The genetic diversity and species-diagnostic markers in the introduced apple snail, Pomacea canaliculata and in the native Thai apple snails; Pila ampullacea, P. angelica, P. pesmei, and P. polita, were investigated by restriction analysis of COI and are reported for the first time. Twenty-one composite haplotypes showing non-overlapping distributions among species were found. Genetic heterogeneity analysis indicated significant differences between species (P < 0.0001) and within P. pesmei (P < 0.0001) and P. angelica (P < 0.0004). No such heterogeneity was observed in Pomacea canaliculata (P > 0.0036 as modified by the Bonferroni procedure), P. ampullacea (P = 0.0824-1.000) and P. polita (P = 1.0000). A neighbor-joining tree based on genetic distance between pairs of composite haplotypes differentiated all species and indicated that P. angelica and P. pesmei are closely related phylogenetically. In addition, the 16S rDNA of these species was cloned and sequenced. A species-specific PCR for P. canaliculata was successfully developed with a sensitivity of detection of approximately 50 pg of the target DNA template. The amplification of genomic DNA (50 pg and 25 ng) isolated from the fertilized eggs, and juveniles (1, 7, and 15 d after hatching) of Pomacea canaliculata was also successful, and suggested that Pomacea canaliculata and Pila species can be discriminated from the early stages of development.
Genetic diversity of the introduced golden apple snail, Pomacea canaliculata (Lamarck, 1822) and four native apple snails; Pila ampullacea (Linneaus, 1758), P. angelica (Annandale, 1920), P. pesmei (Morelet, 1889) and P. polita (Deshayes, 1830) in Thailand were studied by RAPD analysis. Two hundred and two polymorphic fragments (180-1500 bp in length) were generated across overall investigated samples (N = 254) using three informative primers (OPA07, OPB10 and UBC122). The percentages of polymorphic bands were 98.86%, 94.56%, 90.91%, 96.94% and 95.51% for Pomacea canaliculata, P. ampullacea, P. angelica, P. pesmei and P. polita, respectively. This indicated high genetic polymorphism of these taxa. A neighbor-joining tree between pairs of geographic samples within Pomacea canaliculata suggested a lack of phylogeography in this species. Moreover, candidate species-specific RAPD markers (pKUSCARPILA-F/R) found in Pomacea canaliculata (340 bp, OPB10), P. ampullcea (640 bp, OPA07), P. angelica (380 bp, UBC122) and Pila snails (430 bp, OPA07) were cloned and sequenced. Locus-specific primers were designed and tested against the target and nontarget species. A 259 bp SCAR marker was found in 95.0% of Pila apple snails (N = 163) but not in Pomacea canaliculata (N = 30). Therefore, this SCAR marker could be used in coupling with a Pomacea canaliculata-specific RAPD marker to unambiguously differentiate the introduced and native apple snails in Thailand.
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