Molecular Ecology and Evolution
Salas-Leiva, Dayana , Meerow, Alan , Francisco-Ortega, Javier , Griffith, Patrick , Calonje, Michael , Stevenson, Dennis , Nakamura, Kyoko .
Phylogeography and conservation genetics of the Caribbean Zamia clade: Identification of single copy nuclear genes.
Eight representative individuals of the Caribbean Zamia clade (Zamia pumila complex) were used to identify potential single copy nuclear genes for studying the phylogeographic relationships within this complex. Two strategies were used to select target loci: 1) a BlastX search of Arabidopsis conserved ortholog sequence (COS) set against the two Zamia ESTs databases deposited in Genbank and, 2) a BlastX search of APVO SSC genes (Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oriza sativa Shared Single Copy genes) against the two Zamia database in Genebank. For the first strategy, 35 loci from Zamia databases were selected according their Scores, E-value and query coverage. For the second, 16 loci were selected based on length of exons (400-900 bp) and number of exons (1-9). For both strategies, the matching Genbank accessions of Zamia were used as query for retrieving highly similar sequences from Cycas, Picea or Pinus species. After retrieving and aligning all the sequences in each locus, intron predictions were completed and used as reference points for primer design. Degenerate primers (up to 3 bases) were designed manually in regions around the predicted introns taking into account the more conserved regions among different gymnosperm species in the alignments. Initially, only two individuals were used to carry out PCR reactions using a temperature gradient. PCR products were checked using 1.2% agarose gels. Primer combinations with no amplification or more than one band were abandoned. After preliminary amplification, the remaining 6 individuals were also amplified with the successful primer pair combinations per locus in order to assess degree of polymorphism. A total of 35 (69%) loci were successfully amplified as a single band of which, 27 (77%) were single copy loci. Of these, three (11%) were non-variable, 13 (48%) showed low variation and 11 (41%) relatively high variation. A preliminary screening with six polymorphic loci and 258 individuals from Puerto Rico, Dominican Republic and Jamaica was carried out using a phylogenetic network approach. Six loci produced a concatenated matrix of 5758 bp of length and showed 59 polymorphic sites per locus and 46 parsimony informative sites.
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1 - Florida International University, Biological Sciences, OE167 - University Park, Miami, FL, 33199, USA
2 - USDA-ARS-SHRS, UNITED STATES DEPARTMENT OF AGRICULTURE, 13601 Old Cutler Road, Miami, FL, 33158, USA
3 - Montgomery Botanical Center, 11901 Old Cutler Road, Miami, FL, 33156, USA
4 - THE NY BOTANICAL GARDEN, 2900 SOUTHERN BLVD, BRONX, NY, 10458-5126, USA, 718/817-8632
5 - USDA-ARS-SHRS, UNITED STATES DEPARTMENT OF AGRICULTURE, 13601 Old Cutler Road, Miami, FL, 33158, USA
Single/low copy nuclear genes
APVO SSC genes
EST Zamia databases
Zamia pumila complex.
Presentation Type: Oral Paper:Papers for Topics
Location: Union B/Hyatt
Date: Monday, July 9th, 2012
Time: 3:45 PM