Download Selection of primers for ISSR study of conservation genetics Cattleya

57º Congresso Brasileiro de Genética
Resumos do 57º Congresso Brasileiro de Genética • 30 de agosto a 2 de setembro de 2011
Centro de Convenções do Hotel Monte Real Resort • Águas de Lindóia • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
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Selection of primers for ISSR study of
conservation genetics Cattleya granulosa
Lindl.: an endemic and endangered orchid of
Northeastern Brazil
Fajardo, CG1; Brandão, MM2; Marinho, AA3; Rocha, TGF3, Silva, RAR3; Vieira, FA3; Molina, WF1.
1Centro de Biociências, UFRN, Natal, RN, 2 Departamento de Ciências Florestais, UFLA, Lavras, MG, 3Unidade
Acadêmica de Ciências Agrárias, UFRN, Macaíba, RN
[email protected]
Keywords: Cattleya granulosa, ISSR, Genetic Diversity.
Cattleya granulosa Lindl. is a endemic species from Atlantic forest, occurring exclusively in coastal dune forests in the
Northeast, the states of Rio Grande do Norte, Paraiba, Pernambuco, Alagoas and Bahia. The species is threatened
with extinction due to anthropogenic factors that have degraded its habitat throughout its geographic distribution.
The reduction of available habitat for the species has increased the degree of isolation among their populations, probably by modifying the genetic structure. Therefore, it is essential to study the genetic variability in natural populations
of C. granulosa, as subsidies to conservation actions. This study aimed to select the dominant marker primer ISSR
(Inter Simple Sequence Repeats) for analyzing the genetic structure of natural populations of C. granulosa in Rio
Grande do Norte. Leaf samples from five populations located in Natal/RN were obtained and placed in 2ml microtubes containing CTAB (cationic hexadecyl trimethylammonium bromide), and stored in a freezer. The extraction of
genomic DNA was optimized based on the CTAB protocol. After extraction, we carried out DNA quantification in
0.8% agarose gel, by comparing DNA patterns. Five individuals, one from each population were selected to test 20
ISSR primers. Subsequently, DNA was amplified by polymerase chain reaction (PCR) with annealing temperature of
42°C. PCR products were separated by electrophoresis on agarose gels (1.5%), stained with dye GelRed TM, visualized
with ultraviolet light and photographed. The ISSR primers tested that performed better amplification for the species
C. granulosa were UBC 807, UBC 822, UBC 826, UBC 827, UBC 841, UBC 842, UBC 851, UBC 860, UBC 862 and
UBC 898. After the selection of primers, amplifications were performed again to examine the robustness and repeatability of loci and markers. The codification of these loci was the presence or absence of amplified fragments, generating a binary data matrix, which allowed the analysis of repeatability and per locus per primer. The best results were
with primers UBC 826, UBC 827, UBC 842. The primers selected are important for analyzing the genetic structure of
populations of C. granulosa in relation with habitat fragmentation and population reduction as a result of predation.
Financial Support: CAPES.