Conservation Genet Resour (2014) 6:857–859 DOI 10.1007/s12686-014-0224-6

MICROSATELLITE LETTERS

Isolation and characterization of 19 polymorphic microsatellite loci for Atalantia buxifolia (), a traditional medicinal

Miaomiao Shi • Xiaoming Guo • Yizhang Chen • Lianxuan Zhou • Dianxiang Zhang

Received: 9 January 2014 / Accepted: 13 January 2014 / Published online: 23 May 2014 Springer Science+Business Media Dordrecht 2014

Abstract To evaluate genetic diversity and structure of a considered as a close relative of L. (Bayer et al. traditional medicinal plant Atalantia buxifolia, 19 poly- 2009), thus it is valued as rootstocks for grafting com- morphic microsatellite loci were developed and amplified mercially grown Citrus fruit trees. However, due to fre- in four multiplex and two individual reactions. The char- quent human activities, such as over-exploitation and acteristics of these markers were determined in 64 indi- habitat destruction, the populations of A. buxifolia are viduals from four sites in South China. Per locus 4–21 being destroyed. Assessment of genetic diversity by alleles were detected. Observed and expected heterozy- molecular markers is important for efficient management gosities ranged from 0.103 to 0.921 and from 0.483 to and conservation of plant genetic resources. Here, we 0.934, respectively. These markers are useful tools for report the isolation and characterization of the first set of future population genetic studies and conservation of polymorphic microsatellite markers for A. buxifolia and genetic resources of A. buxifolia. expect them to provide a useful tool for population genetic studies. Keywords Atalantia buxifolia Microsatellites Genetic Genomic DNA was extracted from silica gel-dried leaf diversity Medicinal plant Conservation tissue of one individual of A. buxifolia following the ce- tyltrimethyl ammonium bromide (CTAB) method (Doyle Atalantia buxifolia (Poir) Oliv. (Rutaceae) is a spinous 1991). Microsatellite loci were isolated using the Fast shrub, bearing white fragrant flowers and small globose Isolation by AFLP of Sequences COntaining repeats fruits. It is commonly distributed in forests or thickets near (FIASCO) protocol from an enriched (TG)n (Zane et al. ocean in provinces of South China, as well as in Malaysia, 2002). Positive clones were sequenced and primer pairs Philippines and Vietnam (Zhang et al. 2008). The species is were designed using Primer3web version 4.0.0 (http://pri used as a folk medicine for the treatment of malaria, mer3.ut.ee/). After testing on agarose gel and polyacryl- chronic rheumatism, paralysis, and snakebites, as the amide gel, 19 loci could be successfully amplified and extracts from its branch, root or leaves possess various showed some degree of polymorphism. To characterize the bioactivities such as antifeedant, cytotoxic and antibacte- genetic variability of A. buxifolia at the 19 loci, we geno- rial activities (Yang et al. 2012). In addition, A. buxifolia is typed 64 individuals from four sites: Sanya (SY) and Haikou (HK) in Hainan Province, Hebao Island (HB) and Qi’ao Island (QA). Multiplex PCRs were performed in a M. Shi X. Guo Y. Chen L. Zhou D. Zhang (&) total volume of 10 ll, which included 5 ll Master Mix Key Laboratory of Plant Resources Conservation and (Zhuangmeng, Beijing), 0.2 lM of each primer pair and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Xingke Rd. 723, Guangzhou 510650, approximately 10 ng of genomic DNA. All loci were China grouped into four multiplex PCR sets, except that single e-mail: [email protected] PCR was done for Abu48 and Abu119 separately (Table 1). Thermocycler protocol was one cycle of 94 C X. Guo Y. Chen University of Chinese Academy of Sciences, for 4 min, followed by 35 cycles of 30 s at 94 C, 30 s at Beijing 10049, China 58 C (except for Abu48 and Abu119, for which the 123 858 Conservation Genet Resour (2014) 6:857–859

Table 1 Characteristics of 19 microsatellite loci in Atalantia buxifolia

0 0 Locus Primer sequences (5 –3 ) Motif Size (bp) Multiplex NA Ho He

Abu4 F: \FAM[ TGTGTGTGTTGTGGTGTTGG (GA)19 81–93 1 8 0.302 0.641 R: ATAGTTCAGCCGCCCAGAAT Abu7 F: \TAMRA[ ATGATGCGTTAGGGATGGAA (TC)19 120–156 4 13 0.333 0.792 R: TGCAACTTGGAAGCTGAATC Abu16 F: \TAMRA[ TGAAAAATGACAAAGCAAGCA (CT)10 169–173 2 4 0.103 0.612 R: CTTGACTTCGGTCTCTGCAA Abu17 F: \ROX[ TGGGTTCAATAAAGATTACAAGCA (GT)22 187–195 4 12 0.613 0.800 R: CCTCTCTCATCCAGCTCCAG Abu29 F: \FAM[ TCAGACTCAAAGGCCAAGTG (CT)19 192–236 4 21 0.885 0.934 R: GAGGTGTTGGGGATTTCAGA Abu32 F: \FAM[ TGCAGACATGAGATGGCTTT (TC)16 137–163 2 12 0.651 0.871 R: CACAAAGAAGGCGTGTGAGA Abu34 F: \HEX[ ATGCAAGCGTGACAAACAC (AC)12…(AG)13 162–176 3 8 0.597 0.809 R: TATTTTGCGACATCGGATCA Abu37 F: \TAMRA[ TCATTGTCTTTCCGCTCAAAT (TG)12…(AT)8 234–254 3 15 0.371 0.877 R: ACGTGGAAGCTGTTGATTGA Abu40 F: \FAM[ TCTTCTTCCAAACCCTTGCT (CT)14 227–253 3 10 0.236 0.778 R: AGTCATGGAAGCCAAAATGG Abu43 F: \FAM[ TGTCTCTTGCAAGTGCTCGT (AG)13 229–239 2 8 0.359 0.668 R: AACTTTTGGTCCTCCCGATT Abu48 F: \HEX[ TGTGAGGACTGCCACACAGT (AG)19 221–259 3* 16 0.689 0.872 R: CGGCATAATGTGTCCCTATG Abu63 F: \ROX[ AGGGGGTTGTTGTGTGGAGT (GA)20 129–135 1 11 0.450 0.522 R: CTTTGGTTTCAGCGCTTCTC Abu81 F: \HEX[ TCCATCTTCTCTCCAGAAGTGTT (AC)11 150–156 2 6 0.422 0.633 R: TTTAGGCAAAGGCAAGTTGG Abu94 F: \ROX[ TCAGACTCAAAGGCCAAGTG (CT)19 283–325 2 21 0.921 0.927 R: CTTACCGACTCCATCCGAAA Abu97 F: \HEX[ CCGGTAACGTCTGGTTCTGT (TC)11 102–126 1 12 0.700 0.865 R: GCAAAGCCACACAAAGAAGG Abu99 F: \FAM[ GCCGCCAGAAGAGAAGCTAT (TG)22 126–148 3 8 0.557 0.803 R: ATGCTGCCTTCATCATCTCA Abu112 F: \ROX[ AACTGCCCCAAACTGTGATG (TC)11…(CA)6 178–206 3 8 0.492 0.712 R: GGGTGAGGTGGATTGATCTG Abu119 F: \ROX[ TGAGTGAACACGTTGGGAGA (GA)19 140–154 2* 6 0.450 0.483 R: AACAAGCCCCCTGAATTTTT Abu179 F: \FAM[ CCTTACCTCCCTTCGGACTC (CTT)7 168–200 1 6 0.333 0.531 R: TAGCCGCATATGGCATAACA Each forward primer was 50 fluorescently labelled with HEX, FAM, ROX or TAMRA. Multiplex indicates multiplex PCR reaction sets: loci with the same number in this column were amplified with annealing temperature at 58 C and genotyped together, except Abu48 and Abu119 which were individually amplified at 52 C, and then genotyped together with others (*). Locus in bold indicates significant deviation from Hardy– Weinberg equilibrium (P \ 0.05). All the sequences were deposited in GenBank under the accession number KF984527–KF984545

NA number of alleles, Ho observed heterozygosity, He expected heterozygosity annealing temperature was 52 C) and 30 s at 72 C, with a Analyser (Invitrogen) using an internal size standard final extension of 10 min at 72 C. Single PCR products GeneScanTM 500 LIZ. Allele binning and calling were for Abu48 and Abu119 were mixed with the products of done using GeneMarker version 2.4.0 (SoftGenetics LLC, multiplex PCR set 3 and set 2, respectively. Then, PCR State College, Pennsylvania, USA). The number of alleles products were scanned by an ABI PRISM 3100 Genetic (NA), observed (Ho) and expected (He) heterozygosities, as

123 Conservation Genet Resour (2014) 6:857–859 859 well as deviations from Hardy–Weinberg equilibrium were the foundation of Key Laboratory of Plant Resources Conservation estimated using the software CERVUS version 3.0.3. and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences. The characteristics of the loci are shown in Table 1. The number of alleles per locus ranged from 4 to 21 with a mean of 10.8. Ho ranged from 0.103 to 0.921, while He References ranged from 0.483 to 0.934. No significant deviation from Hardy–Weinberg equilibrium was detected for 12 of 19 Bayer RJ, Mabberley DJ, Morton C, Miller CH, Sharma IK, Pfeil BE, loci. Abu4, Abu7, Abu16, Abu40, Abu43, Abu48 and Rich S, Hitchcock R, Sykes S (2009) A molecular phylogeny of Abu179 showed the signals of deviations (P \ 0.05), the orange subfamily (Rutaceae: ) using nine cpDNA sequences. Am J Bot 96:668–685 probably due to the excess of homozygotes. Doyle JJ (1991) DNA protocols for . In: Hewitt GM, Johnston These results confirm that the microsatellite markers A (eds) Molecular techniques in taxonomy. Springer, Berlin, reported here are suitable for population genetic studies of pp 283–293 A. buxifolia. They could be used in future investigation of Yang T, Mei WL, Zeng YB, Zuo WJ, Dong WH, Dai HF (2012) Cytotoxic components from the roots of Atalantia buxifolia spatial genetic structure in general, comparative levels of (Poir.) Oliv. J Trop Subtrop Bot 20:407–412 genetic variation in continental versus insular populations Zane L, Bargelloni L, Patarnello T (2002) Strategies for microsatellite in particular. Furthermore, we expect that these markers isolation: a review. Mol Ecol 11:1–116 will facilitate the efficient conservation and management of Zhang DX, Hartley TG, Mabberley DJ (2008) Rutaceae. In: Wu ZY, Raven PH, Hong DY (eds) Flora of China, vol 11. Science Press, germplasms of this medicinal plant. Beijing, pp 51–98

Acknowledgments This work was supported by Ministry of Sci- ence and Technology of China (Grant Number: 2013FY111200) and

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