For examining within species polymorphism of the plastid genome, 150 grams of fresh leaf tissue was collected and pooled from multiple individuals from four populations of C. americanum (VA, MN, OH, and AL), including the same VA population used for the single population plastid sequencing (Table 1). These populations were chosen because they span the geographic range of C. americanum and were known to differ genetically based on sequencing of individual chloroplast loci (Barnard-Kubow et al., unpublished results). VA individuals were transplanted from the field, while MN, OH, and AL individuals were germinated from field-collected seed in a growth chamber. Plants from all four populations were then grown for several months in the greenhouse with regular watering and fertilization. Intact chloroplasts were isolated from the single population (VA) and pooled samples using a combination of differential centrifugation and separation on a sucrose step gradient [19],[20]. Chloroplasts were then lysed, and DNA was obtained via a phenol-chloroform extraction and ethanol precipitation. The purity of plastid DNA (cpDNA) was confirmed by restriction digestion.
descargar genetica strickberger pdf en 15
2ff7e9595c
Comentários