Extração e amplificação de DNA em Alcantarea nahoumii (Leme) J.R.Grant. e desenho de primers SSR em abacaxizeiro (Ananas comosus (L.) Merr. var. comosus) visando transferibilidade e estudos de diversidade genética

Abstract

The Atlantic Forest is considered one of the richest biomes in terms of biodiversity, at the same time as it is one of the most threatened on the planet. Due to the great alteration of its natural ecosystems and the fragmentation that has been affected, this biome has been considered as one of the priorities in terms of conservation. In view of the presented problem, it is necessary to study the genetic diversity of the populations present in this biome, such as Alcantarea nahoumii; a species belonging to the Bromeliaceae family, which grows on rocky outcrops, causing it to naturally present a very fragmented population structure. For the study of the genetic diversity of the species, at the molecular level, it is necessary to optimize a genomic DNA extraction and amplification protocol using random primers, such as RAPD (Random Amplified Polymorphic DNA). Also, the DNA of A. nahoumii can be amplified by means of specific primers such as SSRs (Simple Sequence Repeats). However, this species still does not have its genome sequenced, which makes it difficult to develop these specific primers. An alternative to solve this problem is transferability, a process that consists of using the genome of a related species to design specific primers. Ananas comosus var. comosus (Pineapple), also belonging to the Bromeliaceae family, is among the most important plant crops in the world, due to its high agroeconomic value, therefore, it already has its genome sequenced and available in databases for SSR primer design. To obtain genomic DNA from A. nahoumii, modifications were made to an already established protocol. Also, two reaction compositions (named A and B) and two PCR conditions (A and B) for DNA amplification were tested. GenBank / NCBI was used to search the genome of A. comosus var. comosus. The Websat software was the tool used to design the SSR primer pairs (forward and reverse primers). The quality of the primer designs was verified using NetPrime software. Through the modifications it was possible to obtain DNA for the species. The reaction (B) and condition (B) were the ones that allowed the amplification of the DNA. Out of a total of 10 tested RAPD primers, 8 showed a good amplification pattern. These primers will be selected for studies on the genetic diversity of the species. A total of 250 microsatellite regions (SSR) were identified and selected. These regions were composed of mono, di, tri tetra, penta and hexanucleotide repeats. For each chromosome, 10 SSR regions were randomly selected to design the SSR primer pairs, totaling 250 primer pairs. The results obtained in this work can be used both for genetic studies in A. nahoumii and in pineapples and others in related species by means of transferability tests.