Genemapper software version 5

The GenElute Blood Genomic DNA kit (Sigma Aldrich, St. Louis, MO, USA) was used to extract the genomic DNA. Sixteen microsatellite loci (Table 1) were selected according to the recommendations of FAO and the International Society for Animal Genetics (ISAG) for genotyping and parentage analyses in goat breeds [21 ]. The markers were selected based on their degree of polymorphism and their position in the goat genome. STR markers were grouped in multiplex PCR according to reaction conditions and expected fragment sizes as reported by [22 (link)]. PCR products were separated by electrophoresis, with an automatic sequencer (ABI PRISM 3130xl, Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Allele sizes were estimated by using the internal size standard GeneScan-400 HD ROX (Applied Biosystems, Foster City, CA). Genotypes were visualized and interpreted with GeneMapper software, version 5.0 (Applied Biosystems, Foster City, CA).

Leaves were sampled from 126 plants known as Manihotirwinii D.J. Rogers & Appan, collected in four localities in the state of Goiás (Table 1). DNA was extracted from silica-dried leaf tissues (Doyle and Doyle 1987 ) and amplified microsatellite markers by polymerase chain reaction (PCR) using primers GA-12, GA-16, GA-21, GA-126, GA-131, GA-134, and GA-136 developed for Manihotesculenta Crantz by Chavarriaga-Aguirre et al. (1998) . The amplification was done in 10 μL reactions containing 3 μL of template DNA (1.5 ng/μL), 3 μL of each primer (0.9 μM), 1 μL 10X enzyme buffer containing 2.5 mM MgCl₂ (500 mM KCl, 100 mg/mL Tris-HCl, pH 8.4, 1% PageBreakTriton X-100), 1.3 μL bovine serum albumin (10 mg/mL), 0.9 μL 2.5 μM dNTP, and 5 units/μL Taq DNA polymerase, completing the volume with 0.65 μL ultrapure deionized water. PCR parameters were: 94°C for 5 min, 30 cycles of 94°C for 1 min, 56°C for 1min, 72°C for 1 min, and 72°C for 45 s. The forward primers were fluorescently labeled for the observation of fragments of DNA using the Applied Biosystems™ 3500 genetic analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA) in two multiplex genotyping systems. The genotypes were determined using GeneMapper® Software Version 5.0 (Applied Biosystems™, Thermo Fisher Scientific Inc., Waltham, MA, USA) with default settings.

DNA was extracted from peripheral blood using QIAamp Blood DNA mini kit (Qiagen, Hilden, Germany). PCR was performed using consensus fluorescent labeled primers to V_H framework region (FR I and II) and joining region(J_H) of the IgH gene. PCR products were separated by capillary electrophoresis on an ABI 3500DX Genetic Analyzer with electropherograms analyzed using GeneMapper software version 5.0 (Applied Biosystems).