Molecular analysis of faecal samples of a red brocket deer (Mazama americana) population for obtaining genetic and ecological information

Author(s)

Márcio Leite de Oliveira

• Document type: Master's Dissertation
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2010-08-30

Examining board members

José Maurício Barbanti Duarte; José Eduardo Garcia; Luciano Martins Verdade

• Advisor: José Maurício Barbanti Duarte

Abstract

Mazama genus is composed by five species in Brazil. All of them are difficult to observe due to their evasive behaviors, what makes the captures and behavioral studies almost impossible. Thus, the use of non invasive methodologies is necessary to study the ecology and genetics of these species. The fecal DNA analysis is one of the most promising techniques for this purpose. This study aimed to genotype a Mazama americana population faecal samples for obtaining genetics and ecological information. For this, 52 deer faecal samples were collected in a 600ha seasonal semideciduos forest fragment (21o20S 47o17W), with the help of a detection dog, stored in ethanol and georeferenced. Of these samples 31% (n=16) was classified as fresh and 69% (n=36) as not fresh. About thirty days after the collection the DNA was extracted using the QIAamp® DNA Stool Mini Kit following the manufacturers instructions. From the 52 samples collected and extracted, 45 were identified by PCR/RFLP as M. americana and the others showed amplification and digestion problems, remaining without identification. Five microsatellite loci were amplified by PCR and the amplification success, visualized in agarose gel, varied with the loco size and age class. The amplifications success occurred in 65% of the fresh samples and in 35% of the non-fresh samples and a negative correlation (R= -0.82) was found between amplification success and loci sizes. It was possible to identify the animal sex in 43% of the samples by the amelogenin gene. The microsatellite loci amplifications were analyzed in an automatic sequencer. The majority of the samples and loci were impossible to genotype because of the quality of the elestroferograms, what made impossible any reliable genetic and ecological analysis. It is evident the difficulty to work with the faecal DNA methodology using field collected forests deer samples for individual and sexual identifications. Some methodological improvements (collect fresh samples, select primers for shorter loci and quantify the extracted DNA by real time PCR) are suggested to increase the genotyping success indexes in future studies (AU)

• FAPESP's process: 08/01377-2 - Ecological and genetics parameters of a red-brocket-deer (Mazama americana) population by non invasive methodology
• Grantee: Márcio Leite de Oliveira
• Support type: Scholarships in Brazil - Master