Identification of Prosopis juliflora and Prosopis - Universidad Pública ...

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8P. pallida and do not correspond to any other species likely to occur in thatarea (Pasiecznik et al. 2001). Seeds of a putative P. chilensis · P. pallida hybridfrom Cape Verde also displayed an amplification pattern similar to that presentedby P. pallida (Figure 2a). The amplification profile corresponding to theputative P. chilensis · P. pallida hybrid showed bands present in P. pallida, andsome bands present in P. chilensis could also be seen (data not shown). Whilethis confirms P. pallida as one of the parents of the putative hybrid, P. chilensiswas not specifically analysed in this study and thus can only be tentativelyconfirmed as the other parent. When DNA derived from seeds of P. julifloracollected in Honduras was analysed it could be seen that different loci wereamplified. Five species-specific molecular markers were found: L04 (410) (Figure3), P08 (440) , P012 (760) , P12 (610) and P14 (745) . A similar profile was obtainedwhen DNA derived from seeds collected in Burkina Faso, presentlyidentified as P. juliflora, was amplified. Thus, the plant material collected inBurkina Faso is very likely to be P. juliflora and differs from P. pallida.When the oligonucleotide L01 was used as primer in amplification reactionsusing as template DNAs derived from plant material collected in Honduras(P. juliflora), Piura and Trujillo (P. pallida), Senegal, Cape Verde, Brazil,Burkina Faso, in the putative hybrid P. chilensis · P. pallida and the materialFigure 2. (a) Amplification of the marker S11 460 , specific to P. pallida, using oligonucleotide S11as primer and the DNAs purified from all accessions analysed in this paper as templates. (b)Southern hybridisation of the cloned marker S11 460 used as probe on membranes containing theamplification products using S11 as oligonucleotide primer and the genomic DNA from allaccessions analysed in this paper as templates. Lane names as Figure 1.
9Figure 3. Amplification of the marker L04 410 using oligonucleotide L04 as primer and the DNAspurified from all accessions analysed in this paper as templates. Note that this oligonucleotideamplifies a low molecular weight marker present in P. pallida accessions. Lane names as Figure 1.classified as P. affinis, it could be seen that a DNA fragment of approximately520 bp was present in all the samples (Figure 4a). This result suggests that theL01 (520) marker could be a P. juliflora–pallida complex-specific marker. DNAhybridisation experiments (see below) confirm this result (Figure 4b).RAPD markers identityThe identification of species on the basis of their RAPD profiles relies on theassumption that bands of the same size appearing in different individuals,populations and/or species correspond to identical sequences and not only to acircumstantial coincidences in size. In order to test this assumption, hybridisationexperiments were performed with cloned RAPD markers.It could be seen that the cloned RAPD marker S11 (460) highlighted a bandthat was present in plant material collected in Trujillo and Piura as well as inthe accessions from Brazil, Cape Verde and Senegal (Figure 2b). This markeralso appeared in samples collected in Brazil and classified as P. affinis as well asin the putative P. chilensis · P. pallida hybrid collected in Cape Verde. Theseresults strongly suggest that the marker S11 (460) identifies populations ofP. pallida. The cloned RAPD marker S11 (460) was absent in sample seeds fromHonduras and Burkina Faso which confirms that neither sample is P. pallida.DNA hybridisation experiments using the cloned marker L01 (520) as a probeshowed that accessions collected in Peru (Piura and Trujillo) and the populationcollected in Honduras belong to the P. juliflora–pallida complex (Figure4b). The presence of this cloned marker in DNA templates derived fromaccessions collected in Brazil and Africa indicate that they also belong to theP. juliflora–pallida complex. Figure 4b also clearly shows the presence ofthe complex-specific marker L01 (520) in the putative P. chilensis · P. pallidahybrid.
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