THE IMPLEMENTATION OF NEXT GENERATION SEQUENCING (NGS) IN MICROSATELLITE MOLECULAR MARKER ASSEMBLY FOR NON MODEL AND THREATENED SPECIES MELANOTAENIA

Rainbowfishes are very famous for ornamental trade since the 1930s (Tappin, 2010), because of their vivid coloration that is reminiscent of a rainbow. They are endemic from New Guinea and Australia. Melanotaenia is the largest genus of the Melanotaeniidae family (Ivantsoff et al., 1991). There are thirty-two species that are endemic from the Vogelkop Province (Nugraha et al., 2015) and eighty species of Melanotaenia in New Guinea and Australia (http://rainbowfish.angfaqld.org.au/).

Considering their increasing economic value and the ever-growing human pressures on their habitats, more research on the genetic variability of these fishes is needed to identify candidate species for aquaculture and for conservation purposes. To address these questions, microsatellite markers were developed, making possible investigation of the genetic variability of Melanotaenia species present in Western New Guinea.  For microsatellite discovery we used and developed from Melanotaenia boesemani Allen and Cross (1980), because the Boesman's rainbowfish Melanotaenia boesemani Allen and Cross (1980) is certainly the most popular species of rainbowfish both in Indonesia and abroad.  This species is very different from most other rainbowfish because it harbors different colors between the anterior and posterior.  

Discovery microsatellite markers were subsequently prepared and sequenced using the 454 GS FLX Titanium (Roche) at Genoscreen (Lille, France). Analysis of raw sequences with the QDD program (Meglecz et al. 2010) resulted in 14,922 sequences harboring microsatellites, and 1,012 markers with validated primer sets. For microsatellite validation markers with mendelian inheritance methode were choosen 12 loci from Sixty-one loci were selected from this list according to the motif length (di-, tri-, tetra-, penta- or hexa-nucleotide). In this research we studied population structure from 31 Melanotaenia population ie: 20 valid species from West Papua, one valid species from central lengguru and three valid species from southern New Guinea. Eight New species of Melanotaenia are described used 12 new microsatellite markers, with total number samples are 878 individus. Number of alleles per locus range from 11-53 and number alleles per species range from 17-133.