Isolation and characterization of microsatellites in selected native and endemic Philippine freshwater fish species [Terminal Report]

Microsatellite markers were developed from selected Philippine endemic and native freshwater fishes. A total of eleven (11) species were collected from Region 4 [Southern Tagalog] including six Gobiidae species (Glossogobius celebius, G. giuris, Rhinogobius sp., Gobiopterus lacustris, Mystichthys luzonensis, and Redigobius dispar); four Cyprinidae species (Rasbora argyrotaenia everetti, Puntius bantolanensis, P. manguaonensis and Nematabramis alestes alestes) and Leiopotherapon plumbeus (Terapontidae). A modified protocol employed for the construction of microsatellite-enriched genomic libraries includes genomic DNA isolation, digestion, adaptor ligation, selective hybridization, cloning and transformation. Recombinant plasmids were sequenced. BLAST analysis of the sequences revealed high similarities to teleosts' DNA and majority were found to contain microsatellite. Dinucleotide is the most frequent repeat motif observed in all families with (CA) sub n being the most prevalent among the gobies and cyprinids while (GA) sub n in L.plumbeus. A total of 42 primer pairs were designed from microsatellite-containing sequences and synthesized, 17 for Gobiidae, 15 for Cyprinidae and 10 for L.plumbeus. Fifteen (88.2%) primers for Gobiidae were able to amplify alleles in two to four goby species while nine (60%) Cyprinidae primers produced PCR products in two to four cyprinids. Successful amplification was observed from nine primer pairs (90%) designed for L. plumbeus, G. celebius, P. bantolanensis, and P. manguonensis each exhibited eight polymorphic luci, ten each for G. giuris and Rhinogobius sp. and seven for G. lacustris. Thirteen Gobiidae primers generated polymorphic PCR products across the species tested, nine for Cyprinidae and eight for L. plumbeus. The PCR results prove the utility of the developed primers. To further validate the efficiency of the developed markers, five goby primers were used to determine the genetic diversity of G. giuris and Rhinogobius sp. populations. The average polymorphism information content (PIC), observed (Ho) and expected heterozygosities (He) are 0.5697, 0.64, and 0.74, respectively in G. giuris. For Rhinogobius sp., PIC is 0.6699, Ho and He 0.82 and 0.76, respectively. Jaccard similarity index values are 0.35 for G. giuris population and 0.44 for Rhinogobius sp. These parameters indicate high degree of diversity within each population. The developed microsatellite markers can be used for species identification, determination of genetic diversity, parentage/hybridity and phylogenetic relationships.