BACKGROUND
microsatellites or short sequence repeats  are relatively small 1– <dig> base-pair  tandem repeats that are found in the genomic dna of pro- and eukaryotes.
although the majority of microsatellites are located in non-coding sequences  <cit>  and considered to be selectively neutral, some microsatellite loci are located in functional regions and involved in chromatin organisation, regulation of gene activity and metabolic processes such as dna replication and recombination  <cit> .
microsatellites exhibit high mutation rates of 10- <dig> to 10- <dig> per locus per generation  <cit> .
the main mutational processes responsible for the variability in microsatellites are considered to be replication-slippage and recombination  <cit> .
both processes change the length of the microsatellite by altering the number of repeats of the microsatellite.
microsatellite variability has been associated with a number of microsatellite characteristics.
mutation rates of microsatellites have been found to be taxon-specific  <cit> .
microsatellite variability covaries with allozyme diversity in a taxon  <cit> .
the number of repeats can predict the variability and stability of a microsatellite motif, with longer loci found to be more variable, but also more unstable than shorter ones  <cit> .
the type of microsatellite motif may affect abundance and variability.
for example, it was found that microsatellites with tri- or hexanucleotide motifs are more frequent in coding than in non-coding regions, possibly because mutations of these microsatellites in coding regions are less likely to result in deleterious frameshift mutations  <cit> .
the high variability of microsatellite markers and their straightforward analysis using the polymerase chain reaction  have led to their frequent application in studies on natural populations.
however, one obstacle to the wider application of microsatellites is the difficulty of developing a sufficient number of suitable markers for any given species.
although microsatellites are ubiquitous in eukaryotic organisms, their abundance varies across different groups  <cit> .
microsatellites are less common, for example, in birds than in other vertebrates  <cit> .
there are two principal strategies to obtain microsatellite markers.
first, microsatellite markers can be developed by screening genomic libraries  <cit> .
success rates differ according to the protocol and taxon, although usually a medium to high number of polymorphic markers can be isolated using this approach.
since microsatellites are present at a relatively low frequency in avian genomes, in this case their isolation is most efficient using enrichment protocols.
this involves many stages, is a skilled and time-consuming process and requires significant funding and a well-equipped molecular laboratory, which are not always available in ecological and conservation research.
the second method makes use of existing microsatellite markers isolated in different species to the species of interest .
for cross-species amplification tests  existing primers developed in related species are tested for amplification and polymorphism in the target species.
one drawback of cross-species amplification is that success rates decline with evolutionary distance between the target species and source species  <cit> .
in birds, most microsatellite markers have been developed for the orders passeriformes and galliformes.
passeriformes is a species-rich relatively recent clade  <cit>  in which more than  <dig> microsatellite markers have been characterised  <cit> .
several studies have successfully identified additional polymorphic loci by cross-species testing in birds.
the development of complete primer sets from cross-species amplification tests has been successful in falconiformes  <cit> , galliformes  <cit>  and passeriformes  <cit> .
however, in many other avian orders fewer microsatellite loci have been isolated and, therefore, the opportunity to develop microsatellite markers by testing loci from other species is limited.
cross-species amplification success varies not only between taxonomic groups, but also among microsatellite loci.
although many markers fail to amplify even in closely related species, some markers have higher utility than others .
a few loci, such as hru <dig>  <cit> , lei <dig>  <cit> , lox <dig>  <cit>  and man <dig>  <cit>  can be almost universally amplified across the avian taxa .
this suggests that some loci are more conserved than others.
since the degree of microsatellite conservation is usually not known at the time of their isolation, identifying conserved primers usually involves extensive primer testing and only a few conserved markers have been identified to date.
the charadriiformes order  is an ancient monophyletic avian order of  <dig> species  <cit>  that probably evolved around 79– <dig> million years ago  <cit> .
recently, the charadriiformes have become the focus of a number of studies in evolutionary biology because they harbour many species with an unusual diversity in mating and parental care strategies, flight metabolism, migratory behaviour and sexual size dimorphism  <cit> .
appropriate genetic markers would help to increase the understanding of, for example, the evolution of breeding systems and the connectivity between populations, but markers are available for fewer than  <dig> charadriiformes species.
additionally, many shorebird populations are declining and genetic markers are needed to monitor and manage their conservation effectively.
in this study, we examine the potential of utilising the available published charadriiformes microsatellite sequences and the sequenced chicken genome to identify conserved charadriiformes-chicken microsatellite loci.
initially, we mapped conserved charadriiformes microsatellites in the chicken genome.
second, we explored their cross-species utility across members of the order charadriiformes.
one concern is that conserved microsatellite loci are located in functional genomic regions and exhibit low or no polymorphism.
therefore, we compared polymorphism and heterozygosity levels across different charadriiform species.
third, we examined correlates of cross-species amplification success and polymorphism to predict the utility of other conserved microsatellite loci.
RESULTS
mapping
sixty-eight charadriiformes microsatellite sequences were assigned to a location on the chicken genome based on sequence homology .
two further sequences  showed homology to an unknown chicken homologue that had not yet been assigned to a chromosomal region.
sixty-four sequences were assigned to fourteen autosomal chicken chromosomes and four to the z chromosome .
the mapping of loci assigned to the z chromosome in chicken was validated in charadriiformes by analysing the genotypes of birds of known sex .
a location on the z was supported if all females were homozygous whilst at least some males exhibited heterozygous genotypes.
this was confirmed for all four loci assigned to the chicken z chromosome: calex- <dig> , bmatatc <dig> , mopl <dig>  and apy <dig> in whiskered auklet .
the microsatellite motif of the charadriiform sequences was not always retained in the homologous microsatellite loci identified in the chicken genome .
a comparison with the chicken genomic sequences revealed that the same microsatellite repeat motif was present in  <dig> sequences , a different microsatellite motif was found in  <dig> sequences  whilst no microsatellite repeat motif was found in  <dig>  of the sequences .
cross-species amplification
in total, we tested  <dig> 'standard primers'  from different conserved microsatellite sequences and  <dig> primers from anonymous microsatellite sequences.
in both groups a similar proportion of microsatellite loci was isolated in each of the three test species representing the suborders charadri, lari and scolopaci .
in  <dig> of  <dig> conserved sequences we obtained a specific product for all three species, whilst we did not obtain specific products for any of the anonymous sequences in all test species.
when we compared the proportion of species in which a primer set amplified a product, primers designed from conserved sequences significantly outperformed primers from anonymous sequences , df =  <dig>  w =  <dig>  p <  <dig> ).
for  <dig> conserved sequences we designed a second, consensus, primer set with primer binding sites in the conserved regions of the flanks.
cross-species amplification rates were higher for consensus primers than standard primers for the same microsatellite sequence .
amplification success increases when the annealing temperature is reduced  <cit> .
hence, the reason for the improvement of amplification could have been that consensus primer were designed and tested at lower annealing temperatures .
however,  <dig> out of  <dig>  consensus primers amplified best at annealing temperatures of 54°c  and the difference in amplification success between consensus and standard primers remained significant  when only the  <dig> loci were analysed.
twenty-three of  <dig> consensus primer pairs exhibited between one and three base-pair mismatches between the chicken and charadriiformes primer binding sites.
each mismatching primer base was replaced by a suitable degenerate base which included both of the possible bases.
the use of degenerate bases will dilute the effective concentration of the primer with the highest affinity to the target, which could potentially reduce amplification efficiency.
however, amplification success was not related to the number of degenerate bases per primer pair , df =  <dig>  χ <dig> =  <dig> , p =  <dig> ).
among conserved sequences, cross-species amplification success was only significantly associated with the e-value of a given sequence .
e-values for loci for which primers amplified in all tested species ranged from e- <dig>  to e- <dig> .
standard primers from sequences with lower e-values amplified in a higher proportion of charadriiform species than those with higher e-values  with binomial error structure: df =  <dig>  b = - <dig> , t = - <dig> , p =  <dig> ).
*model descriptives are given for models after removal of explanatory variables
1based on tests in three charadriiform species: kentish plover charadrius alexandrinus, whiskered auklet aethia pygmaea and ruff philomachus pugnax
2based on proportion of species in which a microsatellite locus was found to be polymorphic when tested in four individuals
3dinucleotide or tetranucleotide repeat motif of microsatellite
4total number of mismatches between chicken and charadriiform sequence for both primer sequences combined
5gga-consensus primer or standard primer design
6observed heterozygosity for source species given in original publication of the microsatellite sequence
7number of mismatches in primer sequence between charadriiform sequence and chicken homologue
8microsatellite sequence interrupted by non-motif base insertions  or not
polymorphism and observed heterozygosity
twenty-three of  <dig> conserved microsatellite loci exhibited two or more alleles in an average of  <dig> of the  <dig> species tested .
there was considerable variation among species in the number of polymorphic markers.
excluding the markers that had been isolated in the target species, we found on average that  <dig> of  <dig> markers per species  were polymorphic when tested in four unrelated individuals from a single population .
none of the  <dig> markers included in polymorphism tests were sex-linked based on the genotypes of known male and female birds  and on their chromosome location predicted from the assembled chicken genome .
the markers were tested in four unrelated individuals from a single population of each of  <dig> species of charadriiformes.
*expected product size in source species based on the sequence of the originally cloned allele.
kpl kentish plover , oys oystercatcher , avo avocet , gsh greater sheathbill , wau whiskered auklet , cpr collared pratincole , bsk brown skua , gbt gull-billed tern , ruf ruff , rnp red-necked phalarope , gsn great snipe , dun dunlin
the proportion of species in which a microsatellite was polymorphic was significantly associated with three factors: i) microsatellite motif, ii) repeat length and iii) whether the microsatellite was interrupted or not .
microsatellite loci with dinucleotide motifs were polymorphic in more species than those consisting of tetranucleotides .
microsatellites with longer repeat regions were polymorphic in more species than those with shorter repeat regions .
interruption of the microsatellite repeat regions reduced the proportion of species in which a locus was polymorphic .
when we examined the variability of loci in the larger kentish plover, whiskered auklet and ruff samples  we found that the mean observed heterozygosities across the three test species were lower than heterozygosities in the species in which a microsatellite had been originally isolated and characterised .
heterozygosity in all three test species declined with increasing genetic distance to the microsatellite source species, and the decline was highly significant in all three species combined .
an alternative model excluding those loci that had been developed in the target species gave the same qualitative results .
n, number of individuals amplified in polymorphism test
k, number of alleles found in test sample
ho, observed heterozygosity
he, expected heterozygosity
* estimated null allele frequency using cervus v.  <dig>
1probability that locus is in hardy-weinberg equilibrium estimated by genepop v  <dig>
all primers were tested in  <dig> unrelated individuals from single populations of each of kentish plover charadrius alexandrinus, whiskered auklet aethia pygmaea and ruff philomachus pugnax.
populations with significant deviation from hardy-weinberg equilibrium are indicated in bold.
discussion
we have shown that sequence information from annotated genomes can be used to identify and map conserved microsatellite loci.
our study has two major findings.
first, primers designed from conserved microsatellite loci amplify across a wider taxonomic range than those derived from anonymous microsatellite loci.
second, when highly conserved regions of the flanks of a microsatellite are used as the primer binding sites amplification success can be further improved.
correlates of cross-species amplification
amplification success was not associated with genetic distance between microsatellite source species and test species in this intraorder analysis of charadriiform microsatellite markers.
the e-values obtained from blast searches served as a better predictor for the width of taxonomic range in which a microsatellite could be amplified.
using  <dig> primers derived from conserved regions of the human genome, housley et al.
<cit>  identified the number of primer mismatches and primer gc content as factors that predicted amplification success in mammals.
in contrast to our study, housley et al.
investigated amplification success using generally conserved sequences in mammals and did not specifically use variable loci such as microsatellite loci.
second, they aligned genomic sequences from human with genomic sequences from dog, rat or mouse to perform intragroup-comparisons of amplification success, whilst we used an outgroup taxon  for sequence alignments and comparisons.
third, our sample size was much smaller than the one used for the intra-mammalian comparison due to the small number of microsatellite loci available from the charadriiformes.
the results of both studies are very similar, despite the large differences in study design.
sequence conservation  was the main predictor for amplification success of a genetic marker.
most loci with an e-value lower than e- <dig> in chicken amplified in all the tested species, suggesting that this could be a critical value that indicates the utility of a marker for cross-species amplification within the order charadriiformes.
consensus primer sets that had been designed to include the smallest number of mismatches between sequences amplified better than standard primers, in which no action to counteract the presence of mismatches had been taken.
amplification success of consensus primers was generally very high across the three suborders of charadriiformes .
the number of mismatches per primer pair did not affect amplification success among the consensus primers for a number of possible reasons.
first, we restricted the sequence mismatches between chicken and charadriiformes for consensus primer pairs to a maximum number of three.
second, we introduced degenerate bases to account for those mismatches.
under our primer design rules we attempted to minimise the number of mismatches and positioned mismatches away from the 3' end for a given primer, although the position of the mismatch/degenerate base did not affect amplification success significantly .
third, inclusion of degenerate bases might have led to amplification failure due to the reduced concentration of suitable primer.
however, we did not observe such failures, probably because the total primer concentration in our tests was relatively high .
thus the amplification success of our consensus primers does not suggest that primers do not have to be a good  match for amplification success.
primmer et al.
<cit>  proposed that the proportion of microsatellite loci that can be amplified declines with increasing genetic distance between source and target species.
our results suggest that the slope of the amplification decline is predominantly locus- specific and will largely depend on the conservation of the sequences flanking the microsatellite repeat.
microsatellite loci with highly conserved flanks can be amplified in more distantly related species whilst those with non-conserved flanks may be useful only in a very narrow taxonomic group.
polymorphism
polymorphism of conserved loci varied greatly between species.
the proportion of species in which a microsatellite exhibited more than one allele was associated with microsatellite repeat length, motif and whether it was interrupted or not.
our results are consistent with previous theoretical and empirical studies that examined the effect of these microsatellite properties on mutation rate  <cit>  and polymorphism  <cit> .
a positive association between repeat length and polymorphism was found empirically in other vertebrates, arthropods and plants  <cit> .
dinucleotide microsatellite loci exhibited higher mutation rates than tetranucleotide microsatellites in mice and yeast  <cit> .
in humans and chimpanzees, microsatellite loci with interrupted repeat regions had a two-fold decrease in the mutation rate, which was interpreted as being due to interruptions reducing the opportunities for replication slippage  <cit> .
although we showed that the amplification success of existing microsatellite markers can be improved by redesigning primer sets, heterozygosity was generally lower in test than source species.
polymorphism declines faster with evolutionary distance than amplification success  <cit> .
a possible explanation is that polymorphism of many loci evolved in the recent evolutionary past and therefore is confined to a phylogenetically narrow range of taxa.
this argument is supported by the findings of other studies in amphibians, birds and mammals  <cit>  that show that the probability of polymorphism drops rapidly with increasing evolutionary distance between source and target species.
however, the steepness of the decline appears to be locus-specific.
there was large variation in the taxonomic range over which a given microsatellite was polymorphic.
for instance, the locus bmatgaa <dig> was polymorphic in only two of twelve species when tested in four unrelated individuals, whilst another locus, bmatatc <dig> isolated in the same species and with a similar repeat length, was polymorphic in seven of twelve species tested.
of the  <dig> charadriiform microsatellite markers we tested, a median of  <dig> markers was polymorphic per species .
five or more polymorphic markers were found in five of the six test species where previously no microsatellite markers had been identified.
for another six species where markers had already been characterised, we found between three and  <dig> new polymorphic markers.
the number of species in which these  <dig> markers are useful is likely to increase because we tested them only in  <dig> of the  <dig> species of charadriiformes.
finally, we assessed the variability of markers only within a single population of each species to make the detected level of polymorphism comparable to the polymorphism in the source population.
some markers that we found to be monomorphic may exhibit population-specific polymorphism or have different alleles fixed in different populations and turn out to be useful to investigate population differentiation.
the observed decline of polymorphism in relation to the genetic distance to the source species could be partly explained by the selection process during the isolation of microsatellite sequences .
during the construction of microsatellite libraries, typically long microsatellite sequences with 10– <dig> repeat units are selected to maximise the probability that a locus is polymorphic in the species in which it is developed.
moreover, only the sequences of the polymorphic loci are normally submitted to sequence databases, which means that monomorphic loci with fewer repeat units in the source species are lost because they are not reported.
however, repeat expansion and microsatellite polymorphism are likely to reflect the recent evolutionary history.
therefore the submission of sequences of monomorphic loci to genomic databases might enable the identification of further conserved markers and the development of useful markers through cross-species amplification.
differences in the degree of microsatellite polymorphism among species are not exclusively attributable to recent divergence in microsatellite evolution.
genetic diversity, which is often reflected by microsatellite polymorphism varies among populations and species.
low microsatellite polymorphism can indicate depleted genetic variability due to bottlenecks, genetic drift or inbreeding.
if genetic diversity for a given population is low, a combination of screening of known microsatellite loci and the development of microsatellite markers using the conventional library approach may be helpful in finding a suitable set of polymorphic markers.
only a handful of shorebird populations have been investigated for genetic diversity.
low genetic variability of both allozymes and of the mitochondrial control region has been found in several species of sandpipers that breed in the high arctic and it has been hypothesized that historical population fluctuations that occurred during and after glaciations are responsible for this low genetic diversity  <cit> .
in our study, the greater sheathbill chionis alba showed the least genetic diversity, being monomorphic at all  <dig> microsatellite loci that we examined .
greater sheathbills breed exclusively in the antarctic, where they live as scavengers close to other bird colonies.
current population estimates give a stable total number of approximately  <dig>  sheathbills  <cit> , but past climatic fluctuations may have led to a small effective population size similar to those of arctic breeders.
thus the low observed microsatellite diversity might reflect a recent population recovery.
alternatively, the evolutionary distance between sheathbills and the source species from which we derived the tested microsatellites is too large, with the microsatellite being lost or all polymorphism being depleted.
different genetic markers, such as markers from the mitochondrial control region, other microsatellite markers or highly variable nuclear genes, such as genes of the major histocompatibility complex, need to be examined to determine whether the low microsatellite variability truly reflects a general low genetic diversity in sheathbills.
contrary to sheathbills, whiskered auklets and kentish plovers showed the highest genetic diversity in our analysis.
in the kentish plover and the whiskered auklet, twelve of the  <dig> microsatellite loci tested were found to be polymorphic when tested in four individuals.
excluding the markers that had been isolated in both species leads to ten  and eleven  newly described polymorphic markers.
both species live in very different habitats and geographical locations.
whiskered auklets are pelagic feeders that inhabit a number of small islands in the northern pacific, whilst kentish plovers are cosmopolitans and found at beaches and saline lakes in temperate and subtropical regions  <cit> .
the high genetic diversity in both species is reflected in the observed heterozygosities at microsatellite loci that had both been identified in these species by cross-species amplification  and isolated from enriched genomic libraries  <cit> .
the high variability of many microsatellite loci in these species suggests that depletion of genetic variation is not a general characteristic of the charadriiformes order, but rather an attribute of certain species or populations due to their historical demography and phylogeography.
the possibilities for the application of conserved markers go beyond examining genetic diversity.
polymorphic conserved markers can be used, for example, to investigate chromosomal organisation by constructing linkage maps  <cit> .
a major advantage of conserved over conventional markers is that the same loci can be used to investigate and so compare chromosomal structure and genomic organisation among several different species  <cit> .
the sequence conservation of flanking regions can be the result of a direct functional role or linkage disequilibrium with functional genomic regions .
selection pressures may affect the variability of a locus by either restricting polymorphism  <cit>  or promoting polymorphism if variability is adaptive  <cit> .
this can be problematic for applications of genetic markers that assume their neutrality.
however, for common applications such as parentage assignment or estimating relatedness such markers will nonetheless be useful.
furthermore, if a marker is found to be associated with a locus that is under selection, its function can be explored and changes or retention of functionality can be compared under different environmental conditions, and across different populations and/or taxa.
the conserved markers we designed and characterised are very convenient to use.
all consensus primer sets for the polymorphic loci amplify under similar pcr conditions , which facilitates i) quick and economical screening for amplification and polymorphism in new target species and ii) efficient processing, since several loci can be run together in a single multiplex pcr.
dealing with null alleles
five of the  <dig> primer sets that we tested for heterozygosity had high estimated null allele frequencies  in one of the three test species .
there was no obvious relationship between the departure from hardy-weinberg equilibrium and the number or position of degenerate bases in each primer pair.
null alleles arise when the primer sequence does not match the target sequence of a given allele and the allele therefore fails to amplify.
if not corrected for, the presence of null alleles may interfere with algorithms to estimate relatedness  <cit> .
sequencing the locus in the study species and redesigning the primers can be used to prevent the occurrence of null alleles.
alternatively, if the proportion of null alleles is low, their impact on relatedness estimates can be reduced by using maximum-likelihood correction methods when computing relatedness relationships  <cit> .
development of conserved markers in other avian groups
charadriiformes, chicken and most other modern birds belong to the neognathae.
recent molecular data suggest that the galliformes, together with the anseriformes, form a sister taxon to the other neognath birds  <cit>  and therefore have the same phylogenetic distance to all neognathae.
flanking regions of about one in seven charadriiform microsatellite loci were found to be conserved in chicken.
since the proportion of microsatellite homologues is likely to be associated with the phylogenetic distance between genomic resource species and source species, we expect a similar proportion of conserved microsatellite loci to be found between chicken and other neognath groups to that observed here between chicken and the charadriiformes.
in fact, for taxa that are more closely related to chicken  we predict an even higher success rate in identifying suitable microsatellite markers through data mining.
genomic sequencing of further organisms will facilitate the use of already- characterised microsatellite loci for designing consensus primer sets.
in birds, the sequenced genome of another neognath bird, the zebra finch  is now available .
the zebra finch is phylogenetically closer to charadriiformes and other neognath birds than is the chicken, hence more microsatellite homologues and conserved markers might now be obtained using zebra finch sequences as a reference.
CONCLUSIONS
we have shown that sequence information available from genomic databases can be used to enhance the utility of microsatellite markers in studies of evolution and conservation, even for taxonomic groups where few sequence data are yet available.
sequence information of translated and untranslated parts of the genome are useful for comparing and designing consensus primers, even when they involve genetically distantly related taxa such as charadriiformes and galliformes.
cross-species amplification tests can be carried out more efficiently by identifying and utilising conserved microsatellite loci that will amplify across a broader taxonomic range.
by selecting highly conserved regions of the microsatellite flanking sequence for primer design, the number of species in which a locus will amplify can be increased even further.
we found that markers derived from conserved loci with an e-value of e- <dig> or lower amplified across the entire charadriiform order.
our findings will facilitate the use of markers in species where no markers have yet been identified and in species where more markers are needed.
to date,  <dig> vertebrate and  <dig> invertebrate genomes have been sequenced and fully assembled .
this number is expected to increase rapidly as sequencing costs decrease.
the methodology we have outlined will make it possible to extend population genetic and evolutionary studies to further non-model species that have been previously neglected because of a lack of sufficient genetic markers.

 microsatellites or short sequence repeats are relatively small 1– base-pair tandem repeats that are found in the genomic dna of pro- and eukaryotes. although the majority of microsatellites are located in non-coding sequences and considered to be selectively neutral, some microsatellite loci are located in functional regions and involved in chromatin organisation, regulation of gene activity and metabolic processes such as dna replication and recombination. microsatellites exhibit high mutation rates of 10- to 10- per locus per generation. the main mutational processes responsible for the variability in microsatellites are considered to be replication-slippage and recombination. both processes change the length of the microsatellite by altering the number of repeats of the microsatellite. microsatellite variability has been associated with a number of microsatellite characteristics. mutation rates of microsatellites have been found to be taxon-specific. microsatellite variability covaries with allozyme diversity in a taxon. the number of repeats can predict the variability and stability of a microsatellite motif, with longer loci found to be more variable, but also more unstable than shorter ones. the type of microsatellite motif may affect abundance and variability. for example, it was found that microsatellites with tri- or hexanucleotide motifs are more frequent in coding than in non-coding regions, possibly because mutations of these microsatellites in coding regions are less likely to result in deleterious frameshift mutations. the high variability of microsatellite markers and their straightforward analysis using the polymerase chain reaction have led to their frequent application in studies on natural populations. however, one obstacle to the wider application of microsatellites is the difficulty of developing a sufficient number of suitable markers for any given species. although microsatellites are ubiquitous in eukaryotic organisms, their abundance varies across different groups. microsatellites are less common, for example, in birds than in other vertebrates. there are two principal strategies to obtain microsatellite markers. first, microsatellite markers can be developed by screening genomic libraries. success rates differ according to the protocol and taxon, although usually a medium to high number of polymorphic markers can be isolated using this approach. since microsatellites are present at a relatively low frequency in avian genomes, in this case their isolation is most efficient using enrichment protocols. this involves many stages, is a skilled and time-consuming process and requires significant funding and a well-equipped molecular laboratory, which are not always available in ecological and conservation research. the second method makes use of existing microsatellite markers isolated in different species to the species of interest. for cross-species amplification tests existing primers developed in related species are tested for amplification and polymorphism in the target species. one drawback of cross-species amplification is that success rates decline with evolutionary distance between the target species and source species. in birds, most microsatellite markers have been developed for the orders passeriformes and galliformes. passeriformes is a species-rich relatively recent clade in which more than microsatellite markers have been characterised. several studies have successfully identified additional polymorphic loci by cross-species testing in birds. the development of complete primer sets from cross-species amplification tests has been successful in falconiformes, galliformes and passeriformes. however, in many other avian orders fewer microsatellite loci have been isolated and, therefore, the opportunity to develop microsatellite markers by testing loci from other species is limited. cross-species amplification success varies not only between taxonomic groups, but also among microsatellite loci. although many markers fail to amplify even in closely related species, some markers have higher utility than others. a few loci, such as hru, lei, lox and man can be almost universally amplified across the avian taxa. this suggests that some loci are more conserved than others. since the degree of microsatellite conservation is usually not known at the time of their isolation, identifying conserved primers usually involves extensive primer testing and only a few conserved markers have been identified to date. the charadriiformes order is an ancient monophyletic avian order of species that probably evolved around 79– million years ago. recently, the charadriiformes have become the focus of a number of studies in evolutionary biology because they harbour many species with an unusual diversity in mating and parental care strategies, flight metabolism, migratory behaviour and sexual size dimorphism. appropriate genetic markers would help to increase the understanding of, for example, the evolution of breeding systems and the connectivity between populations, but markers are available for fewer than charadriiformes species. additionally, many shorebird populations are declining and genetic markers are needed to monitor and manage their conservation effectively. in this study, we examine the potential of utilising the available published charadriiformes microsatellite sequences and the sequenced chicken genome to identify conserved charadriiformes-chicken microsatellite loci. initially, we mapped conserved charadriiformes microsatellites in the chicken genome. second, we explored their cross-species utility across members of the order charadriiformes. one concern is that conserved microsatellite loci are located in functional genomic regions and exhibit low or no polymorphism. therefore, we compared polymorphism and heterozygosity levels across different charadriiform species. third, we examined correlates of cross-species amplification success and polymorphism to predict the utility of other conserved microsatellite loci.  mapping sixty-eight charadriiformes microsatellite sequences were assigned to a location on the chicken genome based on sequence homology. two further sequences showed homology to an unknown chicken homologue that had not yet been assigned to a chromosomal region. sixty-four sequences were assigned to fourteen autosomal chicken chromosomes and four to the z chromosome. the mapping of loci assigned to the z chromosome in chicken was validated in charadriiformes by analysing the genotypes of birds of known sex. a location on the z was supported if all females were homozygous whilst at least some males exhibited heterozygous genotypes. this was confirmed for all four loci assigned to the chicken z chromosome: calex-, bmatatc, mopl and apy in whiskered auklet. the microsatellite motif of the charadriiform sequences was not always retained in the homologous microsatellite loci identified in the chicken genome. a comparison with the chicken genomic sequences revealed that the same microsatellite repeat motif was present in sequences, a different microsatellite motif was found in sequences whilst no microsatellite repeat motif was found in of the sequences. cross-species amplification in total, we tested'standard primers' from different conserved microsatellite sequences and primers from anonymous microsatellite sequences. in both groups a similar proportion of microsatellite loci was isolated in each of the three test species representing the suborders charadri, lari and scolopaci. in of conserved sequences we obtained a specific product for all three species, whilst we did not obtain specific products for any of the anonymous sequences in all test species. when we compared the proportion of species in which a primer set amplified a product, primers designed from conserved sequences significantly outperformed primers from anonymous sequences, df = w = p < ). for conserved sequences we designed a second, consensus, primer set with primer binding sites in the conserved regions of the flanks. cross-species amplification rates were higher for consensus primers than standard primers for the same microsatellite sequence. amplification success increases when the annealing temperature is reduced. hence, the reason for the improvement of amplification could have been that consensus primer were designed and tested at lower annealing temperatures. however, out of consensus primers amplified best at annealing temperatures of 54°c and the difference in amplification success between consensus and standard primers remained significant when only the loci were analysed. twenty-three of consensus primer pairs exhibited between one and three base-pair mismatches between the chicken and charadriiformes primer binding sites. each mismatching primer base was replaced by a suitable degenerate base which included both of the possible bases. the use of degenerate bases will dilute the effective concentration of the primer with the highest affinity to the target, which could potentially reduce amplification efficiency. however, amplification success was not related to the number of degenerate bases per primer pair, df = χ =, p = ). among conserved sequences, cross-species amplification success was only significantly associated with the e-value of a given sequence. e-values for loci for which primers amplified in all tested species ranged from e- to e-. standard primers from sequences with lower e-values amplified in a higher proportion of charadriiform species than those with higher e-values with binomial error structure: df = b = -, t = -, p = ). *model descriptives are given for models after removal of explanatory variables 1based on tests in three charadriiform species: kentish plover charadrius alexandrinus, whiskered auklet aethia pygmaea and ruff philomachus pugnax 2based on proportion of species in which a microsatellite locus was found to be polymorphic when tested in four individuals 3dinucleotide or tetranucleotide repeat motif of microsatellite 4total number of mismatches between chicken and charadriiform sequence for both primer sequences combined 5gga-consensus primer or standard primer design 6observed heterozygosity for source species given in original publication of the microsatellite sequence 7number of mismatches in primer sequence between charadriiform sequence and chicken homologue 8microsatellite sequence interrupted by non-motif base insertions or not polymorphism and observed heterozygosity twenty-three of conserved microsatellite loci exhibited two or more alleles in an average of of the species tested. there was considerable variation among species in the number of polymorphic markers. excluding the markers that had been isolated in the target species, we found on average that of markers per species were polymorphic when tested in four unrelated individuals from a single population. none of the markers included in polymorphism tests were sex-linked based on the genotypes of known male and female birds and on their chromosome location predicted from the assembled chicken genome. the markers were tested in four unrelated individuals from a single population of each of species of charadriiformes. *expected product size in source species based on the sequence of the originally cloned allele. kpl kentish plover, oys oystercatcher, avo avocet, gsh greater sheathbill, wau whiskered auklet, cpr collared pratincole, bsk brown skua, gbt gull-billed tern, ruf ruff, rnp red-necked phalarope, gsn great snipe, dun dunlin the proportion of species in which a microsatellite was polymorphic was significantly associated with three factors: i) microsatellite motif, ii) repeat length and iii) whether the microsatellite was interrupted or not. microsatellite loci with dinucleotide motifs were polymorphic in more species than those consisting of tetranucleotides. microsatellites with longer repeat regions were polymorphic in more species than those with shorter repeat regions. interruption of the microsatellite repeat regions reduced the proportion of species in which a locus was polymorphic. when we examined the variability of loci in the larger kentish plover, whiskered auklet and ruff samples we found that the mean observed heterozygosities across the three test species were lower than heterozygosities in the species in which a microsatellite had been originally isolated and characterised. heterozygosity in all three test species declined with increasing genetic distance to the microsatellite source species, and the decline was highly significant in all three species combined. an alternative model excluding those loci that had been developed in the target species gave the same qualitative results. n, number of individuals amplified in polymorphism test k, number of alleles found in test sample ho, observed heterozygosity he, expected heterozygosity * estimated null allele frequency using cervus v. 1probability that locus is in hardy-weinberg equilibrium estimated by genepop v all primers were tested in unrelated individuals from single populations of each of kentish plover charadrius alexandrinus, whiskered auklet aethia pygmaea and ruff philomachus pugnax. populations with significant deviation from hardy-weinberg equilibrium are indicated in bold. discussion we have shown that sequence information from annotated genomes can be used to identify and map conserved microsatellite loci. our study has two major findings. first, primers designed from conserved microsatellite loci amplify across a wider taxonomic range than those derived from anonymous microsatellite loci. second, when highly conserved regions of the flanks of a microsatellite are used as the primer binding sites amplification success can be further improved. correlates of cross-species amplification amplification success was not associated with genetic distance between microsatellite source species and test species in this intraorder analysis of charadriiform microsatellite markers. the e-values obtained from blast searches served as a better predictor for the width of taxonomic range in which a microsatellite could be amplified. using primers derived from conserved regions of the human genome, housley et al. identified the number of primer mismatches and primer gc content as factors that predicted amplification success in mammals. in contrast to our study, housley et al. investigated amplification success using generally conserved sequences in mammals and did not specifically use variable loci such as microsatellite loci. second, they aligned genomic sequences from human with genomic sequences from dog, rat or mouse to perform intragroup-comparisons of amplification success, whilst we used an outgroup taxon for sequence alignments and comparisons. third, our sample size was much smaller than the one used for the intra-mammalian comparison due to the small number of microsatellite loci available from the charadriiformes. the results of both studies are very similar, despite the large differences in study design. sequence conservation was the main predictor for amplification success of a genetic marker. most loci with an e-value lower than e- in chicken amplified in all the tested species, suggesting that this could be a critical value that indicates the utility of a marker for cross-species amplification within the order charadriiformes. consensus primer sets that had been designed to include the smallest number of mismatches between sequences amplified better than standard primers, in which no action to counteract the presence of mismatches had been taken. amplification success of consensus primers was generally very high across the three suborders of charadriiformes. the number of mismatches per primer pair did not affect amplification success among the consensus primers for a number of possible reasons. first, we restricted the sequence mismatches between chicken and charadriiformes for consensus primer pairs to a maximum number of three. second, we introduced degenerate bases to account for those mismatches. under our primer design rules we attempted to minimise the number of mismatches and positioned mismatches away from the 3' end for a given primer, although the position of the mismatch/degenerate base did not affect amplification success significantly. third, inclusion of degenerate bases might have led to amplification failure due to the reduced concentration of suitable primer. however, we did not observe such failures, probably because the total primer concentration in our tests was relatively high. thus the amplification success of our consensus primers does not suggest that primers do not have to be a good match for amplification success. primmer et al. proposed that the proportion of microsatellite loci that can be amplified declines with increasing genetic distance between source and target species. our results suggest that the slope of the amplification decline is predominantly locus- specific and will largely depend on the conservation of the sequences flanking the microsatellite repeat. microsatellite loci with highly conserved flanks can be amplified in more distantly related species whilst those with non-conserved flanks may be useful only in a very narrow taxonomic group. polymorphism polymorphism of conserved loci varied greatly between species. the proportion of species in which a microsatellite exhibited more than one allele was associated with microsatellite repeat length, motif and whether it was interrupted or not. our results are consistent with previous theoretical and empirical studies that examined the effect of these microsatellite properties on mutation rate and polymorphism. a positive association between repeat length and polymorphism was found empirically in other vertebrates, arthropods and plants. dinucleotide microsatellite loci exhibited higher mutation rates than tetranucleotide microsatellites in mice and yeast. in humans and chimpanzees, microsatellite loci with interrupted repeat regions had a two-fold decrease in the mutation rate, which was interpreted as being due to interruptions reducing the opportunities for replication slippage. although we showed that the amplification success of existing microsatellite markers can be improved by redesigning primer sets, heterozygosity was generally lower in test than source species. polymorphism declines faster with evolutionary distance than amplification success. a possible explanation is that polymorphism of many loci evolved in the recent evolutionary past and therefore is confined to a phylogenetically narrow range of taxa. this argument is supported by the findings of other studies in amphibians, birds and mammals that show that the probability of polymorphism drops rapidly with increasing evolutionary distance between source and target species. however, the steepness of the decline appears to be locus-specific. there was large variation in the taxonomic range over which a given microsatellite was polymorphic. for instance, the locus bmatgaa was polymorphic in only two of twelve species when tested in four unrelated individuals, whilst another locus, bmatatc isolated in the same species and with a similar repeat length, was polymorphic in seven of twelve species tested. of the charadriiform microsatellite markers we tested, a median of markers was polymorphic per species. five or more polymorphic markers were found in five of the six test species where previously no microsatellite markers had been identified. for another six species where markers had already been characterised, we found between three and new polymorphic markers. the number of species in which these markers are useful is likely to increase because we tested them only in of the species of charadriiformes. finally, we assessed the variability of markers only within a single population of each species to make the detected level of polymorphism comparable to the polymorphism in the source population. some markers that we found to be monomorphic may exhibit population-specific polymorphism or have different alleles fixed in different populations and turn out to be useful to investigate population differentiation. the observed decline of polymorphism in relation to the genetic distance to the source species could be partly explained by the selection process during the isolation of microsatellite sequences. during the construction of microsatellite libraries, typically long microsatellite sequences with 10– repeat units are selected to maximise the probability that a locus is polymorphic in the species in which it is developed. moreover, only the sequences of the polymorphic loci are normally submitted to sequence databases, which means that monomorphic loci with fewer repeat units in the source species are lost because they are not reported. however, repeat expansion and microsatellite polymorphism are likely to reflect the recent evolutionary history. therefore the submission of sequences of monomorphic loci to genomic databases might enable the identification of further conserved markers and the development of useful markers through cross-species amplification. differences in the degree of microsatellite polymorphism among species are not exclusively attributable to recent divergence in microsatellite evolution. genetic diversity, which is often reflected by microsatellite polymorphism varies among populations and species. low microsatellite polymorphism can indicate depleted genetic variability due to bottlenecks, genetic drift or inbreeding. if genetic diversity for a given population is low, a combination of screening of known microsatellite loci and the development of microsatellite markers using the conventional library approach may be helpful in finding a suitable set of polymorphic markers. only a handful of shorebird populations have been investigated for genetic diversity. low genetic variability of both allozymes and of the mitochondrial control region has been found in several species of sandpipers that breed in the high arctic and it has been hypothesized that historical population fluctuations that occurred during and after glaciations are responsible for this low genetic diversity. in our study, the greater sheathbill chionis alba showed the least genetic diversity, being monomorphic at all microsatellite loci that we examined. greater sheathbills breed exclusively in the antarctic, where they live as scavengers close to other bird colonies. current population estimates give a stable total number of approximately sheathbills, but past climatic fluctuations may have led to a small effective population size similar to those of arctic breeders. thus the low observed microsatellite diversity might reflect a recent population recovery. alternatively, the evolutionary distance between sheathbills and the source species from which we derived the tested microsatellites is too large, with the microsatellite being lost or all polymorphism being depleted. different genetic markers, such as markers from the mitochondrial control region, other microsatellite markers or highly variable nuclear genes, such as genes of the major histocompatibility complex, need to be examined to determine whether the low microsatellite variability truly reflects a general low genetic diversity in sheathbills. contrary to sheathbills, whiskered auklets and kentish plovers showed the highest genetic diversity in our analysis. in the kentish plover and the whiskered auklet, twelve of the microsatellite loci tested were found to be polymorphic when tested in four individuals. excluding the markers that had been isolated in both species leads to ten and eleven newly described polymorphic markers. both species live in very different habitats and geographical locations. whiskered auklets are pelagic feeders that inhabit a number of small islands in the northern pacific, whilst kentish plovers are cosmopolitans and found at beaches and saline lakes in temperate and subtropical regions. the high genetic diversity in both species is reflected in the observed heterozygosities at microsatellite loci that had both been identified in these species by cross-species amplification and isolated from enriched genomic libraries. the high variability of many microsatellite loci in these species suggests that depletion of genetic variation is not a general characteristic of the charadriiformes order, but rather an attribute of certain species or populations due to their historical demography and phylogeography. the possibilities for the application of conserved markers go beyond examining genetic diversity. polymorphic conserved markers can be used, for example, to investigate chromosomal organisation by constructing linkage maps. a major advantage of conserved over conventional markers is that the same loci can be used to investigate and so compare chromosomal structure and genomic organisation among several different species. the sequence conservation of flanking regions can be the result of a direct functional role or linkage disequilibrium with functional genomic regions. selection pressures may affect the variability of a locus by either restricting polymorphism or promoting polymorphism if variability is adaptive. this can be problematic for applications of genetic markers that assume their neutrality. however, for common applications such as parentage assignment or estimating relatedness such markers will nonetheless be useful. furthermore, if a marker is found to be associated with a locus that is under selection, its function can be explored and changes or retention of functionality can be compared under different environmental conditions, and across different populations and/or taxa. the conserved markers we designed and characterised are very convenient to use. all consensus primer sets for the polymorphic loci amplify under similar pcr conditions, which facilitates i) quick and economical screening for amplification and polymorphism in new target species and ii) efficient processing, since several loci can be run together in a single multiplex pcr. dealing with null alleles five of the primer sets that we tested for heterozygosity had high estimated null allele frequencies in one of the three test species. there was no obvious relationship between the departure from hardy-weinberg equilibrium and the number or position of degenerate bases in each primer pair. null alleles arise when the primer sequence does not match the target sequence of a given allele and the allele therefore fails to amplify. if not corrected for, the presence of null alleles may interfere with algorithms to estimate relatedness. sequencing the locus in the study species and redesigning the primers can be used to prevent the occurrence of null alleles. alternatively, if the proportion of null alleles is low, their impact on relatedness estimates can be reduced by using maximum-likelihood correction methods when computing relatedness relationships. development of conserved markers in other avian groups charadriiformes, chicken and most other modern birds belong to the neognathae. recent molecular data suggest that the galliformes, together with the anseriformes, form a sister taxon to the other neognath birds and therefore have the same phylogenetic distance to all neognathae. flanking regions of about one in seven charadriiform microsatellite loci were found to be conserved in chicken. since the proportion of microsatellite homologues is likely to be associated with the phylogenetic distance between genomic resource species and source species, we expect a similar proportion of conserved microsatellite loci to be found between chicken and other neognath groups to that observed here between chicken and the charadriiformes. in fact, for taxa that are more closely related to chicken we predict an even higher success rate in identifying suitable microsatellite markers through data mining. genomic sequencing of further organisms will facilitate the use of already- characterised microsatellite loci for designing consensus primer sets. in birds, the sequenced genome of another neognath bird, the zebra finch is now available. the zebra finch is phylogenetically closer to charadriiformes and other neognath birds than is the chicken, hence more microsatellite homologues and conserved markers might now be obtained using zebra finch sequences as a reference.  we have shown that sequence information available from genomic databases can be used to enhance the utility of microsatellite markers in studies of evolution and conservation, even for taxonomic groups where few sequence data are yet available. sequence information of translated and untranslated parts of the genome are useful for comparing and designing consensus primers, even when they involve genetically distantly related taxa such as charadriiformes and galliformes. cross-species amplification tests can be carried out more efficiently by identifying and utilising conserved microsatellite loci that will amplify across a broader taxonomic range. by selecting highly conserved regions of the microsatellite flanking sequence for primer design, the number of species in which a locus will amplify can be increased even further. we found that markers derived from conserved loci with an e-value of e- or lower amplified across the entire charadriiform order. our findings will facilitate the use of markers in species where no markers have yet been identified and in species where more markers are needed. to date, vertebrate and invertebrate genomes have been sequenced and fully assembled. this number is expected to increase rapidly as sequencing costs decrease. the methodology we have outlined will make it possible to extend population genetic and evolutionary studies to further non-model species that have been previously neglected because of a lack of sufficient genetic markers. 
to examine the utility of conserved microsatellite loci as genetic markers we collated a database of charadriiformes microsatellites, searched for homologues in the chicken genome and tested conserved markers for amplification and polymorphism in a range of charadriiform species. fifty-five primers designed from conserved microsatellite loci with an e-value of e- or lower amplified across a wider range of charadriiform species than a control group of primers from ten anonymous microsatellite loci. sixty-eight of charadriiform microsatellite loci were assigned to a single location in the chicken genome based on their e-value. despite their popularity, the characterisation of polymorphic microsatellite loci and development of suitable markers takes considerable effort. by maximising primer sequence similarity between source species and database species, markers can be further improved and provide additional markers to study the molecular ecology of populations of non-model organisms. microsatellite markers are popular genetic markers frequently used in forensic biology. newly-available genomic databases make it feasible to identify conserved genetic markers. however, research has been largely restrained by the limited availability of genetic markers. genomic sequence databases are useful tools to identify conserved genetic markers including those located in non-coding regions. we examined the utility and characteristics of conserved microsatellite markers in charadriiformes.
 microsatellite markers are popular genetic markers frequently used in forensic biology. despite their popularity, the characterisation of polymorphic microsatellite loci and development of suitable markers takes considerable effort. newly-available genomic databases make it feasible to identify conserved genetic markers. we examined the utility and characteristics of conserved microsatellite markers in charadriiformes. this order harbours many species with diverse breeding systems, life histories and extraordinary migration biology whose genetics warrant investigation. however, research has been largely restrained by the limited availability of genetic markers. to examine the utility of conserved microsatellite loci as genetic markers we collated a database of charadriiformes microsatellites, searched for homologues in the chicken genome and tested conserved markers for amplification and polymorphism in a range of charadriiform species.  sixty-eight of charadriiform microsatellite loci were assigned to a single location in the chicken genome based on their e-value. fifty-five primers designed from conserved microsatellite loci with an e-value of e- or lower amplified across a wider range of charadriiform species than a control group of primers from ten anonymous microsatellite loci. twenty-three of examined conserved markers were polymorphic, each in on average of species tested.  genomic sequence databases are useful tools to identify conserved genetic markers including those located in non-coding regions. by maximising primer sequence similarity between source species and database species, markers can be further improved and provide additional markers to study the molecular ecology of populations of non-model organisms. 