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Abstract Gubernatrix cristata is a rare and endangered bird species from the South
American Pampas grassland with a restricted and patchy distribution. Here we report
the development of ten microsatellite markers isolated from this species and the
characterization of their allele variability. The number of alleles observed for each locus
ranged from 4 to 14, with an average of 7.5 alleles per locus. The microsatellites proved
to be useful in revealing levels of diversity in G. cristata, and thus can be used to
explore the genetic structure of scattered populations across its present geographic
range.
was linkage disequilibrium among all pairs though this conclusion should be
of loci. MICRO-CHECKER (Van Oosterhout regarded with caution because of the low
et al. 2004) was used to quantify the sample size due to the rarity of the
possible genotyping errors and null species in the wild, and also because of
alleles. Ten SSRs were found to be the low DNA quality in many of these
polymorphic, although four of them samples due to the material collected. In
presented null alleles according to terms of ex situ conservation, these data
MICRO-CHECKER (GcrisC02, GcrisE02, allow us to conclude that creating a G.
GcrisF12, and GcrisH09) and ARLEQUIN cristata captive breeding program would
(Table 1). In the remaining six loci, linkage have a high probability of success since
disequilibrium among any pairs of loci, the species is not suffering from low
genotyping mistakes because of the levels of genetic diversity as shown by
presence of null alleles, allele dominance, the observed heterozygosity. The primers
or stuttering were not detected. The proved to be useful in revealing levels of
number of alleles per locus varied from 4 diversity in G. cristata, and can be used to
to 14 with an average of 7.5 alleles per explore the genetic structure of scattered
locus. The observed heterozygosity (HO) populations across its present geographic
varied from 0.126 to 0.893 and the range.
expected (HE) heterozygosity from 0.130
to 0.930, with means and standard errors Acknowledgements.
of 0.6824 + 0.1144 and 0.7132 + 0.0272
respectively (Table 1) (GenBank accession We thank to Dr. A. P. Souza for the
numbers: GU237061 to GU237070). The training course in microsatellite isolation.
deviation from Hardy-Weinberg We thank to P. Rorato and C. Lopes for
equilibrium presented by 4 loci may be helping us with the software packages
the result of a sampling bias. Since the and valuable discussions. We thank CNPq
species is rare and most of the material for providing the doctorate scholarship to
used was from seizures made by the C. Martins-Ferreira, to CAPES for the
Environmental Police, we cannot Sandwich Scholarship, and to FBPCN for
determine if all of the samples represent financing the project. Laboratory work
the same population. Samples could was funded by NSERC operating grant to
represent several differentiated AJB. We also thank Kleber Valenti Schenk
populations combined that may be the for assistance in the translation the MS
cause of departures from HWE. For the from Portuguese to English.
four loci that had null alleles and
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a b
Locus Primer Sequences Repeat Alleles Size (bp) HO HE PIC values
GcrisC02 F: TCACCCCTTTGGTTGCTTAC (CA)7 4 247 0,267 0,438* 0,373
R: TCAGGGGTGTGTCTCTCCTT
GcrisC08 F: AGCCACTCATCCGATTTGTT (AC)8 6 174 0,911 0,791 0,748
R: AGTCTTGGCTGCCTGTCATT
GcrisE02 F: TTGGAGACCTTGTTGGTGTG (CA)8 4 227 0,229 0,710* 0,648
R: TCACACTGGGCACATGAGAT
GcrisF02 F: TTCACTGGGTTCACAGAAAGG (CA)12 14 190 0,833 0,893 0,873
R: GCTGTCGTCATTGTTCAGGA
GcrisF12 F: AAGATGTGCCTTTGGTCTGG (AC)9 (CA)7 6 230 0,600 0,784* 0,734
R: CCATTGCAGAAATGTCCTGA
GcrisG10 F: CAGGATCCTCTGCCATGTCT (CA)7 4 236 0,130 0,126 0,122
R: TTTTCCCTTTTAACGCCAAG
GcrisH06 F: GAGAGAAACCAGGTGCTTCG (CA)9 10 230 0,735 0,830 0,799
R: GTTTTAAGGCTGGGGACACA
GcrisH07 F: GTGTGACTTGTTCCCCTTCC (CA)7 (AC)5 6 213 0,739 0,755 0,709
R: ACAGGAGCAGCCAGTTGAAT
GcrisH09 F: CAAGGTGTTGTGGAGCCTTT (CA)7 10 236 0,702 0,834* 0,802
R: GCAACCAGCACATGAAATTG
GcrisH12 F: AAGCGTGACCATGAAAAATGT (CA)7 5 249 0,232 0,700 0,637
R: TTCATCTGCCCTCCTTGTTC
a
All forward primers were M13-tailed at the 5' end. Significant departures from HWE: * P < 0.001
b
Polimorphism Information Content
Table 2. Estimates of null allele rates of four loci that departed from HWE using three methods of null allele estimation.