Scrophularia incisa Weinm. (Scrophulariaceae) is a perennial plant inhabiting floodplains, grasslands, and mountain valleys at altitudes between 600 and 3600 m. It presents a belt-like distribution primarily in northern China stretching westward to Central Asia and eastward to Siberia, Russia (Ma et al., 1980; Hong et al., 1998). This species is a traditional Mongolian medicinal herb applied in the treatment of measles, smallpox, chickenpox, and scarlet fever (Ma et al., 1980). According to our field investigations, its current population number and size appears limited, possibly as a consequence of over-exploitation and habitat loss. Therefore, population genetic analyses of S. incisa will be necessary to infer its evolutionary processes and to determine appropriate conservation strategies. Nuclear microsatellites (simple sequence repeats [SSRs]) are highly polymorphic, codominant markers that have been widely applied in assessing population genetic structure and gene flow (Liu et al., 2009). There are hitherto no microsatellite loci available for S. incisa. Hence, development of polymorphic markers is needed. Furthermore, researchers increasingly require universal markers that can readily be transferred between species. Such transferable markers facilitate comparisons among closely related taxa for addressing the mechanisms involved in population divergence and speciation (Noor and Feder, 2006). Scrophularia incisa, S. dentata Royle ex Benth., and S. kiriloviana Schischk. constitute sect. Tomiophyllum of Scrophularia in China. Scrophularia incisa and its allies are morphologically similar and geographically largely separated, presenting a roughly circular geographic pattern on the Qinghai–Tibet Plateau. Scrophularia dentata is distributed in southern and western Tibet, while S. kiriloviana occurs in northern Xinjiang extending to Central Asia (Hong et al., 1998). Thus, transferable markers are critical for comparative studies, even if they only allow investigations in related species. In this sense, they can be used to address whether and which heterogeneous evolutionary processes acted in the same geological time frame in the Qinghai–Tibet Plateau and adjacent regions.
In the current study, we aim to identify polymorphic compound microsatellite markers for S. incisa using a recently developed isolation technique (Lian et al., 2006) to characterize genetic variation of S. incisa populations, and to test their transferability to its close allies, S. dentata and S. kiriloviana. Our developed universal markers should be valuable and robust to address these purposes.
METHODS AND RESULTS
The compound microsatellite marker technique based on a dual-suppression PCR method was applied to develop SSR markers for S. incisa according to Zhai et al. (2010). DNA was isolated from silica gel–dried leaf materials using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle, 1991). First, total DNA of two individuals from a population in Gandi, Qinghai Province, China (population code: GD), were digested by the restriction enzymes HaeIII and SspI (TaKaRa Biotechnology Co., Dalian, China), and the restriction fragments were ligated to an unequal-length adapter using DNA Ligation Kit version 2.0 (TaKaRa Biotechnology Co.). Second, DNA fragments flanked by a microsatellite at one end were amplified from both the HaeIII and SspI libraries using the compound SSR primer (AC)6(AG)5 or (TC)6(AC)5 and an adapter primer AP2 (5′-CTATAGGGCACGCGTGGT-3′). PCR products of 400–1000 bp were purified, inserted, and ligated into PMD18-T vector (TaKaRa Biotechnology Co.) to form a recombinant DNA. Third, the recombinant DNA was transformed into DH5α competent cells (TaKaRa Biotechnology Co.) for culturing, and the clone cells were amplified by an M13 primer to detect the positive clones. Finally, a total of 190 positive clones were obtained and sequenced on an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems, Carlsbad, California, USA).
Characteristics of 12 compound microsatellite loci developed for Scrophularia incisa.
One hundred and ten sequences were found to contain (AC)6(AG)n or (TC)6(AC)n compound SSR motifs, of which 56 fragments possessed sufficient flanking regions for designing specific primers. Sixteen primers were designed using PRIMER version 5.0 (Clarke and Gorley, 2001) following the criteria of Zheng et al. (2012). A total of 78 samples of S. incisa from four populations (Manzhouli, Inner Mongolia, China [MZ]; Gandi, Qinghai, China [GD]; Zhangye, Gansu, China [ZY]; and Qilian, Qinghai, China [QL]) were used to estimate polymorphism. Thirty-five individuals of S. dentata from Xigaze, Tibet, China (RK), and Lhasa, Tibet, China (LS), and 40 individuals of S. kiriloviana from Wensu, Xinjiang, China (WS), and Tashkurgan, Xinjiang, China (TS), were analyzed for cross-species amplification tests. The voucher specimens were deposited in the Herbarium of Zhejiang University (HZU) (Appendix 1).
Results of initial primer screening in four populations of Scrophularia incisa.a
PCRs were conducted in a 15-µL reaction mixture containing 1.5 µL of 10× PCR buffer with MgCl2, 0.75 µL of dNTPs (2.5 mM each), 0.38 µL of each primer (10 µM), 60–100 ng of genomic DNA, 0.5 U of Taq polymerase (TaKaRa Biotechnology Co.), and 0.1 µL of bovine serum albumin (BSA; TaKaRa Biotechnology Co.). PCR amplification conditions were as follows: initial denaturation at 94°C for 5 min, followed by 38 cycles of 30 s at 94°C, 45 s at the optimal annealing temperature (Table 1), 90 s of elongation at 72°C, ending with a 10-min extension at 72°C. PCR amplification products were analyzed on a MegaBACE 1000 autosequencer (GE Healthcare Biosciences, Pittsburgh, Pennsylvania, USA), and alleles were scored by GeneMaker software version 1.97 (SoftGenetics, State College, Pennsylvania, USA). Across these eight populations, the number of observed alleles per locus, as well as observed and expected heterozygosities, were calculated using CERVUS version 3.0.3 (Kalinowski et al., 2007). Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between all these primer pairs were tested using GENEPOP version 4.0.7 (Rousset, 2008).
Results of primer cross-species amplification in Scrophularia dentata and S. kiriloviana.a
Twelve loci could be amplified repeatedly and demonstrated polymorphism, and the remaining four loci could not be amplified reliably. The statistics reported are from the 12 polymorphic loci that could be reliably scored. The mean number of alleles was 19.75 (range: 14–26) for the four S. incisa populations (Table 1); 7.75 (range: 6–11), 8.50 (range: 5–12), 7.75 (range: 6–10), and 7.25 (range: 4–11) for populations MZ, GD, ZY, and QL, respectively (Table 2). The four populations exhibit comparable levels of microsatellite diversity (Table 2). The 12 microsatellite loci developed for S. incisa were successfully transferred in the other two species of sect. Tomiophyllum, S. dentata and S. kiriloviana. All of the SSR markers developed from S. incisa are codominant in S. dentata and S. kiriloviana. Their overall mean numbers of alleles were 5.18 (range: 2–8) and 8.83 (range: 5–13) per locus for S. dentata and S. kiriloviana, and they also exhibit comparable levels of microsatellite diversity (Table 3). We detected deviation from HWE (P ≤ 0.05) at some of the microsatellite loci as a result of heterozygote excess, e.g., three (Scin1, 4, 8), one (Scin7), two (Scin1, 2), and two (Scin7, 9) loci for populations MZ, GD, ZY, and QL, respectively (Table 2); five (Scin1, 2, 3, 8, 9), two (Scin5, 7), and nine (Scin1, 2, 4, 5, 7, 8, 10, 11, 12) loci for populations RK, WS, and TS, respectively (Table 3). No significant LD signal (P ≤ 0.01) was detected for each locus pair across all populations.
The application of these 12 polymorphic microsatellite markers in combination with chloroplast DNA sequences should be robust to reveal geographic patterns of molecular variation in S. incisa, S. dentata, and S. kiriloviana at the population level and across the species ranges in China. From a perspective of comparative phylogeography, these data from such a study system will be substantially valuable to address roles of different evolutionary processes in plants inhabiting the Qinghai–Tibet Plateau and adjacent regions, and to guide appropriate conservation action in the vulnerable ecosystems.
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 The authors thank L. Zheng and Y. P. Zhao for technical assistance with the experiment and suggestions on manuscript preparation. This research was supported by grants from the National Natural Science Foundation of China (no. 1070205); from the opening project of the Key Laboratory of Biological Resources, Talimu University, Xinjiang, China; and from the Key Breeding Project of Zhejiang Province (no. 2012C12912).