Four samples of A. deceptrix were collected in two places, Bosque Protector Cerro Pata de Pájaro canton de Pedernales (N: 00002.609´, W: 079056.706) and the second place was Parque Nacional Machalilla (S: 1034.52,6, W: 80042.30,4) in order to carry out complete sequencing.
Three populations of A. deceptrix were selected to evaluate the diversity in the isolated loci, Agua Blanca; (n=10), Tachina; (n=18) and Humedad; (n=12). Two leaf were collected from each plant and preserved dried in silica gel. The collection of samples of A. deceptrix were authorized by the Ministry of the Environment of Ecuador, number MAAE-CMARG-2021-0442.
Total genomic DNA of A. deceptrix was extracted from young leaves with a modified CTAB (cetyltrimethylammonium bromide) method (Murray and Thompson 1980, Doyle and Doyle 1990). The quality and quantity of isolated DNA was determined using a Nanodrop and agarose gel electrophoresis.
The Nextera Library Preparation Kit (Illumina, San Diego, California, USA) was used to construct the library from 5 ng de DNA (0.5 ng/ul) from four individuals following the manufacturer's protocols applied in the AUSTRAL-omics laboratory, Universidad Austral de Chile.
A total of 200,000 reads pair were obtained, with an average length of 386 bp, generated by three independent runs. The sequences were assembled using the GS De Novo Assembler (V2.9) software. We used MSATCOMANDER (Faircloth 2008) program for locating Simple Sequence Repeat (SSR). The primers were designed using the software PRIMER 3 (Untergasser et al. 2012) according to the parameters: primer size 18–24 bp, Tm (Melting Temperature) of 55–60°C, and GC content of 40–65%. For the amplification of selected SSRs, the PCR was performed following the method developed by Schuelke (2000), which uses three primers: a forward primer with an M13 (-21) tail at its 5' end, a standard reverse primer, and the universal M13 (-21) primer labeled with either 6-FAM, VIC, PET or NED fluorochromes.
The SSR information and GenBank accession numbers are listed in Table 1.
PCR reactions were performed in a 14 μL reaction mixture with 10 ng templates DNA, 0.15 mM of each dNTP; 1× Taq polymerase reaction buffer; 1.5 mM MgCl2; 0.1 μL forward primer; 0.4 μL reverse primer; 0.4 μL M13 primer (FAM/VIC/NED/PET) and 0.05uL Go Taq DNA Polymerase.
PCR amplifications were performed in an Applied Biosystems Veriti thermal cycler (Life Technologies) under the following conditions: initial denaturation at 95°C for 5 min; 10 cycles of 30 s at 95 °C, annealing temperature specific to each primer pair for 45 s, extension at 72 °C for 45 s, followed by 8 cycles of 30 s at 95 °C, annealing at 53 °C for 45 s, extension at 72 °C for 45 s and a final extension at 72 °C for 1 min. The annealing temperature (°C) of the primers is specified in Table 1.
SSR profiles and polymorphisms were resolved as follows: 2 μL of PCR products were mixed with 2 μL del primer M13 VIC, 5 μL del primer M13 NED, 1 μL del primer M13 FAM y 5ul del primer M13 PET and separated by capillary electrophoresis on an ABI 3500 Genetic Analyzer (Life Technologies) in AUSTRAL-omics, Universidad Austral de Chile. Allele sizes were automatically calculated with Geneious V.8.0.4 (Biomatters, http://www.geneious.com).
Allelic richness (A), observed and expected heterozygosity (Ho-He) were estimated using PopGene (Yeh et al. 1999). Deviation from the Hardy–Weinberg equilibrium was determined with GENEPOP (Raymond and Rousset 1995). The inbreeding coefficient (Fis) (Weir and Cockerham 1984) was determined using Genetix (Belkhir et al. 2003). Null alleles were checked using MICRO-CHECKER V 2.2.3 (Van Oosterhout et al. 2004).
The cross-amplification was tested on two threatened species, A. conica and A. manabiensis, endangered species. Primers were considered successful when one clear, distinct band was detected on 2% agarose gel.