Development of 11 Polymorphic Microsatellite Loci from Primula amthystina Franchet. (Primulaceae)

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  • 1 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, China
  • 2 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, China; and Graduate School, Chinese Academy of Sciences, Beijing 100049, China
  • 3 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Lanhei Road 134, Heilongtan, Kunming 650204, Yunnan, China; and College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China

Primula amethystina Franchet. is a beautiful perennial herbaceous plant locally endemic to the alpine area in southwest China. We isolated and characterized 11 polymorphic microsatellite primer pairs from this species. The number of alleles ranged from two to five. The observed and expected heterozygosities (HO and HE) were 0.25 to 0.875 and 0.223 to 0.691, respectively. Six loci were significantly deviated from Hardy-Weinberg equilibrium as a result of the heterozygote deficiency. These markers will have great potential to reveal the genetic population structure and genetic diversity of P. amethystina.

Abstract

Primula amethystina Franchet. is a beautiful perennial herbaceous plant locally endemic to the alpine area in southwest China. We isolated and characterized 11 polymorphic microsatellite primer pairs from this species. The number of alleles ranged from two to five. The observed and expected heterozygosities (HO and HE) were 0.25 to 0.875 and 0.223 to 0.691, respectively. Six loci were significantly deviated from Hardy-Weinberg equilibrium as a result of the heterozygote deficiency. These markers will have great potential to reveal the genetic population structure and genetic diversity of P. amethystina.

Primula amethystina Franchet. is a beautiful perennial herbaceous plant locally endemic to the alpine area in southwest China. It is widely distributed in alpine meadows at altitudes of 3400 to 5000 m in west and northwest Yunnan, southwest Sichuan, over the border into Tibet (Hu and Kelso, 1996; Richards, 2002). P. amethystina subsp. amethystina is confined to the Cangshan Mountains, Dali, west Yunnan, whereas P. amethystina subsp. brevifolia widely spreads through the range. Frequently, P. amethystina subsp. brevifolia is more robust than P. amethystine subsp. amethystina with up to 20 flowers borne on stems to 25 cm. To classify these two species and study the speciation mode of them, their genetic structures need to be understood. Here, 11 polymorphic microsatellite loci of P. amethystina were developed as potential tools to investigate the genetic structures of these species.

Genomic DNA was extracted from leaf tissues using the cetyltrimethyl ammonium bromide (CTAB) method (Milligan, 1992). The isolation of microsatellite loci was performed according to the fast isolation of microsatellite by amplified fragment length polymorphism of sequences containing repeats (FIASCO) (Zane et al., 2002). Approximately 300 ng genomic DNA was completely digested with MseI restriction enzyme (Fermantas). The digested DNA was ligated to MseI adaptor pair (Vos et al., 1995), then 5 μL of the adapter-ligated fragments acted as templates to perform polymerase chain reaction (PCR) in a volume of 20 μL using MseI-N (5′-GATGAGTCCTGAGTAAN-3′) as a primer following the program: 95 °C for 3 min, 30 cycles of 94 °C for 30 s, 53 °C for 60 s, 72 °C for 60 s followed by an elongation step of 5 min at 72 °C.

For enrichment, the PCR products were denatured at 95 °C for 5 min and then hybridized with a 5′-biotinylated probe (AG)15 in 250 μL hybridization solution (20 × SSC, 10% SDS, 100 pmol/μL probe) at 48 °C for 2 h. The DNA hybridized to the probe was separated and captured by streptavidin-coated magnetic beads at room temperature for 20 min followed by two washing steps, including three times in TEN100 for 15 min and three times in TEN1000 for 24 min. The separated single-stranded DNA was subjected to a second round of PCR according to the same procedure as the first round of PCR. The PCR products, after being purified with the E.Z.N.A Gel Extraction Kit (Omega Bio-Tek, Atlanta, GA), were ligated into pMD18-T vector (Takara, Otsu, Shiga, Japan) according to the manufacturer's instructions and then transformed into Escherichia coli strain JM109 (Sangon, Shanghai, China). The positive clones were picked out by blue–white screening and tested by PCR using (AG)10 and M13+/M13 as primers, respectively. Of the 91 clones sequenced, 79 contained potential microsatellite motifs and 42 with unique microsatellites were selected for designing primers using Primer 5.0 (Clarke and Gorley, 2001).

A total of 12 primer sets were abandoned as a result of amplification of multiple bands or unsuccessful amplification of target fragments, and the remaining 30 primer pairs were tested for polymorphisms across 24 individuals from a population of P. amethystina subsp. amethystina and three populations of P. amethystina subsp. brevifolia (Table 1). PCR reaction was done in a 20-μL volume using a PTC0200 thermal cycler (MJ Research, Ashland, OR). Each reaction was performed using 20 ng of genomic DNA, 1 μM of each dNTP, 1 μM of each primer, 1× Taq buffer [100 mm Tris-HCl, pH 8.8, 2.0 mm MgCl2, 200 mm (NH4)2SO4, 0.1% Tween 20], and 1 U of Taq polymerase (Takara). The PCR programs are as follows: initial denaturing step at 95 °C for 5 min, 30 cycles of 94 °C for 30 s, primer-specific annealing temperature 55 to 62 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 8 min. The PCR products were electrophoresed in denaturing 6% polyacrylamide gels using a 25-bp DNA ladder molecular size standard (Fermatas, Ontario, Canada) to estimate allele sizes by silver staining.

Table 1.

Sample information of P. amethystina subsp amethystina and P. amethystine subsp. brevifolia.

Table 1.

Of the 30 new primers, 11 of them displayed polymorphism across populations. The number of alleles per locus, observed (HO) and expected heterozygosity (HE), and deviation form Hardy-Weinberg equilibrium (HWE) were assessed using GENEPOP Version 3.4 (http://wbiomed.curtin.edu.au/genepop/) (Raymond and Rousset, 1995). The number of alleles per locus ranged from two to five with an average of 2.8 (Table 2). The observed and expected heterozygosities (HO and HE) ranged from 0.25 to 0.875 and from 0.223 to 0.691 with averages of 0.508 and 0.442, respectively (Table 2). Among the 11 microsatellite markers, six loci showed significant deviation from HWE (P < 0.01) (Table 2), which was the result of deficiency of heterozygotes or the limitation of sample size. Tests for linkage disequilibrium were run in FSTAT Version 2.9.3.2 (Goudet, 1995). Significance levels were adjusted using sequential Bonferroni corrections (Rice, 1989). No loci showed significant linkage disequilibrium after Bonferroni correction. These polymorphic microsatellite loci presented here would provide a useful tool for studying the population genetic structure and genetic diversity of P. amethystina.

Table 2.

Characteristics of 11 polymorphic microsatellite loci for Primula amethystine Franchet.

Table 2.

Literature Cited

  • Clarke, K.R. & Gorley, R.N. 2001 PRIMER v5: User manual/tutorial PRIMER-E Ltd Plymouth, UK

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  • Goudet, J. 1995 FSTAT (Version 1.2): A computer program to calculate F-statistics J. Hered. 86 485 486

  • Hu, C.M. & Kelso, S. 1996 Primulaceae 147 148 Wu C.Y. & Raven P.H. Flora of China (15) Science Press, Beijing, China, and Missouri Botanical Garden Press Beijing and St. Louis, MO

    • Search Google Scholar
    • Export Citation
  • Milligan, B. 1992 Plant DNA isolation 58 88 Hoelzel A.R. Molecular genetic analysis of populations: A practical approach IRL Press Oxford, UK

  • Raymond, M. & Rousset, F. 1995 GENEPOP version 1.2: Population genetics software for exact tests and ecumenicism J. Hered. 86 248 249

  • Rice, W.R. 1989 Analyzing tables of statistical tests Evolution 43 223 225

  • Richards, A.J. 2002 Primula Timber Press Portland, OR 99

  • Vos, P., Hogers, R., Bleeker, M., Reijans, M., Vandeleet, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. & Zabeau, M. 1995 AFLP: A new technique for DNA fingerprinting Nucleic Acids Res. 23 4407 4414

    • Search Google Scholar
    • Export Citation
  • Zane, L., Bargelloni, L. & Patarnello, T. 2002 Strategies for microsatellite isolation: A review Mol. Ecol. 11 1 16

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Contributor Notes

This work was supported by a project of “Light of Western China” (29O8025813W1), the National Natural Science Foundation (30900090), the Ministry of Science and Technology of China (2008FY110400-2-2 and 2005DK21006), the Ministry of Education of China (B08044 and MUC 985-3-3), and the Japan Society for the Promotion of Science (JSPS/AP/109080).

Dr. Zhikun Wu made contributions in collecting samples from the field.

To whom reprint requests should be addressed; e-mail long@mail.kib.ac.cn.

  • Clarke, K.R. & Gorley, R.N. 2001 PRIMER v5: User manual/tutorial PRIMER-E Ltd Plymouth, UK

    • Export Citation
  • Goudet, J. 1995 FSTAT (Version 1.2): A computer program to calculate F-statistics J. Hered. 86 485 486

  • Hu, C.M. & Kelso, S. 1996 Primulaceae 147 148 Wu C.Y. & Raven P.H. Flora of China (15) Science Press, Beijing, China, and Missouri Botanical Garden Press Beijing and St. Louis, MO

    • Search Google Scholar
    • Export Citation
  • Milligan, B. 1992 Plant DNA isolation 58 88 Hoelzel A.R. Molecular genetic analysis of populations: A practical approach IRL Press Oxford, UK

  • Raymond, M. & Rousset, F. 1995 GENEPOP version 1.2: Population genetics software for exact tests and ecumenicism J. Hered. 86 248 249

  • Rice, W.R. 1989 Analyzing tables of statistical tests Evolution 43 223 225

  • Richards, A.J. 2002 Primula Timber Press Portland, OR 99

  • Vos, P., Hogers, R., Bleeker, M., Reijans, M., Vandeleet, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. & Zabeau, M. 1995 AFLP: A new technique for DNA fingerprinting Nucleic Acids Res. 23 4407 4414

    • Search Google Scholar
    • Export Citation
  • Zane, L., Bargelloni, L. & Patarnello, T. 2002 Strategies for microsatellite isolation: A review Mol. Ecol. 11 1 16

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