor/es:
GRISOLIA, MAURICIO; IMMOBILE MOLARO, GRETA; PESSINO, SILVINA; D'AURIZIO, ROMINA; ROHR, CRISTIAN; BRUTTINI, MARCO
Lugar:
Bahía Blanca
Reunión:
Workshop; V Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis; 2016
Institución organizadora:
CERZOS-CONICET
Resumen:
Apomixis is an asexual mode of reproduction through seeds, resulting from an absent or aberrant meiosis (apomeiosis), fertilization-independent initiation of embryogenesis (parthenogenesis) and the formation of functional endosperm either autonomously or after fertilization (pseudogamy). It is conceived as a deviation from the sexual reproductive pathway, caused by failure or variation of one or a few genetic factors. The ability to produce clonal maternal progeny via seeds is of significant value to agriculture for its power to fix complex favorable hybrid genotypes. In the gramineae, apomixis is genetically controlled by one or a few loci, depending on the species, but expression modulation of some key developmental steps (like gametic fate acquisition and parthenogenesis) could involve epigenetic mechanisms. The objective of this work was to contribute to the study of the apomixis epigenetic control through a comparative analysis of the small RNA transcriptome component in florets of sexual and apomictic P. notatum plants. Small RNAs libraries were produced in triplicate from equitable mixes of spikelets collected at premeiosis, meiosis, postmeiosis and anthesis, as follows: total RNA was extracted with SV Total RNA isolation system (Promega). Samples were initially quantified using the Qubit 2.0 Fluorometer. RNA Integrity Number (RIN) was evaluated using Agilent Bioanalyzer 2100. The Small RNA libraries were prepared in accordance with the Illumina TruSeq Small RNA sample preparation guide. RNA 3´ and RNA 5´ RNA adapters were ligated to each end of sRNA molecules. Reverse transcription followed by PCR was used to create cDNA constructs. PCR was performed with two primers that anneal to the ends of the adapters, one of them containing index sequences. High quality small RNAs were extracted and purified from polyacrylamide gels for subsequent cluster generation. The resulting libraries were validated using the Agilent 2100 Bioanalyzer, to check the size, purity and concentration. For library pooling and sequencing, equal volumes of normalized libraries were combined, denatured and diluted in hybridization buffer. The library pool was combined with 5% PhiX Control to balance the overall lack of sequence diversity. The samples were then sequenced using the Illumina MiSeq in a 1x50bp single read run. The number of reads obtained ranged from 1,558,547 to 2,996,675, depending on the library. Reads quality controls were conducted with FastQC v.0.11.5 and multiQC. Adapters sequences were removed using the software Cut adapt v1.3. Bowtie v. 1.1.2 was used to align reads against reference genomes/transcriptomes. For read counting, the program Subread v 1.5.0-p1 (module feature counts) was used. Small RNAs from 18 to 26 nt were filtered and selected for comparative analysis. Length distribution within this fraction revealed peaks at 21 nt and 24 nt. Differential expression analysis was conducted with the softwares EdgeR and NOISeq. The sRNAs reads were mapped onto a Paspalum notatum 454/Roche floral reference transcriptome. Ninety-two (92) isotigs showed differential association with sRNAs at p < 0.01. Sixty-five of them (70.65%) consisted of ncRNAs. Twenty-seven (29.34%) were protein-coding sequences, out of which 10 corresponded to retrotransposon/transposon proteins. The rest of the protein-coding transcripts (18.47%) were included into the following ontological classes: protein degradation (7), signaling (1), transcription regulation (2), RNA degradation/processing (2), catabolism (2), transport (2), unknown (1). Therefore, 41% of the non-transposon protein-coding genes differentially regulated via sRNA mediated mechanisms corresponded to protein degradation pathways. Mapping onto the Oryza sativa indica databases revealed that 4 miRNA sequences were significantly overrepresented in apomictic libraries. Moreover, using a mirDeep2 customized approach we identified potential novel miRNA precursors among isotigs, predicted their structures through RNAfold and identified potential mature miRNA and star sequences. The following numbers of new miRNA sequences were predicted in the P. notatum sRNA libraries: Apo1: 65; Apo2: 37; Apo3: 41; Sex1: 43; Sex2: 40; Sex3: 24. In further work we will correlate the different libraries data in order to identify unique pre-miRNA predictions and check if some of these novel precursors are differentially active in apomictic and sexual plants.