Publications

2011
Rheindt FE, Edwards SV. Genetic introgression: an integral but neglected component of speciation in birds. The Auk. 2011;128 :620-632. PDF
Alfoldi J, Di Palma F, Grabherr M, Williams C, Kong LS, Mauceli E, Russell P, Lowe CB, Glor RE, Jaffe JD, et al. The genome of the green anole lizard and a comparative analysis with birds and mammals. Nature. 2011;477 :587-591.Abstract

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments(1). Among amniotes, genome sequences are available for mammals and birds(2-4), but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes(2). Also, A. carolinensis mobile elements are very young and diverse-more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds(5). We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.

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Fujita MK, Edwards SV, Ponting CP. Near complete erosion of isochores in the Anolis lizard genome. Genome Biology and Evolution. 2011;3 :974-984.Abstract
Isochores are large regions of relatively homogeneous nucleotide composition and are present in the genomes of all mammals and birds that have been sequenced to date. The newly sequenced genome of Anolis carolinensis provides the first opportunity to quantify isochore structure in a nonavian reptile. We find Anolis to have the most compositionally homogeneous genome of all amniotes sequenced thus far, a homogeneity exceeding that for the frog Xenopus. Based on a Bayesian algorithm, Anolis has smaller and less GC-richisochores compared with human and chicken. Correlates generally associated with GC-rich isochores, including shorter introns and higher gene density, have all but disappeared from the Anolis genome. Using genic GC as a proxy for isochore structure so as to compare with other vertebrates, we found that GC content has substantially decreased in the lineage leading to Anolis since diverging from the common ancestor of Reptilia; 275 Ma, perhaps reflecting weakened or reversed GC-biased gene conversion, a nonadaptive substitution process that is thought to be important in the maintenance and trajectory of isochore evolution. Our results demonstrate that GC composition in Anolis is not associated with important features of genome structure, including gene density and intron size, in contrast to patterns seen in mammal and bird genomes.
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Castoe TA, Bronikowski AM, Brodie III ED, Edwards SV, Pfrender ME, Shapiro MD, Pollock DD, Warren WC. A proposal to sequence the genome of a garter snake (Thamnophis sirtalis). Standards in genomic sciences. 2011;4 :257.Abstract
Here we develop an argument in support of sequencing a garter snake (Thamnophis sirtalis) genome, and outline a plan to accomplish this. This snake is a common, widespread, nonve-nomous North American species that has served as a model for diverse studies in evolutio-nary biology, physiology, genomics, behavior and coevolution. The anole lizard is currently the only genome sequence available for a non-avian reptile. Thus, the garter snake at this time would be the first available snake genome sequence and as such would provide much needed comparative representation of non-avian reptilian genomes, and would also allow critical new in sights for vertebrate comparative genomic studies. We outline the major areas of discovery that the availability of the garter snake genome would enable, and describe a plan for whole-genome sequencing.
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Bonneaud C, Balenger SL, Russell AF, Zhang JW, Hill GE, Edwards SV. Rapid evolution of disease resistance is accompanied by functional changes in gene expression in a wild bird. Proceedings of the National Academy of Sciences of the United States of America. 2011;108 :7866-7871.Abstract

Wild organisms are under increasing pressure to adapt rapidly to environmental changes. Predicting the impact of these changes on natural populations requires an understanding of the speed with which adaptive phenotypes can arise and spread, as well as of the underlying mechanisms. However, our understanding of these parameters is poor in natural populations. Here we use experimental and molecular approaches to investigate the recent emergence of resistance in eastern populations of North American house finches (Carpodacus mexicanus) to Mycoplasma galliseptum (MG), a severe conjunctivitis-causing bacterium. Two weeks following an experimental infection that took place in 2007, finches from eastern US populations with a 12-y history of exposure to MG harbored 33% lower MG loads in their conjunctivae than finches from western US populations with no prior exposure to MG. Using a cDNA microarray, we show that this phenotypic difference in resistance was associated with differences in splenic gene expression, with finches from the exposed populations up-regulating immune genes postinfection and those from the unexposed populations generally down-regulating them. The expression response of western US birds to experimental infection in 2007 was more similar to that of the eastern US birds studied in 2000, 7 y earlier in the epizootic, than to that of eastern birds in 2007. These results support the hypothesis that resistance has evolved by natural selection in the exposed populations over the 12 y of the epizootic. We hypothesize that host resistance arose and spread from standing genetic variation in the eastern US and highlight that natural selection can lead to rapid phenotypic evolution in populations when acting on such variation.

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Kuenstner A, Wolf JBW, Backstroem N, Whitney O, Balakrishnan CN, Day L, Edwards SV, Janes DE, Schlinger BA, Wilson RK, et al. Comparative genomics based on massive parallel transcriptome sequencing reveals patterns of substitution and selection across 10 bird species. (vol 19, pg 266, 2010). Molecular Ecology [Internet]. 2011;20 (13) :2871-2871. Publisher's VersionAbstract
Next-generation sequencing technology provides an attractive means to obtain large-scale sequence
data necessary for comparative genomic analysis. To analyse the patterns of mutation rate variation
and selection intensity across the avian genome, we performed brain transcriptome sequencing using
Roche 454 technology of 10 different non-model avian species. Contigs from
de novo
assemblies
were aligned to the two available avian reference genomes, chicken and zebra finch. In total, we
identified 6499 different genes across all 10 species, with ~1000 genes found in each full run per
species. We found evidence for a higher mutation rate of the Z chromosome than of autosomes (male-
biased mutation) and a negative correlation between the neutral substitution rate (
d
S
) and
chromosome size. Analyses of the mean
d
N
/
d
S
ratio (
ω
) of genes across chromosomes supported the
Hill–Robertson effect (the effect of selection at linked loci) and point at stochastic problems with
ω
as an independent measure of selection. Overall, this study demonstrates the usefulness of next-
generation sequencing for obtaining genomic resources for comparative genomic analysis of non-

model organisms

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Alcaide M, Edwards SV. Molecular Evolution of the Toll-Like Receptor Multigene Family in Birds. Molecular Biology and Evolution [Internet]. 2011;28 (5) :1703-1715. Publisher's VersionAbstract
Toll-like receptors (TLR) are membrane-bound sensors of the innate immune system that recognize invariant and
distinctive molecular features of invading microbes and are also essential for initiating adaptive immunity in vertebrates.
The genetic variation at TLR genes has been directly related to differential pathogen outcomes in humans and livestock.
Nonetheless, new insights about the impact of TLRs polymorphism on the evolutionary ecology of infectious diseases can
be gained through the investigation of additional vertebrate groups not yet investigated in detail. In this study, we have
conducted the first characterization of the entire TLR multigene family (
N
5
10 genes) in non-model avian species. Using
primers targeting conserved coding regions, we aimed at amplifying large segments of the extracellular domains (275–435
aa) involved in pathogen recognition across seven phylogenetically diverse bird species. Our analyses suggest avian TLRs
are dominated by stabilizing selection, suggesting that slow rates of nonsynonymous substitution help preserve biological
function. Overall, mean values of
x
(
5
d
n
/d
s
) at each TLR locus ranged from 0.196 to 0.517. However, we also found
patterns of positive selection acting on specific amino acid sites that could be linked to species-specific differences in
pathogen-associated molecular pattern recognition. Only 39 of 2,875 (
;
1.35%) of the codons analyzed exhibited
significant patterns of positive selection. At least one half of these positively selected codons can be mapped to putative
ligand-binding regions, as suggested by crystallographic structures of TLRs and their ligands and mutagenic analyses. We
also surveyed TLR polymorphism in wild populations of two bird species, the Lesser Kestrel
Falco naumanni
and the House
Finch
Carpodacus mexicanus
. In general, avian TLRs displayed low to moderate single nucleotide polymorphism levels and
an excess of silent nucleotide substitutions, but also conspicuous instances of positive directional selection. In particular,
TLR5 and TLR15 exhibited the highest degree of genetic polymorphism and the highest occurrence of nonconservative
amino acid substitutions. This study provides critical primers and a first look at the evolutionary patterns and implications
of TLR polymorphism in non-model avian species and extends the list of candidate loci for avian eco-immunogenetics
beyond the widely employed genes of the Major Histocompatibility Complex (MHC).
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Bonneaud C, Balenger SL, Russell AF, Zhang J, Hill GE, Edwards SV. Rapid evolution of disease resistance is accompanied by functional changes in gene expression in a wild bird. Proceedings of the National Academy of Sciences of the United States of America [Internet]. 2011;108 (19) :7866-7871. Publisher's VersionAbstract
Wild organisms are under increasing pressure to adapt rapidly to
environmental changes. Predicting the impact of these changes on
natural populations requires an understanding of the speed with
which adaptive phenotypes can arise and spread, as well as of
the underlying mechanisms. However, our understanding of these
parameters is poor in natural populations. Here we use experimen-
tal and molecular approaches to investigate the recent emergence
of resistance in eastern populations of North American house
fi
nches (
Carpodacus mexicanus
)to
Mycoplasma galliseptum
(MG),
a severe conjunctivitis-causing bacterium. Two weeks following an
experimentalinfectionthattookplacein2007,
fi
nchesfromeastern
US populations with a 12-y history of exposure to MG harbored
33% lower MG loads in their conjunctivae than
fi
nches from west-
ern US populations with no prior exposure to MG. Using a cDNA
microarray, we show that this phenotypic difference in resistance
was associated with differences in splenic gene expression, with
fi
nchesfromthe exposedpopulations up-regulatingimmunegenes
postinfection and those from the unexposed populations generally
down-regulating them. The expression response of western US
birds to experimental infection in 2007 was more similar to that
of the eastern US birds studied in 2000, 7 y earlier in the epizootic,
than to that of eastern birds in 2007. These results support the
hypothesis that resistance has evolved by natural selection in the
exposedpopulationsoverthe12yoftheepizootic.Wehypothesize
that host resistance arose and spread from standing genetic varia-
tion in the eastern US and highlight that natural selection can lead
to rapid phenotypic evolution in populations when acting on

such variation

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Janes DE, Chapus C, Gondo Y, Clayton DF, Sinha S, Blatti CA, Organ CL, Fujita MK, Balakrishnan CN, Edwards SV. Reptiles and Mammals Have Differentially Retained Long Conserved Noncoding Sequences from the Amniote Ancestor. Genome Biology and Evolution [Internet]. 2011;3 :102-113. Publisher's VersionAbstract
Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of
noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching
genomes of chicken (
Gallus gallus
), zebra finch (
Taeniopygia guttata
), and green anole (
Anolis carolinensis
), we quantified
the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we
define as sequences
500 bp in length and exhibiting
95% similarity between species. We found 4,294 LCNS shared
between chicken and zebra finch and 574 LCNS shared by the two birds and
Anolis
. The percent of genomes comprised by
LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (
,
0.0003% to
,
0.001%), differences that
we show may be explained in part by differences in genome-wide substitution rates. We reconstruct a large number of LCNS
for the amniote ancestor (ca. 8,630) and hypothesize differential loss and substantial turnover of these sites in descendent
lineages. By contrast, we estimated a small role for recruitment of LCNS via acquisition of novel functions over time. Across
amniotes, LCNS are significantly enriched with transcription factor binding sites for many developmental genes, and 2.9% of
LCNS shared between the two birds show evidence of expression in brain expressed sequence tag databases. These results
show that the rate of retention of LCNS from the amniote ancestor differs between mammals and Reptilia (including birds)

and that this may reflect differing roles and constraints in gene regulation

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Vo A-TE, Bank MS, Shine JP, Edwards SV. Temporal increase in organic mercury in an endangered pelagic seabird assessed by century-old museum specimens. Proceedings of the National Academy of Sciences of the United States of America [Internet]. 2011;108 (18) :7466-7471. Publisher's VersionAbstract

Methylmercury cycling in the Pacific Ocean has garnered significant attention in recent years, especially with regard to rising mercury emissions from Asia. Uncertainty exists concerning whether increases in anthropogenic emissions over time may have caused increased mercury bioaccumulation in the biota. To address this, we measured total mercury and, for a subset of samples, methylmercury (the bioaccumulated form of mercury) in museum feathers from an endangered seabird, the black-footed albatross (Phoebastria nigripes), spanning a 120-y period. We analyzed stable isotopes of nitrogen (δ15N) and carbon (δ13C) to control for temporal changes in trophic structure and diet. In post-1940 and -1990 feathers, we detected significantly higher mean methylmercury concentrations and higher proportions of samples exhibiting above deleterious threshold levels (∼40,000 ng·g−1) of methylmercury relative to prior time points, suggesting that mercury toxicity may undermine reproductive effort in the species. We also found higher levels of (presumably curator-mediated) inorganic mercury in older specimens of albatross as well as two nonpelagic species lacking historical exposure to bioavailable mercury, patterns suggesting that studies on bioaccumulation should measure methylmercury rather than total mercury when using museum collections. δ15N contributed substantially to models explaining the observed methylmercury variation. After simultaneously controlling for significant trends in δ13C over time and δ15N with methylmercury exposure, year remained a significant independent covariate with feather methylmercury levels among the albatrosses. These data show that remote seabird colonies in the Pacific basin exhibit temporal changes in methylmercury levels consistent with historical global and recent regional increases in anthropogenic emissions.

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2010
Castillo-Ramírez S, Liu L, Pearl D, Edwardsm S. V. Bayesian estimation of species trees: a practical guide to optimal sampling and analysis. In: Knowles LL, Kubatko LS Estimating Species Trees: Practical and Theoretical Aspects. New Jersey: Wiley-Blackwell ; 2010. pp. 15-33.
Balakrishnan CN, Y. LJ, Edwards SV. Phylogeography and phylogenetics in the nuclear age. In: Grant P, Grant R Searching for the Causes of Evolution: From Field Observations to Mechanisms. Princeton, NJ: Princeton University Press ; 2010. pp. 65-88.
Organ CL, Rasmussen M, Baldwin MW, Kellis M, Edwards SV. Phylogenomic approach to the evolutionary dynamics of gene duplication in birds. In: Dittmar K, Liberles D Evolution After Gene Duplication. New York: Wiley & Sons ; 2010. pp. 253-267.
Kunstner A, Wolf JBW, Backstrom N, Whitney O, Balakrishnan CN, Day L, Edwards SV, Janes DE, Schlinger BA, Wilson RK, et al. Comparative genomics based on massive parallel transcriptome sequencing reveals patterns of substitution and selection across 10 bird species. Molecular Ecology. 2010;19 :266-276.Abstract

Next-generation sequencing technology provides an attractive means to obtain large-scale sequence data necessary for comparative genomic analysis. To analyse the patterns of mutation rate variation and selection intensity across the avian genome, we performed brain transcriptome sequencing using Roche 454 technology of 10 different non-model avian species. Contigs from de novo assemblies were aligned to the two available avian reference genomes, chicken and zebra finch. In total, we identified 6499 different genes across all 10 species, with similar to 1000 genes found in each full run per species. We found evidence for a higher mutation rate of the Z chromosome than of autosomes (male-biased mutation) and a negative correlation between the neutral substitution rate (d(S)) and chromosome size. Analyses of the mean d(N)/d(S) ratio (omega) of genes across chromosomes supported the Hill-Robertson effect (the effect of selection at linked loci) and point at stochastic problems with omega as an independent measure of selection. Overall, this study demonstrates the usefulness of next-generation sequencing for obtaining genomic resources for comparative genomic analysis of non-model organisms.

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Jaubert-Possamai S, Rispe C, Tanguy S, Gordon K, Walsh T, Edwards O, Tagu D. Expansion of the miRNA Pathway in the Hemipteran Insect Acyrthosiphon pisum. Molecular Biology and Evolution. 2010;27 :979-987.Abstract

The pathways that allow short noncoding RNAs such as the microRNAs (miRNAs) to mediate gene regulation and control critical cellular and developmental processes involve a limited number of key protein components. These proteins are the Dicer-like RNases, double-stranded RNA (dsRNA)-binding proteins, and the Argonaute (AGO) proteins that process stem-loop hairpin transcripts of endogenous genes to generate miRNAs or long dsRNA precursors (either exogenous or endogenous). Comparative genomics studies of metazoans have shown the pathways to be highly conserved overall; the major difference observed is that the vertebrate pathways overlap in sharing a single Dicer (DCR) and AGO proteins, whereas those of insects appear to be parallel, with distinct Dicers and AGOs required for each pathway. The genome of the pea aphid is the first available for a hemipteran insect and discloses an unexpected expansion of the miRNA pathway. It has two copies of the miRNA-specific dicr-1 and ago1 genes and four copies of pasha a cofactor of drosha involved in miRNA biosynthesis. For three of these expansions, we showed that one copy of the genes diverged rapidly and in one case (ago1b) shows signs of positive selection. These expansions occurred concomitantly within a brief evolutionary period. The pea aphid, which reproduces by viviparous parthenogenesis, is able to produce several adapted phenotypes from one single genotype. We show by reverse transcriptase-polymerase chain reaction that all the duplicated copies of the miRNA machinery genes are expressed in the different morphs. Investigating the function of these novel genes offers an exciting new challenge in aphid biology.

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Janes DE, Organ CL, Fujita MK, Shedlock AM, Edwards SV. Genome evolution in Reptilia, the sister group of mammals. Annual Review of Genomics and Human Genetics. 2010;11 :239-64.Abstract

The genomes of birds and nonavian reptiles (Reptilia) are critical for understanding genome evolution in mammals and amniotes generally. Despite decades of study at the chromosomal and single-gene levels, and the evidence for great diversity in genome size, karyotype, and sex chromosome diversity, reptile genomes are virtually unknown in the comparative genomics era. The recent sequencing of the chicken and zebra finch genomes, in conjunction with genome scans and the online publication of the Anolis lizard genome, has begun to clarify the events leading from an ancestral amniote genome--predicted to be large and to possess a diverse repeat landscape on par with mammals and a birdlike sex chromosome system--to the small and highly streamlined genomes of birds. Reptilia exhibit a wide range of evolutionary rates of different subgenomes and, from isochores to mitochondrial DNA, provide a critical contrast to the genomic paradigms established in mammals.

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Warren WC, Clayton DF, Ellegren H, Arnold AP, Hillier LW, Kunstner A, Searle S, White S, Vilella AJ, Fairley S, et al. The genome of a songbird. Nature. 2010;464 :757-762.Abstract

The zebra finch is an important model organism in several fields(1,2) with unique relevance to human neuroscience(3,4). Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken(5)-the only bird with a sequenced genome until now(6). Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes(7). We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

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O'Keefe KJ, Silander OK, McCreery H, Weinreich DM, Wright KM, Chao L, Edwards SV, Remold SK, Turner PE. Geographic differences in sexual reassortment in an RNA phage. Evolution. 2010;64 :3010-3023.Abstract
The genetic structure of natural bacteriophage populations is poorly understood. Recent metagenomic studies suggest that phage biogeography is characterized by frequent migration. Using virus samples mostly isolated in Southern California, we recently showed that very little population structure exists in segmented RNA phage of the Cystoviridae family due to frequent segment reassortment (sexual genetic mixis) between unrelated virus individuals. Here we use a larger genetic dataset to examine the structure of Cystoviridae phage isolated from three geographic locations in Southern New England. We document extensive natural variation in the physical sizes of RNA genome segments for these viruses. In addition, consistent with earlier findings, our phylogenetic analyses and calculations of linkage disequilibrium (LD) show no evidence of within-segment recombination in wild populations. However, in contrast to the prior study, our analysis finds that reassortment of segments between individual phage plays a lesser role among cystoviruses sampled in New England, suggesting that the evolutionary importance of genetic mixis in Cystoviridae phage may vary according to geography. We discuss possible explanations for these conflicting results across the studies, such as differing local ecology and its impact on phage growth, and geographic differences in selection against hybrid phage genotypes.
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Liu L, Yu L, Edwards S. A maximum pseudo-likelihood approach for estimating species trees under the coalescent model. BMC Evolutionary Biology. 2010;10 :302.Abstract

BACKGROUND:Several phylogenetic approaches have been developed to estimate species trees from collections of gene trees. However, maximum likelihood approaches for estimating species trees under the coalescent model are limited. Although the likelihood of a species tree under the multispecies coalescent model has already been derived by Rannala and Yang, it can be shown that the maximum likelihood estimate (MLE) of the species tree (topology, branch lengths, and population sizes) from gene trees under this formula does not exist. In this paper, we develop a pseudo-likelihood function of the species tree to obtain maximum pseudo-likelihood estimates (MPE) of species trees, with branch lengths of the species tree in coalescent units.RESULTS:We show that the MPE of the species tree is statistically consistent as the number M of genes goes to infinity. In addition, the probability that the MPE of the species tree matches the true species tree converges to 1 at rate O(M -1). The simulation results confirm that the maximum pseudo-likelihood approach is statistically consistent even when the species tree is in the anomaly zone. We applied our method, Maximum Pseudo-likelihood for Estimating Species Trees (MP-EST) to a mammal dataset. The four major clades found in the MP-EST tree are consistent with those in the Bayesian concatenation tree. The bootstrap supports for the species tree estimated by the MP-EST method are more reasonable than the posterior probability supports given by the Bayesian concatenation method in reflecting the level of uncertainty in gene trees and controversies over the relationship of four major groups of placental mammals.CONCLUSIONS:MP-EST can consistently estimate the topology and branch lengths (in coalescent units) of the species tree. Although the pseudo-likelihood is derived from coalescent theory, and assumes no gene flow or horizontal gene transfer (HGT), the MP-EST method is robust to a small amount of HGT in the dataset. In addition, increasing the number of genes does not increase the computational time substantially. The MP-EST method is fast for analyzing datasets that involve a large number of genes but a moderate number of species.

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Janes DE, Valenzuela N, Ezaz, Tariq, Amemiya, Chris, Edwards SV. Sex chromosome evolution in amniotes: applications for bacterial artificial chromosome libraries. Journal of Biomedicine and Biotechnology. 2010;2011.Abstract

Variability among sex chromosome pairs in amniotes denotes a dynamic history. Since amniotes diverged from a common
ancestor, their sex chromosome pairs and, more broadly, sex-determining mechanisms have changed reversibly and frequently.
These changes have been studied and characterized through the use of many tools and experimental approaches but perhaps
most effectively through applications for bacterial artificial chromosome (BAC) libraries. Individual BAC clones carry 100–200 kb
of sequence from one individual of a target species that can be isolated by screening, mapped onto karyotypes, and sequenced.
With these techniques, researchers have identified differences and similarities in sex chromosome content and organization across
amniotes and have addressed hypotheses regarding the frequency and direction of past changes. Here, we review studies of sex
chromosome evolution in amniotes and the ways in which the field of research has been affected by the advent of BAC libraries.

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