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 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 intra- and interchromosomal rearrangements in the condor relative to the chicken Z chromosomes. Furthermore, we consider it useful for the future studies to compare Z-linked 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
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
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/
]. 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 ). Finally, there is a web site we have developed for the condor genomics project: https://msu.edu/~romanoff/index2.htm
Representation of avian species in the NCBI databases (as of June 8, 2009).
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
] 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 ; see [60
] 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 2m
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 co-adapted 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 2m
. Thorneycroft [31
] found that chiasma did form between the p-arm of chromosome 2 and one arm of chromosome 2m
, 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 3a
, but not between the q-arms of these chromosomes. Using nine loci within the 2m
rearrangement, Thomas et al. [62
] confirmed that recombination only occurred in the telomere area between the p-arm of chromosome 2 and chromosome 2m
, thus restricting gene flow between the two. In addition, they found that chromosome 2 had five times the nucleotide diversity of chromosome 2m
. Based on these results, Thorneycroft's [31
] meiotic data, and our data on population genotype frequencies (Table ), we predict that chromosome 3a
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 2m
, it is likely that some negative fitness affects are associated with homozygosity; the same is likely true for chromosome 3a
. We did not find 2m
individuals in our karyotypic analyses, nor did we find any 2m
birds in over 546 individuals sampled in the VIP
assay. Thorneycroft [31
] found that parents with a single 2m
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 (2m
/2) pairing with other white birds (2m
/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 ). 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
]. Our comparative analysis of the VIP
] revealed that the Dra
I 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 and ) and other areas of chromosome 2m
suggest that the first inversion predated the divergence of white-throated sparrow from both white-crowned sparrow and Harris's sparrow, originating approximately 2.2 ± 0.3 MYA [62
]. Surprisingly, our comparative data on the VIP
intron suggests that, contrary to Thorneycroft's [31
] expectations, chromosome 2m
(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
], 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