BACKGROUND
precursor mrna  splicing is an important step in eukaryotic gene expression that plays a crucial role in higher eukaryotic developmental regulation and environmental stress response  <cit> .
after precursor mrna splicing, introns are removed and exons are ligated into one or more mature transcripts or isoforms.
since walter gilbert  <cit>  first postulated that alternative splicing  can create different isoforms from a common template, increasing evidence has shown that as widely occurs in animals and plants.
for example, transcripts from 92%–94% of human intron-containing genes and approximately 61% of arabidopsis multiexonic genes can be alternatively spliced  <cit> .
in addition, approximately 63% and 51% of intron-containing genes also undergo as in soybean  <cit>  and maize  <cit> , respectively.
in general, as events can occur via four different mechanisms: exon skipping , alternative donor site , alternative acceptor site , and intron retention .
however, there are also complex as types combined with at least two simple forms or some other low frequent forms such as mutually exclusive exons, alternative transcription start sites, and multiple polyadenylation sites in eukaryotes  <cit> .
however, the frequency of each as event is different and can be gene- and species-dependent.
in animals, es is the most frequent as event and ir is the least frequent; for example, approximately  <dig> % of all as events in humans are caused by by es, but only  <dig> % are caused by ir  <cit> .
in contrast, in arabidopsis  <cit> , maize  <cit> , brachypodium,  <cit>  and cotton  <cit> , ir is the predominant form of as, whereas es only accounts for a small proportion of as events.
as is involved in a wide array of biological functions, particularly in response to biotic and abiotic stress.
in humans, approximately 15% of inherited diseases are caused by mutations that interfere with mrna splicing  <cit> .
in arabidopsis, as is strongly associated with environmental stimuli  <cit> .
for example, isoforms retaining the fourth intron of cca <dig> increased under high light conditions but decreased under low temperature conditions  <cit> .
recent studies found that diverse mrna isoforms produced from a given gene can accelerate genome evolution by generating new functions.
a splice site mutation in the fourth intron of sorghum shattering <dig>  results in the removal of the fourth exon, and this variation underwent selection during the sorghum domestication and improvement  <cit> .
maize , which is a model plant for studying crop domestication, evolved from the annual teosinte zea mays ssp.
parviglumis approximately  <dig>  to  <dig>  years ago and greatly differs in morphology from its progenitors  <cit> .
recently, several key domestication-related genes that are responsible for the morphological changes between maize and teosinte were identified, including tga <dig>  <cit> , tb <dig>  <cit> , ba <dig>  <cit> , ra <dig>  <cit> , and zfl <dig>  <cit> .
dna sequencing advances have led to the discovery of thousands of genes with strong signals of selection  <cit> .
at the transcriptional level, swanson-wagner et al.
<cit>  found hundreds of genes that have altered expression levels or co-expression profiles during domestication.
some of the genes involved in responding to biotic and abiotic stresses were significantly enriched with selective sweeps.
in addition, lemmon et al.
<cit>  suggested that gene expression may be caused by the modification of cis regulatory elements.
they found that approximately 4% of genes showing evidence for consistent cis regulatory divergence that differentiates maize from teosinte were significantly correlated with maize domestication and improvement.
early studies on maize domestication were primarily focused on nucleotide sequence diversity and expression changes between maize and teosinte, but the differences on alternatively spliced genes , alternative splicing types , as events and level between maize and teosinte, and evolutionary role of these differences has been poorly studied.
to better understand the evolutionary role of as in maize domestication, the transcriptome of the seedling stage from six teosinte accessions and ten maize inbred lines were sequenced.
furthermore, genome-wide as profiles were analyzed, and as genes, types, events, and levels were compared between maize and teosinte.
we found that maize had greater as complexity than teosinte, and a subset of as level-altered genes were enriched in transcriptional regulation and stress responses.
this subset was located in regions that have been targets of selection for maize improvement.
RESULTS
transcriptome sequencing and assembly
to assemble the transcriptome of maize and teosinte, cdna libraries of ten maize inbred lines, three zea mays ssp.
parviglumis and three zea mays ssp.
mexicana accessions  were sequenced with the solexa sequencing platform.
in total,  <dig>  million reads  were obtained, with an average of  <dig>  million reads  per sample.
after discarding low quality reads,  <dig>  million high quality reads  were used for further analysis.
without a reference genome, high quality reads of teosinte were first assembled de novo.
after removing redundant sequences,  <dig>  to  <dig>  contigs were reconstructed in six teosinte libraries.
the length of contigs varied from  <dig> to  <dig>  bp, the n <dig> and n <dig> of teosinte transcriptomes ranged from  <dig> to 937 bp and  <dig> to 217 bp, respectively .
for maize inbred lines, transcriptomes were assembled based on b <dig> reference genome:  <dig>  to  <dig>  isoforms were assembled and the n <dig> and n <dig> of maize transcriptomes ranged from  <dig>  to  <dig>  bp and  <dig> to 843 bp, respectively .
for a global view of transcriptomes of the two teosinte subspecies, we assembled pseudo-transcriptomes by clustering contigs from three zea mays ssp.
parviglumis and three zea mays ssp.
mexicana libraries, respectively.
totals of  <dig>  and  <dig>  unique isoforms for zea mays ssp.
parviglumis and zea mays ssp.
mexicana, respectively, were obtained; the longest transcript in zea mays ssp.
parviglumis and in zea mays ssp.
mexicana was  <dig>  and  <dig>  bp, respectively.
under a 95% identity and coverage cutoff, zea mays ssp.
parviglumis and zea mays ssp.
mexicana transcripts could be perfectly matched with  <dig>  and  <dig>  maize reference genes, respectively, and  <dig>  genes were shared by the two teosinte subspecies.
high homology of isoforms between maize and teosinte indicated high conservation of proteins encoded by function genes during maize domestication.
this finding is consistent with the study on expression and cis-regulatory changes between maize and teosinte  <cit> .
variant and genetic diversity characterization
to characterize genetic diversity among all sequenced samples, clean reads from each sample were mapped to the b <dig> reference genome, respectively.
after discarding the low coverage and quality snps and indels, we detected  <dig>  snps from  <dig>  genes with 1– <dig> variant sites in a single gene , and  <dig>  indels with a − <dig> to 65 bp deletion or insertion that was distributed from − <dig> to 20 bp .
of the snps,  <dig>  were transitions and  <dig>  were transversions.
the ratio of transitions to transversions was approximately  <dig>  and likely caused by the sequencing coverage and filtering steps.
moreover,  <dig>  snps were located in exons,  <dig>  were in introns, and  <dig>  were in intergenic regions.
these results indicate that some transcribed sequences may not be annotated in the b <dig> filtered gene set or some unknown fragments were transcribed in our transcriptome data.
phylogenetic analysis using all snps detected in this study showed that maize inbred lines, zea mays ssp.
parviglumis, and zea mays ssp.
mexicana accessions clearly grouped into three clusters.
temperate and tropical maize lines also clearly grouped into different clades .
this result is consistent with previous studies that have used dna markers  <cit> .figure  <dig> phylogenetic relationships among  <dig> sequenced samples.
taxa in the neighbor-joining tree are represented by different shapes.
empty circles denote tropical maize inbred lines; solid circles denote temperate maize inbred lines; empty squares denote accessions of zea mays ssp.
parviglumis; and solid squares denote accessions of zea mays ssp.
mexicana.
the phylogenetic analysis was performed using the neighbor-joining method with  <dig>  bootstrap replicates in the phylip package.
orthologous gene identification
zea mays ssp.
parviglumis and zea mays ssp.
mexicana pseudo-transcriptomes were used to identify orthologous gene pairs between maize and teosinte using the reciprocal best blast hit method.
a total of  <dig>  and  <dig>  high confidence orthologs of maize with an e-value ≤ 1e- <dig> were identified in zea mays ssp.
parviglumis and zea mays ssp.
mexicana, respectively.
of these,  <dig>  orthologs that accounted for  <dig> % of the b <dig> filtered gene set were shared between the two species and distributed across all  <dig> maize chromosomes.
overall, ortholog density was low in centromere-proximal regions and high in chromosome arms .
these shared orthologs were highly conserved during maize domestication; therefore, these shared orthologs were further used for as landscape of maize and teosinte transcriptome analysis.figure  <dig> orthologous gene distribution in the maize genome.
gene density statistics were calculated with a 200-kb sliding-window.
the x-axis denotes the physical position of a chromosome.
the y-axis denotes the number of genes located in this sliding window.
chr = chromosome.
maize and teosinte alternative splicing landscapes
to determine the relationship between sequencing depth and as detection power, the sequencing library  was first used to randomly create sub-libraries to detect as transcripts.
overall, sequencing depth was highly positively correlated with the average coverage of each gene, and positively correlated with the as gene number and as events, in particular, when sequencing depth is less than  <dig>  gb .
however, when sequencing depth was more than  <dig>  gb, the increase of sequencing depth did not significantly increase the number of as genes and all as events .
this result is consistent with liu’s findings in arabidopsis  <cit> .
in this study, sequencing depth of most libraries was more than  <dig>  gb, and implicating our sequencing data was well to support the identification of as transcripts.figure  <dig> relationship between sequencing depth and alternative splicing.
the x-axis denotes the sequencing depth and average gene coverage.
the y-axis denotes the numbe of alternatively spliced genes or number of alternative splicing events.
to characterize the maize and teosinte as landscapes, as events that occurred in all shared orthologs of each sample were evaluated.
in  <dig>  shared genes,  <dig>  genes were annotated with at least two exons  and are potentially subjected to as.
in teosinte,  <dig>  genes, which accounts for  <dig> % of the studied multiexonic genes, were subjected to as.
a total of  <dig>  as events were identified in all teosinte libraries.
after removing the same as events in different libraries,  <dig>  non-redundant as events were detected in teosinte libraries, with an average of  <dig>  as events per gene.
in maize, a total of  <dig>  as events, including  <dig>  non-redundant as events, were identified from  <dig>  genes  in all  <dig> maize libraries, with an average of  <dig>  as events per gene.
among different samples in the same species, the number of as events and as genes varied widely.
in maize,  <dig>  to  <dig>  as events involving  <dig>  to  <dig>  genes were identified.
in teosinte,  <dig>  to  <dig>  as events involving  <dig>  to  <dig>  genes were identified .table  <dig>
number of alternative splicing  events and genes detected in maize and teosinte libraries
as-event
as gene
as events per gene
splicing junction sites  were identified using tophat software  <cit> .
a total of  <dig>  and  <dig>  unique sjs were detected in all maize and teosinte samples.
in maize, sjs were composed of  <dig>   gt-ag,  <dig>   gc-ag, and  <dig>  at-ac.
similarly, in teosinte, sjs were composed of  <dig>   gt-ag,  <dig>   gc-ag, and  <dig>  at-ac.
the number of sjs ranged from  <dig>  to  <dig>  in maize libraries, and from  <dig>  to  <dig>  sjs in teosinte libraries .
in both maize and teosinte, the canonical gt-ag pair represented the highest proportion of all splicing sites, followed by the gc-ag pair.
this result was consistent with previous studies in other species  <cit> .
to identify differences in splice site pairs between maize and teosinte, we compared unique splicing sites identified in the two species.
a total of  <dig>  sjs were identified.
of these,  <dig>   were shared by the two species;  <dig>   and  <dig>   were specifically detected in maize and teosinte, respectively .
we also found that an average of  <dig>  and  <dig>  sjs in maize and teosinte, respectively, were detected for each gene.
more sjs were detected in maize genes, likely because more as transcripts were generated from maize genes relative to their orthologs in teosinte.
this finding may also indicate that maize domestication is accompanied by an increase in the number of sjs rather than a change in splice site pairs.
additionally, the four main as types—ir, es, aa, and ad—were analyzed.
we found that ir accounted for  <dig> % of all as events  and was the most abundant as type in maize, followed by aa , ad , and es .
however, in the six teosinte libraries, aa accounted for  <dig> % of all as events  and was the most common as type, followed by ir , ad , and es  .
these results demonstrate that both ir and aa could contribute to more than 60% of as events and are consistent with findings in other plant species  <cit> .
notably, ir was the most common as type in maize but not in teosinte.table  <dig>
differences in alternative splicing  events
as type
structure
maize events
maize ratio
teosinte events
teosinte ratio
ir: intron retention; es: exon skipping; ad: alternative donor site; aa: alternative acceptor site.
the effect of genomic features on as were evaluated in both maize and teosinte.
the result showed that exon number, gene length, totaling intron length and maximum intron length were highly positively correlated with the unique as events, whereas gc content and exon lengths were negatively correlated with unique as events , indicating occurrence of as may be strongly dependent on genic or genomic features.
a similar result was reported in soybean  <cit> .
furthermore, the sequence length covered by each as type was also analyzed in maize and teosinte.
for ir, the retained intron length ranged from  <dig> to  <dig>  bp in maize, but only  <dig> to  <dig>  bp in teosinte.
although a few of the retained introns were longer in maize than teosinte, the frequency distribution of retained intron length was similar between the two species.
both species had retained introns that were substantially longer than 89 bp, which is longer than those in soybean  <cit> .
for aa, the as sequence length ranged from  <dig> to  <dig>  bp in maize, but  <dig> to  <dig>  bp in teosinte.
both maize and teosinte shared the most frequent aa length .
this result was consistent with previous findings in other species  <cit> .
the most frequent skipped exon  length was 72 bp in maize and 66 bp in teosinte.
moreover, the peaks in the distribution of sequence length for ad were 2 bp in maize and 5 bp in teosinte .
in general, the sequence length covered by each as type did not significantly differ between maize and teosinte.table  <dig>
relationship between genomic feature and unique as events per gene in maize and teosinte
maize
teosinte
features
correlation coefficient
p-value
correlation coefficient
p-value
transposable elements and intron retention
to study the relationship between intron retention and transposable element  insertion,  <dig>  and  <dig>  of the retained introns were retrieved from ir isoforms of maize and teosinte, and then searched against a repetitive dna element database.
however,  <dig>  of the retained introns from  <dig> maize genes contained te sequences that included copia and gypsy  as well as mutator and stowaway  .
similarly, only nine  of the retained introns from teosinte unigenes contained te sequences, which included two lines , as well as helitrons, one en-spm, and one unknown te .
to validate that these as transcripts rose by te insertion, rna sequencing data from kernels   of  <dig> maize inbred lines were used to cross-validate these as transcripts with tes.
consequently,  <dig> of the  <dig> tes inserted in the retained introns were repeatedly detected in the developing kernel , indicating that these as events occurred in seedlings as well as developing kernels.
the low frequency of te sequences that harbor retained introns indicated that te insertion might not be a critical cause of the high frequency of ir events in maize and teosinte.
alternatively, diverse te sequences were detected in  <dig> and nine of the retained introns from maize and teosinte isoforms, respectively.
thus, some genes might be subjected to te insertion, which potentially facilitates the origin of as during maize domestication and improvement.
te insertion in introns may therefore be one of the most important mechanisms of intron retention.table  <dig>
number and type of transposable elements  in retained introns
class of te
type of te
number of tes inserted in maize retained introns
number of tes inserted in teosinte retained introns
hat
helitron
en-spm
stowaway
difference in as levels between maize and teosinte
to compare as levels in maize and teosinte, we identified  <dig>  genes that underwent as in maize but not in teosinte.
as levels of  <dig> genes were four-fold higher in maize than in teosinte, and all  <dig>  genes in maize had increased as levels .
however, only  <dig> genes had decreased as levels in maize compared with teosinte.
a total of  <dig> of these genes lost as isoforms in maize, and the as levels of  <dig> genes were four-fold lower in maize than in teosinte .
the number of as level-increased genes were  <dig> -fold higher than the number of as level-decreased genes in maize.
this finding indicates that more genes have increased the complexity of as but fewer genes have lost the complexity of as during domestication from teosinte to maize.
to gain further insight into the role that these as level-altered gene play, we performed a gene ontology  analysis on the as level-altered genes.
we found that these as level-increased genes were enriched in few biological processes: cellular response to stimuli, response to stress, and dna damage stimuli  and nucleotide excision repair .
as level-decreased genes were only enriched in intracellular signaling cascades and small gtpase-mediated signal transductions  .
as level-increased genes were also enriched in  <dig> molecular function terms, whereas as level-decreased genes were enriched in only five molecular function terms .
interestingly, as level-decreased genes were enriched in vitamin b <dig> binding  and pyridoxal phosphate binding .
vitamin b <dig> is known to be involved in several biological processes, including amino acid metabolism; metabolism of fats and carbohydrates; and the ability to increase biotic and abiotic stresses, photosynthesis, and response to pathogens  <cit> .figure  <dig> gene ontology  enrichment of all alternative splicing level-altered genes.
go enrichment was performed using agrigo.
biological process,  molecular function.
the percentage is the ratio of enriched alternative splicing-altered genes to all genes in a given go term using maize reference genes as background.
the p-value denotes the enriched levels in a go term, which were calculated using fisher’s exact test.
as level-altered genes were mapped to genomic regions experiencing selection during maize domestication and improvement detected by hufford et al.
<cit> .
a total of  <dig> as level-increased genes  and  <dig> as level-decreased genes  were located in genomic regions with strong signals of selection.
perfectly matched potential candidates that were selected during maize improvement were inferred as putatively improved genes.
of these  <dig> genes,  <dig> did not show as in teosinte but generated as isoforms in maize, and  <dig> genes showed an average of a  <dig> -fold increase in as levels in maize relative to teosinte.
in as level-decreased genes,  <dig> genes lost different isoforms in maize, and four genes showed an average of  <dig> -fold higher as level in teosinte than in maize.
these putatively improved genes included some transcription factors, such as nac, zinc finger, wrky, and bzip transcription factor genes, as well as some stress responsive genes, such as heat shock and drought-induced protein encoding genes.
this indicates that these putatively improved genes may be involved in transcription regulation and stress responses.
discussion
since berget et al.
<cit>  discovered intervening sequences, increasing evidence has revealed that as plays an important role in transcription regulation and the origin of the functional diversity of eukaryotic genomes.
as can increase genome complexity without increasing genome content; thus, increasing as levels should be positively correlated with organismal complexity.
using high throughput rna sequencing, we comprehensively explored the number of isoforms and as events, as types, and sjs chosen in highly conserved subsets of genes in maize and teosinte.
we found that the number of sjs was greater in maize than in teosinte, but the difference in the ratio of canonical and non-canonical sjs was not significantly different between the two species.
more genes were subjected to as in maize than in teosinte .
importantly, as levels of orthologous genes greatly diverged between the two species and were higher in maize than in teosinte.
more than  <dig>  genes increased their as levels but only  <dig> genes decreased as levels, indicating that as complexity is increased in modern maize relative to its progenitor.
nevertheless, two transcriptome assembly methods were used in the study, due to the ack of genomic information of teosinte, the difference of assembly methods potentially contributed to the as level difference between maize and tesointe.
a more comprehensive understanding of teosinte as landscape is still dependent on the reference-based transcriptome assembly.
a large-scale study using  <dig> million expressed sequence tags from  <dig> eukaryotic species revealed that the proportion of as genes and the average number of as isoforms per gene  have gradually increased over the past  <dig>  billion years; thus, as complexity can be considered a strong predictor of organismal complexity  <cit> .
a similar phenomenon is also observed in vertebrate species  <cit> , with all of these results indicating that there is a general trend of as increasing levels in eukaryotic evolution.
during crop domestication and improvement, hundreds of genes had altered expression levels and co-expression relationships  <cit> , and dozens of differentially expressed genes show significant enrichment for targets of selection, which indicates that expression level selection of specific sets of genes is an important mechanism in maize evolution.
in this study, we found that  <dig> as level-increased genes were located in regions that were targets of selection during maize improvement and putatively inferred to be improved genes.
however, none were located in potential domestication-related genes detected by genome sequencing  <cit> .
this result demonstrates that the increase in as level might be a result of improving flexibility and the degree of regulation at the transcriptional and post-transcriptional levels during maize improvement.
thus, this as level increase may be an important mechanism of maize evolution.
go enrichment revealed that the  <dig>  as level-increased genes were enriched in a few biological processes and molecular functions .
for example, grmzm2g <dig>  grmzm2g <dig>  grmzm2g <dig>  grmzm2g <dig>  grmzm2g <dig>  and grmzm2g <dig> all encode a group of nac-domain proteins that play important roles in the biotic and abiotic stress response regulation  <cit> .
grmzm2g <dig>  grmzm2g <dig>  and grmzm2g <dig> encode erf transcription factors that are involved in diverse abiotic stress responses and developmental process regulation  <cit> .
grmzm2g <dig> encodes alanine aminotransferase, and both grmzm2g <dig> and grmzm2g <dig> encode aspartate aminotransferases; all are induced by stress factors that facilitate the acquisition of somatic embryogenesis capacities  <cit> .
grmzm2g <dig>  ac <dig> _fg <dig>  and seven other genes encode proteins with an nb-arc domain, which is a central nucleotide-binding domain of a resistance  protein that is involved in pathogen recognition and subsequent innate immune response activation  <cit> .
grmzm2g <dig>  gemzm2g <dig>  grmzm2g <dig>  and  <dig> other heat shock protein-encoding genes were also detected.
all of these genes play potential roles in various biotic and abiotic stress responses.
moreover, grmzm2g <dig>  grmzm2g <dig>  grmzm2g <dig>  and  <dig> other genes are involved in oxygenic photosynthesis; grmzm2g <dig>  grmzm2g <dig>  grmzm2g <dig>  and nine other genes are involved in the calvin–benson–bassham cycle.
some flowering-related genes also showed as level increase in maize, such as grmzm2g <dig> , grmzm2g <dig> , and grmzm2g <dig> .
they were also reported to be involved in flowering regulation in maize .
in particular,  <dig> putative improved genes were enriched in transcription regulation and stress response.
during domestication from teosinte to modern maize, natural and artificial selection occurred in two phases: domestication and improvement.
in the domestication phase, selection focused on making maize cultivatable and improving seed access.
several regulatory genes, such as tga <dig>  <cit> , tb <dig>  <cit> , and ra <dig>  <cit> , are thought to be responsible for the major morphology changes from teosinte to landrace.
in the improvement phase, selection focused on yield, grain quality, and agro-ecosystem adaptations  <cit> .
we suggest that an increase in as complexity as well as changes in the expression and co-expression profile are likely responsible for increased adaptation and organic matter accumulation by modern maize breeding.
CONCLUSIONS
in this study, more than  <dig>  orthologous genes were identified by comparative transcriptome analysis of maize and teosinte, and as profiles of these orthologous genes were then identified.
we found that both maize and teosinte shared similar as mechanisms, but more genes have increased as complexity during domestication from teosinte to maize.
in particular, a subset of as level-increased genes that mainly encode transcription factors and stress-responsive proteins may have been selected during maize improvement.

 precursor mrna splicing is an important step in eukaryotic gene expression that plays a crucial role in higher eukaryotic developmental regulation and environmental stress response. after precursor mrna splicing, introns are removed and exons are ligated into one or more mature transcripts or isoforms. since walter gilbert first postulated that alternative splicing can create different isoforms from a common template, increasing evidence has shown that as widely occurs in animals and plants. for example, transcripts from 92%–94% of human intron-containing genes and approximately 61% of arabidopsis multiexonic genes can be alternatively spliced. in addition, approximately 63% and 51% of intron-containing genes also undergo as in soybean and maize, respectively. in general, as events can occur via four different mechanisms: exon skipping, alternative donor site, alternative acceptor site, and intron retention. however, there are also complex as types combined with at least two simple forms or some other low frequent forms such as mutually exclusive exons, alternative transcription start sites, and multiple polyadenylation sites in eukaryotes. however, the frequency of each as event is different and can be gene- and species-dependent. in animals, es is the most frequent as event and ir is the least frequent; for example, approximately % of all as events in humans are caused by by es, but only % are caused by ir. in contrast, in arabidopsis, maize, brachypodium, and cotton, ir is the predominant form of as, whereas es only accounts for a small proportion of as events. as is involved in a wide array of biological functions, particularly in response to biotic and abiotic stress. in humans, approximately 15% of inherited diseases are caused by mutations that interfere with mrna splicing. in arabidopsis, as is strongly associated with environmental stimuli. for example, isoforms retaining the fourth intron of cca increased under high light conditions but decreased under low temperature conditions. recent studies found that diverse mrna isoforms produced from a given gene can accelerate genome evolution by generating new functions. a splice site mutation in the fourth intron of sorghum shattering results in the removal of the fourth exon, and this variation underwent selection during the sorghum domestication and improvement. maize, which is a model plant for studying crop domestication, evolved from the annual teosinte zea mays ssp. parviglumis approximately to  years ago and greatly differs in morphology from its progenitors. recently, several key domestication-related genes that are responsible for the morphological changes between maize and teosinte were identified, including tga, tb, ba, ra, and zfl. dna sequencing advances have led to the discovery of thousands of genes with strong signals of selection. at the transcriptional level, swanson-wagner et al. found hundreds of genes that have altered expression levels or co-expression profiles during domestication. some of the genes involved in responding to biotic and abiotic stresses were significantly enriched with selective sweeps. in addition, lemmon et al. suggested that gene expression may be caused by the modification of cis regulatory elements. they found that approximately 4% of genes showing evidence for consistent cis regulatory divergence that differentiates maize from teosinte were significantly correlated with maize domestication and improvement. early studies on maize domestication were primarily focused on nucleotide sequence diversity and expression changes between maize and teosinte, but the differences on alternatively spliced genes, alternative splicing types, as events and level between maize and teosinte, and evolutionary role of these differences has been poorly studied. to better understand the evolutionary role of as in maize domestication, the transcriptome of the seedling stage from six teosinte accessions and ten maize inbred lines were sequenced. furthermore, genome-wide as profiles were analyzed, and as genes, types, events, and levels were compared between maize and teosinte. we found that maize had greater as complexity than teosinte, and a subset of as level-altered genes were enriched in transcriptional regulation and stress responses. this subset was located in regions that have been targets of selection for maize improvement.  transcriptome sequencing and assembly to assemble the transcriptome of maize and teosinte, cdna libraries of ten maize inbred lines, three zea mays ssp. parviglumis and three zea mays ssp. mexicana accessions were sequenced with the solexa sequencing platform. in total, million reads were obtained, with an average of million reads per sample. after discarding low quality reads, million high quality reads were used for further analysis. without a reference genome, high quality reads of teosinte were first assembled de novo. after removing redundant sequences, to contigs were reconstructed in six teosinte libraries. the length of contigs varied from to  bp, the n and n of teosinte transcriptomes ranged from to 937 bp and to 217 bp, respectively. for maize inbred lines, transcriptomes were assembled based on b reference genome: to isoforms were assembled and the n and n of maize transcriptomes ranged from to  bp and to 843 bp, respectively. for a global view of transcriptomes of the two teosinte subspecies, we assembled pseudo-transcriptomes by clustering contigs from three zea mays ssp. parviglumis and three zea mays ssp. mexicana libraries, respectively. totals of and unique isoforms for zea mays ssp. parviglumis and zea mays ssp. mexicana, respectively, were obtained; the longest transcript in zea mays ssp. parviglumis and in zea mays ssp. mexicana was and  bp, respectively. under a 95% identity and coverage cutoff, zea mays ssp. parviglumis and zea mays ssp. mexicana transcripts could be perfectly matched with and maize reference genes, respectively, and genes were shared by the two teosinte subspecies. high homology of isoforms between maize and teosinte indicated high conservation of proteins encoded by function genes during maize domestication. this finding is consistent with the study on expression and cis-regulatory changes between maize and teosinte. variant and genetic diversity characterization to characterize genetic diversity among all sequenced samples, clean reads from each sample were mapped to the b reference genome, respectively. after discarding the low coverage and quality snps and indels, we detected snps from genes with 1– variant sites in a single gene, and indels with a − to 65 bp deletion or insertion that was distributed from − to 20 bp. of the snps, were transitions and were transversions. the ratio of transitions to transversions was approximately and likely caused by the sequencing coverage and filtering steps. moreover, snps were located in exons, were in introns, and were in intergenic regions. these results indicate that some transcribed sequences may not be annotated in the b filtered gene set or some unknown fragments were transcribed in our transcriptome data. phylogenetic analysis using all snps detected in this study showed that maize inbred lines, zea mays ssp. parviglumis, and zea mays ssp. mexicana accessions clearly grouped into three clusters. temperate and tropical maize lines also clearly grouped into different clades. this result is consistent with previous studies that have used dna markers.figure phylogenetic relationships among sequenced samples. taxa in the neighbor-joining tree are represented by different shapes. empty circles denote tropical maize inbred lines; solid circles denote temperate maize inbred lines; empty squares denote accessions of zea mays ssp. parviglumis; and solid squares denote accessions of zea mays ssp. mexicana. the phylogenetic analysis was performed using the neighbor-joining method with bootstrap replicates in the phylip package. orthologous gene identification zea mays ssp. parviglumis and zea mays ssp. mexicana pseudo-transcriptomes were used to identify orthologous gene pairs between maize and teosinte using the reciprocal best blast hit method. a total of and high confidence orthologs of maize with an e-value ≤ 1e- were identified in zea mays ssp. parviglumis and zea mays ssp. mexicana, respectively. of these, orthologs that accounted for % of the b filtered gene set were shared between the two species and distributed across all maize chromosomes. overall, ortholog density was low in centromere-proximal regions and high in chromosome arms. these shared orthologs were highly conserved during maize domestication; therefore, these shared orthologs were further used for as landscape of maize and teosinte transcriptome analysis.figure orthologous gene distribution in the maize genome. gene density statistics were calculated with a 200-kb sliding-window. the x-axis denotes the physical position of a chromosome. the y-axis denotes the number of genes located in this sliding window. chr = chromosome. maize and teosinte alternative splicing landscapes to determine the relationship between sequencing depth and as detection power, the sequencing library was first used to randomly create sub-libraries to detect as transcripts. overall, sequencing depth was highly positively correlated with the average coverage of each gene, and positively correlated with the as gene number and as events, in particular, when sequencing depth is less than gb. however, when sequencing depth was more than gb, the increase of sequencing depth did not significantly increase the number of as genes and all as events. this result is consistent with liu’s findings in arabidopsis. in this study, sequencing depth of most libraries was more than gb, and implicating our sequencing data was well to support the identification of as transcripts.figure relationship between sequencing depth and alternative splicing. the x-axis denotes the sequencing depth and average gene coverage. the y-axis denotes the numbe of alternatively spliced genes or number of alternative splicing events. to characterize the maize and teosinte as landscapes, as events that occurred in all shared orthologs of each sample were evaluated. in shared genes, genes were annotated with at least two exons and are potentially subjected to as. in teosinte, genes, which accounts for % of the studied multiexonic genes, were subjected to as. a total of as events were identified in all teosinte libraries. after removing the same as events in different libraries, non-redundant as events were detected in teosinte libraries, with an average of as events per gene. in maize, a total of as events, including non-redundant as events, were identified from genes in all maize libraries, with an average of as events per gene. among different samples in the same species, the number of as events and as genes varied widely. in maize, to as events involving to genes were identified. in teosinte, to as events involving to genes were identified.table number of alternative splicing events and genes detected in maize and teosinte libraries as-event as gene as events per gene splicing junction sites were identified using tophat software. a total of and unique sjs were detected in all maize and teosinte samples. in maize, sjs were composed of gt-ag, gc-ag, and at-ac. similarly, in teosinte, sjs were composed of gt-ag, gc-ag, and at-ac. the number of sjs ranged from to in maize libraries, and from to sjs in teosinte libraries. in both maize and teosinte, the canonical gt-ag pair represented the highest proportion of all splicing sites, followed by the gc-ag pair. this result was consistent with previous studies in other species. to identify differences in splice site pairs between maize and teosinte, we compared unique splicing sites identified in the two species. a total of sjs were identified. of these, were shared by the two species; and were specifically detected in maize and teosinte, respectively. we also found that an average of and sjs in maize and teosinte, respectively, were detected for each gene. more sjs were detected in maize genes, likely because more as transcripts were generated from maize genes relative to their orthologs in teosinte. this finding may also indicate that maize domestication is accompanied by an increase in the number of sjs rather than a change in splice site pairs. additionally, the four main as types—ir, es, aa, and ad—were analyzed. we found that ir accounted for % of all as events and was the most abundant as type in maize, followed by aa, ad, and es. however, in the six teosinte libraries, aa accounted for % of all as events and was the most common as type, followed by ir, ad, and es. these results demonstrate that both ir and aa could contribute to more than 60% of as events and are consistent with findings in other plant species. notably, ir was the most common as type in maize but not in teosinte.table differences in alternative splicing events as type structure maize events maize ratio teosinte events teosinte ratio ir: intron retention; es: exon skipping; ad: alternative donor site; aa: alternative acceptor site. the effect of genomic features on as were evaluated in both maize and teosinte. the result showed that exon number, gene length, totaling intron length and maximum intron length were highly positively correlated with the unique as events, whereas gc content and exon lengths were negatively correlated with unique as events, indicating occurrence of as may be strongly dependent on genic or genomic features. a similar result was reported in soybean. furthermore, the sequence length covered by each as type was also analyzed in maize and teosinte. for ir, the retained intron length ranged from to  bp in maize, but only to  bp in teosinte. although a few of the retained introns were longer in maize than teosinte, the frequency distribution of retained intron length was similar between the two species. both species had retained introns that were substantially longer than 89 bp, which is longer than those in soybean. for aa, the as sequence length ranged from to  bp in maize, but to  bp in teosinte. both maize and teosinte shared the most frequent aa length. this result was consistent with previous findings in other species. the most frequent skipped exon length was 72 bp in maize and 66 bp in teosinte. moreover, the peaks in the distribution of sequence length for ad were 2 bp in maize and 5 bp in teosinte. in general, the sequence length covered by each as type did not significantly differ between maize and teosinte.table relationship between genomic feature and unique as events per gene in maize and teosinte maize teosinte features correlation coefficient p-value correlation coefficient p-value transposable elements and intron retention to study the relationship between intron retention and transposable element insertion, and of the retained introns were retrieved from ir isoforms of maize and teosinte, and then searched against a repetitive dna element database. however, of the retained introns from maize genes contained te sequences that included copia and gypsy as well as mutator and stowaway. similarly, only nine of the retained introns from teosinte unigenes contained te sequences, which included two lines, as well as helitrons, one en-spm, and one unknown te. to validate that these as transcripts rose by te insertion, rna sequencing data from kernels of maize inbred lines were used to cross-validate these as transcripts with tes. consequently, of the tes inserted in the retained introns were repeatedly detected in the developing kernel, indicating that these as events occurred in seedlings as well as developing kernels. the low frequency of te sequences that harbor retained introns indicated that te insertion might not be a critical cause of the high frequency of ir events in maize and teosinte. alternatively, diverse te sequences were detected in and nine of the retained introns from maize and teosinte isoforms, respectively. thus, some genes might be subjected to te insertion, which potentially facilitates the origin of as during maize domestication and improvement. te insertion in introns may therefore be one of the most important mechanisms of intron retention.table number and type of transposable elements in retained introns class of te type of te number of tes inserted in maize retained introns number of tes inserted in teosinte retained introns hat helitron en-spm stowaway difference in as levels between maize and teosinte to compare as levels in maize and teosinte, we identified genes that underwent as in maize but not in teosinte. as levels of genes were four-fold higher in maize than in teosinte, and all genes in maize had increased as levels. however, only genes had decreased as levels in maize compared with teosinte. a total of of these genes lost as isoforms in maize, and the as levels of genes were four-fold lower in maize than in teosinte. the number of as level-increased genes were -fold higher than the number of as level-decreased genes in maize. this finding indicates that more genes have increased the complexity of as but fewer genes have lost the complexity of as during domestication from teosinte to maize. to gain further insight into the role that these as level-altered gene play, we performed a gene ontology analysis on the as level-altered genes. we found that these as level-increased genes were enriched in few biological processes: cellular response to stimuli, response to stress, and dna damage stimuli and nucleotide excision repair. as level-decreased genes were only enriched in intracellular signaling cascades and small gtpase-mediated signal transductions. as level-increased genes were also enriched in molecular function terms, whereas as level-decreased genes were enriched in only five molecular function terms. interestingly, as level-decreased genes were enriched in vitamin b binding and pyridoxal phosphate binding. vitamin b is known to be involved in several biological processes, including amino acid metabolism; metabolism of fats and carbohydrates; and the ability to increase biotic and abiotic stresses, photosynthesis, and response to pathogens.figure gene ontology enrichment of all alternative splicing level-altered genes. go enrichment was performed using agrigo. biological process, molecular function. the percentage is the ratio of enriched alternative splicing-altered genes to all genes in a given go term using maize reference genes as background. the p-value denotes the enriched levels in a go term, which were calculated using fisher’s exact test. as level-altered genes were mapped to genomic regions experiencing selection during maize domestication and improvement detected by hufford et al.. a total of as level-increased genes and as level-decreased genes were located in genomic regions with strong signals of selection. perfectly matched potential candidates that were selected during maize improvement were inferred as putatively improved genes. of these genes, did not show as in teosinte but generated as isoforms in maize, and genes showed an average of a -fold increase in as levels in maize relative to teosinte. in as level-decreased genes, genes lost different isoforms in maize, and four genes showed an average of -fold higher as level in teosinte than in maize. these putatively improved genes included some transcription factors, such as nac, zinc finger, wrky, and bzip transcription factor genes, as well as some stress responsive genes, such as heat shock and drought-induced protein encoding genes. this indicates that these putatively improved genes may be involved in transcription regulation and stress responses. discussion since berget et al. discovered intervening sequences, increasing evidence has revealed that as plays an important role in transcription regulation and the origin of the functional diversity of eukaryotic genomes. as can increase genome complexity without increasing genome content; thus, increasing as levels should be positively correlated with organismal complexity. using high throughput rna sequencing, we comprehensively explored the number of isoforms and as events, as types, and sjs chosen in highly conserved subsets of genes in maize and teosinte. we found that the number of sjs was greater in maize than in teosinte, but the difference in the ratio of canonical and non-canonical sjs was not significantly different between the two species. more genes were subjected to as in maize than in teosinte. importantly, as levels of orthologous genes greatly diverged between the two species and were higher in maize than in teosinte. more than genes increased their as levels but only genes decreased as levels, indicating that as complexity is increased in modern maize relative to its progenitor. nevertheless, two transcriptome assembly methods were used in the study, due to the ack of genomic information of teosinte, the difference of assembly methods potentially contributed to the as level difference between maize and tesointe. a more comprehensive understanding of teosinte as landscape is still dependent on the reference-based transcriptome assembly. a large-scale study using million expressed sequence tags from eukaryotic species revealed that the proportion of as genes and the average number of as isoforms per gene have gradually increased over the past billion years; thus, as complexity can be considered a strong predictor of organismal complexity. a similar phenomenon is also observed in vertebrate species, with all of these results indicating that there is a general trend of as increasing levels in eukaryotic evolution. during crop domestication and improvement, hundreds of genes had altered expression levels and co-expression relationships, and dozens of differentially expressed genes show significant enrichment for targets of selection, which indicates that expression level selection of specific sets of genes is an important mechanism in maize evolution. in this study, we found that as level-increased genes were located in regions that were targets of selection during maize improvement and putatively inferred to be improved genes. however, none were located in potential domestication-related genes detected by genome sequencing. this result demonstrates that the increase in as level might be a result of improving flexibility and the degree of regulation at the transcriptional and post-transcriptional levels during maize improvement. thus, this as level increase may be an important mechanism of maize evolution. go enrichment revealed that the as level-increased genes were enriched in a few biological processes and molecular functions. for example, grmzm2g grmzm2g grmzm2g grmzm2g grmzm2g and grmzm2g all encode a group of nac-domain proteins that play important roles in the biotic and abiotic stress response regulation. grmzm2g grmzm2g and grmzm2g encode erf transcription factors that are involved in diverse abiotic stress responses and developmental process regulation. grmzm2g encodes alanine aminotransferase, and both grmzm2g and grmzm2g encode aspartate aminotransferases; all are induced by stress factors that facilitate the acquisition of somatic embryogenesis capacities. grmzm2g ac _fg and seven other genes encode proteins with an nb-arc domain, which is a central nucleotide-binding domain of a resistance protein that is involved in pathogen recognition and subsequent innate immune response activation. grmzm2g gemzm2g grmzm2g and other heat shock protein-encoding genes were also detected. all of these genes play potential roles in various biotic and abiotic stress responses. moreover, grmzm2g grmzm2g grmzm2g and other genes are involved in oxygenic photosynthesis; grmzm2g grmzm2g grmzm2g and nine other genes are involved in the calvin–benson–bassham cycle. some flowering-related genes also showed as level increase in maize, such as grmzm2g, grmzm2g, and grmzm2g. they were also reported to be involved in flowering regulation in maize. in particular, putative improved genes were enriched in transcription regulation and stress response. during domestication from teosinte to modern maize, natural and artificial selection occurred in two phases: domestication and improvement. in the domestication phase, selection focused on making maize cultivatable and improving seed access. several regulatory genes, such as tga, tb, and ra, are thought to be responsible for the major morphology changes from teosinte to landrace. in the improvement phase, selection focused on yield, grain quality, and agro-ecosystem adaptations. we suggest that an increase in as complexity as well as changes in the expression and co-expression profile are likely responsible for increased adaptation and organic matter accumulation by modern maize breeding.  in this study, more than orthologous genes were identified by comparative transcriptome analysis of maize and teosinte, and as profiles of these orthologous genes were then identified. we found that both maize and teosinte shared similar as mechanisms, but more genes have increased as complexity during domestication from teosinte to maize. in particular, a subset of as level-increased genes that mainly encode transcription factors and stress-responsive proteins may have been selected during maize improvement. 
of these, as level-altered genes that are involved in transcriptional regulation and in stress responses are located in regions that have been targets of selection during maize improvement. importantly, a subset of as level-increased genes that encode transcription factors and stress-responsive proteins may have been selected during maize improvement. this indicates that te insertion might be an important mechanism for intron retention in maize. in this study, six teosinte and ten maize transcriptomes were sequenced to analyze as changes and signatures of selection in maize domestication and improvement. a substantial amount of evidence has demonstrated that as complexity is relevant to eukaryotic evolution, development, adaptation, and complexity.
 alternative splicing is an important regulatory mechanism that greatly contributes to eukaryotic transcriptome diversity. a substantial amount of evidence has demonstrated that as complexity is relevant to eukaryotic evolution, development, adaptation, and complexity. in this study, six teosinte and ten maize transcriptomes were sequenced to analyze as changes and signatures of selection in maize domestication and improvement.  in maize and teosinte, highly conserved genes, including multiexonic genes, were detected. by identifying as isoforms from mutliexonic genes, we found that as types were not significantly different between maize and teosinte. in addition, the two main as types contributed to more than 60% of the as events in the two species, but the average unique as events per each alternatively spliced gene in maize was higher than that in teosinte. moreover, genes generating retained introns with transposable element sequences were detected in maize, which is far more than retained introns with tes detected in teosinte. this indicates that te insertion might be an important mechanism for intron retention in maize. additionally, the as levels of genes were significantly different between maize and teosinte. of these, as level-altered genes that are involved in transcriptional regulation and in stress responses are located in regions that have been targets of selection during maize improvement. these genes were inferred to be putatively improved genes.  we suggest that both maize and teosinte share similar as mechanisms, but more genes have increased as complexity during domestication from teosinte to maize. importantly, a subset of as level-increased genes that encode transcription factors and stress-responsive proteins may have been selected during maize improvement. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords transcriptomealternative splicingzea mays ssp. parviglumiszea mays ssp. mexicanabiotic and abiotic stressesissue-copyright-statement© the author 2015 