Isolation and Characterization of Twenty-four Microsatellite Loci for Rhododendron decorum Franch. (Ericaceae)

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  • 1 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 100039, China
  • 2 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Lanhei Road 132, Kunming 650204, Yunnan, China
  • 3 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, China; and College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China

Rhododendron decorum is a common species in southwest China and northeast Myanmar, in which the flowers have been eaten as a favorite vegetable. We isolated and characterized 24 microsatellite primer pairs from this species. The number of alleles ranged from two to seven. The observed and expected heterozygosities (HO and HE) were 0.3830 to 0.7855 and 0 to 0.7917, respectively. Eleven loci were significantly deviated from Hardy-Weinberg equilibrium as a result of the heterozygote deficiency. Cross-species amplification in another eight Rhododendron species showed their potential use for evolutionary and conservation studied in this genus. These markers will be useful to reveal the genetic population structure and genetic diversity of R. decorum.

Abstract

Rhododendron decorum is a common species in southwest China and northeast Myanmar, in which the flowers have been eaten as a favorite vegetable. We isolated and characterized 24 microsatellite primer pairs from this species. The number of alleles ranged from two to seven. The observed and expected heterozygosities (HO and HE) were 0.3830 to 0.7855 and 0 to 0.7917, respectively. Eleven loci were significantly deviated from Hardy-Weinberg equilibrium as a result of the heterozygote deficiency. Cross-species amplification in another eight Rhododendron species showed their potential use for evolutionary and conservation studied in this genus. These markers will be useful to reveal the genetic population structure and genetic diversity of R. decorum.

Rhododendron L. is a large and diverse genus with a nearly worldwide distribution, and the center of diversity of the genus is in the Himalayas (Kron and Judd, 1990). This genus contains seven subgenus in all, including Hymenanthes, which has 23 subsections with ≈270 species (Fang et al., 2005), and all the species are diploids (2n = 26) (Min and Fang, 1990). Most species in subgenus Hymenanthes are world-famous ornamental plants as a result of the evergreen leaves and large and colorful flowers (Min, 1984). Rhododendron decorum belongs to subgenus Hymenanthes. It is a beautiful evergreen shrub or small tree, commonly distributed in southwest China and northeast Myanmar (Fang et al., 2005). Its flower is a favorite edible vegetable for local ethnic people in southwest China (Yong and Chong, 1980). In common with other species of subgenus Hymenanthes, R. decorum is highly interfertile, which was supported by molecular evidence (Zha et al., 2008; Zhang et al., 2007). However, until now, little research has been conducted related to population genetics of R. decorum. To study more deeply the genetic diversity within this species and the effect of hybridization on the speciation process of this species, we developed 24 microsatellite markers for this species to investigate the genetic diversity and genetic structures among populations and provide a potential tool for studying molecular breeding in R. decorum.

Genomic DNA was extracted from leaf tissues using the cetyltrimethyl ammonium bromide method (Milligan, 1992). Approximately 300 ng genomic DNA was completely digested with MseI restriction enzyme (Fermantas). The digested DNA was ligated to the 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′-GAT GAG TCC TGA GTA AN-3′) as the primer and 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 72 °C for 5 min.

For enrichment, the PCR products were denatured at 95 °C for 5 min, 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) according to the manufacturer's instructions and then transformed into Escherichia coli strain JM109 (Sangon, Shanghai). The positive clone was picked out by blue–white screening and tested by PCR using (AG) 10 and M13+/M13 as primers, respectively. One hundred ten of 292 screened clones contained potential microsatellite motifs.

A total of 50 clones were found to contain simple sequence repeats and then subjected to primer designing using the Primer 5.0 (Clarke and Gorley, 2001). Twenty-four individuals from two wild populations, two semicultivated and cultivated populations, were used to screen polymorphism. PCR reaction was done in 20 μL volume using a PTC0200 thermal cycler (MJ Research, Ashland). Each reaction was performed using 20 ng of genomic DNA, 1 μM of each dNTP, 1 μM 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 took place 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 (Fermantas) to estimate allele size by silver staining.

Of the 50 new primers designed for R. decorum, 32 successfully amplified the target regions and 24 of them displayed polymorphism. 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 seven with an average of 4.1 (Table 1). The observed and expected heterozygosities (HO and HE) ranged from 0.3830 to 0.7855 and from 0 to 0.7917 with averages of 0.6121 and 0.4531, respectively. Among the 24 microsatellite markers, 11 loci show significant deviation from HWE (P < 0.01, Table 1), probably as a result of deficiency of heterozygote 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. For cross-species application, these 24 new primer pairs were tested in the other eight important horticultural species and close taxa such as R. irroratum, R. agastum, R. delavayi, R. araiophyllum, R. molle, R. simsii, R. pachypodu, and R. spinuliferum. Twelve pairs of them can be amplified successfully in all species, whereas the RDW18 failed amplification in all species (Table 2). These polymorphic microsatellite loci presented here would provide a useful tool for studying the population genetic structure and genetic diversity of R. decorum, and it will also be valuable for studying other species in this group.

Table 1.

Characteristics of 24 polymorphic microsatellite loci for Rhododendron decorum.

Table 1.
Table 2.

Cross-species amplification of 24 new microsatellite loci from Rhododendron decorum in other related Rhododendron species.

Table 2.

Literature Cited

  • Clarke, K.R. & Gorley, R.N. 2001 PRIMER v5: User manual/tutorial PRIMER-E Ltd Plymouth, UK 91

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  • Fang, M.Y., Fang, R.Z., He, M.Y., Hu, L.Z., Yang, H.B., Qin, H.N., Min, T.L., David, F., Chamberlain, P.S., Wallace, G.D. & Anderberg, A. 2005 Rhododendron (Ericaceae) 333 Wu Z.Y. & Raven P.H. Flora of China Vol. 14 Science Press and Missouri Botanical Garden Press Beijing, China, and St. Louis, MO

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  • Milligan, B. 1992 Plant DNA isolation. A practical approach 59 88 Hoelzel A.R. Molecular genetic analysis of populations IRL Press Oxford, UK

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    • Export Citation
  • Zha, H.G., Milne, R.I. & Sunday, H. 2008 Morphological and molecular evidence of natural hybridization between two distantly related Rhododendron species from the Sino-Himalaya Bot. J. Linn. Soc. 156 119 129

    • Search Google Scholar
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  • Zhang, J.L., Zhang, C.Q., Gao, L.M., Yang, J.B. & Li, H.T. 2007 Natural hybridization origin of Rhododendron agastum (Ericaceae) in Yunnan, China: Inferred from morphological and molecular evidence J. Plant Res. 120 457 463

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Contributor Notes

This work was supported by the Ministry of Science and Technology of China (2008FY110400-2-2 and 2005DK21006), the Ministry of Education of China (B08044 and CUN-985-3-3), and the Japan Society for the Promotion of Science (JSPS/AP/109080).

Ms. Dong-mei Rui joined in field work to collect samples.

Both authors contributed equally.

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 91

    • Export Citation
  • Fang, M.Y., Fang, R.Z., He, M.Y., Hu, L.Z., Yang, H.B., Qin, H.N., Min, T.L., David, F., Chamberlain, P.S., Wallace, G.D. & Anderberg, A. 2005 Rhododendron (Ericaceae) 333 Wu Z.Y. & Raven P.H. Flora of China Vol. 14 Science Press and Missouri Botanical Garden Press Beijing, China, and St. Louis, MO

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

  • Kron, K.A. & Judd, W.S. 1990 Phylogenetic relationship within the Rhodoreae (Ericaceae) with specific comments on the placement of Ledum Syst. Bot. 15 57 68

    • Search Google Scholar
    • Export Citation
  • Milligan, B. 1992 Plant DNA isolation. A practical approach 59 88 Hoelzel A.R. Molecular genetic analysis of populations IRL Press Oxford, UK

  • Min, T.L. 1984 A revision of subgenus Hymenanthes (Rhododendron L.) in Yunnan and Xizang Acta Botanica Yunnanica 6 141 171

  • Min, T.L. & Fang, R.Z. 1990 The phylogeny and evolution of genus Rhododendron Acta Botanica Yunnanica 12 353 365

  • 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

  • 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
  • Yong, J. & Chong, L.S. 1980 Rhododendrons of China Binford & Mort Portland, OR 307

    • Export Citation
  • Zha, H.G., Milne, R.I. & Sunday, H. 2008 Morphological and molecular evidence of natural hybridization between two distantly related Rhododendron species from the Sino-Himalaya Bot. J. Linn. Soc. 156 119 129

    • Search Google Scholar
    • Export Citation
  • Zhang, J.L., Zhang, C.Q., Gao, L.M., Yang, J.B. & Li, H.T. 2007 Natural hybridization origin of Rhododendron agastum (Ericaceae) in Yunnan, China: Inferred from morphological and molecular evidence J. Plant Res. 120 457 463

    • Search Google Scholar
    • Export Citation
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