Abi prism 3100 avant sequencer

Genomic DNA was extracted from 100 mg of leaf material using the modified CTAB protocol of Lefort and Douglas (1999). Seven cpDNA microsatellite loci were selected and amplified in multiplex polymerase chain reactions (PCR). Two groups of primers were arranged according to allele size and fluorescent labels. The first group was formed by the primer pairs UDT1, UDT3, UCD5, UKK3, and UKK4 previously designed by Deguilloux, Dumolin‐Lapègue, and Gielly (2003). The second group of primers included CMCS6 and CMCS10 previously designed by Sebastiani, Carnevale, and Vendramin (2004). PCRs were performed using the QIAGEN Multiplex PCR kit (QIAGEN) in a volume of 5 µl containing 1X Multiplex PCR Master Mix, 2 µM each primer, dH₂0, and 20 ng template DNA. The thermal cycling conditions consisted of an initial denaturation step for 15 min at 95°C, followed by 40 cycles, each at 95°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min. A final extension at 72°C for 10 min was included. Multiplex PCR products were combined with a GeneScan‐500 LIZ size standard and then run in an ABI‐PRISM 3100‐Avant sequencer (Applied Biosystems). Fragments were analyzed and sized with the Peak Scanner program 1.0 (Applied Biosystems).

Products were sequenced in both directions on Applied Biosystems ABI PRISM 3100-Avant Sequencer (SEQme, the Czech Republic). The nucleotide sequences obtained in this study were edited using DNA Baser Sequence Assembly software (Heracle BioSoft SRL Romania) then aligned with reference sequences of Cryptosporidium spp. and E. bieneusi available in GenBank. Phylogenetic analyses were performed using MEGA6 software (Tamura et al. 2013 (link)). Trees were inferred by neighbor joining (NJ) method based on the Kimura 2-parameter distance model; bootstrapping was performed using 1000 replicates. Sequences from this study have been deposited in GenBank database under the accession numbers KX639723 and KX621279.

DNA from leaves and embryos was extracted using a modified Cetylmethylammonium Bromide (CTAB) protocol [43 ]. Ten microsatellites previously developed for S. purpurea [44 (link)], were amplified via multiplex PCR using QIAGEN Multiplex kit (QIAGEN, Hilden, Germany) in 12 μL reaction volumes. Multiplex PCR amplification conditions followed Cristóbal-Pérez et al., [38 (link)] and fragments were analyzed on an automatic ABI-PRISM 3100-Avant sequencer (APPLIED BIOSYSTEMS, Carlsbad, California, USA), using GeneScan LIZ 600 (APPLIED BIOSYSTEMS) to determine fragment sizes. Alleles were scored manually using GeneMarker Software version 2.6.4 (SOFTGENETICS). To reduce genotyping error, all samples were independently scored by two different researchers to reach a consensus in the final data set. In addition the software MICROCHECKER [45 (link)] was used to detect the presence of null alleles and large allelic dropout across all loci. Three loci (SPUR41, SPUR35 and SPUR39) that were monomorphic in all populations were excluded from further analyses.