Sistemática molecular para ácaros planos do gênero Brevipalpus Donnadieu (Acari: Tenuipalpidae) : utilização de marcadores moleculares mitocondriais e nucleares

Souto-Oliveira. I. , C. Molecular systematics for flat mites of the genus Brevipalpus Donnadieu (Acari: Tenuipalpidae) – using mitochondrial and nuclear molecular markers. 2014, 107P. Dissertation (Master Science), Instituto de Ciências Biológicas, Universidade de Brasília, DF. The genus Brevipalpus is one of the most important in the Tenuipalpidae family. These phytophagous mites can cause damage to their host plants either directly, by feeding on leaves, stems and fruits or indirectly, by transmitting plant virus. Brevipalpus mite taxonomy, particularly of economically important species, represents a challenge for acarologists; numerous synonyms have been established and the occurrence of cryptic species has been confirmed. The accurate identification of these mites is essential to understand the role of each species in the transmission of plant viruses, for listing host plants of each species, and for defining prevention and control strategies. Morphological studies applying different microscopy techniques have enabled a considerable advance in the taxonomy of the group. In an integrative approach, besides the morphological studies is essential to have additional tools for building a consistent systematics for groups of organisms. So far, only one fragment of the Cytochrome Oxidase I (COI) region, of the mitochondrial DNA had been explored in Brevipalpus systematics. Employing a higher number of independent molecular markers is important to give consistency for the studies. The present study aimed to contribute for the advance of Brevipalpus mite molecular systematics by evaluating and optimizing DNA extraction and amplification protocols; comparing phylogenies obtained from sequences of four different genome regions, including mitochondrial– two fragments of the COI region -, and nuclear markers – regions Internal Transcriber Spacer II (ITS2) and the subunit D1-D3 of the gene 28S; presenting intra and interspecific genetic distances; and evaluating the morphological traits in a phylogenetic context. For the study 25 samples of Brevipalpus were obtained, from five countries – Argentina, Brazil, Chile, Spain and Israel, from 22 host plants. Two protocols of DNA extraction were evaluated considering DNA concentration, quality and efficiency for amplification, being one partially destructive and the other one non destructive. Although the DNA concentration for both protocols has been similar, the partially destructive method was more efficient for DNA amplification. For each of the four pairs of primers Polymerase Chain Reactions (PCR) were tested with variations on the components of reaction (MgCl2, BSA, DNTP and primers) as well as in the reactions conditions (amplification temperatures, number and duration of cycles). Reactions were adjusted to make possible DNA amplification from 2μL of DNA, thus allowing amplification of the four markers from DNA of one specimen. For the phylogenetic analyses, in addition to the sequences obtained in this study, Brevipalpus sequences retrieved from Genbank were included in the datasets. The length of the amplified fragment of the COI region using primers DNF & DNR was 373 base pairs (bp) and the final alignment included 190 sequences (92 obtained in this study, 98 retrieved from Genbank), which were classified in 89 haplotypes, clustered in 18 lineages. The length of the amplified fragment of the COI region using primers LCO & HCO was 650bp and the final alignment included 64 sequences (all obtained in this study), which were