GENETIC VARIATION of SAGO PALM (Metroxylon Sagu Rottb.) SAMPLES from MINDANAO, PHILIPPINES USING INTERNAL TRANSCRIBED SPACER ANALYSIS
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Malays. Appl. Biol. (2018) 47(3): 39–48 GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) SAMPLES FROM MINDANAO, PHILIPPINES USING INTERNAL TRANSCRIBED SPACER ANALYSIS ENGUITO, R.Z.C. and NOVERO, A.U.* Department of Biological Sciences and Environmental Studies, College of Science and Mathematics, University of the Philippines Mindanao *E-mail: [email protected] Accepted 6 May 2018, Published online 30 June 2018 ABSTRACT The sago palm is an emerging important food and energy source in the Philippines. There is limited information on the nature of its distribution in the Philippines. This study presents a phylogenetic tree hinting of the possible influence of geographical distribution to Internal Transcribed Spacer (ITS) polymorphism in sago palm. Internal Transcribed Spacer (ITS) Analysis was employed to assess intra-individual heterogeneity and genetic divergence within the sago morphotypes. The variables under observation were armature type (spiny or non-spiny), maturity, and environment, and combination of these variables yielded eight morphotype samples. Deduced gene sequences of the samples showed a relatively low mean GC% of 50.5%, compared to expected 65.8% from the Metroxylon salomonense (Warb.) Becc. outgroup. This phenomenon was attributed to possible cytosine methylation that led to mutability of GC base pair. Furthermore, branching pattern of ITS sequence divergence for both Neighbour-Joining and Maximum Likelihood methods was observed to be influenced by geographic distribution as non-spiny samples collected from Prosperidad clustered into a monophyletic group with the M. salomonense (Warb.) Becc. Outgroup from Papua New Guinea while the spiny samples from Tagum City diverged into a different group. The sago palm spine is a morphological trait that could be used for the differentiation of sago morphotypes within a small population of limited diversity. This trait is of particular interest in breeding due to its possible influence on important agronomic traits such as yield and resistance to pests and diseases. Key words: Sago palm, plant spine, ITS analysis, phylogenetic analysis, Metroxylon sagu Rottb. INTRODUCTION various plant species such as cucumber fruits, wherein spine formation is controlled by two genes Sago palm (Metroxylon sagu Rottb.), a member of (Hutchins, 1940). In a study by Park et al. (2004), the Arecaceae Family is only harvested from wild different genes were observed to be associated with stands in the Philippines. Although well-known and spine formation in Daucus carota hydrolase cultivated in Malaysia and Indonesia for its many associated with the cell wall, tail-fiber assembly uses, sago palm is relatively unheard of in the protein, transcriptional regulatory protein, herberine country. Ehara (2018) commented that sago palm’s bridge enzyme, S-adenosyl methionine synthase increasing potential for use as carbohydrate source (SAMS), transketolase and phenylalanyl tRNA may be inevitable as the palm can grow in poor and synthetase beta chain. Among the genes identified, lowly utilized lands. SAMS would be pivotal to the methylation and M. sagu Rottb. is a potential starch-resource expression of genes, since it acts as methyl donor alternative that would address the continuous that controls gene expression. growth of population and exhaustion of known Spiny and spineless sago palms were originally starch sources. However, armature (presence of classified as different species of Metroxylon, namely spines) poses a problem for the optimum utilization Metroxylon rumphii and Metroxylon sagu, of sago starch. Various studies had identified respectively (Beccari, 1918). Random amplified specific genes that contribute to spine formation in DNA polymorphism (RAPD) analysis performed by Ehara (2009) among sago palm collected from * To whom correspondence should be addressed. Indonesia, Malaysia and Mindanao, Philippines, 40 GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS showed that spiny and spineless sago belong to one among multiple successions of Vigna radiata, and cluster. Zymogram patterns of sago palm in Thailand further analysis showed that variants are hetero- showed no significant difference, concluding that duplex ITS fragments. Wild and cultivated forms of sago palm cultivars are identical (Boonsermsuk et four Vigna species revealed substantial intra-species al., 1995). Furthermore, Amplified Fragment Length divergence from the ten Vigna species under study. Polymorphism (AFLP) analysis employed by Kjær However, among the four species, intra-individual et al. (2004), suggested that variation in armature ITS variation was observed in only one species. The does not correlate with the underlying genetic heterogeneity was further assessed with reverse variation in sago palm. These suggest that M. sagu transcriptase-PCR (RT-PCR) analysis, which showed Rottb. belongs to only one species, wherein that the regulation of loci is controlled by epigenetic spineless palms can produce spiny seedlings gene silencing since rDNA repeat units in certain (Rauwerdink, 1986; Ehara et al., 1998) or vice versa intra-individual ITS variants were transcriptionally (Jong, 1995). High performance liquid chromato- inactive. Intra-species hybridization and slow graphy was also reported in the analysis of DNA ‘molecular drive’, in relation to incomplete methylation, wherein aside from the type of homogenization, are responsible for the observed armature, the condition of the environment and phenomenon (Saini et al., 2008). maturity of the palm was also considered (Novero ITS analysis can also be used in investigating et al., 2012). The results showed significant intraspecies variability among different variants and differences in DNA methylation of spiny and non- cultivars. Freitas et al. (2013) studied genetic spiny sago palms. However, the results did not diversity of Vigna unguiculata (cowpea) cultivars conform to the generally observed inverse using ITS. Significant variability among cultivars relationship of DNA methylation and gene was determined upon amplification and sequencing expression, wherein transcriptionally silent genes of the ITS1 and ITS2 regions. are highly methylated than active genes (Singal ITS analysis was employed to investigate the & Ginder 1999). genetic structure of Acrocomea aculeata (macaw ITS (internal transcribed spacer) refers to a piece palm; Family Arecaceae) groups by analyzing of non-coding RNA situated between structural individual polymorphism of different ITS regions. ribosomal RNAs (rRNA), which are found as Using five ITS primer sets, DNA from 42 leaf samples repeating units that are arranged in tandem arrays confirmed genetic diversity among and within (Liu et al., 2009) and are not included into mature populations (Vieira et al., 2017). ribosomes (Baldwin et al., 1995). Baker et al. (2000) utilized both cpDNA rps16 intron and nrDNA ITS region on the phylogenetic MATERIALS & METHODS reconstruction of the species under subfamily Calamoideae. M. sagu Rottb. was included among Twenty-four sago palm leaf samples, three from each the species subjected to ITS analysis and the data location for the two sago morphotypes (spiny and can be utilized to serve as theoretical results, non- spiny) at different stages of maturity the exploiting it as basis for experimental comparison. juvenile (~2 months old) and mature (~5 years old) The 22 genera investigated indicated that concerted (Table 1), were collected from Agusan del Sur, evolution had not effectively homogenized the ITS Tagum, Davao del Norte and Sta Cruz, Davao del repeats since high levels of intra-individual Sur (Figure 1). Prosperidad, Agusan del Sur, polymorphism was observed. However, no 8.6024°N, 125.8716°E, is a landlocked municipality significant emphasis was given to the polymorphism situated near the Agusan River. It has Type II climate in sago palm armature. Thus, the present study with no dry season. Wet season is characterized by utilized the internal transcribed spacer region to heavy precipitation. Tagum, Davao del Norte, investigate intra-individual heterogeneity of sago 7.4482°N, 125.8094°E, also experiences significant palm morphotypes. rainfall. Its “dry’ months are also beset with rainfall. Fama et al. (2000) reported high level of Sta. Cruz, Davao del Sur, 6.8813°N, 125.3686°E, intra-individual ITS1 polymorphism in Caulerpa is characterized by wet (May to October) and dry racemosa complex. ITS analysis was also employed (November to April) seasons. in the study of intraspecies variation in Dioscorea DNA was extracted using a modified protocol spp. (Munirah et al., 2014) and Fructus evodiae (Liu of Angeles et al. (2005). The reagents for DNA et al., 2009). However results showed no distinct extraction included 200 μL 20% SDS, and 0.1 g of variation of species under consideration. PVP. A freshly prepared 0.4% mercapthoethanol was Saini et al. (2008) showed the extent of intra- added to the solution. About 1 g of the leaf sample individual and intra-species variation among the and the freshly prepared extraction solution were Asiatic Vigna species (subgenus Ceratotropis) using added to a pre-chilled mortar and pestle. The nuclear rDNA ITS. ITS polymorphism was observed homogenized mixture was then transferred to 1.5- GENETIC VARIATION OF SAGO PALM (Metroxylon sagu Rottb.) USING ITS ANALYSIS 41 Table 1. DNA samples of different sago palm morphotypes of different maturity stages as obtained from different environments Designated No. of Armature type Environment Maturity Sampling