Journal of Heredity Advance Access originally published online on June 26, 2008
Journal of Heredity 2008 99(6):688-693; doi:10.1093/jhered/esn052
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brief Communications |
High Degree of Transferability of 86 Newly Developed Zebra Finch EST-Linked Microsatellite Markers in 8 Bird Species
From the Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, PO Box 54 124, Thessaloniki, Macedonia, Greece (Karaiskou); and the Department of Biology, University of Turku, 20014, Turku, Finland (Karaiskou, Buggiotti, Leder, and Primmer)
Address correspondence to Craig Primmer at the address above, or e-mail: craig.primmer{at}utu.fi.
High-resolution analysis for population genetic and functional studies requires the use of large numbers of polymorphic markers. The recent increase of available genetic tools is facilitated by the use of publicly available expressed sequence tag (EST) sequence databases that are a valuable resource for identifying gene-linked markers. In the present study, we applied bioinformatics analyses to identify microsatellite markers present in EST sequences from a zebra finch (Taeniopgia guttata) EST database and we explore the success of cross-species amplification of EST-linked microsatellite markers in 7 passerine and 1 nonpasserine species. Eighty-six zebra finch EST-linked microsatellite loci were screened for polymorphism revealing a high amplification success rate and adequate levels of polymorphism (33.3–51%) for relatively closely related species, whereas success decreased in the most distantly related species to zebra finch. EST-linked microsatellites appear to be more highly transferable between taxa than anonymous microsatellites as they revealed higher amplification and polymorphism success between different families indicating that they will be a useful source of gene-linked polymorphic markers in a broad range of avian species.
Microsatellite markers are a useful resource of variation for population, evolution, and genomic studies. Owing to their codominant and highly polymorphic nature, simple sequence repeats, that is, microsatellites, have increasingly become the marker of choice for these sorts of analyses with a good balance between cost and information content (Hedrick 2001; Sakai et al. 2001). However, the development of microsatellite markers can be tedious and time consuming since it often entails the generation of genomic libraries (Squirrell et al. 2003).
One possible solution to this problem would be to exploit publicly available genomic resources for the development of gene-based microsatellite markers that are more likely to be transferable across taxonomic boundaries (Ellis and Burke 2007). Indeed, microsatellite development is facilitated by the use of expressed sequence tags (ESTs). ESTs are partially sequenced mRNAs and thus are representative of the transcribed portion of the genome and may contain microsatellites. Although EST-linked microsatellites are generally less polymorphic than anonymous ones, their value is enhanced by their transferability across taxa and their applicability as functional markers in defining genes affecting various traits (Gutierrez et al. 2005; Pashley et al. 2006; Slate et al. 2007).
The aim of the present study was to test the transferability of EST-linked zebra finch microsatellite markers in 8 different bird species. We applied bioinformatics analyses to identify microsatellite-containing EST sequences from a zebra finch (Taeniopgia guttata) EST database. A number of primer pairs were designed based on EST-linked microsatellites to test the success of cross-species amplification and the level of polymorphism initially in 7 different passerine birds: pied flycatcher (Ficedula hypoleuca), collared flycatcher (Ficedula albicollis), bluethroat (Luscinia svecica), blue tit (Parus caeruleus), great tit (Parus major), house sparrow (Passer domesticus), and siberian jay (Perisoreus infaustus) and 1 nonpasserine species: tengmalm's owl (Aegolius funereus) aiming to evaluate the transferability and the level of polymorphism of EST-linked markers between different bird species.
 |
Materials and Methods |
|---|
 Top Materials and Methods Results and DiscussionSupplementary MaterialFundingReferences |
|---|
The zebra finch EST sequences used in this study were downloaded from GenBank. All sequences were derived from brain cDNA libraries of late embryos or adult tissue (Replogle et al. 2008). In total, 58,707 zebra finch sequences were screened for di-, tri-, and tetranucleotide microsatellite repeats using the Tandem repeats finder (Benson 1999). The minimum number of mismatches and indels were 7 and 7, respectively. The program cap3 (Huang and Madan 1999) was used to cluster the redundant EST sequences with 40-bp overlap and 95% identity criterion. We attempted to design primers for the majority of EST-linked microsatellites using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). It proved difficult to design primers in many cases because either the microsatellite was located at the border of the EST sequences or it was surrounded by AT-rich sequences. Microsatellite-containing EST sequences were blasted against the Gallus gallus (chicken) Build 2.1 genome assembly database at the National Center for Biotechnology Information (NCBI), and primer pairs were designed in flanking regions either side of the repeat where a higher level of sequence conservation was observed between chicken and zebra finch. The default parameters for primer length (20–24 bp) and product size (100–550 bp) were used. Additionally, a limited number of primers (n = 2) were designed and tested for polymorphism based on the EST microsatellite sequences of G. gallus.
A 5'-GTTT "tail" was added to every reverse primer to improve scoring quality (Brownstein et al. 1996), whereas the forward primer was labeled with 1 of the 4 fluorescent dyes (FAM, NED, PET, and VIC) compatible with the ABI 3130xl Genetic analyzer. Polymerase chain reaction (PCR) amplifications were performed in 10 µl reactions using 50 ng of DNA, 0.3 µM of each primer, 1x reaction buffer, 1.5 mM of MgCl2, 0.25 mM deoxynucleoside triphosphates, and 0.2 units of BioTaq DNA polymerase (Bioline, Taunton, MA). A touchdown PCR amplification protocol (TD:55–45) was initially used. An initial denaturation of 3 min at 94 °C, followed by 19 cycles of 30 s at 94 °C, 30 s at 55 °C, 1 min at 72 °C, with annealing temperature decreasing 0.5 every cycle, followed by 19 cycles of 30 s at 94 °C, 30 s at 45 °C, 1 min at 72 °C, and a final elongation step for 5 min at 72 °C was used. The annealing temperature was lowered, and the PCR amplification protocol TD:50–40 was used for primer pairs with no successful amplification for the 2 flycatcher species. The protocol TD:60–50 was used for only 4 primer pairs. Further cross-species amplification was performed in the other 6 bird species using TD:55–45 protocol. The PCR products were diluted 1/60. Two microliters of each product were mixed with 10 µl of formamide and 1.6 µl of internal size standard GS 600LIZ (Applied Biosystems, Foster City, CA) ranging in size from 20 to 600 bp. The resulting mixture was denatured for 5 min at 100 °C, and electrophoresis was performed for 20 min on an ABI 3130xl Genetic analyzer. Genotypes were scored using Genemapper 4.0 software (Applied Biosystems, Foster City, CA).
Eight unrelated individuals from both pied and collared flycatchers were tested for polymorphism, whereas 4 individuals were scored for all the other studied species except for great tit, where 2 individuals were analyzed. We used GENETIX software package to calculate the observed and the expected heterozygosity values for collared and pied flycatchers, whereas the polymorphic information content (PIC) was calculated using the CERVUS analysis software (Marshall et al. 1998).
|
Results and Discussion |
|---|
|
TopMaterials and MethodsResults and DiscussionSupplementary MaterialFundingReferences |
|---|
The screening of 58,707 EST zebra finch sequences revealed 450 EST-containing microsatellites (di-, tri-, and tetranucleotides). Trinucleotide repeats were the most abundant within zebra finch ESTs, accounting for 226 loci (50%), followed by dinucleotides (36%) and tetranucleotides (14%). The most frequent dinucleotide was AT (60%), the most frequent trinucleotides were GAG (11%) and GAT (10%), whereas 36 different combinations of tetranucleotide repeats were found for the 63 tetranucleotides. The abundance of the 3 different classes of microsatellites present in zebra finch genome was similar to that observed in a recent in silico analysis in ESTs in zebra finch (Slate et al. 2007) with small deviations most likely due to the different search parameters used in each case.
Based on the fact that uninterrupted repeats are more variable than those with interruptions (Petes et al. 1997), we designed primers for 42 EST sequences containing dinucleotide repeats, 39 with trinucleotides, and 7 with tetranucleotide repeats using in each case microsatellites each with at least 8 perfect repeats. The great majority of these sequences gave a single best hit on chicken chromosomes indicating homology (Supplementary Table 1; see Supplementary Material online). In total, 88 primer pairs were designed from nonredundant EST sequences (86 from zebra finch sequences and 2 from chicken) and tested for amplification using the standard touchdown PCR program (55–45: Supplementary Table 1; see Supplementary Material online). The amplification success rate ranged from 68.2% in collared flycatchers to 45.5% in tengmalm's owl (Supplementary Table 1; see Supplementary Material online). Testing of a lower annealing temperature (TD:50–40) in markers that failed to amplify in collared and pied flycatchers increased the amplification success to 85.2% and 80.7%, respectively, indicating that among the other factors the PCR annealing temperature can affect the cross-species amplification success (Primmer et al. 2005). In many cases, considerable differences were observed between the expected and the observed sizes of the amplification products in different species.
|
According to the broad survey of microsatellites conducted in Primmer et al. (2005), the level of polymorphism of microsatellites in birds often increases with increasing microsatellite length and number of repeat units. However, this did not seem to be the case in zebra finch EST-linked microsatellite cross-species amplification analysis because there was no correlation between the number of repeats of zebra finch EST-linked microsatellites and the polymorphism level revealed (data not shown). Thus, it was not possible to predict the minimum number of tandem repeats in zebra finch EST-linked microsatellites required for detecting polymorphism in cross-amplified species.
As far as the level of polymorphism is concerned for zebra finch EST-linked microsatellite loci, 28 out of 59 were polymorphic for collared flycatcher (47.5%) ranging from 2 to 6 alleles and 20 out of 52 EST-linked microsatellite loci for pied flycatcher (38.5%) ranging from 2 to 7 alleles. Seven additional polymorphic markers where revealed for the 2 species when lowering the annealing temperature. For the other 6 studied species, 51% of the markers were polymorphic for house sparrow, 47.8% for bluethroat, 44.8% for blue tit, 33.3% for great tit, 18.5% for siberian jay, and 15.4% for tengmalm's owl. Additionally, 1 chicken EST-linked locus was polymorphic for 5 of the tested species (Table 1). Based on previous studies (e.g., Primmer et al. 1996), the success rate of cross-species amplification and polymorphism in birds is negatively related to the phylogenetic distance between species. The decrease in the amplification/polymorphism success of zebra finch EST-linked microsatellite loci with the increase of cytb divergence distances was also verified in the present study when comparing data of the 8 species using the same annealing temperature (Figure 1). The level of polymorphism is relatively high for all passerida species (33.3–51%) and decreases in siberian jay and even lower in tengmalm's owl, which are both the most distantly related to zebra finch (cytb genetic distances 0.178 and 0.28, respectively).
|
Based on the linear trend lines for EST and anonymous microsatellite data (cross-species amplification data taken from Primmer et al. [2005]) given in Figure 1, EST-linked markers appear to be more highly transferable between taxa as they revealed higher amplification and polymorphism success than anonymous microsatellites between different families, indicating that although evolutionary and functional constraints can limit microsatellite repeat expansion in the expressed portion of the genome (Hancock 1996; Dokholyan et al. 2000; Metzgar et al. 2000), they still reveal sufficient levels of variation to be applied for population and functional genetics. Indeed, 34 of the 88 loci tested amplified in at least 7 of the 8 species tested (87.5%) and 14 loci revealed polymorphism in at least 5 of the 8 (62.5%) species tested (Table 1). These markers can be recommended as a good starting point for cross-species testing when resources are limited. In addition, the fact that just 2 of the 88 markers tested failed to amplify in any species indicates that EST-linked microsatellites will provide a useful source of gene-linked polymorphic markers for, for example, linkage mapping (Hansson et al. 2005; Backstrom et al. 2006) or identification of signatures of selection (Vasemägi et al. 2005) in a broad range of avian species.
|
Supplementary Material |
|---|
|
TopMaterials and MethodsResults and DiscussionSupplementary MaterialFundingReferences |
|---|
Supplementary material can be found at http://www.jhered.oxfordjournals.org/.
|
Funding |
|---|
|
TopMaterials and MethodsResults and DiscussionSupplementary MaterialFundingReferences |
|---|
Academy of Finland (Centre of Excellence in Evolutionary Genetics and Physiology).
|
Acknowledgments |
|---|
We thank Hans Ellegren, Niclas Bäckström, Jan Lifjeld, Arild Johnsen, Paula Lehtonen, Jon Brommer, and Erkki Korpimäki for supplying DNA and Niina Wahlroos and Mirkka Heinonen for excellent technical assistance.
|
Footnotes |
|---|
Corresponding Editor: Rob Fleischer
Received November 19, 2007
Accepted May 22, 2008
|
References |
|---|
|
TopMaterials and MethodsResults and DiscussionSupplementary MaterialFundingReferences |
|---|
-
Backstrom NM, Brandstrom L, Gustafsson A, Qvarnstrom H, Cheng H, Ellegren H. Genetic mapping in a natural population of collared flycatchers (Ficedula albicollis): conserved synteny but gene order rearrangements on the avian Z chromosome. Genetics (2006) 174:377–386.
Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res (1999) 27:573–580.
Brownstein MJ, Carpten JD, Smith JR. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques (1996) 20:1004–1010.[Web of Science][Medline]
Dokholyan NV, Buldyrev SV, Havlin S, Stanley HE. Distributions of dimeric tandem repeats in non-coding and coding DNA sequences. J Theor Biol (2000) 202:273–282.[CrossRef][Web of Science][Medline]
Ellis JR, Burke JM. EST-SSRs as a resource for population genetic analyses. Heredity (2007) 99:125–132.[CrossRef][Web of Science][Medline]
Gutierrez MV, Patto MCV, Huguet T, Cubero JI, Moreno MT, Torres AM. Cross-species amplification of Medicago truncatula microsatellites across three major pulse crops. Theor Appl Genet (2005) 110:1210–1217.[CrossRef][Web of Science][Medline]
Hancock JM. Simple sequences and the expanding. Bioessays (1996) 18:421–425.[CrossRef][Web of Science][Medline]
Hansson B, Akesson M, Slate J, Pemberton JM. Linkage mapping reveals sex-dimorphic map distances in a passerine bird. Proc R Soc Lond B Biol Sci (2005) 272:2289–2298.[Medline]
Hedrick PW. Conservation genetics: where are we now? Trends Ecol Evol (2001) 16:629–636.[CrossRef]
Huang XQ, Madan A. CAP3: a DNA sequence assembly program. Genome Res (1999) 9:868–877.
Marshall TC, Slate J, Kruunk LEB, Pemberton JM. Statistical confidence for likelihood-based paternity inference in natural population. Mol Ecol (1998) 7:639–655.[CrossRef][Medline]
Metzgar D, Bytof J, Wills C. Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome Res (2000) 10:72–80.
Pashley CH, Ellis JR, McCauley DE, Burke JM. EST databases as a source for molecular markers: lessons from Helianthus. J Hered (2006) 97:381–388.
Petes TD, Greenwell PW, Dominiska M. Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae. Genetics (1997) 146:491–498.[Abstract]
Primmer CR, Moller AP, Ellegren H. A wide-range survey of cross-species microsatellite amplification in birds. Mol Ecol (1996) 5:365–378.[CrossRef][Medline]
Primmer CR, Painter J, Koskinen M, Palo J, Merila J. Factors affecting avian cross species microsatellite amplification. J Avian Biol (2005) 36:348–360.[CrossRef]
Replogle KL, Arnold AP, Ball GF, Band M, Bensch S, Brenowitx EA, Dong S, Drnevich J, Ferris M, George JM, et al. The songbird neurogenomics (SoNG) initiative: community based tools and strategies for study of brain gene function and evolution. BMC Genomics (2008) 9:131–201.[CrossRef][Medline]
Sakai AK, Allendorf FW, Holt JS, Lodge DM, Molofsky J, With KA. The population biology of invasive species. Annu Rev Ecol Syst (2001) 32:305–332.[CrossRef][Web of Science]
Slate J, Hale MC, Birkhead TR. Simple sequence repeats in zebra finch (Taeniopygia guttata) expressed sequence tags: a new resource for evolutionary genetic studies of passerines. BMC Genomics (2007) 8:52.[CrossRef][Medline]
Squirrell J, Hollingsworth PM, Woodhead M, Russell J, Lowe AJ, Gibby M. How much effort is required to isolate nuclear microsatellites from plants? Mol Ecol (2003) 12:1339–1348.[CrossRef][Medline]
Vasemägi A, Nilsson J, Primmer CR. Expressed sequence tag-linked microsatellites as a source of gene associated polymorphism for detecting signatures of divergent selection in Atlantic salmon (Salmon salar L.). Mol Biol Evol (2005) 22:1067–1076.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||