Isolation and characterization of microsatellite DNA markers from the Lanyu scops owl (Otus elegans botelensis)

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Molecular Ecology Notes (2003) 3, 595–597 doi: 10.1046/j.1471-8286.2003.00523.x

P R I M E R N O T E

Isolation and characterization of microsatellite DNA

markers from the Lanyu scops owl (

Otus elegans botelensis

)

Y U - C H E N G H S U ,*† L U C I A L . S E V E R I N G H A U S ,‡ Y A O - S U N G L I N * and S H O U - H S I E N L I §

*Laboratory of Biodiversity, Institute of Zoology, National Taiwan University, Taipei 106 Taiwan, ROC; ‡Laboratory of Ornithology, Institute of Zoology, Academia Sinica, Taipei 115 Taiwan, ROC; §Laboratory of Genetic Diversity, Department of Biology, National Taiwan Normal University, Taipei 116 Taiwan, ROC

Abstract

From a genomic library enriched for GATA /CTAT and GAAA /CTTT repeats, 12 polymorphic

microsatellite markers were developed for the Lanyu scops owl (Otus elegans botelensis).

Polymorphism of these loci was evaluated in a sample of 58 adult individuals of unknown relationship. The allele numbers of each locus were from five to 25 and the observed heterozygosity of each locus ranges from 0.707 to 0.914. When using these loci in parentage assignment, the probability of false parent exclusion is greater than 0.999. All loci conformed to Hardy–Weinberg expectations.

Keywords: Otus elegans botelensis, Lanyu scops owl, microsatellites, strigidae, enrichment, genotyping

Received 18 June 2003; revision received 12 August 2003; accepted 12 August 2003

Lanyu scops owl (Otus elegans botelensis) is a subspecies of

elegant scops owl (O. elegans) and was listed as endangered

in the Red Data Book (King 1981). It is found only on Lanyu

Island (22° N, 121°5′-E), a 45.7-km2_{island 60 km southeast}

of Taiwan. A long-term study on the behaviour, ecology and population dynamics of this species has been conducted since 1985 (e.g. Severinghaus 2000; Severinghaus & Rothery 2001) making it one of the few tropical owl species intensively studied. Extra-pair copulations were observed occasionally during the breeding season (Severinghaus, personal observa-tion). To further investigate the population structure and genetic mating system of this species, highly polymorphic genetic markers, such as microsatellites, are needed. In this study, we constructed a microsatellite-enriched genomic DNA library of Lanyu scops owl to screen for highly poly-morphic microsatellite DNA loci.

Genomic DNA from an adult owl was used to construct

a library. After digestion with HaeIII, RsaI and AluI

restric-tion enzymes, the fragments were ligated with SNX linker

(Hamilton et al. 1999). These ligates were first amplified

using polymerase chain reaction (PCR) at 94 °C for 5 min,

30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 70 °C for

2 min then 70 °C 5 min, followed by a solid phase

hybrid-ization with 3′-biotin-labelled (GAAA)₁₀ or (GATA)₁₀

oligo-nucleotides to select for microsatellite-containing DNA fragments. The biotin-labelled DNAs were eluted using Dynalbead streptavidin M280 (Dynal®) according to the manufacturer’s instruction. These microsatellite-enriched

DNAs were PCR-amplified (Li et al. 1997) then ligated into

vectors and transformed to JM109 competent cells, using pGEM®-T Easy vector system II (Promega). Transformed cells were grown on LB agar plates containing ampicil-lin, X-gal and IPTG for blue/white selection of positive

clones at 37 °C for 16 h. A total of 1500 positive clones were

lifted to Hybond-N+_{membranes (Amersham Pharmacia}

Biotech) and hybridized with [r32P] ATP end-labelled

(GAAA)₈ or (GATA)₈ oliginucleotides, then 192 positive

inserts were selected to extract their plasmid DNA, using the Montage Plasmid Miniprep kit (Millipore). These plasmid DNAs were sequenced using DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBACE on MegaBACE 1000 autosequencer (Amersham Bioscience).

Sequences were edited in sequencher software (V4.0.5,

Gene Codes corporation) and 22 primer pairs were designed from 54 sequences containing tandem repeats and suitable

flanking sequences, using fastPCR 2.3.10 program which

is available at http://www.biocentre.helsinki.fi/bi/bare-1_html/download.htm.

Correspondence: Shou-Hsien Li. †Present address: Department of Biology, National Taiwan Normal University, Taipei 116 Taiwan, ROC. Fax: 886-2-29312904; E-mail: t43028@cc.ntnu.edu.tw

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Table 1 Characterization of 12 microsatellite loci in the Lanyu scops owls

Locus Repeat motif Primer sequences (5′–3′)

Size of cloned allele

No. of

alleles Ta (°C) MgCl2 (mM) HO HE P(E)(1) P(E)(2)

GenBank accession no.

Oe3–7 (GATA)12 F-GTGGGTTTATTGCCCCCTCG 121 7 52 2.0 0.776 0.765 0.367 0.545 AY312418

R-CAGATGAATGAATGGATAGATGG

Oe3–21 (GATA)15 F-GATTAGAGACCCGATTCCACA 139 5 52 2.0 0.845 0.741 0.326 0.501 AY312419

R-TTATCTGAGTGGAAGGGTAGTGC

Oe2–57 (GAAA)19 F-TTTATCTTGTATTTTTCATCACTG 197 25 55 1.5 0.914 0.899 0.647 0.785 AY312420

R-CCAACTAAACACTATCTTTCTCC

Oe022 (GATA)12 F′-ATCTGATTCAGTGTCTCCATCCATG 260 6 64 2.5 0.741 0.725 0.579 0.736 AY312421

R′-CCCAGGAATCTCTCCCAATGTT

Oe045 (GATA)13GATTA(GATA)10 F-GTATGTTCTACGTTGGATTTCCA 181 16 58 2.5 0.845 0.830 0.237 0.411 AY312422

R-AAACCTGGCAAGTGCTGTT

Oe050 (GATA)11 F-AGAGTTGTCCTTGGTTGG 136 12 57 2.5 0.862 0.874 0.308 0.484 AY312423

R-TTCTAGTAACCTCATCTGC

Oe053 (GATA)13 F-CTCTGCATCTTAACGCACAGGAC 181 7 64 2.5 0.707 0.645 0.631 0.774 AY312424

R′-CCTCCAAGTGGACAGGAAAAGC

Oe054 (GATA)10 F-TCAGAAAGAAAACTTCAGCAACC 256 20 64 2.5 0.828 0.869 0.332 0.515 AY312425

R-CATATATGTATACACAGGCACATGC

Oe092 (GATA)12GAAGGAAAGATA F-CTCACTGCCTCTGAGCAGAAG 233 14 65 1.5 0.828 0.894 0.507 0.678 AY312426

GAT(GATA)9AATA(GATA)3 R-AGCCAAGGTCAAACCACCAC

Oe128 (GATA)13 F′-CGTTGTAAATGATGAATCGCCTAGTGC 297 8 64 2.5 0.759 0.735 0.585 0.739 AY312427

R′-ATGCATGTATACATACAAACCTGG

Oe142 (GATA)14 F-TGAATCAGCAAACCTGTGCCTG 293 7 65 2.0 0.810 0.808 0.429 0.606 AY312428

R-AGCTAACCTAGAGTCAGCCAGC

Oe171 (GATA)12 F-TTTTACAAACTACTAGTGCATGTCTCC 245 7 64 2.5 0.776 0.782 0.389 0.568 AY312429

R-AGATGTTGTATTTCAGTGTCAG

Overall — — — — — 0.808 0.797 0.999412 0.999994

Ta: annealing temperature; HO: observed heterozygosity; HE: expected heterozygosity; P(E)(1): exclusionary power of false parent when neither parent is unknown; P(E)(2): exclusionary

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P R I M E R N O T E 597

Nineteen of the primer pairs showed a PCR product with expected sizes and 12 of them were selected to screen for polymorphism. The forward primers of these 12 loci were labelled with HEX, FAM or TAMRA fluorescent dyes. For the characterization of polymorphism of these loci, 58 adult individuals with unknown relationship were genotyped. PCR were set up in 10 µL reaction volume

con-taining 30 ng genomic DNA, 0.3 µm each primer, 0.5 mm

dNTP, 10 mm Tris-HCL, pH 9.0, 50 mm KCL, 0.01% (w/V) gelatin, 0.1% Triton X-100, 0.4 U Pro Taq DNA polymerase

(Protech) and 1.5–2.5 mm MgCl₂ (see Table 1). The PCR

condition was as follows: Firstly, denaturation at 94 °C for

3 min, then 34 cycles of 94 °C 30 s, 30 s at the optimal

annealing temperature of each primer pair (Table 1) and 72 °C for 30 s, followed by a final extension at 72 °C for 3 min. All of the PCR in this study were conducted using iCycler thermal cycler (Bio-Rad). The PCR products were electrophoresed in megabace 1000 autosequencer (Amer-sham Biosciences). Sizing of alleles was analysed with the software genetic proflier 2.0 (Amersham Biosciences).

Table 1 summarized the polymorphism of the 12 micro-satellite loci. The observed and expected heterozygosities, probability of false parent exclusion and conformation to Hardy–Weinberg expectations were calculated using pro-gram cervus 2.0 (Marshall et al. 1998). No significant devi-ation from Hardy–Weinberg equilibrium was detected across all loci. Overall, the mean number of alleles per locus was 11.17 and the mean expected heterozygosity was 0.797. When applying these loci to paternity analysis, more

than 99.9% of false putative parents can be excluded. The highly polymorphic nature of these microsatellite loci makes it an ideal genetic marker system to study the population genetics and mating system of the Lanyu scops owl.

Acknowledgements

This work was funded by the National Science Council to LLS (NSC 90–2313-B-001–036) and by the Council of Agriculture to SHL [COA 90AT-1.3.3-F-R1(5)]. We thank Dr Y. J. Huang and members of Genetic Diversity Laboratory for the help in lab work and all assistants of Ornithology Laboratory for collecting blood samples.

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