TECHNICAL NOTE

A new set of microsatellite loci for Leptonycteris yerbabuenaeand cross species amplification with other glossophagines

Jose Antonio Romero-Meza • Stacey L. Lance •

Jorge Ortega

Received: 2 September 2011 / Accepted: 5 September 2011 / Published online: 15 September 2011

� Springer Science+Business Media B.V. 2011

Abstract A technique based on 454 sequencing of an

enriched library was used to construct genomic libraries

highly enriched for microsatellite loci for Leptonycteris

yerbabuenae. Twenty-four polymorphic microsatellites

were developed and tested as markers in the target species.

We probed ascertainment bias in 5 different glossophagines

(L. nivalis, Glossophaga leachii, G. soricina, Anoura geoff-

royi, and Choeronycteris mexicana), indicating their potential

utility for suitable studies of population genetics and other

related analyses. Levels of expected heterozygosity were

medium–low for all markers (mean HE = 0.147, range

0.008–0.46).

Keywords 454 Sequencing � Ascertainment bias �Glossophagines � Leptonycteris yerbabuenae �Microsatellites

Leptonycteris yerbabuenae is a bat restricted to the tropical

and dry areas from south US to Central America. This bat

species is listed as vulnerable by the IUCN and critically

vulnerable in some states of US and Mexico. This bat

migrates following the flowering season of nectaring plants

of the xeric habitats. One individual may visit as many as

100 flowers per night and are specialist pollinators of night

flowering plants. A previous set of microsatellite loci was

recently developed for the target species (Ramırez et al.

2011). Here we present a new set of microsatellite loci

specially developed for Leptonycteris yerbabuenae by using

a 454 sequencing protocol.

We collected tissue samples from two vouchered spe-

cies of L. yerbabuenae. Wing tissue was kept in alcohol

(96%) until DNA extraction following the Universal Salt-

Extraction Protocol (Aljanabi and Martınez 1997). DNA

was then serially enriched twice for microsatellites using 3

probe mixes following Glenn and Schable (2005) with the

changes described in Lance et al. (2010). There were two

primary changes to the Glenn and Schable (2005) protocol.

First, a different linker was used (SimpleX-3 Forward 50-AAAA CGTGCTGCGGAACT-30 and SimpleX-3 Reverse

50-pAGTTCCGCAGCACG-30). Second, the enriched

libraries were sequenced on a 454 using titanium chemistry

following standard Roche 454 library protocols (454 Life

Sciences, a Roche company, Branford CT). All methods

for sequencing, microsatellite identification, and primer

design, are as described in Faircloth (2008) and Lance et al.

(2010) with the exception that the universal CAG tag was

not used.

We recovered 118 unique loci (61 di, 4 tri, and 53 tet-

ranucleotide), but only 24 were chosen for polymerase

chain reaction (PCR) trials. We directly labelled forward

primers (FAM or HEX) for each of the chosen loci. PCR

reactions were performed in a 15 lL volume containing

30 ng of DNA, 0.3 mM of dNTP’s, 0.5 lM of each primer,

19 Taq buffer (2.0 lM of MgCl2, 10 mM of Tris–HCl,

50 mM of KCl), 2.59 of BSA, and 1.0 U of FlexiTaq

polymerase (PROMEGA). PCR cycling conditions were as

follows: initial denaturation at 94�C for 10 min, followed

by 35 cycles of 92�C for 1 min, gradient temperature for

J. A. Romero-Meza � J. Ortega (&)

Laboratorio de Ictiologıa y Limnologıa, Posgrado en Ciencias

Quimicobiologicas, Departamento de Zoologıa, Escuela

Nacional de Ciencias Biologicas, Instituto Politecnico Nacional,

Prolongacion de Carpio y Plan de Ayala s/n, Col. Sto. Tomas,

11340 Mexico, DF, Mexico

e-mail: artibeus2@aol.com

S. L. Lance

Savannah River Ecology Laboratory, University of Georgia,

Aiken, SC 29803, USA

123

Conservation Genet Resour (2012) 4:291–294

DOI 10.1007/s12686-011-9527-z

Table 1 Primer sequences and characteristics of 24 microsatellite loci isolated from Leptonycteris yerbabuenae

Genebank No. Locus Repeat motifs Sequences 50–30

JN598904 LepyA1 (GTT)9 F:TET-AAACAGCATGACCTCTGTGC

R:CGAGCGAGCGAAGTACTC

JN598903 LepyA2 (ACA)9 F:TET-AACCTATCTGTGGGCATGGG

R:TGTGCATCTGATAGGTTGGC

JN598902 LepyA3 (CAT)6 F:HEX-TCCCTACTGTGCGATGTTGC

R:GTGCTGCGGAACTACTCTTC

JN598901 LepyA4 (ACA)7 F:TET-GCGACATTGTTCCTTCTCGG

R:TCCGTTTCTCCTTGTTTGTAGC

JN598900 LepyA5 (CATA)1 CACAGATA(CATA)8 F:TET-GGTTAGCAATGCCTCCTCAC

R:CTACGAGTCTGGTCCTTCGC

JN598899 LepyA6 (CTAT)9 F:TET-GTTCCTTGTGCTGTTCTGGG

R:TGGGAGGTGTTGTATTTGGTAG

JN598898 LepyA7 (TATC)9 F:TET-TGTACATTCCAGTCCACTCTCC

R:CCCTGCCTTCTATCCTGAGG

JN598897 LepyA8 (CTAT)10CCAT(CTAT)2 F:HEX-CGTAAAGGACCAGGGAGTAGAG

R:CATGCAATGACCCTCAGCTG

JN598896 LepyA9 (CA)10 F:TET-ACAGCCAGGAAAGGTAAATGG

R:CGGAACTACTTGAAAGACTGCG

JN598895 LepyA10 (GA)10 F:TET-AGTCTGATTCACTGTGTTGGC

R:GAGATTGTATGTCGCCGCTC

JN598894 LepyA11 (CA)7TA(CA)10 F:TET-ACCAGCCAGACCCATTTCTC

R:GTTGTCGTAGTGTGTCTCCTG

JN598893 LepyA12 (CA)1C(CA)2TT(CA)12 F:HEX-ACCATCCCTCACACTGCATG

R:AGAGAAAGAAGGAGGGTCGC

JN598892 LepyA13 (CA)2GAGA(CA)1TAGA(CA)9 F:TET-AGATGCAAGTGAGGGTCCAG

R:CCATGCCTCTCTCCTAGCTG

JN598891 LepyA14 (CA)11 F:TET-TGTTTCAGGCCCTCTCCATC

R:CTGCGGAACTACACTCAGGG

JN598890 LepyA15 (CA)34 F:TET-CTGCAGAGACCATGAGAAGC

R:ATGAGCTAGGGTGTTGGGTG

JN598889 LepyA16 (GTAT)8 F:TET-TAGTAGCTGGTCCTTCGCAG

R:TTCCCACTGGCCTAAACTGG

JN598888 LepyA17 (GGAA)3GAA(GGAA)7G(GGAA)2 F:TET-TGTGTAGGTAGTGGTGCTCC

R:AAATGCAGTGAATCAGGGCC

JN598887 LepyA18 (GATA)8 F:TET-ACCCAAGTTAACACATCTTCCC

R:CCATCTCACACTCTGGTCCC

JN598886 LepyA19 (GT)11 F:TET-ATGCCTAGGTTGTGAGCCTG

R:TGTCTGTCCAAAGGCTACCC

JN598885 LepyA20 (CT)11 F:TET-GACTACGTGTGGGAAGCAAC

R:TCCAGGGCATCAGTGATACG

JN598884 LepyA21 (CA)15CT(CA)5 F:TET-ACAATGCAGGCCTGATTTCC

R:CGAGCGGAACTACATGCATG

JN598883 LepyA22 (CTAT)9 F:HEX-TCCTACGGCCTGAGACAATG

R:TGGATCTGGATTGTCTCAGGTG

JN598882 LepyA23 (TGAA)7 F:TET-GGTGCTCCTAACCCAGAAAG

R:AAACACAGACTGAGCACCTG

JN598881 LepyA24 (GATA)4CATA(GATA)8 F:TET-TTGGCCTCCCTGAACATTTG

R:GGAGTTTCTGCCATTCCCAC

292 Conservation Genet Resour (2012) 4:291–294

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1 min (range from 49 to 60�C), and 72�C for 60 s, and

ending with 72�C for 1 min. Exact annealing temperatures

for each primer are given of Table 1. We visualized by

electrophoresis on 1.5% agarose gel the expected size of

the PCR product. Markers were tested for amplification

success, polymorphism and specificity in 14 individuals of

L. yerbabuenae from distinct locations.

The results of the microsatellite profiles were examined

using GENEMAPPER (Applied Biosystems) and peaks

were scored by hand. We estimated the proportion of

polymorphic loci and the average of number of alleles per

locus by using the GDA software (Lewis and Zaykin

2001), and the observed (HO) and the expected heterozy-

gosity (HE) using POPGENE (Yeh and Boyle 1997), we

also calculated linkage disequilibrium and Hardy–Wein-

berg proportions. MICROCHECKER was used to screen

null alleles in each loci (van Oosterhout et al. 2004).

All 24 loci were polymorphic for L. yerbabuenae, with

an average of 10.12 alleles per locus (range 6–12 alleles

per locus). The high polymorphism observed is likely

due the loci test in 16 individuals from different parts of

its distributional range. Our results suggest that these

markers would be useful for population genetic studies on

L. yerbabuenae, specially conducted on large spatial scales.

Hardy–Weinberg equilibrium or linkage disequilibrium

was not detected by using POPGENE software. Levels of

expected heterozygosity were medium–low for all markers

(mean HE = 0.147, range 0.008–0.46), and mean for

observed heterozygosity was HO = 0.20. MICROCHE-

KER detected significance evidence of null alleles at locus

Lepy06, but we could not find evidence of mistakes on the

scoring or large allele dropout. Large scale population

genetic studies and landscape studies are necessary for this

migrate bat species to assess the influence of the traveling

behavior on the local genetic structure of its populations in

the wild.

Ascertainment bias was tested in 5 different glosso-

phagines (L. nivalis, Glossophaga leachii, G. soricina,

Anoura geoffroyi, and Choeronycteris mexicana) related

to L. yerbabuenae, to probe wherever polymorphic loci

isolated from the source species are less variable in close

related species (Hogan et al. 2009). A positive control

containing DNA from L. yerbabuenae was added to each

PCR reaction. Almost all of the tested loci were very

similar in size and some of them showed specific mono-

morphisms. Ascertainment bias resulted in a better

Table 1 continued

No. alleles Range (bp) Pair product size Ho HE HW P value Annealing

temperature

10 172–235 241 0.2982 0.2048 0.0978 59

11 132–271 274 0.0204 0.0083 0.7041 59

10 139–169 169 0.2308 0.1367 0.4414 60

11 149–179 182 0.1228 0.0542 0.4034 59

10 81–138 138 0.2593 0.1733 0.3385 60

11 236–272 272 0.1071 0.0476 0.5331 59

6 89–282 286 0.25 0.0889 0.436 59

8 88–191 191 0.25 0.1875 0.1919 60

11 143–157 143 0.0909 0.0389 0.5869 60

9 119–129 119 0.4444 0.32 0.0541 59

12 275–287 265 0.2245 0.175 0.99 59

12 162–176 162 0.1538 0.12 0.7613 60

10 244–260 234 0.2308 0.1367 0.4228 59

10 248–272 244 0.2727 0.2222 0.204 60

10 370–390 378 0.2 0.1 0.6131 60

10 266–282 266 0.2453 0.1917 0.4309 59

11 179–195 175 0 0.0083 0.9442 60

10 341–357 337 0.2157 0.1458 0.5568 60

12 107–129 117 0.0526 0.0375 1 59

10 141–155 141 0.2308 0.1367 0.4307 60

9 289–299 291 0.434 0.4667 0.1529 59

11 234–254 234 0 0.0375 0.9427 59

9 198–210 198 0.3617 0.3689 0.5901 59

10 227–247 219 0.2453 0.1917 0.4288 60

Conservation Genet Resour (2012) 4:291–294 293

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amplification and success in the most related species

(L. yerbabuenae and L. nivalis—Fig. 1), and transferability

of amplification was low in the rest of the glossophagines.

Our markers can be suitable for population genetics studies

because showed successful amplification and limited utility

in sister species of the target bat.

Acknowledgments Financial support was provided by FOMIX-

Campeche (CONACyT) 95900. Tissue samples were obtained from

L. Leon (Museo de Zoologıa, Facultad de Ciencias, UNAM), A.

Rendon (Instituto Tecnologico de la Cuenca del Papaloapan), C.

Lopez (CIIDIR-Durango, IPN), and O. Gaona/R. S. Galicia (Instituto

de Ecologıa, UNAM). We thank the field assistance provided by

I. Campos and A. Hernandez-Davila. Manuscript preparation was

partially supported by the DOE under Award Number DE-FC09-

07SR22506 to the University of Georgia Research Foundation.

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