RNA-seq and de novo assembly of sexual and apomictic floral transcriptomes of Paspalum notatum

JUAN PABLO A. ORTIZ; SANTIAGO REVALE; SIENA, LORENA ADELINA; PODIO, MARICEL; DELGADO LUCIANA; JULIANA STEIN; OLIVIER LEBLANC; PESSINO, SILIVINA CLAUDIA

Bahía Blanca

Workshop; V Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis; 2016

CERZOS-CONICET

Apomixis is a natural form of asexual reproduction by seeds present inmore than 35 flowering plant families. Progenies derived from apomictic plantsare genetically identical to the maternal parent. This type or reproductionoriginates after bypass thefundamental aspects of plant sexual reproduction: meiosis and fertilization. Apomicticplants are able to form either embryos or female gametes from unreduced somaticcells. The mechanisms involved are diverse, but all of them are considered deviationsfrom the typical sexual reproductive pathway, probably caused by genetic and/orepigenetic factors affecting one or few genes. This trait has significant valuefor agriculture. Transferring apomixis into economically important crops could allowthe fixation of hybrid combinations, the generation of new intergeneric andinterspecific hybrids adapted to local environments and the utilization ofseeds in crops that are propagated by vegetative organs. Paspalum is one of the largest genus of the Poaceae family. It includes severalspecies that are important natural forage resources for the tropical andsubtropical region of South America. Over the past five decades, a wealth of information regarding thebiology, genetic and reproductive behavior of many Paspalum species has been produced. Particularly,P. notatum and P. simplex became valuable models for the study of apomixis because they concurrentlyrepresent systems for mining candidate gene(s) and important forage crops. Inboth species, a number of genes associated with the trait have been identified bygenetic and/or expression analyses. However,apomictic races arepolyploid, highly heterozygous and have a relatively large DNAcontent. Therefore, the identification of fundamental geneshas been difficult. Moreover, no reference genome is still available, and thenumber of characterized sequences deposited in databanks is still limited. Inthis context, global transcriptomic approaches can contribute significantly tothe elucidation of the molecular bases of the trait. RNA-Seq technology offers several key advantagesover other existing methodologies for characterizing transcriptomes. It isparticularly attractive for non-model organisms with genomic sequences that areyet to be determined. The use of Next-Generation Sequencing (NGSs) technologiesdoes not require neither cloning libraries nor any prior knowledge of thespecies genome. Particularly, 454/Roche has become a method of choice foranalyzing transcriptomes of non-model organisms, because of its long-readcapacity, which makes data more amenable to denovo assembly and annotation. The objectives of this work were: 1) to producerobust reference transcriptome datasets from flowers of sexual and apomicticgenotypes, and 2) to deliver a list of transcripts with differentialrepresentation between sexual and apomictic types. A long-read 454/Roche FLX+sequencing strategy was used for database construction. Raw data originatedfrom sexual and apomictic flowers collected from premeiosis to anthesis wasused to assembly three libraries: i) sexual, ii) apomictic and iii) global(including both reads). The global assembly produced 48,842 floral transcripts,including 67,617 allelic variants. A group of physically-supported mRNA and ESTsequences was matched with high level of confidence to both sexual andapomictic libraries. Dozens of molecular functions and biological pathways operatingin P. notatum flowers wereidentified. A preliminary trial allowed discovery of the whole set of putativealleles/paralogs corresponding to 23 previously identified apomixis-associatedcandidate genes. Moreover, a list of 3,732 genes and several co-expressionnetworks differentially represented between both plant types were detected. Thereference floral transcriptomes reported here will be of interest to plantreproductive research and Paspalumbreeding projects.