BACKGROUND
cotton produces the world’s most utilized natural fiber, making it a staple of the textile and garment industries.
in  <dig> cotton fiber production in the us was valued at approximately  <dig>  billion dollars, with an additional  <dig> million dollars derived from cotton seed extracts  <cit> .
this places cotton among the top five most valuable us crops.
accordingly, it is important to understand cotton’s gene repertoire and its evolutionary history.
with the vast majority of cotton production being derived from the allotetraploid species gossypium hirsutum, followed by smaller contributions from a second domesticated allotetraploid, g. barbadense, an appreciation of cotton biology in the context of its allotetraploid history has become a major research focus for the improvement of this crop.
here we present a comprehensive assembly of approximately  <dig>  million est sequences from these two allotetraploid species and from the diploid species representing the progenitor genomes of allotetraploid cotton.
we illustrate how this assembly facilitates diagnosis of the genome-of-origin for what otherwise would have been ambiguously derived transcripts, and demonstrate the utility of this assembly for addressing questions pertaining to the phenomenon of allopolyploidy and cotton transcriptome evolution.
the cotton genus comprises about  <dig> species, divided into eight monophyletic diploid genome groups , and a clade of five allotetraploid species, the latter containing a genome from both the a and d diploid genome groups .
the cytological and phylogenetic history of this genus is well-resolved , and data demonstrate that the best living models of the progenitor diploids of the a- and d-genomes of modern allotetraploid cotton are the diploid species g. arboreum and g. raimondii, respectively.
in addition, extensive seed collections are available and include both wild progenitor forms along with their domesticated descendants.
collectively, these resources provide a useful system for studying the genomic and phenotypic changes that accompany domestication.
embedding these resources within their phylogenetic context results in a powerful framework for the study of polyploidy and domestication, and consequently cotton has emerged as a model system in these areas of research.
recent examples of the utility of the cotton system in this regard include detailed analyses of the cotton fiber transcriptome during development  <cit> , as well as explorations of the mutational and expression changes that accompany allopolyploidization  <cit> .
these types of studies, as well as those aimed at crop improvement, are enriched or facilitated by the availability of cotton est collections, underscoring the utility and importance of developing a rich and deep database of est resources.
prior to this study, the community-driven global assembly of cotton ests included ~  <dig>  sanger sequences, with ~  <dig>  g. arboreum , ~  <dig>  g. raimondii , and ~  <dig>  g. hirsutum  sequences assembled into a single reference collection yielding  <dig>  contigs  <cit> .
because this assembly  contained sequences from both model diploid parents and a natural allotetraploid representing their combination, it could be used to identify a- and d-genome-specific snps, which can in turn be used to assign genomic origin to homoeologous transcripts in allotetraploid cotton .
implementation of this est collection strategy has enabled several research possibilities, including monitoring genome-specific expression in natural allotetraploids  <cit>  and the quantification of the extent of nonreciprocal homoeologous recombination among natural allotetraploid species   <cit> .
these efforts have elucidated the dynamics of gene expression evolution and genic content variation that accompanied allotetraploid formation and evolution in cotton.
here we present a vastly expanded cotton est assembly, which contains approximately  <dig>  million sanger and next-generation  transcripts.
like previous assemblies  <cit> , this one incorporates ests from both the a- and d-genome diploid progenitors, along with allotetraploid ests from two species of allotetraploid cotton, g. barbadense and g. hirsutum.
the  <dig>  contigs obtained from this assembly represent a vastly expanded representation of the genic content of cotton.
to add additional depth to the assembly, we also generated ~ <dig> million 82 bp illumina reads, representing the fiber transcriptome of diploid a- and d-genome cotton as well as the allotetraploids g. barbadense and g. hirsutum.
together these resources allow us to detect  <dig>  genome-specific snps, which in turn can be used to distinguish the a- and d-genome homoeologs found in the allotetraploid cotton genome.
we describe this collection and document its utility for genome-specific transcriptome analysis in allotetraploid cotton.
we also present a characterization of the functional properties of the cotton transcriptome and analyses of molecular evolution following the most recent whole genome duplication that accompanied allotetraploid formation 1– <dig> million years ago <cit> .
at the time of writing, allotetraploid cotton is now among the most important crops lacking a whole genome sequence, but as progress is made in this regard, the est assembly and genome-specific snp resources presented here will be of use in assembling and annotating the cotton genome.
RESULTS
est assembly characteristics
the cotton assembly presented here contains est sequences  from g. arboreum , g. barbadense , g. hirsutum , and g. raimondii  .
ests from these species were assembled jointly in a multispecies assembly.
the justification for this multispecies assembly stems from the fact that the transcripts from resident a- and d-genomes in the allotetraploid nucleus are more closely related to their diploid homologs than they are to each other .
thus, combining diploid est sequences with the ests of two tetraploid species does not introduce appreciable amounts of additional sequence divergence.
from a total of approximately  <dig>  million gossypium ests,  <dig> , <dig> were assembled into contigs, while  <dig>  ests remained unassembled .
the assembly includes previously published sanger sequences  as well as approximately  <dig>  million new  <dig> sequences .
the assembled collection of ests produced  <dig>  contigs with an average length of  <dig> .5 bp .
a study that validates the quality of this assembly is described in the materials and methods section, wherein we find high nucleotide similarity between our assembled contigs and cloned and sequenced cotton genes.
this suggests that the sequence quality and assembly routine accurately recapitulate the structure and mutational history found in well-characterized cotton genes.
1accession unknown or derived from numerous accessions.
references for sanger est collections can be found in udall et al.
<cit> .
among the contigs,  <dig>  include a predicted coding region, with an average coding length of  <dig>  bp.
this estimated coding sequence length for cotton is a little less than the average coding sequence length of some angiosperms with sequenced genomes.
for example, the average coding sequence lengths of a. thalianapopulus trichocarpa , and vitis vinifera  are  <dig> ,  <dig> , and  <dig>  bp, respectively .
if we assume that the true coding sequence length in cotton is comparable to the average length for these species , we estimate that our est assembly provides coverage of about 76% of the coding sequence for the average contig.
in addition to breadth of coverage, the assembly achieves a new level of depth of coverage; the median number of ests per contig was  <dig>  with a range from  <dig> to  <dig> , and the average depth of coverage for each assembled nucleotide was  <dig> .
these results mark a significant improvement over previous cotton transcriptome assemblies, where the average contig length was 791 bp, the median number of ests per contig was  <dig>  and the average depth of coverage for each assembled nucleotide was  <dig>   <cit> .
to assess our expectation of co-assembly of expressed sequences from multiple cotton genomes, species membership was compiled for each contig of the assembly .
the most common class of contigs  are those that contain ests from all four species , indicating that genic conservation is substantial among these species and that we have achieved sequence coverage sufficient to reveal this.
<dig>  contigs were represented by sequences from both g. arboreum  and g. raimondii , whereas  <dig>  contigs contained sequences derived from both allotetraploids.
interestingly, we observed  <dig>  contigs for which sequences were lacking only for g. arboreum and only  <dig> contigs constructed entirely from g. arboreum ests .
these findings together suggest that the g. arboreum ests collection represents a significantly narrower pool of genes relative to the other species.
although it is possible that some g. arboreum orthologs are missing because of gene loss or gene silencing, there is substantial evidence that g. arboreum has not lost a large portion of its transcriptome or experienced massive global shifts in gene expression  <cit> .
we note that g. arboreum was the only species lacking  <dig> sequences generated by titanium chemistry, having instead only  <dig> flx reads.
in support of this point, we do observe a 13 % enrichment of  <dig> titanium reads from g. barbadenseg.
hirsutum, and g. raimondii in the contigs missing only g. arboreum when compared to their proportions in all other contigs .
our best hypothesis is that technical differences between these two sequencing chemistries, including sequencing bias, differences in library construction, and assembly characteristics underlie the unexpectedly narrow breadth of the g. arboreum est collection.
assembly annotation
sequence similarity searches  to the refseq  database were used to provide an approximation of gene function.
the contigs and singletons  were compared to annotated proteins from refseq with a minimum e-value score of 1e- <dig>   <dig>  contig sequences had a high similarity  to refseq sequences and  <dig>  contig sequences had a match less than 1e- <dig>  the contig sequences were also compared to the annotated genomes of carica papaya and a. thaliana.
when only the top match was considered,  <dig>  contigs had a near identical match to an a. thaliana gene  and  <dig>  contigs had top matches with an e-value lower than 1e- <dig>  more than  <dig>  contigs had a top match to an a. thaliana gene with an e-value lower than 1e- <dig>  when only the top c. papaya match was considered,  <dig>  contigs had a near identical match to a c. papaya gene  and  <dig>  had top matches with an e-value lower than 1e- <dig>  more than  <dig>  contigs had a top match to a c. papaya gene with an e-value lower than 1e- <dig>  the similar results obtained with both arabidopsis and carica lend confidence that this set of annotations is accurate.
identification of protein coding domains
among the  <dig>  contigs in the assembly,  <dig>  had a predicted translational product , and among this subset  <dig>  protein products had at least one annotated pfam domain or could be assigned to a pfam family.
not surprisingly, the most abundant domain was the pentatricopeptide repeat .
this domain is among the most numerous in the angiosperms and may be associated with rna stabilization and processing  <cit> .
other highly prevalent pfam annotations include the wd <dig>  protein kinase, and myb dna binding domains, as well as the leucine rich repeat and rna recognition motifs.
like the ppr repeat, these domains are also abundant in other angiosperm genomes.
to better assess the functional characteristics of the cotton transcriptome assembly, we converted pfam annotations into go categories.
the relative abundance of various high-level go categories is shown in figure  <dig>  these data indicate that our current cotton est assembly includes a large and diverse set of genes.
this broad sampling of annotated genes should be useful for enrichment-based tests among differentially expressed genes as determined by future microarray, rna-seq, or similar experiments.
we anticipate this set of genes will serve as a useful transcriptome model until empirically derived transcripts can be paired with gene predictions from a de novo cotton genome sequence.
homoeologous snps
because our cotton est assembly includes a large number of sequences from a- and d-genome diploid progenitors as well as ad-genome allotetraploid derived ests, it can be used to diagnose homoeologs and homoeolog-specific snps.
in a strategy similar to buggs et al.,  <cit> , we identified homoeolog-specific snps directly from the 454/sanger assembly and supplemented this assessment with  <dig> , <dig> illumina sequences, also derived from the a- and d-genome diploid progenitors and ad-genome allotetraploids, and mapped to the de novo 454/sanger est assembly.
this strategy leverages the strengths of both the  <dig> and illumina next-generation platforms.
the  <dig>  reads are longer and assemble into contigs more readily, while the illumina platform offers deep sequence coverage that can be used to confirm the 454/sanger snps and find new snps in regions with low 454/sanger read coverage.
in total, we identified  <dig>  snps between the diploid a- and d-genomes, including  <dig>  that were exclusive to the de novo 454/sanger assembly,  <dig>  exclusive to the illumina reference mapping, and  <dig>  common to both strategies .
the intersection of both snp pools is only a fraction of the total.
however, to identify homoeologous snps in the 454/sanger assembly it is necessary to have ests from the a- and d-genome diploids present, as is the case for  <dig>  contigs .
furthermore, within these contigs the a- and d-genome diploid sequences must overlap one another.
this occurs for approximately 29 % of all available nucleotide positions within the eligible contigs .
finally, cross-validating a snp with both the 454/sanger assembly and the illumina reads requires an overlap between both diploid parents from both sources of ests .
this occurs for about 23% of all sites from the  <dig>  eligible contigs .
the diminishing effect these requirements have on snp identification likely explains the modest proportion of snps that intersect between both platforms.
among those genome-specific snps found in both the 454/sanger and illumina data sets, only  <dig>  were in disagreement about the nucleotides representing the a- and d- genomes, leaving  <dig>   in agreement .
this last class of snps represents a high confidence category, though the snps identified only in the 454/sanger or illumina data sets have considerable internal support and also are likely reliable.
for the cross-platform verified snps, we confirmed the presence of all a- and d-genome diploid snp variants within the allotetraploid illumina reads.
this additional constraint of no further nucleotide evolution subsequent to polyploidization in either the diploid or allotetraploid lineages ensures that these snps almost certainly occurred prior to the formation of the allotetraploids.
these snps can be used to diagnose both diploid homologs and allotetraploid homoeologs.
detection of nonreciprocal homoeologous recombination
genes doubled by allopolyploidy may, in principle, evolve independently or they may interact, thereby evolving in a “concerted” fashion, mediated by gene conversion or other forms of duplicate gene sequence homogenization.
salmon et al.
<cit> , studied this phenomenon in allotetraploid cotton using an earlier release of the cotton transcriptome assembly.
following the methods outlined in salmon et al.
<cit> , we report an updated account of nrhr.
this updated account includes a richer est assembly, and also differs in that the current assembly includes sequences from a second allotetraploid species, g. barbadense.
because the two allotetraploid species used in this study  share a common ancestor  <cit> , detected nrhr can be mapped onto their phylogeny.
through this process we partitioned exchanges that occurred in only one allotetraploid lineage from those that occurred in their common ancestor .
a total of  <dig>  unique nrhr were detected in the new assembly.
of these, approximately 46 % and 31 % are found only in g. hirsutum and in g. barbadense, respectively, whereas about 23% of the exchanges were found in both species .
interestingly, the unique set of  <dig>  nrhr belong to only  <dig>  contigs.
contigs with multiple exchanges may have experienced independent nrhr events, or alternatively, they may represent only one complex event, with several internal exchanges, that we have been unable to bridge together and resolve.
this finding was initially reported by salmon et al.
<cit> , and its reiteration here with a much larger sample size provides evidence that certain duplicated genes or regions of the allotetraploid genome may harbor lengthy complex nrhr or could be “hotspots” of repeated exchange.
molecular evolution in diploid and allotetraploid cotton
the perspective provided by previous taxonomic and phylogenetic work  <cit>  combined with the est assembly generated here offers an unparalleled opportunity to explore the molecular evolutionary properties of genes in diploids and their allotetraploid derivatives.
to assess the potential impact of polyploidy we compared sequence evolution between a- and d-genome orthologs in diploid cottons to that of a- and d-genome homoeologs in both species of allotetraploid cotton .
this was achieved by first isolating the a- and d-genome homoeologous reads from both allotetraploids, g. barbadense and g. hirsutum,  using est sequences from the a- and d-genome diploid species as a guide .
for each inter-  or intra-genomic  comparison we were able to study thousands of contigs and millions of nucleotides , providing exquisite detail on the amount of divergence that has accumulated since the two diploids last shared a common ancestor, and since allotetraploid formation.
1see molecular evolution section in the materials and methods for further detail about contig selection.
we isolated the coding regions  from which we calculated the rates of synonymous  and nonsynonymous  divergence within and between species .
these two metrics can also be combined in as a ratio , which gives an indication of the selection pressures operating on the protein composition of a gene.
elevated dn/ds ratios  may be indicative of positive selection, though with low overall levels of divergence, as is seen in cotton, this interpretation must be viewed with caution as stochastic processes operating on a small number of total mutations can lead to spurious inferences of selection.
nonetheless, gene and genome duplication are thought to provide the opportunity for rapid gene evolution of one duplicate, so long as the other duplicate maintains sufficient ancestral functionality  <cit> .
in the present context, we might expect to find an elevated dn/ds ratio between cotton at and dt homoeologs, when compared to the dn/ds ratio of their a <dig> and d <dig> diploid progenitors.
as shown in table  <dig>  we estimated dn and ds for anywhere from  <dig>  to  <dig>  contigs in pairwise comparisons among genomes.
these values fall short of the total available contig counts listed in figure  <dig> because some contigs lack a predicted protein-coding region or because the species in question fail to overlap one another within the contig alignment.
several conclusions are evident from these data, mirroring but also vastly extending similar results published by senchina et al.
<cit>  for a sample of only  <dig> genes: first, because the dataset is based on thousands of alignments in each comparison, it provides an accurate depiction of the ancestry of polyploid cotton.
in this regard, the a <dig> diploid genome has a mean synonymous distance  of  <dig>  or  <dig>  from the two at-genomes whereas the comparable figure is  <dig>  or  <dig>  for d <dig> versus the two dt genomes.
from this we infer that modern g. arboreum is a better model  of the actual genome donor of allotetraploid cotton than is g. raimondii, consistent with previous suggestions based on diverse data sources  <cit> .
second, though there is little protein evolution, that which exists is approximately twice as high  in inter-genomic contrasts  genomes as it is for intra-genomic contrasts  or d <dig> vs. dt ), as expected from the longer time period since a- and d-genome divergence  than that between diploids and their corresponding genomes in the allotetraploid   <cit> .
we also note that the approximately 50% increase in dn for d <dig> vs. dt as compared to a <dig> vs. at mirrors the results for synonymous substitutions.
finally, we compared the mean dn/ds ratio for a <dig> vs. d <dig> relative to the two allotetraploid species .
the mean dn/ds ratio for a <dig> vs. d <dig> is  <dig> , while the two tetraploid species have at vs. dt dn/ds ratios of  <dig>  and  <dig> , for g. barbadense and g. hirsutum, respectively .
in addition, wilcoxon signed-rank tests on the dn/ds ratios show that g. hirsutum is significantly greater than either g. barbadense or a <dig> vs. d <dig>  while g. barbadense or a <dig> vs. d <dig> are statistically equivalent .
these data demonstrate that in the case of cotton, there is evidence for positive selection at the protein level following polyploidy in g. hirsutum, but not in g. barbadense.
of course, our data do not exclude the possibility that positive selection has occurred for individual genes based on functional advantages provided by amino acid substitutions in specific proteins in g. barbadense.
we note that to the extent that such selection has existed, its footprints are most likely to be evident in the genes exhibiting high dn/ds ratios.
to facilitate future exploration, we provide dn, ds, and dn/ds ratios at both the diploid and polyploid levels in additional file  <dig>  we also note the caveat that there is limited divergence experienced by cotton homoeologs, which results in stochasticity among dn/ds ratios derived from these small values.
given the many factors that influence the estimation of these ratios, as well as small sample size per taxon and the relatively minor difference in dn/ds ratios, it may be premature to conclude that the g. hirsutum truly has experienced accelerated protein evolution.
the overall low dn/ds ratios described above suggest considerable evolutionary constraint operating on both diploid and polyploid cotton genes.
however, when comparing rates of sequence evolution between diploid and polyploid a- and d-genomes much of the evolutionary history is shared between parental species  and their corresponding polyploid homoeologs .
to assess molecular evolution in the absence of this dependency, we phylogenetically polarized all nucleotide substitutions and filtered out only those that are specific to one lineage .
though there are relatively few overall lineage-specific substitutions, we observe an increased level of dna substitutions among both homoeologous genomes of both allotetraploid species relative to their diploid progenitors .
the same overall pattern was found by senchina et al.
<cit> , though their results were not statistically significant due to a much smaller gene sample size.
in conclusion, polyploidy in cotton has resulted in an increased level of dna substitution in both allotetraploid genomes , but this has only translated into an increased proportion of nonsynonymous mutations in g. hirsutum .
discussion
the cotton est assembly
in the introduction we noted that because of cotton’s economic significance, it is important to understand both its transcriptome and its evolutionary history.
here we illustrate the interconnectedness of these two aspects of gossypium, showing how insight into the transcriptome of allotetraploid cotton requires a sound phylogenetic and molecular evolutionary framework.
it has become clear from gossypium and investigations of other allotetraploids that genome doubling often entails violations of the assumption of additivity, i.e., the allotetraploid genomes do not represent a simple combination of their diploid parents in their gene expression or content.
for example, microarray analyses in allotetraploid cotton have shown that various tissues exhibit a pattern of parental genome expression described as “genomic dominance”   <cit> .
additionally, our analyses and those of salmon et al.
<cit>  have catalogued a surprising amount of nrhr in cotton allotetraploids that exhibit exclusive bivalent pairing at meiosis with no obvious cytogenetic hints at homoeologous exchanges.
in light of these findings, it is important to understand the gene content similarities and differences among diploid and allotetraploid cotton.
toward that end, we present here a combined diploid and allotetraploid cotton transcriptome assembly, and utilize this assembly to assess broad patterns of genic evolution prior to and following polyploidy.
the cotton transcriptome assembly presented here contains  <dig>  contigs.
this assembly offers only a slight increase in contig number over the previous iteration  <cit> , which had  <dig>  contigs, but increases nucleotide coverage by nearly 7-fold.
this expanded coverage makes it much easier to identify nucleotide substitutions.
we used this added power and further supplementation by illumina reads to document approximately  <dig>  genome-specific snps and demonstrate the utility of these snps in diagnosing genic evolution and nrhr and in cotton allotetraploids.
we expect that this collection of genome-specific snps will have broad utility in a diverse array of future studies spanning the full breadth of investigations in cotton that are enriched by genomic insight, including myriad applied objectives where snp information will facilitate breeding, for example, or in fundamental investigations of genome-specific expression.
a surprising level of non-independent evolution among homoeologs
with respect to the concept that allopolyploidy is accompanied by novel genetic or genomic phenomena of possible evolutionary significance, we build upon the approach of salmon et al.
<cit>  to show the extent of non-independent evolution among genic homoeologs in gossypium.
for comparison, in an initial survey of nrhr, salmon et al.
<cit> , attempted to determine the age of these events using a phylogenetic approach involving the  <dig> natural gossypium allotetraploid species.
among six nrhr events, they found that five occurred in only one species, with the sixth event being ancestral.
here we extend this approach to examine tens of thousands of genes for conserved nrhr from two allotetraploid species .
based on these data, we report the surprising result that about 7% of all contigs in the assembly show evidence of nrhr in at least one of the two polyploidy species.
as discussed by salmon et al.
<cit> , bioinformatic inferences of nrhr are subject to several possible sources of false positives, including assembly artifacts, sequencing error, and autapomorphic homoplasy subsequent to polyploid formation.
however, salmon et al.
<cit>  established a 70% rate of validation using independent laboratory methods , thus applying this false-positive rate we infer a true rate of nrhr in cotton to be approximately 5% of all contigs.
this high frequency of homoeologous contact is an astonishing result given the absence of prior cytogenetic observations that would have suggested this possibility, and apparently complete bivalent formation at meiosis.
furthermore, about one-quarter of all detected events are shared by both allotetraploids, indicating that they likely occurred in a common ancestor and have been retained to the present.
the remaining exchanges occurred more recently, since the divergence of the two allotetraploid lineages, indicating that homoeologous exchange need not be restricted to the initial stages of allopolyploidization, during which time bivalent formation may not yet have been evolutionarily stabilized, but that it arises persistently on an evolutionary time scale.
we also uncover approximately 50% more nrhr events in g. hirsutum compared to g. barbadense.
the detection of nrhr relies on diagnostic reads, and thus detection should increase with deeper read sampling.
interestingly, g. hirsutum is only represented by about 7% more reads than g. barbadense in our assembly.
for this reason, the increase in identified nrhr in g. hirsutum likely does not stem from greater read sampling alone.
instead, our results suggest that the rates of nrhr in the g. hirsutum and g. barbadense lineages have diverged following speciation.
equally interesting is the possibility that nrhr events are not randomly distributed, instead that some genes maybe “hotspots” for nrhr.
it will be fascinating to explore the genomic distribution of these hotspots once genome sequences become available.
this approach might lead to insights into the mechanistic underpinnings of this form of “illegitimate intergenomic contact” following allotetraploidy in cotton.
in addition, experimental analyses are required to assess whether any of the detected nrhr events have had physiological consequences and hence may be adaptively relevant.
molecular evolution in diploids and allotetraploids
the data set generated here provides an exceptionally detailed view of genic evolution in diploid and allotetraploid cotton.
one of the key biological insights of the present study emerges from the observation of evidence for an increased rate of nonsynonymous to synonymous substitution between the a- and d-genomes in g. hirsutum when compared to the diploid a- and d-genomes or those in g. barbadense.
we reiterate, however, the caveats that there is a limited timeframe for substitutions to have accumulated, which would tend to make these ratios sensitive a small number of substitutions.
on the other hand, we do find strong evidence for a generalized increase in the nucleotide substitution rate in both allotetraploid genomes relative to their diploid progenitors.
this same data set also provides a rich quantitative depiction of the genomic composition and history of allotetraploids, revealing the evolutionary relationships among the a <dig>  d <dig>  at and dt genomes.
to assess whether the data could be mined to detect genic targets of selection, we estimated the rates of nonsynonymous to synonymous  mutation between the diploid a <dig> and d <dig> genomes and the allotetraploid at and dt genomes.
overall, the vast majority of these dn/ds values are less than one.
regarding outliers, we find  <dig> contigs have a value greater than one between the a- and d-genome diploids,  <dig> between the at and dt genomes in g. barbadense, and  <dig> between the at and dt genomes in g. hirsutum.
these contigs have experienced a greater amount of amino acid substitution than silent substitution , a hallmark of positive selection, but we note again  the high likelihood of false positives for dn/ds ratios when dealing with small numbers of substitutions.
among the genes that might be explored for selection, the largest outliers in each lineage are a cupin domain containing protein, a conserved plant protein of unknown function, and a putative phytocyanin protein .
none of these rapidly evolving genes have yet been linked to phenotypic traits in cotton, but this is not surprising as relatively few cotton genes have a demonstrated phenotypic effect.
CONCLUSIONS
in this manuscript we produce a new transcriptome assembly from diploid and allotetraploid cotton species and demonstrate several biological insights that can be derived from this resource.
the large number of est sequences generated for this study offer the most comprehensive view of the cotton transcriptome to date, and possibly the largest examination of the transcriptome from a polyploid plant and its diploid progenitors.
by sequencing extant a- and d-genome diploids along with a- and d-genome containing allotetraploids, we are able to identify a vast number of snps that differentiate the allotetraploid a- and d-genomes.
these snps can be used to explore molecular evolution within and between these genomes in both the presence and absence of polyploidy.
moreover, though cotton is a major crop, its genome is unsequenced.
our characterization of the transcriptome sheds light on the functional properties of the cotton genome.
our results reveal an increased rate of nucleotide substitution in both polyploid lineages, though this increase only impacts the nonsynonymous to synonymous substitution ratio in g. hirsutum.
we also uncover a surprising level of nonreciprocal homoeologous recombination  between the allotetraploid a- and d-genomes.
from these two findings we can conclude that genome duplication has impacted the mutation rate, and through long-term co-residency between the cotton a- and d-genomes, resulted in frequent illegitimate contact, resulting in homoeologous sequence conversion through nonreciprocal exchange.

 cotton produces the world’s most utilized natural fiber, making it a staple of the textile and garment industries. in cotton fiber production in the us was valued at approximately billion dollars, with an additional million dollars derived from cotton seed extracts. this places cotton among the top five most valuable us crops. accordingly, it is important to understand cotton’s gene repertoire and its evolutionary history. with the vast majority of cotton production being derived from the allotetraploid species gossypium hirsutum, followed by smaller contributions from a second domesticated allotetraploid, g. barbadense, an appreciation of cotton biology in the context of its allotetraploid history has become a major research focus for the improvement of this crop. here we present a comprehensive assembly of approximately million est sequences from these two allotetraploid species and from the diploid species representing the progenitor genomes of allotetraploid cotton. we illustrate how this assembly facilitates diagnosis of the genome-of-origin for what otherwise would have been ambiguously derived transcripts, and demonstrate the utility of this assembly for addressing questions pertaining to the phenomenon of allopolyploidy and cotton transcriptome evolution. the cotton genus comprises about species, divided into eight monophyletic diploid genome groups, and a clade of five allotetraploid species, the latter containing a genome from both the a and d diploid genome groups. the cytological and phylogenetic history of this genus is well-resolved, and data demonstrate that the best living models of the progenitor diploids of the a- and d-genomes of modern allotetraploid cotton are the diploid species g. arboreum and g. raimondii, respectively. in addition, extensive seed collections are available and include both wild progenitor forms along with their domesticated descendants. collectively, these resources provide a useful system for studying the genomic and phenotypic changes that accompany domestication. embedding these resources within their phylogenetic context results in a powerful framework for the study of polyploidy and domestication, and consequently cotton has emerged as a model system in these areas of research. recent examples of the utility of the cotton system in this regard include detailed analyses of the cotton fiber transcriptome during development, as well as explorations of the mutational and expression changes that accompany allopolyploidization. these types of studies, as well as those aimed at crop improvement, are enriched or facilitated by the availability of cotton est collections, underscoring the utility and importance of developing a rich and deep database of est resources. prior to this study, the community-driven global assembly of cotton ests included ~  sanger sequences, with ~   g. arboreum, ~  g. raimondii, and ~   g. hirsutum sequences assembled into a single reference collection yielding contigs. because this assembly contained sequences from both model diploid parents and a natural allotetraploid representing their combination, it could be used to identify a- and d-genome-specific snps, which can in turn be used to assign genomic origin to homoeologous transcripts in allotetraploid cotton. implementation of this est collection strategy has enabled several research possibilities, including monitoring genome-specific expression in natural allotetraploids and the quantification of the extent of nonreciprocal homoeologous recombination among natural allotetraploid species. these efforts have elucidated the dynamics of gene expression evolution and genic content variation that accompanied allotetraploid formation and evolution in cotton. here we present a vastly expanded cotton est assembly, which contains approximately million sanger and next-generation transcripts. like previous assemblies, this one incorporates ests from both the a- and d-genome diploid progenitors, along with allotetraploid ests from two species of allotetraploid cotton, g. barbadense and g. hirsutum. the contigs obtained from this assembly represent a vastly expanded representation of the genic content of cotton. to add additional depth to the assembly, we also generated ~ million 82 bp illumina reads, representing the fiber transcriptome of diploid a- and d-genome cotton as well as the allotetraploids g. barbadense and g. hirsutum. together these resources allow us to detect genome-specific snps, which in turn can be used to distinguish the a- and d-genome homoeologs found in the allotetraploid cotton genome. we describe this collection and document its utility for genome-specific transcriptome analysis in allotetraploid cotton. we also present a characterization of the functional properties of the cotton transcriptome and analyses of molecular evolution following the most recent whole genome duplication that accompanied allotetraploid formation 1– million years ago. at the time of writing, allotetraploid cotton is now among the most important crops lacking a whole genome sequence, but as progress is made in this regard, the est assembly and genome-specific snp resources presented here will be of use in assembling and annotating the cotton genome.  est assembly characteristics the cotton assembly presented here contains est sequences from g. arboreum, g. barbadense, g. hirsutum, and g. raimondii. ests from these species were assembled jointly in a multispecies assembly. the justification for this multispecies assembly stems from the fact that the transcripts from resident a- and d-genomes in the allotetraploid nucleus are more closely related to their diploid homologs than they are to each other. thus, combining diploid est sequences with the ests of two tetraploid species does not introduce appreciable amounts of additional sequence divergence. from a total of approximately million gossypium ests,, were assembled into contigs, while ests remained unassembled. the assembly includes previously published sanger sequences as well as approximately million new sequences. the assembled collection of ests produced contigs with an average length of.5 bp. a study that validates the quality of this assembly is described in the materials and methods section, wherein we find high nucleotide similarity between our assembled contigs and cloned and sequenced cotton genes. this suggests that the sequence quality and assembly routine accurately recapitulate the structure and mutational history found in well-characterized cotton genes. 1accession unknown or derived from numerous accessions. references for sanger est collections can be found in udall et al.. among the contigs, include a predicted coding region, with an average coding length of  bp. this estimated coding sequence length for cotton is a little less than the average coding sequence length of some angiosperms with sequenced genomes. for example, the average coding sequence lengths of a. thalianapopulus trichocarpa, and vitis vinifera are,, and  bp, respectively. if we assume that the true coding sequence length in cotton is comparable to the average length for these species, we estimate that our est assembly provides coverage of about 76% of the coding sequence for the average contig. in addition to breadth of coverage, the assembly achieves a new level of depth of coverage; the median number of ests per contig was with a range from to, and the average depth of coverage for each assembled nucleotide was. these results mark a significant improvement over previous cotton transcriptome assemblies, where the average contig length was 791 bp, the median number of ests per contig was and the average depth of coverage for each assembled nucleotide was. to assess our expectation of co-assembly of expressed sequences from multiple cotton genomes, species membership was compiled for each contig of the assembly. the most common class of contigs are those that contain ests from all four species, indicating that genic conservation is substantial among these species and that we have achieved sequence coverage sufficient to reveal this. contigs were represented by sequences from both g. arboreum and g. raimondii, whereas contigs contained sequences derived from both allotetraploids. interestingly, we observed contigs for which sequences were lacking only for g. arboreum and only contigs constructed entirely from g. arboreum ests. these findings together suggest that the g. arboreum ests collection represents a significantly narrower pool of genes relative to the other species. although it is possible that some g. arboreum orthologs are missing because of gene loss or gene silencing, there is substantial evidence that g. arboreum has not lost a large portion of its transcriptome or experienced massive global shifts in gene expression. we note that g. arboreum was the only species lacking sequences generated by titanium chemistry, having instead only flx reads. in support of this point, we do observe a 13 % enrichment of titanium reads from g. barbadenseg. hirsutum, and g. raimondii in the contigs missing only g. arboreum when compared to their proportions in all other contigs. our best hypothesis is that technical differences between these two sequencing chemistries, including sequencing bias, differences in library construction, and assembly characteristics underlie the unexpectedly narrow breadth of the g. arboreum est collection. assembly annotation sequence similarity searches to the refseq database were used to provide an approximation of gene function. the contigs and singletons were compared to annotated proteins from refseq with a minimum e-value score of 1e- contig sequences had a high similarity to refseq sequences and contig sequences had a match less than 1e- the contig sequences were also compared to the annotated genomes of carica papaya and a. thaliana. when only the top match was considered, contigs had a near identical match to an a. thaliana gene and contigs had top matches with an e-value lower than 1e- more than contigs had a top match to an a. thaliana gene with an e-value lower than 1e- when only the top c. papaya match was considered, contigs had a near identical match to a c. papaya gene and had top matches with an e-value lower than 1e- more than contigs had a top match to a c. papaya gene with an e-value lower than 1e- the similar results obtained with both arabidopsis and carica lend confidence that this set of annotations is accurate. identification of protein coding domains among the contigs in the assembly, had a predicted translational product, and among this subset protein products had at least one annotated pfam domain or could be assigned to a pfam family. not surprisingly, the most abundant domain was the pentatricopeptide repeat. this domain is among the most numerous in the angiosperms and may be associated with rna stabilization and processing. other highly prevalent pfam annotations include the wd protein kinase, and myb dna binding domains, as well as the leucine rich repeat and rna recognition motifs. like the ppr repeat, these domains are also abundant in other angiosperm genomes. to better assess the functional characteristics of the cotton transcriptome assembly, we converted pfam annotations into go categories. the relative abundance of various high-level go categories is shown in figure these data indicate that our current cotton est assembly includes a large and diverse set of genes. this broad sampling of annotated genes should be useful for enrichment-based tests among differentially expressed genes as determined by future microarray, rna-seq, or similar experiments. we anticipate this set of genes will serve as a useful transcriptome model until empirically derived transcripts can be paired with gene predictions from a de novo cotton genome sequence. homoeologous snps because our cotton est assembly includes a large number of sequences from a- and d-genome diploid progenitors as well as ad-genome allotetraploid derived ests, it can be used to diagnose homoeologs and homoeolog-specific snps. in a strategy similar to buggs et al.,, we identified homoeolog-specific snps directly from the 454/sanger assembly and supplemented this assessment with, illumina sequences, also derived from the a- and d-genome diploid progenitors and ad-genome allotetraploids, and mapped to the de novo 454/sanger est assembly. this strategy leverages the strengths of both the and illumina next-generation platforms. the reads are longer and assemble into contigs more readily, while the illumina platform offers deep sequence coverage that can be used to confirm the 454/sanger snps and find new snps in regions with low 454/sanger read coverage. in total, we identified snps between the diploid a- and d-genomes, including that were exclusive to the de novo 454/sanger assembly, exclusive to the illumina reference mapping, and common to both strategies. the intersection of both snp pools is only a fraction of the total. however, to identify homoeologous snps in the 454/sanger assembly it is necessary to have ests from the a- and d-genome diploids present, as is the case for contigs. furthermore, within these contigs the a- and d-genome diploid sequences must overlap one another. this occurs for approximately 29 % of all available nucleotide positions within the eligible contigs. finally, cross-validating a snp with both the 454/sanger assembly and the illumina reads requires an overlap between both diploid parents from both sources of ests. this occurs for about 23% of all sites from the eligible contigs. the diminishing effect these requirements have on snp identification likely explains the modest proportion of snps that intersect between both platforms. among those genome-specific snps found in both the 454/sanger and illumina data sets, only were in disagreement about the nucleotides representing the a- and d- genomes, leaving in agreement. this last class of snps represents a high confidence category, though the snps identified only in the 454/sanger or illumina data sets have considerable internal support and also are likely reliable. for the cross-platform verified snps, we confirmed the presence of all a- and d-genome diploid snp variants within the allotetraploid illumina reads. this additional constraint of no further nucleotide evolution subsequent to polyploidization in either the diploid or allotetraploid lineages ensures that these snps almost certainly occurred prior to the formation of the allotetraploids. these snps can be used to diagnose both diploid homologs and allotetraploid homoeologs. detection of nonreciprocal homoeologous recombination genes doubled by allopolyploidy may, in principle, evolve independently or they may interact, thereby evolving in a “concerted” fashion, mediated by gene conversion or other forms of duplicate gene sequence homogenization. salmon et al., studied this phenomenon in allotetraploid cotton using an earlier release of the cotton transcriptome assembly. following the methods outlined in salmon et al., we report an updated account of nrhr. this updated account includes a richer est assembly, and also differs in that the current assembly includes sequences from a second allotetraploid species, g. barbadense. because the two allotetraploid species used in this study share a common ancestor, detected nrhr can be mapped onto their phylogeny. through this process we partitioned exchanges that occurred in only one allotetraploid lineage from those that occurred in their common ancestor. a total of unique nrhr were detected in the new assembly. of these, approximately 46 % and 31 % are found only in g. hirsutum and in g. barbadense, respectively, whereas about 23% of the exchanges were found in both species. interestingly, the unique set of nrhr belong to only contigs. contigs with multiple exchanges may have experienced independent nrhr events, or alternatively, they may represent only one complex event, with several internal exchanges, that we have been unable to bridge together and resolve. this finding was initially reported by salmon et al., and its reiteration here with a much larger sample size provides evidence that certain duplicated genes or regions of the allotetraploid genome may harbor lengthy complex nrhr or could be “hotspots” of repeated exchange. molecular evolution in diploid and allotetraploid cotton the perspective provided by previous taxonomic and phylogenetic work combined with the est assembly generated here offers an unparalleled opportunity to explore the molecular evolutionary properties of genes in diploids and their allotetraploid derivatives. to assess the potential impact of polyploidy we compared sequence evolution between a- and d-genome orthologs in diploid cottons to that of a- and d-genome homoeologs in both species of allotetraploid cotton. this was achieved by first isolating the a- and d-genome homoeologous reads from both allotetraploids, g. barbadense and g. hirsutum, using est sequences from the a- and d-genome diploid species as a guide. for each inter- or intra-genomic comparison we were able to study thousands of contigs and millions of nucleotides, providing exquisite detail on the amount of divergence that has accumulated since the two diploids last shared a common ancestor, and since allotetraploid formation. 1see molecular evolution section in the materials and methods for further detail about contig selection. we isolated the coding regions from which we calculated the rates of synonymous and nonsynonymous divergence within and between species. these two metrics can also be combined in as a ratio, which gives an indication of the selection pressures operating on the protein composition of a gene. elevated dn/ds ratios may be indicative of positive selection, though with low overall levels of divergence, as is seen in cotton, this interpretation must be viewed with caution as stochastic processes operating on a small number of total mutations can lead to spurious inferences of selection. nonetheless, gene and genome duplication are thought to provide the opportunity for rapid gene evolution of one duplicate, so long as the other duplicate maintains sufficient ancestral functionality. in the present context, we might expect to find an elevated dn/ds ratio between cotton at and dt homoeologs, when compared to the dn/ds ratio of their a and d diploid progenitors. as shown in table we estimated dn and ds for anywhere from to contigs in pairwise comparisons among genomes. these values fall short of the total available contig counts listed in figure because some contigs lack a predicted protein-coding region or because the species in question fail to overlap one another within the contig alignment. several conclusions are evident from these data, mirroring but also vastly extending similar results published by senchina et al. for a sample of only genes: first, because the dataset is based on thousands of alignments in each comparison, it provides an accurate depiction of the ancestry of polyploid cotton. in this regard, the a diploid genome has a mean synonymous distance of or from the two at-genomes whereas the comparable figure is or for d versus the two dt genomes. from this we infer that modern g. arboreum is a better model of the actual genome donor of allotetraploid cotton than is g. raimondii, consistent with previous suggestions based on diverse data sources. second, though there is little protein evolution, that which exists is approximately twice as high in inter-genomic contrasts genomes as it is for intra-genomic contrasts or d vs. dt ), as expected from the longer time period since a- and d-genome divergence than that between diploids and their corresponding genomes in the allotetraploid. we also note that the approximately 50% increase in dn for d vs. dt as compared to a vs. at mirrors the results for synonymous substitutions. finally, we compared the mean dn/ds ratio for a vs. d relative to the two allotetraploid species. the mean dn/ds ratio for a vs. d is, while the two tetraploid species have at vs. dt dn/ds ratios of and, for g. barbadense and g. hirsutum, respectively. in addition, wilcoxon signed-rank tests on the dn/ds ratios show that g. hirsutum is significantly greater than either g. barbadense or a vs. d while g. barbadense or a vs. d are statistically equivalent. these data demonstrate that in the case of cotton, there is evidence for positive selection at the protein level following polyploidy in g. hirsutum, but not in g. barbadense. of course, our data do not exclude the possibility that positive selection has occurred for individual genes based on functional advantages provided by amino acid substitutions in specific proteins in g. barbadense. we note that to the extent that such selection has existed, its footprints are most likely to be evident in the genes exhibiting high dn/ds ratios. to facilitate future exploration, we provide dn, ds, and dn/ds ratios at both the diploid and polyploid levels in additional file we also note the caveat that there is limited divergence experienced by cotton homoeologs, which results in stochasticity among dn/ds ratios derived from these small values. given the many factors that influence the estimation of these ratios, as well as small sample size per taxon and the relatively minor difference in dn/ds ratios, it may be premature to conclude that the g. hirsutum truly has experienced accelerated protein evolution. the overall low dn/ds ratios described above suggest considerable evolutionary constraint operating on both diploid and polyploid cotton genes. however, when comparing rates of sequence evolution between diploid and polyploid a- and d-genomes much of the evolutionary history is shared between parental species and their corresponding polyploid homoeologs. to assess molecular evolution in the absence of this dependency, we phylogenetically polarized all nucleotide substitutions and filtered out only those that are specific to one lineage. though there are relatively few overall lineage-specific substitutions, we observe an increased level of dna substitutions among both homoeologous genomes of both allotetraploid species relative to their diploid progenitors. the same overall pattern was found by senchina et al., though their results were not statistically significant due to a much smaller gene sample size. in conclusion, polyploidy in cotton has resulted in an increased level of dna substitution in both allotetraploid genomes, but this has only translated into an increased proportion of nonsynonymous mutations in g. hirsutum. discussion the cotton est assembly in the introduction we noted that because of cotton’s economic significance, it is important to understand both its transcriptome and its evolutionary history. here we illustrate the interconnectedness of these two aspects of gossypium, showing how insight into the transcriptome of allotetraploid cotton requires a sound phylogenetic and molecular evolutionary framework. it has become clear from gossypium and investigations of other allotetraploids that genome doubling often entails violations of the assumption of additivity, i.e., the allotetraploid genomes do not represent a simple combination of their diploid parents in their gene expression or content. for example, microarray analyses in allotetraploid cotton have shown that various tissues exhibit a pattern of parental genome expression described as “genomic dominance”. additionally, our analyses and those of salmon et al. have catalogued a surprising amount of nrhr in cotton allotetraploids that exhibit exclusive bivalent pairing at meiosis with no obvious cytogenetic hints at homoeologous exchanges. in light of these findings, it is important to understand the gene content similarities and differences among diploid and allotetraploid cotton. toward that end, we present here a combined diploid and allotetraploid cotton transcriptome assembly, and utilize this assembly to assess broad patterns of genic evolution prior to and following polyploidy. the cotton transcriptome assembly presented here contains contigs. this assembly offers only a slight increase in contig number over the previous iteration, which had contigs, but increases nucleotide coverage by nearly 7-fold. this expanded coverage makes it much easier to identify nucleotide substitutions. we used this added power and further supplementation by illumina reads to document approximately genome-specific snps and demonstrate the utility of these snps in diagnosing genic evolution and nrhr and in cotton allotetraploids. we expect that this collection of genome-specific snps will have broad utility in a diverse array of future studies spanning the full breadth of investigations in cotton that are enriched by genomic insight, including myriad applied objectives where snp information will facilitate breeding, for example, or in fundamental investigations of genome-specific expression. a surprising level of non-independent evolution among homoeologs with respect to the concept that allopolyploidy is accompanied by novel genetic or genomic phenomena of possible evolutionary significance, we build upon the approach of salmon et al. to show the extent of non-independent evolution among genic homoeologs in gossypium. for comparison, in an initial survey of nrhr, salmon et al., attempted to determine the age of these events using a phylogenetic approach involving the natural gossypium allotetraploid species. among six nrhr events, they found that five occurred in only one species, with the sixth event being ancestral. here we extend this approach to examine tens of thousands of genes for conserved nrhr from two allotetraploid species. based on these data, we report the surprising result that about 7% of all contigs in the assembly show evidence of nrhr in at least one of the two polyploidy species. as discussed by salmon et al., bioinformatic inferences of nrhr are subject to several possible sources of false positives, including assembly artifacts, sequencing error, and autapomorphic homoplasy subsequent to polyploid formation. however, salmon et al. established a 70% rate of validation using independent laboratory methods, thus applying this false-positive rate we infer a true rate of nrhr in cotton to be approximately 5% of all contigs. this high frequency of homoeologous contact is an astonishing result given the absence of prior cytogenetic observations that would have suggested this possibility, and apparently complete bivalent formation at meiosis. furthermore, about one-quarter of all detected events are shared by both allotetraploids, indicating that they likely occurred in a common ancestor and have been retained to the present. the remaining exchanges occurred more recently, since the divergence of the two allotetraploid lineages, indicating that homoeologous exchange need not be restricted to the initial stages of allopolyploidization, during which time bivalent formation may not yet have been evolutionarily stabilized, but that it arises persistently on an evolutionary time scale. we also uncover approximately 50% more nrhr events in g. hirsutum compared to g. barbadense. the detection of nrhr relies on diagnostic reads, and thus detection should increase with deeper read sampling. interestingly, g. hirsutum is only represented by about 7% more reads than g. barbadense in our assembly. for this reason, the increase in identified nrhr in g. hirsutum likely does not stem from greater read sampling alone. instead, our results suggest that the rates of nrhr in the g. hirsutum and g. barbadense lineages have diverged following speciation. equally interesting is the possibility that nrhr events are not randomly distributed, instead that some genes maybe “hotspots” for nrhr. it will be fascinating to explore the genomic distribution of these hotspots once genome sequences become available. this approach might lead to insights into the mechanistic underpinnings of this form of “illegitimate intergenomic contact” following allotetraploidy in cotton. in addition, experimental analyses are required to assess whether any of the detected nrhr events have had physiological consequences and hence may be adaptively relevant. molecular evolution in diploids and allotetraploids the data set generated here provides an exceptionally detailed view of genic evolution in diploid and allotetraploid cotton. one of the key biological insights of the present study emerges from the observation of evidence for an increased rate of nonsynonymous to synonymous substitution between the a- and d-genomes in g. hirsutum when compared to the diploid a- and d-genomes or those in g. barbadense. we reiterate, however, the caveats that there is a limited timeframe for substitutions to have accumulated, which would tend to make these ratios sensitive a small number of substitutions. on the other hand, we do find strong evidence for a generalized increase in the nucleotide substitution rate in both allotetraploid genomes relative to their diploid progenitors. this same data set also provides a rich quantitative depiction of the genomic composition and history of allotetraploids, revealing the evolutionary relationships among the a d at and dt genomes. to assess whether the data could be mined to detect genic targets of selection, we estimated the rates of nonsynonymous to synonymous mutation between the diploid a and d genomes and the allotetraploid at and dt genomes. overall, the vast majority of these dn/ds values are less than one. regarding outliers, we find contigs have a value greater than one between the a- and d-genome diploids, between the at and dt genomes in g. barbadense, and between the at and dt genomes in g. hirsutum. these contigs have experienced a greater amount of amino acid substitution than silent substitution, a hallmark of positive selection, but we note again the high likelihood of false positives for dn/ds ratios when dealing with small numbers of substitutions. among the genes that might be explored for selection, the largest outliers in each lineage are a cupin domain containing protein, a conserved plant protein of unknown function, and a putative phytocyanin protein. none of these rapidly evolving genes have yet been linked to phenotypic traits in cotton, but this is not surprising as relatively few cotton genes have a demonstrated phenotypic effect.  in this manuscript we produce a new transcriptome assembly from diploid and allotetraploid cotton species and demonstrate several biological insights that can be derived from this resource. the large number of est sequences generated for this study offer the most comprehensive view of the cotton transcriptome to date, and possibly the largest examination of the transcriptome from a polyploid plant and its diploid progenitors. by sequencing extant a- and d-genome diploids along with a- and d-genome containing allotetraploids, we are able to identify a vast number of snps that differentiate the allotetraploid a- and d-genomes. these snps can be used to explore molecular evolution within and between these genomes in both the presence and absence of polyploidy. moreover, though cotton is a major crop, its genome is unsequenced. our characterization of the transcriptome sheds light on the functional properties of the cotton genome. our results reveal an increased rate of nucleotide substitution in both polyploid lineages, though this increase only impacts the nonsynonymous to synonymous substitution ratio in g. hirsutum. we also uncover a surprising level of nonreciprocal homoeologous recombination between the allotetraploid a- and d-genomes. from these two findings we can conclude that genome duplication has impacted the mutation rate, and through long-term co-residency between the cotton a- and d-genomes, resulted in frequent illegitimate contact, resulting in homoeologous sequence conversion through nonreciprocal exchange. 
molecular evolutionary analyses of protein-coding regions demonstrate that the rate of nucleotide substitution has increased among both allotetraploid genomes relative to the diploids, and that the ratio of nonsynonymous to synonymous substitutions has increased in one of the two polyploid lineages we sampled. in this paper we present a comprehensive est assembly derived from diploid and model allotetraploid cottons and demonstrate several evolutionary inferences regarding genic evolution that can be drawn from these data. we also use these snps to show that a surprisingly high percentage of duplicate genes show a signature of non-independent evolution in the allotetraploid nucleus, having experienced one or more episodes of nonreciprocal homoeologous recombination. we demonstrate that cotton genes have experienced an increased rate of molecular evolution following duplication by polyploidy, and that polyploidy has enabled considerable levels of nonreciprocal exchange between homoeologous genes. in this study we characterize the functional and mutational properties of the cotton transcriptome, produce a large genome-specific snp database, and detect illegitimate genetic exchanges between duplicate genomes sharing a common allotetraploid nucleus. diploid a- and d-genome species can be compared to the a- and d-genomes found within these allotetraploids to make evolutionary inferences about polyploidy.
 modern allotetraploid cotton contains an “a” and “d” genome from an ancestral polyploidy event that occurred approximately 1– million years ago. diploid a- and d-genome species can be compared to the a- and d-genomes found within these allotetraploids to make evolutionary inferences about polyploidy. in this paper we present a comprehensive est assembly derived from diploid and model allotetraploid cottons and demonstrate several evolutionary inferences regarding genic evolution that can be drawn from these data.  we generated a set of cotton expressed sequence tags, comprising approximately million sanger and next-generation transcripts supplemented by approximately million illumina reads from diploid and allotetraploid cottons. from the est alignments we inferred genome-specific single nucleotide polymorphisms. molecular evolutionary analyses of protein-coding regions demonstrate that the rate of nucleotide substitution has increased among both allotetraploid genomes relative to the diploids, and that the ratio of nonsynonymous to synonymous substitutions has increased in one of the two polyploid lineages we sampled. we also use these snps to show that a surprisingly high percentage of duplicate genes show a signature of non-independent evolution in the allotetraploid nucleus, having experienced one or more episodes of nonreciprocal homoeologous recombination.  in this study we characterize the functional and mutational properties of the cotton transcriptome, produce a large genome-specific snp database, and detect illegitimate genetic exchanges between duplicate genomes sharing a common allotetraploid nucleus. our findings have important implications for our understanding of the consequences of polyploidy and duplicate gene evolution. we demonstrate that cotton genes have experienced an increased rate of molecular evolution following duplication by polyploidy, and that polyploidy has enabled considerable levels of nonreciprocal exchange between homoeologous genes. cottonduplicate gene evolutiongene conversiongossypiumhomoeologous recombinationpolyploidytranscriptome 