Conservation Genet Resour (2013) 5:417–420 DOI 10.1007/s12686-012-9817-0

TECHNICAL NOTE

Development of 24 new microsatellite markers in the Crested Serpent ( cheela hoya)

Hsin-Hsin Hsu • Shih-Torng Ding • Yi-Ying Chang • Ming-Chieh Chao • Hsien-Shao Tsao • Fang-Tse Chan • Chi-Chen Hsu • Hsiao-Wei Yuan • Pei-Hwa Wang

Received: 10 October 2012 / Accepted: 3 November 2012 / Published online: 18 November 2012 Ó Springer Science+Business Media Dordrecht 2012

Abstract Twenty-four microsatellite markers were Keywords (Spilornis cheela) developed for the Crested Serpent Eagle (Spilornis cheela Genetic structure Selective hybridization hoya) composing a new marker set. The markers were Microsatellite markers tested on 61 Crested Serpent Eagle individuals. The num- ber alleles ranged from 2 to 8 per locus (average = 3.8), and the effective number of alleles ranged from 1.13 to The Crested Serpent Eagle (Spilornis cheela), also known 6.07 (average = 2.34). In the new marker set, there were as eagle, and serpent eagle, is a raptor dis- 11 markers with high polymorphism (PIC [ 0.5), and the tributed mostly over the Indian subcontinent and the South average HE and HO over all loci was 0.50 and 0.49, , Taiwan, and part of the Southeast (between respectively. The results showed that the new marker set latitude 35°N and 9°S). According to morphological vari- was highly polymorphic as an individual genetic marker ation and habitat, the basic taxon generally can be divided compared to the existing cross- markers previously into 13 (Del Hoyo et al. 1992; Ferguson-Lees used for population genetic structure monitoring of the and Christie 2001). The subspecies inhibited in Taiwan is Taiwan Crested Serpent Eagle. In conclusion, the micro- Spilornis cheela hoya. Although being assigned to the satellite marker set developed can be applied as a molec- Least Concern category in IUCN Red List (IUCN 2009), ular tool to investigate the genetic structure or phylogeny the Crested Serpent Eagle is classified as critically endan- of the Crested Serpent Eagle. gered species in the Japanese Red Data Book (Ministry of the Environment 2010). It is a rare and valuable protected raptor in Taiwan, because of natural habitat destruction, H.-H. Hsu and S.-T. Ding contributed equally to this paper. and poaching. Therefore, development of a reliable and highly polymorphic microsatellite marker set for analyzing H.-H. Hsu S.-T. Ding Y.-Y. Chang C.-C. Hsu P.-H. Wang (&) their genetic structure is important for the intended con- Department of Science and Technology, National servation work. Taiwan University, No. 50, Lane155, Sec. 3, Keelung Rd., Microsatellite markers have been frequently used to Taipei 10673, Taiwan study the genetic structure of many raptors, such as the e-mail: [email protected] Madagascar sea eagle (Haliaeetus vociferoides) (Johnson M.-C. Chao H.-S. Tsao et al. 2009), the Harpy eagle (Harpia harpyja) (Banhos Taipei Zoo, No. 30, Sec. 2, Xinguang Rd., Taipei 11656, Taiwan et al. 2008), and the Northern goshawk (Accipiter gentilis) (Takaki et al. 2009). The cross-species microsatellite F.-T. Chan Endemic Species Research Institute, Council of Agriculture, markers selected from a related family (), No. 1, Ming-shen East Rd., Chichi Township, White-tailed Sea Eagle (Haliaeetus albicilla) (Hailer et al. Nantou County 552, Taiwan 2005) and Spanish imperial eagle (Aquila adalberti) (Martinez-Cruz et al. 2002), were tested in the Crested H.-W. Yuan School of Forestry and Resource Conservation, National Taiwan Serpent Eagle. However, the genotyping results suggested University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan that the information may be insufficient to understand the 123 418 Conservation Genet Resour (2013) 5:417–420

Table 1 Characterization of 24 developed microsatellite loci in 61 Crested Serpent

0 0 Locus Primer sequences F/R (5 ? 3 ) Repeat motif Allele size (bp) Na Ne HE HO PIC

CSE74 F: TCCAAAATATATACCCAAGCCTCT (AC)6 211–213 2 1.99 0.50 0.57 0.37 R: GCTTGCCAGTTAGGTCCTTG

CSE76 F: TTCATCCCATTTCCCATTGT (AC)7 140–146 3 1.76 0.44 0.41 0.36 R: GCTTGTTTGAAAGTTGCTGCT

CSE137 F: TCAACTTTCACAGGCCACTAA (AC)15 294–302 4 3.22 0.70 0.72 0.63 R: TGGGTCCAGAAGTTCACATAAA

CSE354 F: ATTATGTTTGAAGGCAGTTTT (AC)15 395–399 3 1.31 0.24 0.27 0.21 R: CTCAAGAAAACTGTTCTTTG

CSE560 F: TCCCAAACTTTCTGGTGCAT (AGAT)7 145–155 5 2.95 0.67 0.62 0.62 R: TCTTGCCAAAAGACAAGGTG

CSE782* F: AAAAGCCCCCTCTGCTTAAT (AC)13 146–148 2 1.26 0.20 0.13 0.18 R: TCTGAAACAGTTTTCACCTCCA

CSE896 F: TGGGCTCATAAAGGCAGTTC (ATCT)6 263–291 5 2.22 0.55 0.52 0.51 R: TAACCCATGCGCTGGTTAAT

CSE898 F: CGGTTGTCACCACAACCATA (CA)9 193–207 4 2.15 0.54 0.54 0.49 R: CTCATCCCCATTGTCAGCTT

CSE942 F: TCTTTCTTACTTCCCAGAGTTGGT (AC)13 240–251 4 3.77 0.74 0.84 0.69 R: TCATCATCAAACGCATCAGA

CSE1030* F: GTGTAAGTGGGCTCCGTGAT (TA)4(CA)8(TA)4 241–267 6 2.82 0.65 0.46 0.58 R: TGAAAAGGTGGCCTGATTTC

CSE1090 F: CTTGCTGACTGGGTGAAACA (CA)14 336–362 7 2.83 0.65 0.66 0.62 R: CGGGAGTCTTCTCAGTGACC

CSE1155 F: CTTCCACCTAGGTTAATTGTAATGTCT (AC)5ATGC(AC)8 272–298 3 1.72 0.44 0.43 0.37 R: CCAAGGTACAAAGATTGAAAGGA

CSE1157 F: TTGTGCTTTTCTCTGCTTGG (CA)11 328–338 5 3.58 0.73 0.79 0.67 R: AGCTTCCTGTGTGGGTAGAAA

CSE1160 F: TCCTGCTGCATGCTCATTAC (CA)10 237–239 2 1.13 0.11 0.12 0.11 R: AACAAGGAAGGTTGATTTGACTG

CSE1349 F: CCCTGTTTTGCCTTACCCTA (AC)12 284–306 8 6.07 0.84 0.74 0.81 R: GCAAGCACATATCAATCACCA

CSE1371 F: TCTCCTGTTACTATTTCTCTTCTGC (AT)8(AC)6(AT)7 297–305 4 1.57 0.36 0.33 0.34 R: GCAGATACAGCCTGCAAAAA

CSE1447 F: CAAGCAGTTAGGACAACCATCT (CA)9TAC(AT)4 283–291 3 1.29 0.24 0.24 0.21 R: GACTGCAGCCAAAACTTTCC

CSE1449 F: GAGTTGTCCCTTAAAACATCTCC (CA)14 376–382 2 1.48 0.33 0.34 0.27 R: TTGGCTTCCTTCTGGTCTGT

CSE1453 F: GTCCTCTCGTCTTTCCCTGA (CA)11(AT)8 198–206 4 2.79 0.65 0.60 0.57 R: TTCTTGTTCAAGGAGGCACA

CSE1602* F: AACATCACTCAGCACATTATCAAA (AT)5(AC)9(AT)4 192–198 4 2.28 0.57 0.44 0.51 R: TTGTGAAGCCATCTTGAAACA

CSE1685* F: CCTCTAGTCTCCCCAACCAA (AC)11 178–188 5 3.07 0.68 0.69 0.61 R: GAAGCATGCCGTCTAGAAAAA

CSE1712 F: ACCATCAAGCAGATGTGCAA (AC)10 292–298 2 1.81 0.45 0.53 0.35 R: CATGCAGGACAGGAAAACAA

CSE1780 F: CCCATGTCCCTCAAGCTATT (ATCT)6 253–257 2 1.33 0.25 0.25 0.22 R: CCCTTTCATTCTTTTCCCTCA

CSE1805 F: ACAGCGATGCAGTTTTTCAA (CA)2GA(CA)8 171–181 3 1.69 0.42 0.46 0.34 R: TGCTCTGCAGGTGAAGTAGG

123 Conservation Genet Resour (2013) 5:417–420 419

Table 1 continued

0 0 Locus Primer sequences F/R (5 ? 3 ) Repeat motif Allele size (bp) Na Ne HE HO PIC

Mean 3.8 2.34 0.50 0.49 0.44

Na Number of alleles per locus, Ne number of effective alleles, HE expected heterozygosity, HO observed heterozygosity, PIC polymorphism information content * Exact test of Hardy–Weinberg equilibrium showed significant heterozygote deficits in loci (P \ 0.05) genetic structure of the Crested Serpent Eagle due to a low MSATCOMMANDER (Faircloth 2008) in 148 sequences amount of polymorphism observed in the microsatellite which had more than 10 dinucleotide or 6 tetranucleotide markers of cross-species (unpublished data). It became repeats. The primer sets for 46 microsatellite loci were then imperative that species-specific microsatellite markers are designed base upon the sequencing results using Primer 3 required for studying the genetic structure of the Crested (Rozen and Skaletsky 2000). A CAG tag (50-CAG- Serpent Eagle. TCGGGCGTCATCA-30) was added at the 50 end of each Samples were obtained from (1 male and 1 female) forward primer. kept in the Taipei Zoo (Taipei, Taiwan) and Endemic Twenty-four of the tested microsatellite markers were Species Research Institute in Taiwan (Nantao, Taiwan). isolated based on allele numbers (n32). The new micro- Blood samples were drawn from the brachial vein into a satellite marker set was used to genotype life birds in the Ò BD Vacutainer with K3EDTA (BD, Franklin Lakes, Taipei Zoo (n = 11), the Endemic Species Research USA). Genomic DNA was extracted and purified from Institute (n = 42), as well as the specimen from National blood samples of two individuals by a standard phenol– Taiwan Museum (n = 8). The characteristics and geno- chloroform protocol (Sambrook et al. 1989). Microsatellite typing results using the 24 new microsatellite markers were markers were developed using a selective hybridization calculated and analyzed using ARLEQUIN 3.0 (Excoffier method described previously by Glenn and Schable (2005). et al. 2005) for heterozygosities (Ho, HE, and PIC), and the The pooled DNA was digested with RsaI and XmnI number of alleles (Na); POPGENE version 1.32 (Yeh et al. (New England Biolabs, UK) at 60 °C for 4 h, and digested 1997) for the number of effective alleles (Ne); and fragments were ligated to SuperSNX linkers (Chang et al. GENEPOP 4.0.10 (Raymond and Rousset 1995) for

2012). Biotinylated (CA)12 and (AGAT)8 oligonucleotides Hardy–Weinberg equilibrium test. The statistical results probes (Invitrogen Taiwan Ltd., Taiwan) were used in were summarized in Table 1. an enrichment hybridization, and the repeat sequences The newly developed microsatellite marker set obtained were selectively captured by streptavidin-labeled magnetic in this study was more polymorphic than existing cross- beads (Dynabeads Streptavidin M-280, Prod. No. 112.05). species markers, and it also had better performance in The microsatellite enrichment fragments eluted from beads characterizing individual Crested Serpent Eagles. This were amplified with PCR using the SuperSNX24 linker as a marker set can be used to investigate the genetic structure primer. The PCR were performed in a final volume of of populations of Spilornis species and the Crested Serpent

25 lL, containing 1 9 PCR buffer (1.5 mM MgCl2), Eagle (Spilornis cheela hoya) subspecies, and subse- 0.5 lM SuperSNX24 forward primer, 0.15 mM dNTP, quently improving their breeding and conservation plan- 0.03 U/lL Taq DNA polymerase (Takara BIO Inc., Japan), ning. Furthermore, it also has the potential for studying and 2 lL DNA fragments. The thermocycling condition phylogenetics in related raptors. was same as described previously (Chang et al. 2012). The amplified enriched DNA was ligated into pGEM-TÒ Easy Acknowledgments We thank Taipei Zoo, Endemic Species Vector (Promega, Madison, USA) and transformed into Research Institute and National Taiwan Museum for supplying Crested Serpent Eagle samples. Our gratitude also goes to Dr. Harry HIT DH5a competent cells (United Bioinformatica, Can- Mersmann for his comments and English editing on the manuscript, ada). Plasmids from 384 clones from a total 1,920 positive and to Hung-Wen Lin and Yi-Hui Wang for technical assistance. This colonies were extracted for sequencing. Additional 1,536 work was supported in part by grants from Taipei Zoo (Grant No. colonies were screened by dot-blotting and hybridized 98-AR-6 and 99-AR-9). to the probes (CA)12 and (AGAT)8 radiolabelled with P32-cATP. The positive colonies were rescreened and reconfirmed through sequencing. Vector and linker References sequences were identified and removed using Phred soft- ware (Ewing and Green 1998; Ewing et al. 1998), and Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410 the sequence duplicates were removed using BLAST Banhos A, Hrbek T, Gravena W, Sanaiotti T, Farias I (2008) Genomic (Altschul et al. 1990). Repeat motifs were detected with resources for the conservation and management of the Harpy

123 420 Conservation Genet Resour (2013) 5:417–420

eagle (Harpia harpyja, falconiformes, accipitridae). Genet Mol Johnson J, Tingay RE, Culver M, Hailer FM, Clarke L, Mindell DP Biol 31:146–154 (2009) Longterm survival despite low genetic diversity in the Chang YY, Chao MC, Ding ST, Lin E-C, Tsao HS, Yuan HW, Wang critically endangered Madagascar fisheagle. Mol Ecol 18:54–63 PH (2012) Development of microsatellite markers in an ungulate Martı´nez-Cruz B, David VA, Godoy JA, Negro JJ, O’Brien SJ, mammal, the Formosan serow (Capricornis swinhoei). Conserv Johnson WE (2002) Eighteen polymorphic microsatellite mark- Genet Resour 4:755–757 ers for the highly endangered Spanish imperial eagle (Aquila Del Hoyo J, Elliott A, Sargatal J, Cabot J (1992) Handbook of the adalberti) and related species. Mol Ecol Notes 2:323–326 birds of the world, Vol. 2. Lynx Edicions, Barcelona, pp 52–205 Ministry of the Environment (2010) Ministry of the Environment, Ewing B, Green P (1998) Base-calling of automated sequence traces government of Japan, http://www.env.go.jp/press/press.php? using phred. II. Error probabilityies. Genome Res 8:186–194 serial=7849 Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of Raymond M, Rousset F (1995) GENEPOP Version 1.2: population automated sequence traces using phred. I. Accuracy assessment. genetics software for exact tests and ecumenicism. J Hered Genome Res 8:175–185 86:248–249 Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: an Rozen S, Skaletsky HJ (2000) Primer 3 on the WWW for general integrated software package for population genetics data anal- users and for biologist programmers. In: Krawetz SA, Misener S ysis. Evol Bioinform Online 1:47–50 (eds) Bioinformatics methods and protocols: methods in molec- Faircloth BC (2008) MSATCOMMANDER: detection of microsat- ular biology. Humana Press, Totowa, pp 365–386 ellite repeat and automated, locus-specific primer design. Mol Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a Ecol Resour 8:92–94 laboratory manual. Cold Spring Harbor Laboratory Press, Cold Ferguson-Lees J, Christie DA (2001) Raptors of the world. Houghton Spring Harbor Mifflin Harcourt, Boston, pp 457–460 Takaki Y, Kawahara T, Kitamura H, Endo K, Kudo T (2009) Genetic Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci. diversity and genetic structure of Northern goshawk (Accipiter Meth Enzymol 395:202–222 gentilis) populations in eastern Japan and central asia. Conserv Hailer F, Gautschi B, Helander B (2005) Development and multiplex Genet 10:269–279 PCR amplification of novel microsatellite markers in the Yeh FC, Yang RC, Boyle TBJ, Ye ZH, Mao JX (1997) POPGENE, Whitetailed Sea Eagle, Haliaeetus albicilla (Aves: Falconifor- the user-friendly shareware for population genetic analysis. mes, Accipitridae). Mol Ecol Notes 5:938–940 Molecular biology and biotechnology centre. University of IUCN (2009) IUCN red list categories and criteria. IUCN Species Alberta, Canada Survival Commission. IUCN, Gland

123