Poliploidia em Lippia alba (MILL.) N. E. Brown (Verbenaceae): origem e consequências na expressão gênica

Abstract

Polyploidy, or whole-genome duplication (WGD), is widely recognized as the major evolutionary force of plants. Changes in genome size may result in wide phenotypic plasticity. The taxonomy of Lippia presents a controversial history and may have been influenced by the occurrence of polyploidization in the genus. In this particular, Lippia alba is the only species so far considered a well documented polyploid complex. The species has a remarkable chemical variation associated with ploidal levels. The present work aimed to contribute to elucidate the evolutionary history of Lippia alba in an integrated perspective to the emergence of the genus Lippia. In addition, we intended to contribute to understand of the genetic relationships between the accessions of Lippia alba and the terpene biosynthetic pathways associated with each ploidal level. Four approaches were employed: (1) We investigated 38 species of Lippia and 68 accessions of L. alba using nuclear and plastid DNA sequences, Bayesian analysis and molecular dating. (2) The genetic relationship was estimated in 98 accessions of Lippia alba by microsatellite and ITS and trnL-F EF sequences. The genetic distance and structure was estimated from SSR data. Nucleotide variation was visually observed selecting conserved blocks of ITS and trnL-F EF gene alignments. The haplotype networks were constructed using cloned ITS region. (3) In an attempt to investigate the relationship among the accessions based on gene expression related to the chemical constitution of the essential oil, the stability of nine candidate reference genes in Lippia alba was evaluated. (4) Orthologous genes were identified from the newly assembled RNA-seq (Illumina Miseq with paired-end libraries) of a triploid accession of L. alba. Estimates of gene expression related to terpene biosynthetic pathways were obtained by real-time quantitative PCR (RT-qPCR). It was possible to identify that the genus Lippia arose in the Late Miocene, which coincides with the emergence of the Cerrado, where the genus may have expanded and adapted. The polyploidy in Lippia alba occurred more recently during the Pleistocene. Genetic distance analysis and gene sequence data showed that the accessions were grouped by ploidal level and some recurrent groups were found in Lippia alba. There is a distinct lineage of diploids unrelated to other ploidal levels. Triploids formed a well-defined group that originated from a single group of diploids. The tetraploids and the hexaploid grouped together, suggesting the contribution of the tetraploids to the hexaploid origin. The G6I gene (glucose-6-phosphate isomerase) has been identified as a good reference gene for RT-qPCR studies in Lippia alba, given its stable expression in different cytotypes. Gene expression analysis did not show significant differences in expression levels among ploidal levels. Aspects related to the activation of the terpene biosynthetic pathway, gene identification and the exact gene copy responsible to terpene biosyntheses, the complexity network of gene regulation in polyploids and the conserved biosynthesis terpene pathway in autopolyploids might explain the results.