Taxus L. and Pseudotaxus W. C. Cheng are two closely related sister genera with similar appearance in Taxaceae (Fu et al., 1999). Their only distinction is the difference in color in the stomatal bands and aril (Fu et al., 1999). Both T. chinensis (Pilg.) Rehder var. mairei (Lemée & H. Lév.) W. C. Cheng & L. K. Fu and P. chienii (W. C. Cheng) W. C. Cheng are coniferous species endemic to China. Taxus chinensis var. mairei, in particular, has a high medicinal value because it contains the anticancer agent taxol (Li et al., 2008). Pseudotaxus chienii, the sole species in the monotypic genus, is an evergreen shrub or small tree with an average height of 4 m (Su et al., 2009). Due to overexploitation and human activities, the population size of P. chienii is shrinking. The species is categorized as an endangered species in the Red List of Endangered Plants in China (Fu and Jin, 1992). As an “old rare species,” P. chienii has adapted well to habitat fragmentation and ecological heterogeneity across a wide range of habitats and is found in Zhejiang, Jiangxi, Hunan, and Guangxi provinces (Deng et al., 2013). The previous nuclear inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers have revealed that P. chienii possesses high genetic diversity, which provides a large pool of raw material for adaptive evolution (Su et al., 2009; Deng et al., 2013). However, the level of genetic variation in the P. chienii chloroplast genome is unknown.

Chloroplast simple sequence repeat (cpSSR) markers, which have been extensively used in population genetics, possess important and unique characteristics such as haploidy, nonrecombination, uniparental inheritance, and a low nucleotide substitution rate (Ebert and Peakall, 2009). cpSSR loci are generally distributed throughout the noncoding regions with higher sequence variations and have conservative flanking regions (Huang et al., 2015). In particular, the chloroplast genome retains ancient genetic patterns and can therefore provide unique insight into evolutionary processes (Provan et al., 2001). Therefore, cpSSR markers can be used to investigate genetic variation in small, fragmented populations and can be transferred to related species (Schaal et al., 1998; Petit et al., 2005; Pan et al., 2014). More important, because cpSSRs are paternally inherited in gymnosperms, they can be used to assess pollen-mediated gene flow, population genetic variation, and phylogeographic patterns. Information revealed by cpSSRs is complementary to that obtained from nuclear SSRs (Powell et al., 1996; Provan et al., 2001). Although no chloroplast genome sequences of P. chienii have been reported, the complete chloroplast genome sequence of T. chinensis var. mairei is available in the National Center for Biotechnology Information's GenBank (accession no. NC_020321.1). Thus, here we first isolated 18 cpSSRs in T. chinensis var. mairei, then applied 10 polymorphic markers to evaluate the genetic diversity of P. chienii. These markers will be further applied to survey the chloroplast genetic variation in P. chienii.

Table 1.

Characteristics of 18 cpSSR markers developed for Taxus chinensis var. mairei.

METHODS AND RESULTS

In this study, a total of 109 individuals from four populations of P. chienii were collected throughout its natural distribution range, including Shuimenjian (Zjsmj) in Zhejiang Province, Zhangjiajie (Hnzjj) in Hunan Province, Zizhuba (Jxzzb) in Jiangxi Province, and Damingshan (Gxdms) in Guangxi Zhuang Autonomous Region, China (Appendix 1). One T. chinensis var. mairei population was gathered from Fenshui (Jxfs) in Jiangxi Province. Due to its rare and endangered properties, only 11 individuals were sampled. Young leaves were collected and dried in silica gel immediately. Genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987).

From the complete chloroplast genome sequence for T. chinensis var. mairei (GenBank accession no. NC_020321.1), 32 cpSSR loci were identified with the repeat threshold settings of 10 repeats for mononucleotides and five repeats for di-, tri-, tetra-, penta-, and hexanucleotide cpSSRs. Based on their flanking regions, we designed 27 primers using Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, California, USA). One individual (SMJ27) from Zjsmj population for P. chienii and one individual (FS7) from Jxfs population for T. chinensis var. mairei were selected to screen these primers. PCR was performed in a total volume of 20 µL containing 20 ng of genomic DNA, 1× PCR buffer, 5 mM MgCl₂, 0.2mM dNTPs mixture, 0.25 µM of each primer, and 1 unit Taq polymerase (TaKaRa Biotechnology Co., Dalian, China). Reaction conditions included initial denaturation at 94°C for 3 min; followed by 35 cycles at 94°C for 1 min, annealing temperature for 1 min, and 72°C for 1 min; with a final extension at 72°C for 10 min. The annealing temperature was optimized by gradient PCR (Table 1). Amplified products were separated by 6% denaturing polyacrylamide gel electrophoresis and visualized by silver staining. The allele sizes were estimated with a 50-bp DNA ladder (TaKaRa Biotechnology Co.) as size standard. Eighteen of 27 primers (approximately 67%) could produce clear bands in both P. chienii and T. chinensis var. mairei. The 18 cpSSRs were divided into three categories in terms of motif structure: 15 perfect, one imperfect, and two compound repeats. The high frequency of perfect repeats was in accordance with Ebert's description (Ebert and Peakall, 2009).

The utility of these 18 cpSSR primers was further examined in 109 and 11 individuals of P. chienii and T. chinensis var. mairei, respectively. The PCR reactions were conducted as described above. Among these loci, 10 (PTC-cp02, PTC-cp03, PTC-cp04, PTC-cp05, PTC-cp08, PTC-cp13, PTC-cp15, PTC-cp21, PTC-cp23, and PTC-cp28) showed polymorphisms in P. chienii, whereas six (PTC-cp03, PTC-cp05, PTC-cp08, PTC-cp09, PTC-cp15, and PTC-cp28) were polymorphic in T. chinensis var. mairei (Table 1). The genetic parameters, including the number of alleles (A), haploid diversity (h), and unbiased haploid diversity (h_unb) for each population, were evaluated with GenAlEx version 6.41 (Peakall and Smouse, 2006). Ten polymorphic cpSSR loci for P. chienii and six polymorphic loci for T. chinensis var. mairei were used. For P. chienii, A was between one and four, h ranged from 0.000 to 0.620, and h_unb varied from 0.000 to 0.641 (Table 2). Population Zjsmj revealed obviously higher diversity than other populations. For T. chinensis var. mairei, A, h, and h_unb were one to three, 0.000–0.496, and 0.000–0.545, respectively (Table 2).

Table 2.

Genetic properties of 10 polymorphic cpSSR loci for Pseudotaxus chienii and six polymorphic loci for Taxus chinensis var. mairei.^a

Analysis of molecular variance (AMOVA) was performed to measure genetic differentiation and the ratio of genetic variations within and among P. chienii populations in Arlequin version 3.5 (Excoffier and Lischer, 2010). The results revealed significant difference in partitioning of variation among and within populations (29.03% and 70.97%, respectively; Table 3) and uncovered significant genetic differentiation among all populations (F_ST = 0.2903).

CONCLUSIONS

The polymorphic chloroplast SSR loci developed from T. chinensis var. mairei in this study were verified to be reliable for assessing genetic variation of P. chienii populations. Combined with the nuclear SSR loci previously developed (Deng et al., 2013), the 18 cpSSRs will contribute to further exploration of whether the adaptation of P. chienii to environmental heterogeneity is driven through nuclear or chloroplast loci. In addition, the conservative nature of cpDNA may allow these markers to be used in other conifers.

Table 3.

The analysis of molecular variance (AMOVA) within and among populations based on 10 polymorphic cpSSRs in Pseudotaxus chienii.