Isolation and Characterization of Microsatellite Markers in the Endangered Species Taxus wallichiana Using the FIASCO Method

The Himalayan yew, Taxus wallichiana Zucc., is an endangered species with a scatted distribution in the Eastern Himalayas and southwestern China. In the present study, 10 microsatellite markers from the genome of T. wallichiana were developed using the protocol of fast isolation by amplified fragment length polymorphism of sequences containing repeats (FIASCO). Polymorphism of each locus was assessed in 28 samples from four wild populations of the Himalayan yew. The allele number of the microsatellites ranged from two to five with an average of 2.9 per allele. The observed and expected heterozygosity varied from 0.00 to 1.00 and from 0.3818 to 0.7552, respectively. Cross-species amplification in another two yew species showed eight of them holding promise for sister species. Two of the 10 loci (TG126 and TC49) significantly deviated from Hardy-Weinberg expectations. No significant linkage disequilibrium was detected between the comparisons of these loci. These polymorphic microsatellite markers would be useful tools for population genetics studies and assessing genetic variations to establish conservation strategy of this endangered species. The Himalayan yew, Taxus wallichiana Zucc., is an endangered, dioecious, and windpollinated tree species, which is restricted to the Eastern Himalayas and southwestern China (Fu et al., 1999; Möller et al., 2007; Shah et al., 2008). This species usually grows scattered in the understory of montane broadleaved, coniferous, and mixed forests between 2000to 3500-m altitudes (Fu et al., 1999; Li and Fu, 1997). Yew is economically important as the source of paclitaxel, a cancerinhibiting compound first found in the bark of the Pacific yew, Taxus brevifolia (Wani et al., 1971) and later found in T. wallichiana as well (Appendino, 1993; Mukherjee et al., 2002). Currently, taxol is difficult to synthesize on an industrial scale (Kikuchi and Yatagai, 2003) and still relies heavily on natural resources (Denis et al., 1988; Schippmann, 2001; Takeya, 2003). This has led to an overexploitation of the natural resources of T. wallichiana along the Himalayas and in southwestern China and the natural resources have become scarce and the plants seriously threatened with extinction in this region (Gao et al., 2007; Schippmann, 2001). Consequently, T. wallichiana was listed in Appendix II of CITES in 1995 and listed as the first class-protected wild species in China (The State of Council of the People’s Republic of China, 1999). As a result of these developments, the plants are in danger of genetic erosion and increased inbreeding (by consanguineous mating, i.e., mating among closely related individuals; see Zhang et al., 2009). To quantify the effects of these stochastic genetic events, accurate estimations of the levels of genetic diversity are required. This provides baseline data for future conservation programs. In previous studies, random amplified polymorphic DNA (RAPD), intersimple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) data (Mohapatra et al., 2008, 2009; Saikia et al., 2000; Zhang et al., 2009) have been used to investigate the genetic diversity of T. wallichiana. However, the dominant nature of these genetic markers can lead to an underestimation of the recessive allele frequency in a population causing bias in the estimate of genetic diversity and genetic differentiation (Nybom, 2004). This problem does not occur with microsatellite markers. Compared with other, random fingerprinting techniques such as RAPDs, ALFPs, and ISSRs, microsatellites show numerous advantages because they are locus-specific, codominant, highly reproducible and usually highly polymorphic (Powell et al., 1996). The development of microsatellite makers of congeneric species was recently reported for Taxus sumatrana (Miq.) de Laub. (Huang et al., 2008), T. yunnanensis W.C. Cheng & L.K. Fu (Miao et al., 2008), and T. baccata L. (Dubreuil et al., 2008) although no nuclear microsatellite primers hitherto have been reported for T. wallichiana Zucc. Therefore, we set out to develop microsatellite markers (simple sequence repeat) for this species using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol of Zane et al. (2002). We describe the development of 10 microsatellite loci for T. wallichiana. DNA was extracted from dried leaf tissues and ground in liquid nitrogen using a modified CTAB method (Doyle and Doyle, 1987). Total genomic DNA ( 500 ng) was completely digested with MseI and then ligated to an MseI AFLP adaptor. A diluted digestion– ligation mixture (1:10) was amplified with an adaptor-specific primer (5#-GATGAGTCCT GAGTAAN-3#). Amplified DNA fragments, with a size range of 200 to 800 bp, were enriched for repeats by magnetic bead selection with a 5#-biotinylated (AC)15 and (AG)15 and (AAG)10 probe. The enriched fragments were amplified again with the previously mentioned adaptor-specific primer. Polymerase chain reaction (PCR) products were purified using an EZNA Gel Extraction Kit (Omega Bio-Tek, Guangzhou, China). The purified DNA fragments were ligated into the pGEM-T vector (Promega, USA) and transformed into Dh5a cells (TaKaRa, Dalian, China). Positive clones were tested by PCR using (AC)10 /(AG)10/(AAG)7 and T7/Sp6 as primers. In total, 425 clones with positive inserts were sequenced on an ABI PRISM 3730xl DNA sequencer (Applied Biosystems Inc., USA) according to the manufacturer’s protocols. A total of 183 (42%) sequences were found to contain microsatellite repeats, and 80 of them were suitable for designing locus-specific primers using the primer 5.0 program (Clarke and Gorley, 2001). The levels of polymorphisms of all the 64 microsatellite loci were assessed in 28 DNA samples from four natural populations of T. wallichiana. The PCR reactions were Received for publication 29 July 2009. Accepted for publication 18 Sept. 2009. This study was supported by the National Natural Sciences Foundation of China (grant no. 30700042), the Natural Sciences Foundation of Yunnan (grant no. 2007C088M), the Knowledge Innovation Program of the Kunming Institute of Botany of CAS (540806321211) and GBOWS, the West Light Foundation of The Chinese Academy of Sciences (9223111W1), and the Talent Project of the Yunnan Province of China (grant no. 2008PY064). We are grateful to J. Yang and Y. Zhao for their help with laboratory work and data analyses. We also thank Dr. Michael Möller, Royal Botanic Garden Edinburgh, for improving English. To whom reprint requests should be addressed; e-mail [email protected]. HORTSCIENCE VOL. 44(7) DECEMBER 2009 2043 performed in a total volume of 15 mL containing, 30 to 50 ng genomic DNA, 0.6 mM of each primer pair (Table 1), 7.5 mL 2 · Taq PCR MasterMix [Tiangen (Tiangen Biotech Co., Ltd, Beijing, China); 0.1 U Taq polymerase/mL, 0.5 mM dNTP each, 20 mM Tris-HCl (pH 8.3), 100 mM KCl, 3 mM MgCl2]. PCR amplifications were conducted on an MJ PTC-200 Thermal Cycler (Waltham, MA) under the following conditions: 97 C for 3 min followed by 30 to 36 cycles at 94 C for 30 s followed by the annealing temperature for each specific primer (Table 1) for 30 s, 72 C for 1 min, and a final extension step at 72 C for 7 min. The PCR products were separated on 8% polyacrylamide denaturing gels using a 20-bp or 10-bp ladder molecular size standard (OrangeRuler ; Fermentas, Shenzhen, China) visualized by silver staining. In the end, 10 of the 64 screened primer pairs displayed polymorphisms. The sequences of the 10 microsatellite markers are submitted to GenBank (Table 1). Standard genetic diversity parameters, Hardy– Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between pairs of loci were estimated using GENEPOP Version 3.4 (Raymond and Rousset, 1995; http:// genepop.curtin.edu.au/). The number of alleles per locus (A) was two to five with an average of 2.9. The values for observed heterozygosity (HO) and expected heterozygosity (HE) ranged from 0.00 to 1.00 and from 0.3818 to 0.7552 with averages of 0.4782 and 0.5685, respectively (Table 1). Two of the 10 loci (TG126 and TC49) significantly deviated from HWE proportions. This might have been the result of the small population sizes and isolated samples used in this survey. No significant LD was detected between the loci (P < 0.001) in our analysis. To test for cross-species/variety application, these 10 primer pairs were used on samples of Taxus fuana and T. chinensis var. mairei. Eight of the 10 primer pairs successfully amplified fragments in these two taxa, also showing microsatellite polymorphisms (data not shown). The microsatellite markers isolated here detected polymorphisms even in our small set of DNA samples. They will be very useful for further investigating the spatial genetic structure, genetic diversity, and gene flow of T. wallichiana, which will provide additional insight into population genetics and help to develop viable strategies for the conservation and management of this threatened medicinal plant. The promising cross-taxa applicability indicates their usefulness in allied taxa of this genus.