African Journal of Agricultural Research Vol. 6(20), pp. 4760-4768, 26 September, 2011 Available online at http://www.academicjournals.org/AJAR DOI: 10.5897/AJAR10.611 ISSN 1991-637X ©2011 Academic Journals Full Length Research Paper Genetic diversity and molecular discrimination of the closely related Taiwanese Ulmaceae species Celtis sinensis Persoon and Celtis formosana Hayata based on ISSR and ITS markers Shih-Chieh Lee 1, Chi-Feng Chang 2 and Kuen-Yih Ho 2* 1Department of Bio-Industry Technology, Da-Yeh University, Changhua, 51591, Taiwan. 2Department of Forestry and Nature Resources, National Chiayi University, Chiayi, 60054, Taiwan. Accepted 14 September, 2010 Celtis sinensis Persoon and Celtis formosana Hayata belong to the Ulmaceae family. These closely related species are native to Taiwan. In the present study, 120 samples from 18 natural habitats in Taiwan were studied. The genetic diversity of these two species was determined by comparing the inter-simple sequence repeat (ISSR) and internal transcribed spacer (ITS) regions; these data were also used for the molecular identification of each species. Among the 71 bands amplified by PCR using nine ISSR primers, 51 exhibited polymorphism (71.8%). The population genetic variation analysis (POPGENE) revealed a genetic differentiation (Gst) of 0.3814 and a gene flow (Nm) of 0.8110. AMOVA showed that interspecies differences accounted for 57.38% of the variance (p < 0.0001). Despite their high morphological similarity, C. sinensis and C. formosana can be discriminated and classified into two independent species at the molecular level. Key words: Celtis , genetic diversity, molecular discrimination, inter-simple sequence repeat (ISSR), internal transcribed spacer (ITS). INTRODUCTION Celtis sinensis Persoon grows in the piedmont plain side; the petioles are about 1 cm long (Liu, 1985). This regions throughout the entire island of Taiwan. This plant tree species is commonly planted near beaches or grows rapidly in a variety of soil types under various around villages to provide shade and wind resistance. conditions, from moist, fertile areas to hot, dry locations in The bark and leaves of C. sinensis can be used as the full sun. C. sinensis Persoon is wind and drought medicine to treat various maladies, including urticaria and tolerant once established. It may be propagated through lumbago (Liu et al., 1994). Celtis formosana grows in seeds or cuttings. C. sinensis is a large deciduous tree both the lowlands and highlands of Taiwan and is found in that can grow up to 20 m in height; its bark is smooth and mainland China, the Ryukyu Islands, India, and Indochina gray. This species features chartaceous leaves that are (Liu et al., 1994). This species variant survives easily in ovate to ovate-oblong, obtuse to acute, obliquely broad- hot, humid environments and tolerates drought and salt. It cuneate at their base, and crenated-serrated toward the is very similar to the model species C. sinensis, except base. The leaves are dark green and smooth above and that C. formosana has a smoother bark surface, a are glabrous and slightly glaucous beneath. There are tapering leaf apex, and a fruit diameter of 6 to 9 mm. Both three nerves at the base, and three-four lateral veins per species are used in similar ways (Yang and Lu, 1996). The ISSR (inter-simple sequence repeat) molecular fingerprinting technology developed by Zietkiewicz et al. (1994) utilizes random priming to amplify chromosomal *Corresponding author. E-mail: [email protected]. Tel: DNA fragments that can then be used as molecular +886-5-2717463. Fax: +886-5-2717467. markers. This method can discriminate between particular Lee et al. 4761 Table 1. The taxon, collection site, elevation, and source codes of C. sinensis and C. formosana samples collected from 18 natural habitats in Taiwan. Taxon Collection site Elevation (m) Temp./ Year (°C) Rainfall/year (mm) Source code Zhongli (Taoyuan County) 151 21.6 1820 S-jl1 − S-jl5 Xinfeng (Hsinchu County) 25 21.8 1845 S-sf1 − S-sf10 Huoyenshan (Miaoli County) 250 22.0 1813 S-hy1 − S-hy5 Xinshe (Taichung County) 613 22.2 1742 S-ss1 − S-ss7 C. sinensis Pakuashan (Changhua County) 180 22.6 1820 S-bg1 − S-bg6 Ssuhu (Yunlin County) 23 23.5 2305 S-shu1 − S-shu7 Potzu (Chiayi County) 33 23.1 2200 S-pt1 − S-pt5 Xinhua (Tainan County) 60 24.2 2030 S-sh1 − S-sh5 Tashan (Kinmen County) 48 22.6 1825 S-ts1 − S-ts10 Fuxing (Taoyuan County) 482 22.4 2987 F-fs1 − F-fs10 Wufeng (Hsinchu County) 1290 19.3 2745 F-wf1 − F-wf10 Henglungshan (Miaoli County) 850 20.3 2697 F-hl1 − F-hl5 Kukuan (Taichung County) 1045 18.9 2953 F-gg1 − F-gg5 C. Jenlun (Nantou County) 1001 18.7 3126 F-rl1 − F-rl5 formosana Shifmengku (Chiayi County) 1750 17.4 3522 F-sm1 − F-sm5 Tatungshan (Chiayi County) 1241 18.5 3342 F-dd1 − F-dd10 Nantzuhsienhsi (Kaohsiung County) 1850 19.6 3523 F-nt1 − F-nt5 Lilungshan (Pingtung County) 724 20.6 3207 F-ll1 − F-ll5 genotypes and directly reflects the genetic differences MATERIALS AND METHODS between organisms. Furthermore, a considerable number of gene loci can be studied due to the large numbers of Plant materials fragments that are amplified by these primers. Therefore, Samples of C. sinensis Persoon and C. formosana Hayata were this method has been widely used for species gathered from 27 sites across Taiwan and its adjacent islands identification and phylogenetic analyses as well as in (Figure 3). Between 20 and 40 specimens of each species were studies of plant diversity (Petros et al., 2008; Zong et al., sampled for leaf morphology analysis (Table 2), and 20 to 60 2008). The internal transcribed spacers in ribosomal DNA specimens of each species were sampled for molecular marker analysis (ITS and ISSR) (Table 2). In addition, 5 to 10 mature, non- (rDNA ITS) have been used as molecular markers to diseased leaf blade samples were dehydrated, aliquoted into air- study the genetic relationships among closely related permeable bags, and stored in dry, sealed containers. Sample taxonomic groups by examining the diversity of ITS collection sites and DNA sample codes are listed in Table 1. The fragments. Bellarosa et al. (2005) investigated the genetic identification of all of the leaves was authenticated by Dr. Fu-Yuan relationships among the subgenera in the genus Quercus Lu in the Department of Forestry and Nature Resources at National spp. of the Fagacease family using ITS. Won and Renner Chiayi University. Voucher specimens were deposited in the Department of Forestry and Nature Resources at National Chiayi (2005) studied 25 species from the genus Gnetum in University. South America, Africa, and Asia through an analysis of ITS diversity. Traditional plant taxonomy has been based on DNA extraction differences in external morphology. However, plant morphology might be affected by changes in the external DNA was extracted using the CTAB extraction method published by Kobayashi et al. (1998), with slight modifications. DNA was environment and by endogenous factors, such as visualized by agarose gel electrophoresis followed by staining with genetics. The interplay between environmental and ethidium bromide (Sambrook et al., 1989). Known amounts of genetic factors often causes difficulties in taxonomic lambda DNA (MBI, Fermentas, Hanover, MD, USA) were included studies. The present study aimed to investigate the on the gel to facilitate quantification of the DNA. genetic diversity in natural populations of C. formosana and C. sinensis in Taiwan and to use molecular data (that ISSR amplification is, ISSR and ITS sequences) to discriminate between these two closely related species. One hundred random primers (UBC 801-900, Canada) were 4762 Afr. J. Agric. Res. Table 2. The nine UBC primers and the number of corresponding fragments used in the ISSR analysis. Number of Number of Percentage of Annealing UBC primer No. Sequence 5’- 3’) polymorphic monomorphic polymorphic ((( temperature(°C) fragments fragments fragments (%) UBC 823 TCTCTCTCTCTCTCTCC * 52 6 3 66.7 UBC 825 ACACACACACACACACT 53 5 2 71.4 UBC 834 AGAGAGAGAGAGAGAGYT 56 9 0 100 UBC 886 VDVCTCTCTCTCTCTCT 57 5 2 71.4 UBC 887 DVDTCTCTCTCTCTCTC 56 8 0 100 UBC 888 BDBCACACACACACACA 56 5 2 71.4 UBC 889 DBDACACACACACACAC 56 7 3 70.0 UBC 890 VHVGTGTGTGTGTGTGT 56 3 4 42.9 UBC 891 HVHTGTGTGTGTGTGTG 56 3 4 42.9 *B = ( C, G, T ); D = ( A, G, T ); H = ( A, C, T ); V = ( A, C, G ); Y = ( C, T ). screened for PCR. Among them, a total of nine optimal primers the gel were visualized and photographed using the EZlab Uni- were selected for use in amplification (Table 2). Each of the ISSR photo system. PCR reaction mixture contained 16.5 µl ddH 2O, 2.5 µl 10× PCR buffer, 1.6 µl 2.5 mM dNTPs, 2 µl primers (10 ng/ µl), 0.4 µl Taq DNA polymerase (1 U), and 2 µl DNA template (20 ng/ µl) in a total Data analysis based on ISSR marker profiles volume of 25 µl. The template DNA was denatured at 94°C for 5 min and then subjected to 35 cycles of 94°C for 30 s, 52 to 56°C for The pairwise similarities between samples were calculated by the 50 s, and 72°C for 2 min. The final cycle included an extension at simple matching formula in NTSYS (Rohlf, 1993) to establish the 72°C for 10 min. similarity matrix. POPGENE (Yeh et al., 1997; Yeh et al., 1999) was used to calculate the H values of genetic diversity between different populations (Nei, 1973), the G st values of genetic differentiation ITS amplification (Nei, 1973), and the genetic distance (Nei, 1978). The G st values were used to estimate the Nm values, which represent gene flow Five samples each from C.