Enzyme with the pair HindIII/BamHI for BSOLV and SpeI/DraI

(2009, 2011) and Hippolyte et al. (2012) depending on SSR marker evaluation. As this phylogeny was not representative of M. balbisiana diversity, we genotyped 23 seedy M.Enzyme from the pair HindIII/BamHI for BSOLV and SpeI/DraI for BSGFV and BSIMV. Digested 1479-5868-9-35 DNA was then purified by dialysis on a cellulose membrane (Millipore, Molsheim, France), separated by electrophoresis on 1 agarose gels run for 16 h at 40 V in 0??TBE and transferred to positively charged Hybond N?nylon membrane (GE BAY 11-7083 custom synthesis Healthcare, Velizy-Villacoublay, France) in accordance with the man?ufacturer's instructions. Membranes were hybridized overnight at 65 C with radiolabelled probes containing the complete genome of BSOLV, BSGFV or BSIMV (Chabannes et al., 2013), and after that treated in accordance with the manufacturer's guidelines. Autoradiography was performed employing a phosphorimager (Typhoon FLA 9000, GE Healthcare) following 24 h of exposure.PCR and Southern blot-based information analysisWe carried out PCR and derived cleaved amplified polymorphic sequences (dCAPS) evaluation for the 3 BSVs studied. The PCR and dCAPS data had been scored in line with the presence or absence of fragments for every single accession. We score as present only CAPS displaying PKW expected results. Hybridization patterns resulting from Southern blots had been analysed applying the computer software ImageQuant TL (GE Healthcare). This computer software permits automatic fragment detection on the membrane image and calculates their size by referring to the ladder and constructive controls present on each and every membrane. A visual manage of final hybridization patterns was performed to validate data obtained by implies from the software. We utilized two pairs of enzymes to digest DNA for all samples as described above. Every single sample pattern was recorded based on the presence or absence of expected bands/fragments obtained following separate hybridization using the three BSV probes by comparison with each the PKW and also the allelic BAC clone 369158 patterns. For every single banana accession, we obtained a final picture of the eBSV structure depending on the presence/absence of fragments for each and every BSV (Fig. 2A ).A Musa phylogeny representative in the diversity of each wild and cultivated M. acuminata (denoted A)- and M. balbisiana (denoted B)-derived genotypes was proposed by Perrier et al. (2009, 2011) and Hippolyte et al. (2012) depending on SSR marker analysis. As this phylogeny was not representative of M. balbisiana diversity, we genotyped 23 seedy M. balbisiana diploids too as interspecific hybrids, diploid (ABB) or haploid (AAB) for the B genome. Combining the two datasets, we performed a novel microsatellite-based evaluation on a total of 567 accessions. From this general phylogeny (information not shown), we extracted a sub-tree corresponding to our 77 accessions constituting the sample and rooted on the outgroup accessions (OG) (Fig.Enzyme from the pair HindIII/BamHI for BSOLV and SpeI/DraI for BSGFV and BSIMV. Digested 1479-5868-9-35 DNA was then purified by dialysis on a cellulose membrane (Millipore, Molsheim, France), separated by electrophoresis on 1 agarose gels run for 16 h at 40 V in 0??TBE and transferred to positively charged Hybond N?nylon membrane (GE Healthcare, Velizy-Villacoublay, France) in line with the man?ufacturer's instructions. Membranes were hybridized overnight at 65 C with radiolabelled probes containing the full genome of BSOLV, BSGFV or BSIMV (Chabannes et al., 2013), after which treated according to the manufacturer's directions.