Scientific Papers Series B Horticulture

ibosomal to investigate 13 species ofMangifera (Yonemori et al., 2002); Usingamplified fragment length polymorphism(AFLP) information by Yamanaka (Yamanakaet al., 2006); using trnL-F gene sequence toanalysis four species of Mangifera (Fitmawatiand Hartana, 2010); also phylogenetic anddiversification of Mangifera from Indonesianand Thailand by Hidayat (Hidayat et al., 2011).All molecular phylogenetic researches ofMangifera were to analysis the phylogeneticand phyletic original of ancestor. So that, it isquite important to construct phylogenetic treeand analyze phylogenetic of Mangifera usingdifferent molecular marker, especially based onrbcL gene sequence as marker in plant. rbcL isgene for coding ribulose-1,5-bisphospatecarboxylase (RuBisCO).All kind of plants have this gene with moderatemutation. Mutation in rbcL has positivecorrelation with species diversification inAngiosperm (Barraclough et al., 1996), so it isexpected will be able to give phylogeneticinformation closer to the real condition.MATERIALS AND METHODS16 samples of Mangifera, 13 samples of leafare collected from Indonesia (Kebun RayaBogor) and three samples from Thailand(Forestry Departemnt of Kasetsart University,Bangkok). Two Outgroup, they are Boueamacrophylla from Bogor and Anacardiumoccidentale. The last outgroup is taken fromNCBI genebank (Aguilar and Sosa, 2004).Three main steps of research are rbcL primerdesign, DNA genome isolation from Mangiferaleaf and rbcL amplification, and the last isphylogenetic tree construction.Template of rbcL gene was retrieved fromMangiferaindicarbcL gene in NCBI (Gadek, etal., 1996). That sequence was used fordesigning primer both rbcL-F and rbcL-R byGenamicsExpression software and confirmedwith primer blast at NCBI.DNA Genome was extracted from Mangiferaleaf using CTAB method protocol (Porebski etal., 1997) with modification. Then, rbcL genewas amplified by PCR and sequenced inMacrogen Inc (Korea) with the same primer.For constructing Phylogenetic tree, all thesequences were edited and performedalignment by Bioedit and ClustalX program(Thompson et al., 1997)Phylogenetic tree constructed with maximumparsimony (MP) and neighbour joining (NJ)using PAUP* 40.b10 (Swofford, 2002).Appearance the phylogenetic tree use tree viewwin 32 software (Roderic, 2001).Table 1. Plant material and originNumber NAME OF SPECIES ORIGIN1 Mangifera caesia Jack Java, Indonesia2 Mangifera similis Auet Sumatera, Indonesia3 Mangiferamacrocarpa Blume Java, Indonesia4 Mangifera laurina Blume Java, Indonesia5 Mangiferagedebe Miquel Sumatra, Indonesia6 Mangifera indica Lin Java, Indonesia7 Mangifera sp Borneo, Indonesia 8 Mangiferaapplanata Kosterm Borneo, Indonesia9 Mangiferacasturi Kosterm Borneo, Indonesia10 Mangiferaodorata Griff Java, Indonesia11 Mangiferafoetida Lour Java, Indonesia12 Mangiferaaltissima Blanco Java, Indonesia13 Mangiferalongipes Griff Java, Indonesia14 M. cochinchinensis Engler Thailand15 Mangiferalaurina Blume Thailand16 Mangifera macrocarpa Blume Thailand17 Bouea macrophylla Griff * Java, Indonesia18 Anacardium ocidentale Lin* * Accession number in NCBI: AY462008.1 = outgroup* = outgroup which taken from NCBI236