Title : Standardization of a microsatellite PCR protocol for enterolobium cyclocarpum populations in Costa Rica.
Abstract:
The Guanacaste tree or Enterolobium cyclocarpum is native to tropical and subtropical America, is a species used in the reforestation and restoration of degraded areas and is considered the national tree of Costa Rica. Because of its economic and cultural importance, it is necessary to determine the genetic conservation status of the natural populations of E. cyclocarpum in Costa Rica. However, there are few studies on the genetic diversity of this species and the use of molecular techniques such as microsatellites, so this work aims to standardize and validate the PCR protocol in individuals from 90 populations of E. cyclocarpum located in different areas of Costa Rica. From the location of these populations, fruits and seeds were taken and germinated at the “Instituto de Investigación y Servicios Forestales” of the “Universidad Nacional”, when the plants were approximately 30cm high, four leaflets were taken and stored at -20°C for later use. Two extraction protocols were evaluated. For PCR, different changes in MgCl2 concentrations, primers, and DNA concentration were evaluated, as well as different programs in the thermal cycler cycles. Once the extraction was standardized, the PCR protocol was validated with 100 individuals of E. cyclocarpum. The best extraction protocol was determined to be DNA precipitation with 5M NaCl and 95% ethanol. A 25µl final volume reaction was validated with 1X PCR buffer, 2.5mM MgCl2, 0.2mM dNTPs, 2 units of Taq polymerase, 0.4mg bovine albumin serum, 2% DMSO and 80ng DNA. The results of these protocols generate enough polymorphic alleles to characterize the populations and determine genetic diversity parameters such as the number of alleles per locus, and expected and observed heterozygosity, among others. We expect to carry out the entire diversity analysis of the 90 populations located throughout the Costa Rican territory.
Audience Take Away:
- The audience will gain insights into the genetic conservation status of Enterolobium cyclocarpum (Guanacaste) populations in Costa Rica through the standardized PCR protocol. This knowledge is applicable for researchers, conservationists, and policymakers involved in the management of forest genetic resources. Understanding the genetic diversity of Guanacaste is crucial for informed decision-making related to conservation, reforestation, and sustainable use.
- For researchers and scientists, the standardized PCR protocol provides a reliable and reproducible tool for assessing the genetic diversity of Guanacaste populations. Conservationists can utilize the information to develop targeted strategies for preserving genetic variability in natural populations. Policymakers may find the results instrumental in formulating policies for sustainable forest management.
- This research offers a foundation for other faculty members to expand their studies in forest genetics. The standardized protocol for assessing genetic diversity in E. cyclocarpum populations can be applied to similar studies on other tree species, contributing to a broader understanding of forest ecosystem dynamics. Additionally, the methodology employed in this research may serve as an educational resource for teaching molecular biology and genetics.
- Facilitates informed decision-making in conservation and reforestation efforts, contributes to the sustainable management of forest genetic resources, establishes a reliable methodology for large-scale population studies, enhances the reproducibility of genetic diversity assessments, provides a foundation for further studies on tree species diversity and ecology
