INVESTIGADORES
PESSINO Silvina Claudia
congresos y reuniones científicas
Título:
Construction of reference floral transcriptome databases for apomictic and sexual Paspalum notatum
Autor/es:
PESSINO SC; SIENA LA; SARTOR M; PODIO M; DELGADO L; ORTIZ JPA
Lugar:
Bahía Blanca
Reunión:
Workshop; IV Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis en gramíneas forrajeras; 2014
Institución organizadora:
CERZOS-CONICET
Resumen:
Apomixis is an asexual reproductive mode via seeds, which probably arose from a few alterations in genes controlling plant sexuality. This project was aimed at generating floral reference transcriptomes from tetraploid Paspalum notatum genotypes Q4117 (apomict) and C4-4x (sexual). Flowers at different developmental stages were collected and equitatively mixed. Total RNA was extracted using the SV RNA Total Isolation Kit (Promega). Sequencing was carried out at INDEAR (Instituto de Agrobiotecnología de Rosario, Rosario, Argentina). Samples were quantified with the Quant-iT RiboGreen RNA Reagent and Kit (Invitrogen). Messenger RNAs were purified and quantified with Dynabeads (Invitrogen) and Quant-iT RiboGreen RNA (Invitrogen), respectively. Libraries were prepared following instructions described at the cDNA Rapid Library Preparation Method Manual. An RNA 6000 Pico Chip (Agilent Bioanalyzer 2100) and a High Sensitivity DNA Chip (Agilent Bioanalyzer 2100) were used to control the quality of the fragmentation and libraries, respectively. Then, libraries were quantified using the Kapa Library Quantification Kit 454 Lib-L (Roche), as indicated by the manufacturers. A large-scale emulsion PCR was done with the GS Titanium LV-emPCR Kit Lib-L v2 (Roche), as indicated in the emPCR Amplification Method Manual?Lib L LV. Each library (sexual or apomictic) was sequenced by using the 454 GS FLX+ Roche method in a complete Titanium plate, according to the protocol described at the Sequencing Method FLX+ Roche Manual. The sexual and apomictic libraries produced 1,367,227 and 1,378,523 reads with an average size of 470,21 and 494,88, summing up 642,887,313 and 682,198,061 sequenced base pairs (bp), respectively. The quality and quantity of the reads outweighed the expected values. Sequences corresponding to rRNAs were removed using HMM. Low quality reads, adapters and primers were removed by using PRINSEQ. Assemblies carried out with NEWBLER_v2.8 with the urt option showed 35,430 or 37,124 isogroups, 31,551 or 47,642 contigs and 43,888 or 47,569 isotigs, for the sexual or apomictic libraries, respectively. The proportion of isogroups including more than one isotig resulted significantly higher in the apomictic library (P: 0.1613; 95% CI: 0.1576-0.1651) with respect to the sexual one (P: 0.1380; 95% CI: 0.1344-0.1416), revealing a higher level of variation that could be associated with the presence of a larger number of splicing variants/alleles/paralogs. Trancripts were assembled using TRINOTATE. The reference floral transcriptomes reported here will be of interest to Paspalum research and molecular breeding projects currently conducted in Argentina.