Abstract
Characterization of populations by means of DNA techniques provides a tool for precise identification and a quantitative estimate of genetic diversity, crucial in evaluation of genetic fragmentation within and among populations. NBS profiling are PCR-based approaches that sample genetic variation in resistance genes (R-gene), and R gene analogs (RGA). To date, myb patterns have not been used for evaluating genetic diversity in other species. NBS primers are homologous to the conserved sequences in the Nucleotide-Binding-Site of the NBS-LRR class of R-genes. A total of 12 populations from five Campanula species (C. barbata L., C. latifolia L., C. rapunculoides L., C. spicata L. and C. trachelium L.), autochthonous of the West Italian Alps, were genotyped via nucleotide-binding site (NBS) and myb gene profiling. The selected markers produced a total of 361 bands, showing high levels of polymorphism. Genetic diversity among and within species and population structure was evaluated by different statistical analyses performed using TREECON software, Mantel Nonparametric Test, NTSYS package, AMOVA and STRUCTURE. The correlation between genetic variability and geographical location suggests that the five Campanula species have been subjected to long-term evolutionary processes consistent with the natural fragmentation of continuous mountains areas.
Highlights
Molecular studies may prove useful in improving spatial genetic variation knowledge and for delineating evolutionary genetic processes.[16,17,18,19] Neutral markers such as amplified fragment length polymorphisms (AFLP20) or simple sequence repeats (SSR21,22) are commonly used to screen collections
Species have been subjected to long-term evo- Recently, nucleotide-binding site (NBS) and and myb profiling in Campanula as tools for lutionary processes consistent with the natural myb gene profiling techniques have been genetic diversity studies, which we used to fragmentation of continuous mountains areas. assessed in various genetic approaches
Cluster analysis performed on the single myb gene profiling confirmed a C. trachelium cluster (Group 2); within the C. rapunculoides species genetic variability as high as in the NJ tree based on the combined markers (Group 3) were not revealed
Summary
Total genomic DNA was extracted from freeze-dried leaves using the Dneasy Plant Mini Kit (QIAGEN, Santa Clarita, California, CA, USA) according to the manufacturer’s instructions. The NBS profiling was applied according to the protocol described by van der Linden et al.[23] with some modifications. Restriction digestion and adaptor ligation were performed in a single reaction by incubating 400 ng of DNA overnight at 37°C. Amplification of NBS-specific fragments was achieved through a two-step PCR as described in the original protocol.[23] The first (linear) PCR reaction (5 μL of restriction-ligation template, 2.5 μL of PCR buffer, 1 μL of 5mM dNTP, 0.08 μL of HotstarTaq polymeraseQiagen, 2 μL of 10 pmol/uL NBS-specific primer and adapter primer in a final volume of 25 μL)
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