RNA-seq and de novo assembly of sexual and apomictic floral transcriptomes of Paspalum notatum. A key database for studying apomixis candidate genes

ORTIZ JPA; REVALE, S; SIENA LA; PODIO M; DELGADO L; STEIN J; LEBLANC O; PESSINO SC

Bahía Blanca

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

Grupo apomixis Argentina

RNA-seq and de novo assembly of sexual and apomictic floral transcriptomes of Paspalum notatum. A key database for studying apomixis candidate genesJuan Pablo A. Ortiz1, Santiago Revale2, Lorena A. Siena1, Maricel Podio1, Luciana Delgado1, Juliana Stein1, Olivier Leblanc3, Silvina C. Pessino11Instituto de Investigaciones en Ciencias Agrarias de Rosario (IICAR), CONICET-UNR/Laboratorio de Biología Molecular, Facultad de Ciencias Agrarias, UNR, Campo Experimental Villarino, (S2125ZAA), Zavalla, Santa Fe, Argentina; 2Instituto de Agrobiotecnología de Rosario (INDEAR), Ocampo 210 bis, (2000) Rosario, Santa Fe, Argentina; 3Institut de Recherche pour le Développement, ERL 5300 IRD/CNRS, UMR 232 IRD, 911 Avenue Agropolis, Montpellier, France. Email: jortiz@unr.edu.arApomixis is a natural form of asexual reproduction by seeds present in more than 35 flowering plant families. Progenies derived from apomictic plants are genetically identical to the maternal parent. This type or reproduction originates after bypass the fundamental aspects of plant sexual reproduction: meiosis and fertilization. Apomictic plants are able to form either embryos or female gametes from unreduced somatic cells. The mechanisms involved are diverse, but all of them are considered deviations from the typical sexual reproductive pathway, probably caused by genetic and/or epigenetic factors affecting one or few genes. This trait has significant value for agriculture. Transferring apomixis into economically important crops could allow the fixation of hybrid combinations, the generation of new intergeneric and interspecific hybrids adapted to local environments and the utilization of seeds in crops that are propagated by vegetative organs. Paspalum is one of the largest genus of the Poaceae family. It includes several species that are important natural forage resources for the tropical and subtropical region of South America. Over the past five decades, a wealth of information regarding the biology, 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 concurrently represent systems for mining candidate gene(s) and important forage crops. In both species, a number of genes associated with the trait have been identified by genetic and/or expression analyses. However, apomictic races are polyploid, highly heterozygous and have a relatively large DNA content. Therefore, the identification of fundamental genes has been difficult. Moreover, no reference genome is still available, and the number of characterized sequences deposited in databanks is still limited. In this context, global transcriptomic approaches can contribute significantly to the elucidation of the molecular bases of the trait. RNA-Seq technology offers several key advantages over other existing methodologies for characterizing transcriptomes. It is particularly attractive for non-model organisms with genomic sequences that are yet to be determined. The use of Next-Generation Sequencing (NGSs) technologies does not require neither cloning libraries nor any prior knowledge of the species genome. Particularly, 454/Roche has become a method of choice for analyzing transcriptomes of non-model organisms, because of its long-read capacity, which makes data more amenable to de novo assembly and annotation. The objectives of this work were: 1) to produce robust reference transcriptome datasets from flowers of sexual and apomictic genotypes, and 2) to deliver a list of transcripts with differential representation between sexual and apomictic types. A long-read 454/Roche FLX+ sequencing strategy was used for database construction. Raw data originated from sexual and apomictic flowers collected from premeiosis to anthesis was used 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 EST sequences was matched with high level of confidence to both sexual and apomictic libraries. Dozens of molecular functions and biological pathways operating in P. notatum flowers were identified. A preliminary trial allowed discovery of the whole set of putative alleles/paralogs corresponding to 23 previously identified apomixis-associated candidate genes. Moreover, a list of 3,732 genes and several co-expression networks differentially represented between both plant types were detected. The reference floral transcriptomes reported here will be of interest to plant reproductive research and Paspalum breeding projects.