1 Research Techniques in Molecular Biology I UPLB Graduate School BIO 206 S1-1L 2 nd semester, A.Y. 2014-15 Cloning and Characterization of Ribulose-1,5-Bisphosphate Carboxylase/ Oxygenase Large Chain (rbcL) Gene in Sweet Potato (Ipomoea batatas Lam.) CECILLE ANN L. OSIO 1 , CARLO VICTOR CUSTODIO 1 , ANNA MAE DELOS REYES 2 , MELISSA S. DICHOSO 1 , JAN B. DIVINA 1 , EMIL C. LAT 2 , GRACETINE MAGPANTAY 1 , JOHN CARLO MARASIGAN 1 , FERGIE ANN QUILLOY 1 , ROWENA RAMOS 2 , NICHELLE VILLARTA 1 , MA. CARMINA C. MANUEL 1 1 Genetics and Molecular Biology Division (GMBD), 2 Microbiology Division (MCBD), Institute of Biological Sciences (IBS), University of the Philippines Los Baños (UPLB) Key words: sweet potato, Ipomoea batatas, PCR, RE digestion, cloning, ligation, transformation, sequence analysis INTRODUCTION Sweet potato (Ipomoea batatas (L.) Lam.) is a clonally propagated hexaploid crop native to the Americas. is a member of the family Convolvulaceae (morning glory family) consisted mostly of tropical and some temperate species valuable as ornamentals, medicinals, and food crops. It is the world’s second most important root crop, the sixth most important crop for food and industrial after rice, wheat, potato, maize, and barley, and the fifth most important crop in many developing countries, such as Southeast Asian countries including the Philippines. It is a cash crop with high yield, drought, tolerance, and wide adaptability to various climates and farming systems. Recently, studies on increasing biomass production by improving photosynthesis are becoming numerous since sweet potato is capable of growing even on areas not suitable for corn, which is a major source of starch and ethanol in some countries (Huaman, 1999; Stefanovic, et al., 2002; Loebenstein, 2009;Roullier, et al., 2013; Yusong, et al., 2013). Genetic studies on sweet potato always involved techniques related to evolutionary genetics, phylogenetics, marker-assisted breeding and crop improvement. One particular study employed the use of the rbcL gene found in the chloroplast genome. This gene encodes the large subunit of ribulose-1,5- bisphosphate carboxylase/oxygenase (RUBISCO). It has been widely sequenced from numerous plant taxa and the resulting database has significantly aided plant phylogeny studies in inferring genetic relationships from the genus up to the order level, estimates of genetic diversity, identification of varieties and cultivars, and analysis of plant populations. They are, however, unsuccessfully used ABSTRACT The ribulose-1,5-bisphosphate carboxylase/oxygenase large chain (rbcL) gene is an important marker for genetic relationship and phylogenetic studies. This gene is isolated and characterized in this study through PCR and RE digestion. The nucleotide and amino acid sequences from the three sweet potato cultivars analyzed using bioinformatics tools showed high confidence prediction and homology to the known rbcL gene sequence isolated from the chloroplast genome of Ipomoea batatas cultivar Xushu 18. Protein structure predictions revealed that samples M1 and M2 models have more similar and are larger compared to the M3 model. The rbcL sequences obtained from this study may be utilized for the screening of population, species diversity estimates, and vertical identification of related taxa locally found here in the Philippines that may aid in genetic engineering, plant breeding, and crop improvement studies regarding sweet potato.