e167

American Journal of Botany: e167–e169. 2012.

American Journal of Botany: e167–e169, 2012; http://www.amjbot.org/ © 2012 Botanical Society of America

Diplopanax stachyanthus Hand.-Mazz. is a relict tree within the Araliaceae, and is mainly distributed in southern China and northern Vietnam ( Zhu et al., 1998 ). Recent studies have sug-gested the placement of this species within the Cornaceae (e.g., Zhu et al., 1998 ). It is medicinally and phylogenetically impor-tant ( Khac et al., 1990 ; Wu, 1997 ). However, largely due to the low seed yield and germination rates, low seedling survival rate, and anthropogenic disturbance, most of its natural popula-tions have decreased ( Wang et al., 2010 ). Diplopanax stachyan-thus has long been listed among the Grade II Rare and Endangered Plants in China ( Wu, 1997 ). A good estimate of the genetic diversity of this species is fundamental to formulate ef-fective conservation strategies, and such information can only be obtained by detailed population genetic analyses ( Hamrick and Godt, 1996 ).

Here, we present a novel set of microsatellite loci in D. stachyanthus for the analysis of its genetic diversity. Their cross-taxa transferability was also examined in four distantly related species. All voucher specimens of the investigated spe-cies are stored in the herbarium of the School of Biological Science and Technology, Central South University, China (Appendix 1).

METHODS AND RESULTS

The development of microsatellite markers followed a slight modifi cation of the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) proto-col as described by Zane et al. (2002) . Total genomic DNA was extracted from leaf tissues using the 2 × cetyltrimethylammonium bromide (CTAB) method of Wang and Fang (1998) . Genomic DNA from one individual of D. stachyanthus (DS10GX02) was digested with Mse I, and then ligated to the Mse I AFLP adapt-ers (5 ′ -TACTCAGGACTCAT-3 ′ /5 ′ -GACGATGAGTCCTGAG-3 ′ ) using T4 DNA ligase (New England Biolabs, Boston, Massachusetts, USA). The diges-tion – ligation mixture was diluted (1 : 10) and amplifi ed with the adapter-specifi c primer Mse I-N (5 ′ -GATGAGTCCTGAGTAAN-3 ′ ) using the following PCR program: initial denaturation of 94 ° C for 3 min; followed by 30 cycles of 94 ° C for 30 s, 53 ° C for 30 s, and 72 ° C for 60 s; and a fi nal extension of 72 ° C for 10 min. To enrich repeat motifs, the PCR products were denatured at 95 ° C for 5 min, and then hybridized to 5 ′ -biotinylated probes (AG) 15 and (AC) 15 , re-spectively, in a 250- µ L hybridization solution (4 × SSC + 0.1% SDS) at 48 ° C for 2 h. Streptavidin-coated magnetic beads (Promega, Madison, Wisconsin, USA) were used to separate and capture the DNA fragments hybridized to the probes at room temperature for 20 min. Finally, the target fragments were washed by two steps: three times in TEN 100 for 8 min and three times in TEN 1000 for 8 min, and amplifi ed with the primer Mse I-N. PCR products ranging from 400 to 800 bp were purifi ed using the AxyPrep DNA Gel Extraction Kit (Axygen Scientific, Silicon Valley, California, USA), ligated into pMD18-T vector (TaKaRa Biotechnology Co., Dalian, China), and then transformed into Escherichia coli strain JM109 (TaKaRa Biotechnology Co.) following the manu-facturer ’ s instructions. The positive clones were screened using (AG) 10 /(AC) 10 and M 13 universal primers, respectively.

A total of 51 clones were unidirectionally sequenced by Shanghai Sangon Biological Engineering Technology Service Co., Ltd. (Shanghai, China), using an ABI PRISM 3730 Genetic Analyzer (Applied Biosystems, Foster City, Cali-fornia, USA). Of the 51 obtained sequences, 34 were selected for primer design using Primer Premier version 5.0 (PREMIER Biosoft International, Palo Alto, California, USA). These primer pairs were then evaluated for their utilities us-ing 25 individuals from the same population of D. stachyanthus (DS10GX02). PCR amplifi cations were conducted in the thermal cycler PTC-100 (MJ Re-search, Waltham, Massachusetts, USA) using the following protocol: a fi nal volume of 10 µ L containing 50 ng DNA template, 1 × PCR buffer, 1.5 mM

1 Manuscript received 18 September 2011; revision accepted 7 December 2011.

The authors thank the editor and an anonymous reviewer for their critical comments and constructive suggestions on an earlier version of the manuscript. This study was fi nancially supported by the National Natural Science Foundation of China (grant no. 30800088) and the Key Project of the Cutting-Edge Research Program of CSU (grant no. 2177-721500011).

4 Author for correspondence: csu.wangli@gmail.com

doi:10.3732/ajb.1100476

AJB PRIMER NOTES & PROTOCOLS IN THE PLANT SCIENCES

ISOLATION AND CHARACTERIZATION OF MICROSATELLITE LOCI IN THE ENDANGERED TREE DIPLOPANAX STACHYANTHUS

(ARALIACEAE) 1

LI WANG 2,4 , MIN WU 2 , ZHI WEN 2 , QIU-PING XIA 2 , HAI-FEI YAN 3 , AND ZHI-YONG LUO 2

2 School of Biological Science and Technology, Central South University, Changsha 410013, People ’ s Republic of China; and 3 South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People ’ s Republic of China

• Premise of the study: Microsatellite markers were developed in Diplopanax stachyanthus to investigate the population genetics of this endangered tree.

• Methods and Results: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, 15 microsatellite markers were developed in D. stachyanthus and evaluated for their variability in 25 samples from a natural population. For the 11 polymorphic loci, the number of alleles ranged from two to eight, while the observed and expected heterozygosities ranged from 0.5200 to 0.7600 and 0.4200 to 0.7813, respectively. Their cross-taxa transferability was also examined in Acanthopanax gracilistylus , Tetrapanax papyrifer , Cornus controversa , and Dendrobenthamia japonica var. chinensis , and four to 15 loci proved amplifi able in these species.

• Conclusions: These microsatellite markers could be employed to investigate the population genetics of D. stachyanthus , and may potentially be applicable to other related species.

Key words: Araliaceae; Diplopanax stachyanthus ; endangered species; microsatellites.

e168 AMERICAN JOURNAL OF BOTANY [Vol. 0

for 10 min. The PCR products were separated in 10% nondenaturing polyacryl-amide gels and then visualized by silver staining. The 20 bp DNA ladder (TaKaRa Biotechnology Co.) was used as DNA molecular weight markers. Allele sizes were automatically determined using Bio-Rad Gel Documentation Sys-tem (Bio-Rad Laboratories, Hertfordshire, United Kingdom) and manually

MgCl 2 , 50 mM KCl, 0.25 mM each of dNTPs, 0.25 U Taq polymerase (Fer-mentas, Burlington, Ontario, Canada), and 0.6 µ M of each primer. The initial denaturation was set at 94 ° C for 4 min; followed by 28 cycles of denaturation at 94 ° C for 30 s, appropriate annealing temperature (see Table 1 for details) for 40 s, and extension at 72 ° C for 30 s; and followed by a fi nal extension of 72 ° C

TABLE 1. Characterization of 15 microsatellite loci in Diplopanax stachyanthus .

Locus Primer sequences (5 ′ – 3 ′ ) Repeat motif T a ( ° C) Size range (bp) GenBank accession no.

Dst003 F: GTGAATTGAGAAGACAAAAGAGG ( GA ) 31 ( TG ) 8 ( GA ) 5 55 202 – 222 JN657178 R: TTACGTGGCAGCCCAATTAT

Dst008 F: CTTTGCTGGGACTTCTTTGG ( AT ) 7 ( AC ) 9 ( AG ) 6 60 182 – 192 JN657179 R: TGGATTGGCTTCATCCACTT

Dst026 F: GGCAAACATACCCCCTGATA ( AG ) 21 ( TA ) 5 55 126 – 148 JN657180 R: CCACATCAAAGCCACAATGA

Dst032 * F: TTGCAGCTATCAAACCATGC ( GT ) 6 ( GA ) 7 60 229 JN989544 R: ATTGCCGGTATTTTGGGAAG

Dst043 F: CCAAACTGCTGCAAATCAAA ( AG ) 14 60 232 – 296 JN657181 R: CAACACACAACCCACATCACT

Dst052 * F: GCTACGAGTTCGTTCCCTCA ( AC ) 10 AT ( AG ) 15 60 253 JN989545 R: AAAGTTGGTTTGGGGTCGTT

Dst056 F: TCCAAATTGAAATCCAAACCA ( AG ) 8 60 223 – 263 JN657182 R: CACAAACACATCATTTCTCTCTCTC

Dst061 F: GCCTTCACCGAGTGGATAAA ( GA ) 12 62 186 – 206 JN657183 R: TTGCTGCAGCTGTATTCGAG

Dst068 F: CCAACATGACCACGCATAGA ( AG ) 17 60 187 – 197 JN657184 R: AAGCAGCTGTGGTTGAAGGT

Dst089 F: TGGGTAATGGGTAATGGGTTA ( TG ) 7 ( GT ) 5 ( GA ) 9 60 206 – 240 JN657185 R: TCCTTTATCCAACACCAATGTG

Dst091 F: TGCTTCCCCCTTCACATATT ( GA ) 29 55 271 – 301 JN657186 R: GCTTGAGACGTGGAAGACATC

Dst103 F: CGTTCTGCGTTACGTATCCT ( GA ) 13 60 228 – 286 JN657187 R: GACCCGTGGAGGACTGATAA

Dst121 * F: CAACCACACATCTTCAAACCA ( AC ) 8 55 201 JN989546 R: GCAAAACCGTGAGCTTTAGG

Dst123 F: TTTCCTGGAGGGAGTTCTGA ( AG ) 19 55 237 – 285 JN657188 R: TGATTTTATGGGGGTTTTGG

Dst126 * F: GGTGGAACAGATCGGTCAAA ( AG ) 10 55 288 JN989547 R: CTGCTCGGTGTTGCTACAAA

Note : T a = annealing temperature.* Monomorphic loci.

TABLE 2. Genetic variability of the 15 microsatellite loci based on 25 individuals of Diplopanax stachyanthus , and their cross-taxa transferability in four other species.

Locus N a H o H e

Cross-taxa transferability ( n = 4)

Agra Tpap Ccon Djap

Dst003 5 0.5200 0.7240 3 — — — Dst008 5 0.5600 0.5736 2 1 — — Dst026 5 0.5200 0.6560 1 — — — Dst032* 1 0.0000 0.0000 2 1 1 2 Dst043 7 0.7500 0.7813 3 — 2 2 Dst052* 1 0.0000 0.0000 1 — — — Dst056 6 0.6471 0.7785 2 2 — 2 Dst061 4 0.6000 0.6863 1 — — — Dst068 4 0.5500 0.6750 1 1 1 — Dst089 2 0.6000 0.4200 1 — — 1 Dst091 5 0.6800 0.6472 2 — — 2 Dst103 6 0.7200 0.7456 2 2 — — Dst121* 1 0.0000 0.0000 1 2 2 1 Dst123 8 0.7600 0.7736 1 — — — Dst126* 1 0.0000 0.0000 2 1 — 1

Note : — = failed amplifi cation; Agra = Acanthopanax gracilistylus ; Ccon = Cornus controversa ; Djap = Dendrobenthamia japonica var. chinensis ; H e = expected heterozygosity; H o = observed heterozygosity; n = number of individuals assayed for each species; N a = number of alleles; Tpap = Tetrapanax papyrifer .

* Monomorphic loci.

e169April 2012] AJB PRIMER NOTES & PROTOCOLS — DIPLOPANAX STACHYANTHUS MICROSATELLITES

adjusted if necessary. Following the discarding of loci due to poor amplifi ca-tion, 15 loci that yielded clear bands were obtained, with 11 of them being polymorphic ( Table 1 ).

The program PowerMarker version 3.0 ( Liu and Muse, 2005 ) was employed to estimate various parameters for the polymorphic loci: the number of alleles per locus ( N a ), observed ( H o ) and expected ( H e ) heterozygosities, linkage dis-equilibrium (LD), and departure from Hardy – Weinberg equilibrium (HWE) ( Table 2 ). N a varied between two ( Dst089 ) and eight ( Dst123 ). H o and H e ranged from 0.5200 ( Dst003 & Dst026 ) to 0.7600 ( Dst123 ) and from 0.4200 ( Dst089 ) to 0.7813 ( Dst043 ), respectively. Following sequential Bonferroni correction with a level of signifi cance of 0.05, neither LD nor departure from HWE was detected in any of these loci.

These microsatellite markers were also examined for their cross-transferabil-ity in Acanthopanax gracilistylus W. W. Sm. (Araliaceae) (AG11HN01), Tetrapa-nax papyrifer (Hook.) K. Koch (Araliaceae) (TP11HN01), Cornus controversa Hemsl. (Cornaceae) (CC11HN01), and Dendrobenthamia japonica (Siebold & Zucc.) Hutch . var. chinensis (Osborn) W. P. Fang (Cornaceae) (DJ11HN01). Us-ing the same PCR conditions as for D. stachyanthus , all loci were successfully amplifi ed in A. gracilistylus ; by contrast, only seven, four, and seven loci proved amplifi able in the other three species, respectively ( Table 2 ).

CONCLUSIONS

These microsatellite markers could be employed to investi-gate the population genetics of D. stachyanthus . A good knowl-edge of its genetic diversity and structure is essential to formulate effective conservation strategies for this endangered

tree species. In addition, they may potentially be applicable to other related species.

LITERATURE CITED

HAMRICK , J. L. , AND M. J. W. GODT . 1996 . Conservation genetics of endemic plant species. In J. C. Avise and J. L. Hamrick [eds.], Conservation genetics: Case histories from nature, 281 – 304. Chapman and Hall, New York, New York, USA.

KHAC , D. D. , S. TRAN-VAN , A. M. CAMPOS , J.-Y. LALLEMAND , AND M. FETIZON . 1990 . Ellagic compounds from Diplopanax stachyanthus. Phytochemistry 29 : 251 – 256 .

LIU , K. , AND S. V. MUSE . 2005 . PowerMarker: An integrated analysis envi-ronment for genetic marker analysis. Bioinformatics (Oxford, England) 21 : 2128 – 2129 .

WANG , G.-L. , AND H.-Y. FANG . 1998 . Plant gene engineering: Principles and techniques. Science Press, Beijing, China.

WANG , L. , Z.-P. YOU , Y. XU , AND D.-H. ZHANG . 2010 . Research status and endangered causes of Diplopanax stachyanthus. Bulletin of Botanical Research 30 : 344 – 348 .

WU , S.-G. 1997 . A living fossil plant: Diplopanax stachyanthus Hand.-Mazz. Plants 97 : 23 .

ZANE , L. , L. BARGELLONI , AND T. PATARNELLO . 2002 . Strategies for micro-satellite isolation: A review. Molecular Ecology 11 : 1 – 16 .

ZHU , W. , Q. XIANG , X. GAN , J. CHEN , AND B. XIAO . 1998 . Wood anatomy of Diplopanax stachyanthus Hand.-Mazz. and its systematic implica-tion. Journal of Sichuan Agricultural University 16 : 106 – 109 .

APPENDIX 1. Sampling details of the plant materials used in this study. All voucher specimens of the investigated species are stored in the herbarium of the School of Biological Science and Technology, Central South University (Changsha, China).

Species Sampling localities Latitude ( ° N) Longitude ( ° E) Altitude (m) Voucher ID Collector

Acanthopanax gracilistylus W. W. Sm. Hengshan, Hunan Province, China

27 ° 17 ′ 37 ″ 112 ° 42 ′ 33 ″ 840 AG11HN01 Li Wang

Cornus controversa Hemsl. Hengshan, Hunan Province, China

27 ° 13 ′ 46 ″ 112 ° 38 ′ 27 ″ 720 CC11HN01 Li Wang

Dendrobenthamia japonica (Siebold & Zucc.) Hutch. var. chinensis (Osborn) W. P. Fang

Hengshan, Hunan Province, China

27 ° 16 ′ 36 ″ 112 ° 40 ′ 24 ″ 1060 DJ11HN01 Li Wang

Diplopanax stachyanthus Hand.-Mazz. Jinxiu, Guangxi Province, China

23 ° 57 ′ 37 ″ 110 ° 06 ′ 44 ″ 1902 DS10GX02 Li Wang and Min Wu

Tetrapanax papyrifer (Hook.) K. Koch Hengshan, Hunan Province, China

27 ° 16 ′ 29 ″ 112 ° 12 ′ 46 ″ 435 TP11HN01 Li Wang