Exploring the causes of heterozygosity-fitness correlations in the blue tit

We have characterized a set of 106 microsatellite markers in 26-127 individual blue tits (Cyanistes caeruleus), and assigned their location on the zebra finch (Taeniopygia guttata) and on the chicken (Gallus gallus) genome based on sequence homology. Thirtyone markers are newly designed from zebra finch EST sequences, 22 markers were developed by others from EST sequences using different methods, and the remaining 53 loci were previously designed or modified passerine markers. The 106 microsatellite markers are distributed over 26 and 24 chromosomes in the zebra finch and in the chicken genome, respectively, and the number of alleles varies between two and 49. Eight loci deviate significantly from Hardy-Weinberg equilibrium and show a high frequency of null alleles, and three pairs of markers located in the same chromosome appear to be in linkage disequilibrium. With the exception of these few loci, the polymorphic microsatellite markers presented here provide a useful genome-wide resource for population and evolutionary genetic studies of the blue tit, in addition to their potential utility in other passerine birds. Published as: Olano-Marin J, Dawson DA, Girg A, Hansson B, Ljungqvist M, Kempenaers B, Mueller JC (2010) A genome-wide set of 106 microsatellite markers for the blue tit (Cyanistes caeruleus). Molecular Ecology Resources 10:516-532. Heterozygosity-fitness correlations in the blue tit 18 Research on passerine birds has provided important insights to evolutionary biology. For instance, quantitative genetic studies on this group have enhanced our knowledge on the associations between heritability, selection and microevolution of fitness-related traits (Merilä et al. 2001; MacColl and Hatchwell 2003; Raberg et al. 2003; Charmantier et al. 2004; Garant et al. 2005; Postma and van Noordwijk 2005). Recent advances in avian genomics, in particular the release of the genome sequence assembly of the red jungle fowl Gallus gallus (International Chicken Genome Sequencing Consortium 2004), and the zebra finch Taeniopygia guttata genome assembly (Warren et al. 2010), are promising for the development of genetic and genomic resources for passerines. In this context, the design of a large set of genome-wide distributed polymorphic markers, which are anchored in the avian reference genomes, would be of particular interest for commonly studied passerine species. Such a marker set opens up the possibility to build linkage maps, conduct comparative genomics, map quantitative trait loci (QTL), understand heterozygosity–fitness correlations and reveal the underlying genetic basis of phenotypic variation and adaptive evolution. In the past few years, EST (Expressed Sequence Tags) collections of model organisms have been used as a source to develop molecular markers and to explore the genome of related non-model species of interest to ecologists and evolutionary biologists (Kantety et al. 2002; Rexroad et al. 2005; Karaiskou et al. 2008; reviewed in Bouck and Vision 2007). The mining of SSR (Simple Sequence Repeats) from EST sequence databases has proven to be a useful, inexpensive and fast approach to identify microsatellite loci in several species (e.g. Perez et al. 2005; Kong et al. 2007; Slate et al. 2007; Kim et al. 2008; Tang et al. 2008). Another advantage of EST-SSRs over markers designed by cloning and sequencing, is their higher transferability between species, and even between genera when compared to anonymous microsatellites (e.g. Cordeiro et al. 2001; Bouck and Vision 2007; Karaiskou et al. 2008; Dawson et al. 2010). An additional valuable resource for the development of markers in an array of bird species, is the high numbers of passerine microsatellite loci deposited on public sequence databases (e.g. EMBL, GenBank). Details for 550 passerine microsatellite loci were compiled by Dawson et al. (2006), and the Passerine BIRDMARKER Database presents data of the cross-species utility of a large number of markers in a wide range of passerine species (http://www.shef.ac.uk/misc/groups/molecol/deborah-dawson-birdmarkers.html). Many of these markers are polymorphic only in the species where they were isolated, Chapter 1: Microsatellite markers 19 although cross-species amplification success generally increases as the genetic distance between the species decreases (Primmer et al. 1996; Dawson et al. 2000; Galbusera et al. 2000). Indeed, microsatellites originally isolated in a diverse set of bird species were used for the construction of the first linkage map in any passerine, the great reed warbler Acrocephalus arundinaceus (Hansson et al. 2005). The blue tit, Cyanistes caeruleus, is a common European passerine bird that has been the focus of numerous ecological and behavioral studies in the wild (e.g. Doutrelant et al. 1999; Foerster et al. 2003; Tremblay et al. 2003; Valcu and Kempenaers 2008). Around 20 polymorphic microsatellite markers have been previously described for this species, either by isolation from blue tit genomic libraries (Dawson et al. 2000), by testing markers originally isolated in other bird species (Primmer et al. 1996; Galbusera et al. 2000; Richardson et al. 2000; Johannessen et al. 2005; Poesel et al. 2006; see also Passerine BIRDMARKER Database http://www.shef.ac.uk/misc/groups/molecol/deborah-dawsonbirdmarkers.html), or by searching for potentially functional polymorphisms in candidate genes (Johnsen et al. 2007; Steinmeyer et al. 2009). In the present study, we present a set of 106 polymorphic microsatellite markers for the blue tit, and anchor them to the zebra finch and the chicken genome. We designed 31 new markers from zebra finch EST-SSR. The other loci tested were developed by others from EST-SSR sequence using different methods or previously described for other bird species, including 10 markers that were originally isolated in the blue tit (Table 1.2). Approximately 60% of the 53 previously described loci had been assigned a location in the chicken genome based on sequence homology (Dawson et al. 2006). In order to assign chromosome locations for as many loci as possible, we compared all sequences (and/or the zebra finch homologs of these sequences) against the zebra finch and chicken genomes (following Dawson et al. 2006; Dawson et al. 2007). This large set of in silico mapped polymorphic markers complements the limited genetic resources available for the blue tit, and constitutes a potential resource of molecular markers for other passerine birds. Heterozygosity-fitness correlations in the blue tit 20 Material and Methods Microsatellite markers In a previous study, Slate et al. (2007) identified simple sequence repeats in a collection of zebra finch EST sequences deposited in GenBank, and predicted their location in the chicken genome. We aligned these zebra finch EST sequences with homologous sequences of multiple species, and then designed conserved primer pairs flanking the repeat region. First, we aligned the zebra finch EST bearing SSR with its chicken genome sequence homolog and, if available, with other bird species’ sequence homolog. Homologs were identified using the BLAT search function at the University of California Santa Cruz (UCSC) Chicken Genome Browser (http://genome.ucsc.edu/cgibin/hgGateway), and/or the BLAST search function against “Aves” sequences on the nucleotide collection (nr/nt) or the non-human, non-mouse ESTs (est_others) databases of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). Second, we designed primers with the program PrimaClade (http://www.umsl.edu/services/kellogg/primaclade.html): primer length was between 17-27 bp, with a maximum of 3 degenerated positions, and with an expected product size between 100-400 bp. We tested the primers in one zebra finch and 7-14 presumably unrelated blue tit individuals from a breeding population at Kolbeterberg, Vienna (Austria). Genomic DNA was extracted from blood with the GFX Genomic Blood DNA Purification Kit (GE Healthcare Europe, Freiburg, Germany), following the manufacturer’s protocol. Each 10 μl PCR reaction contained 1x PCR buffer (Fermentas), 2mM MgCl2, 0.2mM dNTPs, 0.5 μM of each primer, 0.25 U Taq DNA Polymerase (recombinant, Fermentas), and between 20–40 ng of genomic DNA. We used a touchdown PCR program as follows: 94°C for 5 min; 8 cycles of 94°C for 30 sec, annealing temperature (Ta, Table 1.2) + 4°C (reducing 1°C per cycle) for 1 min, and 72°C for 1 min; 22 cycles of 94°C for 30 sec, Ta for 1 min, and 72°C for 1 min; and 70°C for 15 min. The products were visualized on a 4.5–10% native polyacrylamide gel stained with ethidium bromide. When bands of different sizes were observed, we confirmed the presence of a polymorphism by separating the fragments on an ABI 3130 xl Genetic Analyzer with the GeneScanTM 500 LIZ® Size Standard (Applied Biosystems), using fluorescently labeled primers in the PCR reactions. Raw data were analyzed with GeneMapper 4.0. Chapter 1: Microsatellite markers 21 Additionally, to make the list of markers more exhaustive, we also tested and validated all markers known to be of utility in blue tit and other passerine species. We tested 80 loci including many for which conserved primer sets had been developed and proven to be of high utility in a wide range of passerine and non-passerine birds (Dawson 2007; Dawson et al. 2010 & unpublished data; G.N. Hinten, unpublished data). We checked that the microsatellite loci listed in Table 1.2 were not represented multiple times due to description of the same repeat region in different species and/or via different methods. To identify redundant sequences we used a similar approach to the one described in Dawson et al. (2006): a file of the compiled sequences of all the microsatellite loci was compared against itself using the NCBI specialized BLAST to align two (or more) sequences (bl2seq) (http://blast.ncbi.nlm.nih.gov/Blast.cgi), with the sequence accession records of all microsatellites as both the input file and the reference database. A hit was defined as significant with an E value < 1 x 10-10 and an alignment score > 180.