臺灣地區巢鼠的微隨體基因座

(1)

Taiwania, 54(2): 118-121, 2009

118

Polymorphic Microsatellite Markers for the Harvest Mouse (Micromys

minutus) in Taiwan

Show-Yueh Jiang(1) and Y. Kirk Lin(1,2*)

1. Institute of Ecology and Evolutionary Biology, National Taiwan University, No. 1, Sect. 4, Roosevelt Rd., Taipei 10617, Taiwan. 2. Department of Life Sciences, National Taiwan University, No. 1, Sect. 4, Roosevelt Rd., Taipei 10176, Taiwan.

* Corresponding author. Tel: +886-2-33664534; Email: kirklin@ntu.edu.tw

(Manuscript received 24 January 2009; accepted 9 April 2009)

ABSTRACT: Seven polymorphic microsatellite loci are described for the harvest mouse, Micromys minutus in Taiwan. For a panel of 38 individuals with unknown relationship, the numbers of alleles per locus ranged from 3 to 17. The observed and expected heterozygosities averaged 0.433 and 0.656, respectively. These seven markers should offer potentials to investigate the harvest mouse population genetic structure.

KEY WORDS: harvest mouse, Micromys minutus, microsatellite.

INTRODUCTION

The harvest mouse (Micromys minutus) is widely distributed across much of Eurasia. The species inhabits grassy vegetation, including grassland, reeds, cereal crops, roadside verges, and salt marshes, throughout its range (Churchfield et al., 1997). Populations of the harvest mouse could fluctuate dramatically, both within and between years (Trout, 1976). In Taiwan, the harvest mouse (Fig. 1) is often found in habitats at early succession stages, such as grassy fields developed after fire or cultivation disturbance. A harvest mouse population inhabits the salt marsh in the Guandu Nature Park at suburban Taipei, Taiwan. The salt marsh underwent rapid succession as indicated by aerial photos during 2000-2004. Particularly, suitable habitats (dense vegetation) for the harvest mouse seemed to have declined, and become fragmented (Fig. 2). Such changes would affect genetic structure of the local harvest mouse population (Layme et al., 2004; Kearney et al., 2007). We developed microsatellite markers with an aim to study the effects of succession on population genetic structure of harvest mice.

MATERIALS AND METHODS

The DNA was extracted according to the standard phenol-chlorophorm extraction procedures described in Sambrook et al. (1989). Genomic DNA was digested with Sau3AI and fractioned on a 1% agarose gel. DNA of size range 300-1200bp was eluted, purified with

GFXTM _{Band Purification Kit (Amersham) and ligated}

into plasmids PUC118/BamHI/BAP (TaKaRa) according to manufacturer’s protocols. Ligated plasmids

were transformed into the competent ECOS 101 cells (Yeastern Biotech). Recombinant clones containing

inserts were transferred to Hybond-N+_{nylon membranes}

(Amersham), which were hybridized to a set of oligonucleotide probes, including (AC)15, (AT)15, (AG)15,

(AAT)10, (AAG)10, and (GATA)6. Probes were labeled

with Digoxigenin (DIG) Oligonucleotide 3’-End Labeling Kit (Roche). Hybridization was performed at 50-53℃ for 16 hours in a standard hybridization buffer, consisting of 5X SSC, 0.1% Sodium N-lauroylsarcosine, 0.02% SDS, and 1% Blocking Reagent (Roche). The membranes were washed twice, each for 5 min at 45℃ with a solution of 2X SSC, 0.1% SDS, and then twice, each for 15 min at 65℃ with a solution of 0.1X SSC, 0.1% SDS. Chemiluminescent detection was performed with DIG Luminescent Detection Kit (Roche). A total of 64 positive clones were sequenced using a MegaBACE 1000 automated sequencer. Twenty-two clones containing repeat motifs with more than 6 repeats and sufficient flanking region were selected to design primers. About 4% of screened clones yielded positives clones, which was higher than the average of 2-3% in many other taxa (Zane et al., 2002).

Primers were designed with the on-line program Primer 3.0 (Rozen and Skaletsky, 2000) and FastPCR 1.2 (Kalendar, 2007). Polymerase chain reaction (PCR) conditions were optimized for each primer pair. Each PCR reaction mixture (10 μL) contained 50-100 ng template DNA, 0.5 units of Taq DNA polymerase (Bioman, Taipei,

Taiwan), 2.0 mM of Mg2+_{, 0.2 mM dNTP, 10X buffer (20}

mM of Tris-HCl (pH8.8), 10 mM KCl, 10 mM (NH4)2SO4,

and 0.1% Triton X-100, Bioman), and 0.25 μM primer, with the forward or reverse primer being end-labeled with

(2)

June, 2009 Jiang & Lin: Microsatellite markers for the harvest mouse

119

fluorescent dyes (FAM, HEX or TAMRA). Amplification was carried out by the thermal profile: 94℃ 5 min, followed by 40 cycles of 94℃ 30 s, optimal annealing temperature for 30 s, 72℃ for 30 s, and a final extension step at 72℃ for7 min. PCR products were electrophoresised on a MegaBACE 500 automated sequencer with ET-400 Size Standard (Amersham). Individual genotypes were determined and individuals with ambiguous genotypes or homozygote were amplified and scored at least twice to determine the allele sizes.

Fig. 1. The harvest mouse (Micromys minutus) is the smallest rodent in Taiwan, as well as many parts of the world. (Photo taken by Mr. Shao-Min Yang)

RESULTS AND DISCUSSION

Seven microsatellite loci were polymorphic among thirty-eight M. minutus individuals (Table 1). The number of alleles averaged 5.75 (3-10). The observed and expected heterozygosity averaged 0.6693 and 0.6936, respectively (Table 1). Hardy-Weinberg expectation and linkage disequilibrium for each locus were tested with the program GENEPOP 4.0 (Rousset, 2008) and FSTAT 2.9.3 (Goudet, 2001), respectively. Large allele drop out and error due to stutter were tested with MICROCHECKER (Van Oosterhout et al., 2004). There was no evidence of deviation from Hardy-Weinberg equilibrium or linkage (P > 0.001786, after Bonferroni correction). No evidence of large allele drop and error due to stutter. The levels of polymorphism uncovered at these loci suggested that they should be useful to study population structure of harvest mice (Micromys minutus).

ACKNOWLEDGEMENTS

We thank the many undergraduate and graduate students in the College of Life Sciences who assisted with the field work, especially all members in YKL’s research group. We are deeply indebt to Dr. Alex H-T Yu and his research group, especially Yi-hui Chen, En-Min You, Huang-Chi Chen, and Hsiao-pei Lue for assistance in molecular technique. Financial support for this research came from the Taiwan National Science Council (NSC95-2621-B-002-004) to YKL.

Fig. 2. An aerial photo showing habitat mosaics of the study site, a salt marsh in the Guandu Nature Park at suburban Taipei, Taiwan. (Curtsey of the Spacial Ecology Laboratory of the National Taiwan University)

(3)

Taiwania Vol. 54, No. 2

120

LITERATURE CITED

Brownstein M. J., J. D. Carpten and J. R. Smith. 1996. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. BioTech. 20: 1004-1010.

Churchfield, S., J. Hollier and V. K. Brown. 1997. Community structure and habitat use of small mammals in grasslands of different successional age. J. Zool. 242: 519-530.

Goudet, J. 2001. FSTAT--A program to estimate and test gene diversities and fixation indices, version 2.9.3. Http://www.unil.ch/izea/softwares/fstat.html

Haim, A. and I. Izhaki. 1994. Changes in rodent community during recovery from fire - relevance to conservation. Biodivers. Conserv. 3: 573-585.

Ishiwaka, R. and T. Mori. 1999. Early development of climbing skills in harvest mice. Anim. Behav. 58: 203-209. Janova, E, M. Heroldova and J. Bryja. 2008. Conspicuous

demographic and individual changes in a population of the common vole in a set-aside alfalfa field. Annal Zool. Fenni. 45: 39-54.

Kalendar, R. 2007. FastPCR: a PCR primer and probe design and repeat sequence searching software with additional tools for the manipulation and analysis of DNA and protein. Kearney. N, K. Handasyde, S. Ward and M. Kearney. 2007.

Fine-scale microhabitat selection for dense vegetation in a heathland rodent, Rattus lutreolus: Insights from intraspecific and temporal patterns. Austr. Ecol. 32: 315-325.

Layme, V. M. G, A. P. Lima and W. E. Magnusson. 2004. Effects of fire, food availability and vegetation on the distribution of the rodent Bolomys lasiurus in an Amazonian savanna. J. Trop. Ecol. 20: 183-187.

Masters, P. 1993. The effects of fire-driven succession and rainfall on small mammals in Spinifex grassland at Uluru National Park, Northern-Territory. Wildl. Res. 20: 803-813. Rousset, F. 2008. GENEPOP 2007: a complete

re-implementation of the GENEPOP software for Windows and Linux. Mol. Ecol. Res. 8: 103-106.

Rozen, S. and H. Skaletsky. 2000. Primer 3 on the WWW for general users and for biologist programmers. In: Krawetz, S. and S. Misener (eds.), Bioinformatics Methods and Protocols. Humana Press, Totowa, NJ, USA. pp. 365-386. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular

cloning: a laboratory manual. 2nd_{ed. Cold Spring Laboratory} Press, New York, USA.

Tallmon, D. A., H. M. Draheim, L. S. Mills and F. W. Allendorf. 2002. Insights into recently fragmented vole populations from combined genetic and demographic data. Mol. Ecol. 11: 699-709.

Trout, R. C. 1976. An ecological study of populations of wild Harvest mice (Micromys minutus soricinus Hermann). Ph.D. thesis, University of London.

(4)

June, 2009 Jiang & Lin: Microsatellite markers for the harvest mouse

121 Van Oosterhout, C., W. F. Hutchinson, D. P. M. Wills and

P. Shipley. 2004. MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Res. 4: 535-538.

Yarnell, R. W., D. M. Scott, C. T. Chimimba and D. J.

Metcalfe. 2007. Untangling the roles of fire, grazing and rainfall on small mammal communities in grassland ecosystems. Oecol. 154: 387-402.

Zane, L., L. Bargelloni and T. Patarnello. 2002. Strategies for microsatellite isolation: a review. Mol. Ecol. 11: 1-16.