The value of avian genomics to the conservation of wildlife

Background Genomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution. Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care). In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes. Results Genotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current population's genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology. Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m form, originally thought to be the derived condition, might actually be the ancestral one. Conclusion Mining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease. In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology.


Introduction
Genomic studies in a variety of mammalian taxa have contributed to the development of more comprehensive plans for their conservation, as well as to our understanding of the generation and maintenance of genetic diversity in general (e.g., [1][2][3]). With the advances of genomic resources for other species, it is now feasible to expand investigation to additional taxonomic realms.
Non-domesticated species of birds are emerging as new animal models with great potential to advance comparative avian genomics and contribute to conservation efforts for threatened species. Birds show incredible diversity in morphology, physiology, and behavior -all of which are analogous to phenotypic variation encountered in other species. With the completion of the chicken genome, as well as significant advances in turkey and zebra finch genomic resources, it is now possible to examine the genetic bases of complex traits using "tools" borrowed from these species. There is a special need for genomic studies in endangered birds to secure their recovery in natural habitats and to increase their resistance to potential threats (e.g., disease, anthropogenic effects). In addition, because many avian species are relatively easy to observe in the wild, they have been intensely studied for decades and, as a result, several long-term studies of their behavior, ecology, and evolution now exist (e.g., the pied flycatcher, Ficedula hypoleuca, the great tit, Parus major, and the blue tit, Parus caeruleus; see for review [4,5]), making it possible to examine adaptive genetic variation. Finally, many avian species are the proverbial "canary in the coal mine", providing us with indicators of the health of an ecosystem and sentinels for the impacts of environmental changes.
In this paper, we review the recent genomics advances in two model avian species, the California condor (Gymnogyps californianus) and the white-throated sparrow (Zonotrichia albicollis). The critically endangered condor has been affected by habitat destruction and an extreme bottleneck. The white-throated sparrow is an indicator species for the Northern Boreal forest, an essential habitat for avian biodiversity and a key carbon sink [6]. Through studies of their genomes, we will develop a deeper understanding of the genetic mechanisms affecting variation, adaptation, evolution, and behavior in these species, thereby obtaining new information relevant to conservation management of wild populations.

California condor
The management of wild avian species, including those that are critically threatened and/or endangered, has greatly benefited from the recent advances in genetics and genomics. Among the most striking examples of genetic application to conservation of an endangered species is the California condor (Gymnogyps californianus). The condor is an avian icon of California that belongs to the New World vulture family (Cathartidae). Over the last two centuries, there has been a rapid decline in the condor population as their natural habitat dramatically shrank. In the 1950s, wild condors were thought to be restricted to Los Padres National Forest in central California. By the early 1980's, a California condor rescue and restoration program was set in motion. Captive propagation was quite successful, and condors have been reintroduced to central and northern California (including Los Padres National Forest and Monterey County), the Grand Canyon area in Arizona, and Baja California del Norte, Mexico [7].
Captive breeding necessitated the initiation of genetic studies to assess the diversity of the remaining population. Early genetic studies of the condor were done at the San Diego Zoo's Institute for Conservation Research, and included the genotyping of the original population with RFLPs [8] and mtDNA [9] analyses ( Figure 1). Within the founder population, three unrelated groups (clans) of closely related individuals were identified based on the analysis of multi-locus RFLP fingerprinting [8]. Using mtDNA markers, four distinct maternal lineages were then identified [9]. Finally, karyotype and molecular techniques were developed for sexing condors, involving first an RFLP approach and later utilizing conserved avian sex chromosome sequences for PCR primers [9]. Together, these findings were used to recreate the hypothetical founding generation of California condors, an essential step in developing an effective population management strategy [10].
Genetically small populations are at increased risk of impacts of deleterious mutations as a result of founder effect and genetic drift. A lethal mutation was identified in the expanding pedigree of California condors that negatively affected limb development [11]. The mutant phenotype, called chondrodystrophy, is similar to genetic disorders observed in the chicken (nanomelia) [12], turkey [13], Japanese quail [14], mouse [15,16], and human [17]. Ralls et al. [11] found that the embryonic lethal condition of chondrodystrophy segregates in the affected ped-California condor pedigree of 69 individuals used for genetic studies Figure 1 California condor pedigree of 69 individuals used for genetic studies. A pedigree includes founders and early generation offspring used for the DNA fingerprint [8], mtDNA [9] and microsatellite (msat; current study) analyses. All individuals are designated using their studbook numbers.
igree as a Mendelian character in a manner consistent with an autosomal recessive mode of inheritance.
With the advent of the genomic era, a detailed comparative molecular cytogenetic analysis of condor chromosomes (GCA) was carried out using chicken macrochromosome (GGA1-GGA9, GGAZ, and GGAW) paints, revealing that the condor has 80 chromosomes [18], comparable to the chicken and turkey karyotypes. This study showed a great homology of chicken and condor macrochromosomes, except for GGA4 that corresponds to GCA4 and GCA9, and suggested an incomplete differentiation of the condor sex chromosomes in the evolution [18].
These studies laid the groundwork for application of genomic technologies in support of California condor conservation efforts. Construction of a genetic map of the California condor and identification of linked markers for the chondrodystrophy mutation were primary goals. The availability of the chicken genome sequence [19] and a large-insert BAC library that was generated at the BACPAC Resources Center, Children's Hospital Oakland Research Institute (CHORI) [20] were crucial components of these efforts. The BAC library consists of 89,665 clones and provides approximately 14-fold coverage of the condor genome. Based upon 172 probes, mostly available from the chicken genome project, we screened the library and produced a first-generation BAC-based chicken-condor comparative map that contained 93 loci.
Among the genes identified in the condor BAC library, several are candidates for chondrodystrophy because they are involved in bone and tissue formation. One such candidate is aggrecan (ACAN), which is an integral part of the extracellular matrix [21]. The aggrecan molecule consists of three globular domains, two chondroitin sulfate domains, and one keratin region. Aggrecan molecules attach to a long hyaluronan molecule and form aggregates. Moreover, this functional candidate gene was found to cause skeletal dysplasia in model organisms [12,13,[15][16][17]. We detected two BAC clones positive for the condor ACAN gene, one of which was sequenced. As expected, the condor gene sequence showed a great similarity to the chicken homolog [20]. Building on these data, we have continued to investigate the genetic basis of disease in this species. The development and application of practical approaches for management of deleterious genes identified in small populations, including diagnosis of risk factors, identification of unaffected carriers, and breeding strategies to retain genetic variation in such populations with segregating lethal mutations will find precedent in these initial studies.

White-throated sparrow
The white-throated sparrow (Zonotrichia albicollis; Family Emberizidae) is a socially monogamous passerine that breeds almost exclusively (>85% of the breeding population) in the North American boreal forest. With large declines in species abundances, increasing evidence for global warming and acid rain, and with the realization that the amount of boreal forest currently protected will not sustain migratory bird populations [6], conservation efforts have begun to focus on such key areas before it is too late. An essential component of a valid conservation plan will include the study of the genomes of boreal species, such as the white-throated sparrow, for the amount and kind of adaptive genetic variation they actually possess, and its distribution across varying habitats.
The white-throated sparrow has several important characteristics that make it an ideal model for the study of conservation genomics. The species is polymorphic, and both sexes occur as either tan (T) or white (W) morphs based on the color of their crown stripes ( Figure 2). In addition to morphology, behavioral and life-history characteristics differ between the morphs as well [22][23][24][25] (Table 1). In general, white birds are more aggressive than tan birds, yet white are not dominant to tan. Males of the two morphs employ alternative reproductive strategies based on the tradeoffs between current and future reproduction. White males invest heavily in securing matings through song, territorial intrusion, bigamy, and the pursuit of promiscuity (i.e. extra-pair copulations) at the expense of mateguarding and paternal care [22,23,[26][27][28]. White males have higher levels of circulating testosterone than tan males early in the breeding season [29], most likely because they tend to establish territories in areas of high Morphs of the white-throated sparrow Figure 2 Morphs of the white-throated sparrow. Photographs of the morphs of the white-throated sparrow; A) white morphs are heterozygous for a chromosomal rearrangement on autosome 2, while B) tan morphs are homozygous for the non-inverted form of autosome 2.

B) A)
density where encounters with conspecifics are frequent [24]. By contrast, tan males invest in monogamy and high levels of parental care, and tend to establish territories in more isolated areas. White and tan females seem to exhibit similar trade-offs between investment in parental effort and investment in reproductive effort.
Interestingly, morph was absolutely correlated with a chromosomal polymorphism resulting from what was believed to be a pericentric inversion of the 2 nd largest autosome [30,31]. White birds are heterozygous for the rearrangement (i.e. 2 m /2, where 2 m represents the inverted chromosome 2), and tan birds are homozygous non-carriers (i.e. 2/2). The autosomal inversion segregates in a Mendelian fashion [31]. White-throated sparrows mate disassortatively with respect to the polymorphism [22,23,31], which maintains both morphs in the population [31] and results in parental "phenotypes" that differ in the amount and type of care provided. These parental types respond differently to environmental conditions (e.g., predation, disease, and long-term climatic factors) by altering the condition, sex, and morph of their own offspring through mechanisms that persist across generations.
Results gathered from detailed cytogenetic and molecular analyses in the white-throated sparrow can be combined with long-term data on behavior, physiology, and ecology to reveal the bases of adaptive variation in a natural population. Morphs of the sparrow are somewhat analogous to natural "inbred" lines, where selective factors affecting fitness can be linked to specific genes. In this species, we have the unique opportunity to transcend from genotype to ecosystem. Such results can transform our understanding of evolutionary and conservation biology. In addition, results can be generalized to endangered and at-risk species, providing conservation biologists with a strong foundation with which to build their captive and reintroduction plans.

DNA samples
A condor female named Molloko, studbook (SB) #45, the first ever condor chick hatched in captivity, was selected as the DNA source for constructing the condor microsatellite and BAC libraries. Sequences of microsatellite and BAC clones from Molloko have been deposited in the GenBank and Trace Archive.
For genetic diversity and linkage studies, a set of 121 condor DNA samples including that of Molloko was chosen. These samples were previously isolated for fingerprinting studies and/or gender determination and represent a pedigree of related individuals from four generations and seven families (Additional file 1, created using a computer program Pedigraph, Version 2.4 [32]). This pedigree serves as a condor resource population for further microsatellite and linkage analyses, and also involves three chondrodystrophic individuals, SB #60 and eggs #1405 and #2537.

Microsatellite-based analyses
A California condor microsatellite-enriched library was created at Genetic Identification Services, following the company's protocol, and includes eight sublibraries, A to H, with enrichments for repeat motifs CA, ATG, TACA, TAGA, AAG, AAT, AAAT, and CATC, respectively. For marker design, approximately 180 random clones were sequenced. Dye-labeled primers for a set of 17 informative microsatellite markers identified by Genetic Identification Services (Table 2) were used to amplify condor DNA. These included 13 tetra-, two tri-and two dinucleotide repeat loci. Three more avian microsatellite markers, FhU2, HrU2, and HrU6 (Accession Numbers X84361, X84087, and X84091) [33,34], were tested using Schuelke's [35] procedure for the fluorescent labeling of PCR fragments with the M13(-21) universal primer.
The PCR products were run on an ABI 3100, ABI 3130 or ABI 3730 Genetic Analyzer instruments (Applied Biosystems), the fragments or alleles generated were verified (Figure 3), and the data analyzed for population genetic parameters and linkage relationships. The following computer programs were used for estimating population genetic statistics: Microsatellite Tools for Excel, Version 3.1 [36], and POPGENE, Version 1.31 [37]. A preliminary linkage analysis was done using Locusmap, Version 1.1 [38].
Additionally, over 1,900 random clones from the same microsatellite-enriched library were bi-directionally sequenced at High-Throughput Sequencing Solutions (University of Washington, Seattle). These were then used to develop additional polymorphic microsatellite markers using the Primer3 online program [39] for primer design. All sequences have been deposited in the GenBank (Accession Numbers DQ471953, DQ483109 -DQ484036, EF108178 -EF108181, EF116886 -EF116903) and analyzed for the presence of repetitive elements using a package of NCBI BLAST programs [40] and RepeatMasker http://www.repeatmasker.org/ [41].

BAC library screen
A second library screening was carried out using the CHORI protocol http://bacpac.chori.org/overgohyb.htm and a four-dimensional filter hybridization procedure [42], with modifications [20]. The latter, in brief, included the simultaneous 32 P-labeling of 216 overgo probes (Additional file 2) and their hybridization to BAC filters, with variable 36 overgo pools per filter per dimension. This strategy allowed the completion of a total of 24 hybridizations in a relatively short amount of time. The overgo probes were available from the previous chicken and zebra finch genome projects [43,44] or newly designed on the basis of available condor and chicken sequences (Additional file 2). Other screening procedure details and overgo sequences can be provided by the authors upon request.

Cytogenetic techniques
Avian fibroblast cell lines from feather pulp were established at the San Diego Zoo's Institute for Conservation Research as described in [46]; these cell lines were used for karyotyping and fluorescence in situ hybridization (FISH).
Metaphase chromosomes were obtained by arresting cell division with colcemid (Gibco BRL), hypotonic treatment with 0.075 M KCl, and fixation in 3:1 methanol:acetic acid. To assess chromosome number, we stained chromosome preparations with 5% Giemsa (Sigma) in 0.07 M phosphate buffer (pH 6.8). To examine banding patterns, some slide preparations were trypsin G-banded following [47].
BAC DNA was used as FISH probes and extracted following standard procedures. After extraction from a BAC clone, DNA was purified and labeled with a fluorochrome according to the manufacturer's guidelines (Vysis, Abbott Laboratories).
An example of microsatellite genotyping profile at the loci C5 and D6

Fragment and sequence analysis
Fragments from coding regions of the ACAN candidate gene were PCR amplified and sequenced using an ABI 3100 or ABI 3130 Genetic Analyzer. Sequencher (Gene Codes) was employed for post-sequencing analysis.
A condor fibroblast cell line was utilized for mRNA extraction and consequent generation of cDNA sequences at Washington University Genome Sequencing Center using the 454 sequencing technology [48]. The total RNA was used to prepare a polyT-primed cDNA library by an inhouse version of the Clontech SMART protocol without normalization [49]. The 454 sequence data set has been deposited in Trace Archive.
For analyses of the white-throated sparrow morph, we modified the Michopoulos et al. [50] protocol to amplify a portion of the vasoactive intestinal peptide (VIP) gene located on chromosome 2 that either has a DraI restriction site (the "2 m " state) or lacks it (the "2" state). Our modifications included fluorescently labeling both the forward and reverse primers so that all products could be visualized on an ABI PRISM 310 Genetic Analyzer, and reducing PCR reactions to a final volume of 10 μl. Figure 4 shows the genetic analyzer output for white and tan morphs. To examine evolutionary history, we used this same "morph" assay on DNA samples collected from several related passerine species.

California condor studies Genetic diversity and linkage analysis
For genetic diversity assessment, linkage analysis and construction of the condor genetic map, we chose 121 related individuals that formed a condor resource population. Their genotyping at 17 microsatellite loci resulted in the estimates of within-population genetic variation shown in the A preliminary linkage analysis suggested that among the 17 microsatellite loci we may expect a linkage between loci D10 and D6 (LOD score = 21.67), as well as between A20 and D9 (LOD score = 5.12) and between B7 and H238 (LOD score = 5.12).
To expand the microsatellite mapping subproject, we tested three more avian microsatellite markers, FhU2, HrU2, and HrU6 [34,35] that previously showed satisfactory amplification results in a wide range of other birds, including some New World vultures. We were able to amplify the condor FhU2 and HrU2 PCR fragments, while HrU6 did not work on condor DNA in our hands.
Additionally, we obtained over 900 clone sequences from the microsatellite-enriched library and derived from them around 300 new markers for genotyping the resource population at the next project stage.
In silico mapping In total, we obtained approximately 1000 short genomic sequences from the microsatellite-enriched library. In the course of in silico mapping, we found that 30% of these are homologous to chicken sequences across almost all chicken chromosomes (GGA1-GGA15, GGA17, GGA19-GGA21, GGA23, GGA24, GGA26-GGA28, GGAZ, and UN) and some other avian sequences. Many of these sequences contain microsatellites and other repetitive ele-Molecular assay for morph determination Figure 4 Molecular assay for morph determination. Molecular assay for morph determination in the white-throated sparrow, modified in the Tuttle laboratory for use on automated sequencers from primers described in [50]. Panel A shows a tan morph with a single peak at 284 bp amplified from chromosome 2. Panel B shows a white morph with a band for chromosome 2 (at 284 bp) and two additional bands at 88 bp and 189 bp, which occur when PCR product amplified from chromosome 2 m is digested with restriction endonuclease DraI. If they occur in nature, we assume that birds homozygous for 2 m ("super whites") would produce a pattern showing only the 88 bp and 189 bp bands. This technique has been verified in adults of known plumage. Values along the y-axis represent the intensity of fluorescence (a.u.), values along the x-axis mark the size of the DNA fragments (a function of time).

A) B)
ments (LINEs, retroviral LTRs, and satellites) known in chicken. We also found satellite sequences previously detected by Keyser et al. [51] that only occur in New World vultures.

BAC library screening
The second screen of a 2.8× subset of the condor BAC library was done using 216 overgo probes. The latter were mostly available from the zebra finch and chicken genome projects [20] or derived from the condor microsatellite library clone sequences (Additional file 2). As a result, we added 100 more loci onto the BAC-based chicken-condor comparative map, and it currently contains 199 loci (Additional file 3). At present, the total number of condor BAC-gene or other sequence assignments is almost 670 (Additional file 4).

FISH mapping
To verify the efficiency of cross-species hybridization of the condor BAC library and to build the condor cytogenetic map, we performed FISH using more than 50 condor BAC clones that were positive for targeted condor genes ( Figure 5). Most BACs were found to be orthologous to the appropriate chicken genes and chromosomes (Additional file 5). We identified one intrachromosomal rearrangement on chromosome 4 (the FGF2 locus), with more intrachromosomal rearrangements on the Z chromosome.

Candidate gene hunting
Among the positive clones for almost 200 genes identified in the condor BAC library, we selected and sequenced one that harbors the ACAN gene (CH262-21P20, Acc. No. AC171743). Based on the sequence information, we designed PCR primers to re-sequence the entire gene in affected chicks, their parents, as well as unaffected nonrelatives. After sequencing coding regions of the gene, we have yet to detect SNPs that could be associated with the chondrodystrophy.

Comparative human-condor mapping
To date, NISC has generated sequence of 13 condor BACs, each 100-200 kb in size. These sequences are orthologous to human chromosome 7 and six chicken chromosomes. Based on the sequences, a condor-human comparative physical map for a region on HSA7 was designed and is available online through the NISC web site (Additional file 6). After comparing against the chicken whole genome sequence using BLAST, the 13 sequenced BACs were also added onto the BAC-based chicken-condor comparative map (Additional file 3).

sequencing
At the Washington University Genome Sequencing Center, almost 440,000 cDNA sequences were generated from a fibroblast cell line using the 454 technology. The total length of these short transcripts (about 100 bp each) is 43,379,069 bp. We found that 78% transcripts were homologous to chicken genes, while being distributed across almost all chicken chromosomes ( Figure 6). After assembly and clustering, we were left with around 15,000 contigs that contained about 190,000 reads.
Surprisingly, for a number of genes expressed in this cell line, we had unusually high numbers of reads, with the most extreme transcript having over 55,000 reads (Additional file 7). Amongst the most abundant transcripts (Additional file 8), there were several proteins that are expected to be expressed in fibroblast cell lines, like proteoglycans, a keratin associated protein, fibroblast growth factor receptor 1 and alpha-actin, and other housekeeping proteins. However, one protein, the mitotic arrest deficient-like 1 (MAD1L1), had especially higher levels of expression in this cell line.

White-throated sparrow studies Chromosomal studies
To confirm previous studies and as a necessary first step in genomic studies, we established eight white-throated California condor FISH mapping Figure 5 California condor FISH mapping. An example of FISH using metaphase chromosomes of the California condor and a biotinylated BAC clone containing the ACVR2B gene located on GCA2.
sparrow cell lines (six white, and two tan; KB15792 -KB15799) accessioned in the Frozen Zoo ® at the Zoological Society of San Diego. Using standard Giemsa staining, we confirmed that there is a total diploid chromosome number of 82, including the Z and W sex chromosomes ( Figure 7). G-banding comparisons indicate that the rearrangements distinguishing 2 m from 2 and 3 a from 3 are complex. The banding differences in each pair involve the centromere and cannot be resolved by inverting only one segment of the chromosome and thus are likely the result of more than one inversion on each chromosome ( Figure  8).

Comparative analyses
The VIP assay for morph is based on the fact that there is a SNP on chromosome 2 m that forms a restriction site for DraI, whereas the VIP fragment on chromosome 2 does not contain that site [50]. In an assay of 546 whitethroated sparrows (Table 4), 50.9% were 2 m /2 (white) and 49.1% were tan (2/2); no individuals were 2 m /2 m ("superwhites"). When we examined this site in the four other species of Zonotrichia (Z. atricapilla, Z. querula, Z. leucophrys, Z. capensis), only the North American species of Zonotrichia show polymorphism for the VIP fragment suggesting that the common ancestor of all four had the polymorphism (Table 4; Figure 9). All 10 rufous-collared sparrows (Z. capensis) tested were monomorphic for the DraI restriction site. In addition, other passerine birds tested (three subspecies of junco, including Junco hyemalis hyemalis, J. h. oreganos, and J. h. caniceps, swamp sparrow (Melospiza georgiana), song sparrow (M. melodia), and purple finch (Carpodacus purpureus)) were also monomorphic for the DraI restriction site (Table 4). Preliminary sequence analysis of the VIP intron indicates that in the Distribution of the condor 454 transcripts across the chicken chromosomes rufous-collared sparrow, the junco, the song sparrow, and the swamp sparrow, the SNP is the same as that found in chromosome 2 m of the white-throated sparrow ( Figure  10).

California condor genetics and genomics
Genetic management of California condors aims to maximize the retention of genetic diversity by minimizing mean kinship within the population. We used a series of mostly tetranucleotide repeat markers to evaluate genetic diversity among individuals obtained from the wild and ascertain linkage relationships between microsatellite loci. The observed level of heterozygosity (0.45) found in the resource population could be considered as a good sign of a successful captive propagation program and population recovery. On the other hand, observation of Hardy-Weinberg equilibrium deviations at eight out of 17 loci might suggest that the current condor population is impacted by inbreeding. These results can be used for validating genetic management recommendations currently used for this endangered species.
Taking advantage of recent progress in genome studies of the domestic chicken, we are developing a genetic map for the California condor. Among the 17 microsatellite loci already used, there are three potential linkages established. Approximately 300 more markers have been designed and, after validation, will be used for genotyping the resource population. This would be the first attempt to make a genetic linkage map for condors, which will be later supplemented with SNP markers to locate and characterize candidate loci for the chondrodystrophy mutation, to identify carriers of the chondrodystrophy allele and to improve genetic management of this and other heritable disease risk-factor loci.
The conventional re-sequencing of ACAN, a likely chondrodystrophy candidate gene, was complicated by the compound structure of this gene, including the large number of exons (18 in the chicken) and a complex repetitive structure of its longest exon that encodes for chondroitin sulfate attachment domain. The latter contains in the chicken a variable repeat region that includes 19 repeats, each of 60 nucleotides, with the reported nanomelic stop codon [12]. Although we have not yet been able to identify a causative SNP in coding regions of the condor ACAN gene, we continue this investigation in both exonic and intronic sequences as well as other regulatory regions that could also be associated with changes in the ACAN regulation. We plan to use a high throughput 454 sequencing technology that may identify or eliminate this locus as the locus of the chondrodystrophy mutation. Construction of the condor linkage map will bring us closer to the mapping of the chondrodystrophy locus and it will enable confirmation of a role for the ACAN gene or other (marker) loci linked to the disease. In either scenario, we will be able to develop a molecular assay for heterozygous carriers of the chondrodystrophy mutation.
The 454 cDNA sequence data reported here provides a first insight into the condor transcriptome. Interestingly, ACAN was found to be expressed in this cell line. We expect that establishment of fibroblast cell lines for normal and affected chicks will enable direct comparison of full-length cDNA sequences to significantly assist in the identification of the potential chondrodystrophy mutation in ACAN or other candidate genes. Such data will provide one more functional genomics approach to pinpoint the disorder mutation.
MAD1L1 was shown to be highly overexpressed in the condor fibroblast cell line analyzed. MAD1L1 is a component of the mitotic spindle-assembly checkpoint, and its mutations are suggested to play a role in the pathogenesis of various types of human cancer (e.g., [52]). Abnormal overexpression of MAL1L1 gene may be linked to a particular phenotype observed in this condor cell line. The cell G-banded chromosomes 1-9 and Z from the sparrow line has a continuous long-term proliferation and heteroploid features, an indication that the line is transformed possibly due to a mutation affecting MAD1L1 expression. As MAD1L1 is known to be involved in neoplasia, further investigation of this cell line can provide a source of comparative studies and shed light on de-regulation of cell proliferation in avian species.
We were able to obtain dense coverage of the Z chromosome (Additional file 3) with the second BAC library screen. We chose to cytogenetically assign the Z-linked BACs to condor chromosomes using FISH because preliminary indications suggested that there could be intraand interchromosomal rearrangements in the condor relative to the chicken Z chromosomes. Furthermore, we consider it useful for the future studies to compare Zlinked gene expression in condor males and females to see if there is any difference or if we have a dosage compensation effect similar to what was shown for the zebra finch and chicken [53,54].
Overall, we detected a few changes in gene location and order between chicken and condor chromosomes, indicating a high degree of conserved synteny. Our data support a higher similarity of the cathartid genome to the avian ancestral karyotype and a more basal position of Cathartidae as compared to Accipitridae and Falconidae [55], as well as the general stability of avian genomes over the course of evolution [56]. Another piece of evidence in support of those ideas comes from the fact that the chicken and condor genomes demonstrate a high degree of sequence homology and share many common avian repetitive elements including LINEs, retroviral LTRs, and satellites. However, the presence of satellite sequences specific to New World vultures [51] suggests that some distinctive genomic features have emerged in the cathartids over almost the 100-million years of evolution in this avian lineage. We hypothesize that the specific satellite sequences inherent to the Cathartidae may represent unique genomic signatures that could help resolve the long disputed taxonomic position of this avian family (e.g., [55,57,58]).
The 454 cDNA sequence data, as well as condor microsatellite library clone sequences, have been added to a new avian genomic database Gallus GBrowse that includes the whole genome sequence of the chicken as a reference http://birdbase.net/cgi-bin/gbrowse/gallus08/ [59]. Along with sequence information for turkey and zebra finch, this database serves as a new powerful tool for avian comparative genomics. All the sequence information available for the condors is also being deposited in GenBank and Trace Archive, adding the condor to the ever-expanding list of avian genomes (Table 5). Finally, there is a web site we have developed for the condor genomics project: https:// msu.edu/~romanoff/index2.htm.

White-throated sparrow as a behavioral model
Advances in white-throated sparrow genomics have begun at an obvious point -the characterization of the chromosomal rearrangement affecting morphic differences. Thorneycroft [30,31] first karyotyped the species, reporting a diploid chromosome number of 82 or 84, and based on centromere position, presumed pericentric inversions in both chromosome 2 and 3. Here, our karyotypic analyses confirm a diploid number of 82, with the typical avian pattern of approximately 30 pairs of microchromosomes ( Figure 7; see [60,61] for reviews of avian microchromosomes). G-banding analyses suggest that the rearrangements in chromosomes 2 and 3 are not simple, but instead consist of multiple inversions involving the centromere. These results are consistent with those reported in Thomas et al. [62], who used comparative cytogenetic mapping to show that 2 m and 2 differ by a pair of pericentric inversions spanning at least 98 Mb or greater than 86% of the chromosome. Chromosomal inversions are often adaptive because they can result in coadapted gene complexes [63], therefore further investigation of gene structure in this species is warranted. However, to be valuable to evolutionary and conservation biology, such studies must be done within the context of the ecological and social environments that selected for the maintenance of polymorphism. In the white-throated sparrow, recombination is suppressed in heterozygotes for chromosome 2 m . Thorneycroft [31] found that chiasma did form between the p-arm of chromosome 2 and one arm of chromosome 2 m , but they did not form between the q-arm of chromosome 2 and chromosome 2 m. In addition, chiasma formed between the p-arm of chromosome 3 and the p-arm of chromosome 3 a , but not between the q-arms of these chromosomes. Using nine loci within the 2 m rearrangement, Thomas et al. [62] confirmed that recombination only occurred in the telomere area between the p-arm of chromosome 2 and chromosome 2 m , thus restricting gene flow between the two. In addition, they found that chromosome 2 had five times the nucleotide diversity of chromosome 2 m . Based on these results, Thorneycroft's [31] meiotic data, and our data on population genotype frequencies (Table 3), we predict that chromosome 3 a has also evolved under limited recombination and reduced gene flow.
Chromosomal inversions may be disadvantageous if they foster the accumulation of suites of deleterious alleles within the area of low recombination. Since the inversion in the white-throated sparrow is relatively large and covers at least 86% of chromosome 2 m , it is likely that some negative fitness affects are associated with homozygosity; the same is likely true for chromosome 3 a . We did not find 2 m /2 m or 3 a /3 a individuals in our karyotypic analyses, nor did we find any 2 m /2 m birds in over 546 individuals sam-pled in the VIP assay. Thorneycroft [31] found that parents with a single 2 m chromosome passed this on to half of their offspring however, parents with a single chromosome 3 passed this on to only a quarter of their offspring. Given his results, we would expect that white birds (2 m /2) pairing with other white birds (2 m /2) would result in up to 25% lethal or semi-lethal homozygotes. It is surprising then that in our study, the frequencies of particular genotypes differed in the sexes and between the two disassortative pair types (Table 3). Certain combinations of chromosomes 2 and 3 also appeared more viable than others. Together, these factors suggest non-Mendelian transmission, interchromosomal linkage and pleiotropy, and a strong interaction between autosomal genotype and sex. Our laboratory is continuing to investigate the relationship between chromosomes 2 and 3 using a combination of genomics techniques and population data.
Comparative analyses between the white-throated sparrow and its relatives can be quite revealing about genomic evolution. In the rufous-collared sparrow (Zonotrichia capensis), Rocha et al. [64] reported polymorphisms in chromosomes 3 and 5 -also presumably due to pericentric inversions. The closely related junco exhibits polymorphisms for pericentric inversions in chromosomes 2 and 5 [65] and the white-crowned sparrow (Z. leucophrys) has centric rearrangements in chromosomes 3, 5, and 12 [66]. Thus, chromosomal inversions in Zonotrichia and its congeners seem to be relatively common [65,66]. Our comparative analysis of the VIP intron [50] revealed that the DraI polymorphism likely existed in the common ancestor of all four North American species of Zonotrichia, but not in the South American species, Z. capensis. In accordance with these results, sequence data from VIP (Figures 9  and 10) and other areas of chromosome 2 m suggest that VIP sequence for several passerine species Figure 10 VIP sequence for several passerine species. Sequence alignment for an intron of the vasoactive intestinal peptide (VIP) gene (primers described in [50] [62]. Surprisingly, our comparative data on the VIP intron suggests that, contrary to Thorneycroft's [31] expectations, chromosome 2 m (or at least a portion of it) might actually be "ancestral". We are undertaking further comparative mapping to resolve these alternatives.
In the past few years, genomic resources for avian species have advanced by leaps and bounds, making it now possible to use comparative genomics to determine the genes responsible for differences in white and tan morphology, behavior, and physiology. Chicken chromosome paints revealed that white-throated sparrow chromosome 2 was analogous to chicken chromosome 3 [62], now allowing us to pinpoint candidate genes associated with phenotypic differences. In addition, major advances in the zebra finch genome [67] as well as a white-throated sparrow BAC library (CHORI-264; http://bacpac.chori.org/ library.php?id=469), mean that detailed mapping is now possible. Over 1000 microsatellite markers, developed for parentage and population genetics analyses in songbirds [68,69], have been used to construct a predicted passerine genome map based on sequence similarity to the chicken genome. Using microsatellite markers that cross-amplify in the Zonotrichia with the detailed pedigrees of over 350 families (data not shown) we will be able to generate linkage maps for this species. Finally, in order to advance conservation and evolutionary genomics in this species, it will be important to identify adaptive genetic variation and the role that the environment has had in selecting for the evolution of alternative genotypes. We have also established a website for the sparrow project: http:// www.whitethroatedsparrow.org.

Conclusion
The retention and capacity for generation of novel adaptive genetic variation in a population is key to conservation efforts. Without maintaining this diversity, given the increased anthropogenic impacts of populations and their habitats, many species will face increased vulnerability to extinction in the near future [70,71]. Conservation genetics has traditionally relied on assays of neutral genetic variation, assuming that it accurately reflects adaptive variation [72]. However, the correlation between variation in adaptive and non-adaptive genetic elements can sometimes be low [72][73][74], and so a true understanding of the interplay between genes and biodiversity will benefit from the additional data that a broader genomics approach can provide. Here we show the utility of applying such a "genomics approach" to study of two key avian species.
Conservation efforts in the endangered California condor will benefit from the characterization and deeper understanding of the segregating lethal chondrodystrophy mutation. Management of the species' genetic diversity under circumstances of maintaining a viable, self-sustaining, managed population that provides birds for reintroduction, and genetic and demographic augmentation of newly established wild California condor populations in the United States and Mexico is a conservation paradigm that merits the fullest support of scientific prowess in genomics, the health sciences, and behavioral ecology, as well as through public policy, public education and law enforcement. Results gathered from the condor studies will help identify recovery options for other at-risk species as their populations continue to decline and experience genetic bottlenecks.
Genomic studies in the white-throated sparrow are still in their infancy although the potential benefits of this avenue of study are great. Morphs of the white-throated sparrow provide a novel intraspecific comparison of selective pressures, where factors affecting fitness can be linked to specific genes -in other words, we have the opportunity to directly assess adaptive variation. In addition, genomic studies in the sparrow will contribute to our understanding of chromosomal inversions, where suites of genes are often inherited as co-adapted gene complexes and the "success" of a phenotype is highly dependent on orchestrated gene cascades and environmental effects.
Birds are highly influential model organisms in conservation and evolutionary research, and the foundation of work on many avian species is already strong. With the advances of genomic resources for chicken, turkey, zebra finch, and now, the California condor and white-throated sparrow, it is now feasible to expand genomics studies to other avian groups.
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