Construction of reference floral transcriptome databases for apomictic and sexual Paspalum notatum

PESSINO, SILIVINA CLAUDIA; SIENA, LORENA ADELINA; SARTOR, MARÍA E.; PODIO, MARICEL; DELGADO LUCIANA; ORTIZ, JUAN PABLO AMELIO

Bahía Blanca

Encuentro; IV Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis en gramíneas forrajeras; 2014

Cerzos-CONICET

Apomixis is an asexual reproductive mode via seeds,which probably arose from a few alterations in genescontrolling plant sexuality. This project was aimed atgenerating floral reference transcriptomes from tetraploidPaspalum notatum genotypes Q4117 (apomict) andC4-4x (sexual). Flowers at different developmental stageswere collected and equitatively mixed. Total RNA wasextracted using the SV RNA Total Isolation Kit (Promega).Sequencing was carried out at INDEAR (Instituto deAgrobiotecnología de Rosario, Rosario, Argentina).Samples were quantified with the Quant-iT RiboGreenRNA Reagent and Kit (Invitrogen). Messenger RNAs werepurified and quantified with Dynabeads (Invitrogen) andQuant-iT RiboGreen RNA (Invitrogen), respectively.Libraries were prepared following instructions described atthe cDNA Rapid Library Preparation Method Manual. AnRNA 6000 Pico Chip (Agilent Bioanalyzer 2100) and aHigh Sensitivity DNA Chip (Agilent Bioanalyzer 2100)were used to control the quality of the fragmentation andlibraries, respectively. Then, libraries were quantifiedusing the Kapa Library Quantification Kit 454 Lib-L(Roche), as indicated by the manufacturers. A large-scaleemulsion PCR was done with the GS Titanium LV-emPCRKit Lib-L v2 (Roche), as indicated in the emPCRAmplification Method Manual?Lib L LV. Each library(sexual or apomictic) was sequenced by using the 454 GSFLX+ Roche method in a complete Titanium plate,according to the protocol described at the SequencingMethod FLX+ Roche Manual. The sexual and apomicticlibraries produced 1,367,227 and 1,378,523 reads with anaverage size of 470,21 and 494,88, summing up642,887,313 and 682,198,061 sequenced base pairs(bp), respectively. The quality and quantity of the readsoutweighed the expected values. Sequencescorresponding to rRNAs were removed using HMM. Lowquality reads, adapters and primers were removed byusing PRINSEQ. Assemblies carried out withNEWBLER_v2.8 with the urt option showed 35,430 or37,124 isogroups, 31,551 or 47,642 contigs and 43,888or 47,569 isotigs, for the sexual or apomictic libraries,respectively. The proportion of isogroups including morethan one isotig resulted significantly higher in theapomictic 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 thatcould be associated with the presence of a larger numberof splicing variants/alleles/paralogs. Trancripts wereassembled using TRINOTATE. The reference floraltranscriptomes reported here will be of interest toPaspalum research and molecular breeding projectscurrently conducted in Argentina.