ГЕНЕТИКА, 2012, том 48, № 10, с. 1237-1240
КРАТКИЕ СООБЩЕНИЯ
УДК 575.174.5
ISOLATION AND CHARACTERIZATION OF MICROSATELLITE MARKERS IN CHINESE HERB OF Dendrobium loddigesii
© 2012 G. Ding1, D. Zhang2, X. Ding3
1 Chemical and Biological Engineering College, Yancheng Institute of Technology, Yancheng 224003, China
e-mail: dingzyc@yahoo.com.cn
2 Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Normal
University, Yancheng 224003, China
3 Jiangsu Provincial Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China Received January 10, 2012
As an invaluable herb, Dendrobium loddigesii is widely used in Chinese medicinal field. In order to develop a convenient and efficient identification method and investigate the genetic diversity and structure of this species, twelve microsatellite loci were isolated from two microsatellite-enriched libraries. Twenty-six individuals from Baise population were analysed. The observed and expected heterozygosities ranged from 0.174 to 0.617 and from 0.182 to 0.608, respectively. These microsatellite loci characterized from D. loddigesii will contribute to research on the individual authentication, population structure, genetic diversity and conservation of this species and its similar species.
In China, "Herba Dendrobii Shihu" is an important traditional Chinese medicine and it has commonly been used as a tonic in many Asian countries for centuries [1]. "Herba Dendrobii Shihu" was divided into two groups as "Huangcao Shihu" and "Fengdou Shihu" according to their different morphological characters and processing methods [2]. Dendrobium loddigesii is one of the five species listed in the Chinese Pharmacopoeia and it is suitable for processing into "Fengdou Shihu" [3]. It benefits human health in many ways, such as nourishing yin and clearing away unhealthy heat, benefiting the stomach, promoting the production of body fluid, resisting cancer and prolonging life [3].
In recent years, adulterants made up of other stems of Dendrobium are sold in markets and used clinically as the stems of D. loddigesii because of similar appearance and tissue structure [4, 5]. The general approaches to herbal identification, such as morphological, anatomic and chemical analyses, are often affected by environmental and developmental factors during plant growth [6—8].
Natural populations of D. loddigesii have been destroyed for human overexploitation enticed by high profits of"Fengdou". Knowledge of the levels and distribution of genetic variation is essential to understand the population evolution and help present effective conservation strategies [9, 10]. Simple sequence re-
peats have been widely used as DNA markers in population genetic studies because of their high level of polymorphism [11]. Microsatellite markers can also be used to authenticate D. loddigesii from its adulterant Dendrobium species.
Twenty-six individuals of D. loddigesii were collected from Baise population in Guangxi. They were all with the same morphological type. (CA)n and (GA)n-enriched libraries were constructed. Genomic DNA was extracted with the QIAGEN DNeasy plant mini kit (Qiagen) according to the manufacturer's protocol. The genomic DNA was digested with Sau3AI (TaKaRa). The 200—800 bp DNA fragments were extracted and purified using the QIAquick gel extraction kit (QIAGEN). Purified DNA fragments were ligated with a double-strand linker, and then polymerase chain reaction (PCR) was performed using one strand of the linker as the primer. Two biotinylated probes (GA)15 and (CA)15 were hybridized to the PCR products. Afterwards, the DNA fragments hybridized to bi-otinylated probes were isolated using streptavidin-coated magnetic beads (Promega).
The selected fragments were amplified by poly-merase chain reaction (PCR) and the enriched microsatellite fragments were ligated into pMD18-T vector (TaKaRa) and transformed into competent Escherichia coli DH5a cells. With these, the microsatellite-enriched genomic library was constructed. As a result,
Characteristics of twelve polymorphic microsatellite loci in Dendrobium loddigesii
Locus Primer sequence (5'—3') Repeat motif Ta, °C Size, bp Na Ho He GenBank Accession no.
FH-01 F: GAACCTCTTGAGGGAGGTCTTG R: CCCTTGTGAATCCCCTCACAAA (AG)g 55 125- -137 4 0.423 0.385 JN986794
FH-02 F: GCATTTGGAGATAGATAGGG R: TCTGCTTACATGTCTGGCAG (GT)3GC(GT)8(GA)15 51 167- -181 4 0.302 0.311 JN986795
FH-03 F: CTTCACTAGTTCCATGATAA R: GAAGCTTGATTGTTCTGATC (GT)30-(GT)10 48 293- -313 10 0.564 0.597 JN986796
FH-04 F: CGAACACATATGGATCACAG R: TCTCACATACACCAAGAGTC (GT)3AT(GT)4TG(GT)5 51 173- -181 3 0.225 0.258 JN986797
FH-05 F: CCGACTAAAGGAAAGATTCATC R: CGACTGTCATCGTCTCAAAGCT (TC)23(TG)29 51 198- -226 11 0.617 0.608 JN986798
FH-06 F: AGGTCCGAAAGGTGGTACAC R: GCCAAGTGCGCTCGACGAAG (TG)5A(GT)10 56.5 194- -202 6 0.242 0.261 JN986799
FH-07 F: ACTTCTTTGAGGGAGCTTGGTG R: AAACTCCCCCTCACAAGACTCC (GA)3TT(GA)9 55 103- -113 7 0.274 0.306 JN986800
FH-08 F: GAGAAACAACATGCAGACTAACC R: AAGCTCTCTCTCAGTACAACCC (CT)9 54 120- -132 5 0.239 0.275 JN986801
FH-09 F: GAGAGTCTTCTTAGAACCTCTTG R: TCACAAACTCCCCCTCATAAGAC (GA)7 54 123- -137 3 0.251 0.267 JN986802
FH-10 F: GATTACACCAATACAAGACACC R: GTAAAGAATTTGGCTCCTTGG (AC)g 50 107- -115 4 0.406 0.424 JN986803
FH-11 F: GAGATTATACGGTATGATCAG R: GGTCCAGATTAGGGTTGAGTCT (TG)n 50 182- -190 5 0.174 0.182 JN986804
FH-12 F: TGGCCGTTGGCATAGGGTTA R: CATGCATAAAACATAAATAGG (TG)3TT(TG)6(TTTG)3 51 206- -224 4 0.418 0.432 JN986805
Notes: Ta, annealing temperature; Na, number of alleles; Hq, observed heterozygosity; i/g, expected heterozygosity.
ISOLATION AND CHARACTERIZATION OF MICROSATELLITE MARKERS
1239
80 positive clones were selected to be sequenced using the M13 universal primers by ABI 3730XL DNA Analyzer with BigDye Terminator Cycle Sequencing Ready Reaction Kit version 3.1(Applied Biosystems).
GenePop version 3.4 [12] was used to calculate Hardy—Weinberg equilibrium (HWE) and linkage disequilibrium (LD) in the polymorphic loci. The polymorphism analysis including allele diversity, observed (HO) and expected (HE) heterozygosity were calculated using Arlequin version 3.1 [13].
Thirty-six sequences (45%) including microsatellite repeat motifs were obtained and twenty primer pairs were designed from the flanking sequences using oligo 5.0 software. PCRs were carried out in 10-|l volume with the following components: 10 ng of template DNA, 0.4 |M of each primer, 0.2 mM dNTP (TaKaRa), 0.5 U TaqDNA polymerase (TaKaRa), 10x PCR buffer. The conditions for amplifications were 96°C for 3 min, followed by 30—35 cycles at 95°C for 30 s, annealing temperature (detailed in table) for 30 s, 72°C for 30 s, and then a final extension at 72°C for 5 min. PCR products were separated on a 6% polyacry-lamide gel and visualized by silver-staining. Product sizes were estimated by comparison with a 25-bp DNA ladder (Promega).
Twelve highly polymorphic loci were successfully amplified with twenty-six individuals from Baise population. We observed that the twelve microsatellite loci showed polymorphic with three to 11 alleles per locus, with a mean number of 5.5 (table). Observed heterozygosity of the 12 loci ranged from 0.174 to 0.617 with a mean value of 0.345, while expected heterozygosity ranged from 0.182 to 0.608 with a mean value of 0.359 as calculated according to Nei [14] (table). No significant (P < 0.05) departures from HWE expectations were observed. These data showed that the 12 markers have a potential to be utilized in the population level.
These results suggest that the twelve microsatellite markers here developed will be useful for the study of individual authentication, population structure, genetic diversity and conservation of D. loddigesii and its similar species.
ACKNOWLEDGMENTS
This work was supported by the grant from National Science Foundation of China (No. 31000142), Natural Science Foundation of Jiangsu Province (No. BK2011421) and the Foundation of Yancheng Insititute of Technology (XKR2010003).
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