Isolation and characterization of 18 polymorphic microsatellites in

Isolation and characterization of 18 polymorphic microsatellites in the grasshoppers Chorthippus
group binotatus (Orthoptera: Acrididae) species complex
Víctor Noguerales1, Conchi Cáliz-Campal1, Pedro J. Cordero1 and Joaquín Ortego1,2
Grupo de Investigación de la Biodiversidad Genética y Cultural, Instituto de Investigación en
Recursos Cinegéticos - IREC (CSIC, UCLM, JCCM), Ronda de Toledo s/n, E-13071 Ciudad Real, Spain
Department of Integrative Ecology, Estación Biológica de Doñana, EBD-CSIC, Avda. Américo Vespucio
s/n, E-41092 Seville, Spain
Author for correspondence (Víctor Noguerales)
Grupo de Investigación de la Biodiversidad Genética y Cultural
Instituto de Investigación en Recursos Cinegéticos - IREC (CSIC, UCLM, JCCM)
Ronda de Toledo s/n, E-13071 Ciudad Real, Spain
The Chorthippus group binotatus (Orthoptera: Acridae) is a species complex of grasshoppers
distributed in southwest Europe and north Africa. They often form small and fragmented populations
due to human-driven habitat destruction and/or the patchy distribution of their natural montane
habitats. Here, we describe 18 novel polymorphic microsatellite markers obtained from the nominal
subspecies Chorthippus binotatus binotatus. For this subspecies, the number of alleles ranged from 4
to 16 and their observed heterozygosity ranged from 0.21 to 0.94. Most of the loci also amplified and
were polymorphic across all species/subspecies of the complex. These markers will be used to
investigate the consequences of fragmentation and climate change on the demographic and
evolutionary trajectories of these taxa of great conservation concern.
Keywords Chorthippus binotatus · Landscape genetics · Microsatellite · Population fragmentation
The Chorthippus group binotatus (Orthoptera: Acridae) is a complex of montane grasshoppers that
includes at least two species and seven subspecies distributed through southwest Europe (France,
Spain and Portugal) and north of Africa (Morocco) (Defaut 2011). All taxa of this group are
oligophagus and feed exclusively on grasses or certain bush legumes (tribe Genisteae) (Defaut 2011).
These grasshoppers generally form small and highly fragmented populations due to human-driven
habitat destruction, which has resulted in the nominal subspecies C. binotatus binotatus is included
as threatened in the French Red List (Pratz and Cloupeau 2010). Some narrow endemic taxa within
this group also show highly fragmented populations due to the patchy distribution of their natural
habitats, which are often restricted to small and isolated areas located in different mountain ranges
(Defaut 2011). Here, we report the development of 18 polymorphic microsatellite loci from C.
binotatus binotatus and test their functionality in the remaining species/subspecies of the group.
Genomic libraries were developed by GenoScreen ( using DNA extracted
from eight individuals collected in different populations of C. binotatus binotatus (Table S1). Libraries
were generated using a microsatellite enrichment method and sequenced using a 454 GS-FLX
Titanium pyrosequencer (Malausa et al. 2011). A total of 398 primer pairs were designed and 66
were tested using 20 individuals of C. binotatus binotatus collected in Jaén, Spain (Table S2). We
discarded 36 loci because they failed to amplify or produced inconsistent and multiple bands in
agarose gels. Primers producing products of expected size were labelled with fluorescent dyes (6FAM, PET, NED or VIC) to allow analysis on an automated DNA sequencer and determination of levels
of polymorphism. We selected 18 polymorphic markers and the rest were discarded because they
were monomorphic or produced non-resolvable electropherograms. Finally, the 18 polymorphic
markers were tested for cross-amplification using 20-22 individuals from one location of each of the
species/subspecies within the complex (Table S2).
Amplifications were conducted in 10-μL reaction volumes containing 5 ng of genomic DNA,
1X reaction buffer (EcoStart Reaction-Buffer, Ecogen), 2 mM MgCl2, 0.2 mM of each dNTP, 0.15 μM of
each primer and 0.1 U of Taq DNA EcoStart-Polymerase (Ecogen). The PCR programme used was 9
min denaturing at 95ºC followed by 40 cycles of 30 s at 94ºC, 45 s at the annealing temperature
(Table 1) and 45 s at 72ºC, ending with a 10 min final elongation stage at 72ºC. Amplification
products were run on an ABI 310 Genetic Analyzer (Applied Biosystems) and genotypes were scored
using GENEMAPPER 3.7 (Applied Biosystems). Calculation of observed and expected heterozygosities
and tests for departure from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD)
were performed in ARLEQUIN 3.5 (Excoffier and Lischer 2010).
We found no evidence of genotypic linkage disequilibrium at any pair of loci. For the nominal
subspecies, five loci significantly deviated from HWE and MICRO-CHECKER analyses (Van Oosterhout et
al. 2004) indicated that this could be due to the presence of null alleles (Table S3). The number of
alleles per locus (NA) ranged from 4 to 16 and observed (HO) and expected (HE) heterozygosity per
locus ranged from 0.21 to 0.94 and from 0.66 to 0.92, respectively. Most of the loci also amplified
and were polymorphic in the other taxa of the species complex (Table S3). Overall, the panel of
microsatellites obtained constitutes a basic genetic tool to study the genetic diversity, demographic
changes and genetic structure in the different species and subspecies of the C. binotatus group,
many of them of great conservation concern.
Acknowledgements This work received financial support from the projects POII10-0197-0167 and
CGL2011-25053. During this work V.N. was supported by a FPI pre-doctoral fellowship (BES-2012053741). J.O. was supported by a Severo Ochoa (EBD) (SEV-2012-0262) research contract.
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