BACKGROUND
olive  has long been among important topics of agricultural research due to its well - known nutritional and health value.
therefore, numerous studies on physiological  <cit>  phytochemical  <cit> , molecular systematic  <cit>  and molecular genetics / genomics  <cit>  aspects of olive have been reported.
further genetic studies involving molecular mechanism of fruit set, fruit development, fruit detachment and alternate bearing in olive have not been widely reported though there are reports on various aspects of alternate bearing such as endogenous and environmental factors  <cit> .
while the idea of generating a genetically modified olive tree has not been welcome, it is possible to explore molecular mechanisms of the common problems of olive through molecular genetics approaches.
identifying transcription factors specific for important genes of fruit senescence only, for instance, can lead to subsequent steps of controlling these molecules for potentially getting a relatively more uniform harvest.
one of the first steps to achieve such long term aims is constructing and characterizing cdna libraries to identify genes that are specific to certain tissues and / or developmental stages.
furthermore, since the olive genome has not yet been sequenced, olive molecular genetics studies mostly depend on cdna libraries to identify novel genes, or genes associated with certain processes such as fruit development and senescence.
although numerous cdna libraries of various plant tissues and organs under very specific conditions such as phosphorus stressed roots  <cit> , root hairs  <cit> , glucose stressed root tips  <cit> , nodules  <cit> , ripe fruit detachment tissues  <cit>  and leaves  <cit>  are available, olive cdna libraries have largely been restricted to fruit libraries  <cit> .
hence, reports such as comparison of olive genes expressed in leaves of “on year” and that of “off year”, or comparison of olive genes expressed in fruited leaves and non-fruited leaves are rare.
we have recently reported micro rnas  <cit>  and global transcripts  <cit>  associated with alternate bearing in olive.
in this study, olive leaves of trees in “on year” and in “off year” in july  and in november  were harvested into liquid nitrogen and used to construct cdna libraries to identify cdnas specific for each time and condition.
additionally, a fruit cdna library was also constructed to further confirm the specificity of the cdnas obtained from each library.
analyses of the results revealed cdnas specific for each library, and hence, a number of candidate cdnas associated with alternate bearing were identified.
additionally, bioinformatics tools such as detailed blast searches, genonthology and kegg analyzes on the obtained sequences were applied to extract further information about these cdna molecules.
RESULTS
selection of the reference gene
using the appropriate reference gene in quantitative real-time pcr  to normalize the initial total rna template amounts is one of the most important factors affecting the reliability of the qrt-pcr results.
that is why commonly used reference genes should be tested first in the organism in use to accurately pick the one that has no variation based on changes because of diverse factors  <cit> .
since reference genes in olive have not widely been reported for qrt-pcr, expression levels of seven commonly used reference genes  in various plants were determined via qrt-pcr  and gapdh was decided to be an appropriate reference gene to use with olive.
brief overview of the cdnas obtained from the libraries
genbank homologous records for each insert sequence obtained through blastn search  <cit>  at ncbi, revealed 11% - 16% protein coding gene homologs and 3% unknown cdnas while the remaining 84% to 89% constituted non-coding rna molecules including rrnas and trnas .
all the cdna sequences  had similarity to cdna records of other plants previously registered in the genbank databases  while jnf <dig>  jnf <dig> and jnf <dig> had no similarity for any records in any available database.
in each library, more than half of the cdnas were new for olive genome .
* cdnas that are new for olive.
** obtained from genbank for the sequences generated through this study.
* cdnas that are new for olive.
** obtained from genbank for the sequences generated through this study.
* cdnas that are new for olive.
** obtained from genbank for the sequences generated through this study.
* cdnas that are new for olive.
** obtained from genbank for the sequences generated through this study.
* cdnas that are new for olive.
** obtained from genbank for the sequences generated through this study.
cdna contents and qrt-pcr validation of the individual libraries
in july, “on year” leaves , 4% of the protein coding gene homologs was detected as p <dig> monooxygenases  while homologs of menthone:neomenthol reductase  <dig>  a transcription factor  and a protease  were represented by 2% .
the remaining five cdnas were represented with 1% .
qrt-pcr confirmed the abundance of p <dig> monooxygenases mentioned in jf library, and revealed embryo defective binding / small gtpase regulator  to be specific to “on year” leaves in july .
in july “off year” leaves , the predominating cdnas were  again homologs of p <dig> monooxygenase  that was also confirmed by qrt-pcr .
the second most abundant cdnas were homologs of ethylene responsive protein , wound stress protein , a gene complex of multiple constitutive proteins and intergenic spacers  and ribosomal protein l <dig> .
only in this library  were detected cdnas  that were completely novel to any available nucleotide database.
qrt-pcr revealed wound stress protein , one of the abundant cdnas in jnf library, as a specific cdna to “off year” leaves in july.
although the abundance of the cdnas detected in november, “on year” leaves  were the least  among all libraries generated , nf <dig> , nf <dig>  and a cdna from jnf library  appeared to express at very high levels .
homologs of cryptochrome  <dig>  mrna, dnaj heat shock family protein , and a calmodulin binding protein  were detected as 2%  but their abundance was not confirmed by qrt-pcr.
a cdna that is homologous to both ariadne  and a zinc finger family protein  was the most abundant cdna of the remaining two libraries .
it was detected at a rate of 3% in each library .
qrt-pcr analysis revealed this cdna’s expression as  <dig> fold to  <dig> fold more in f library than in any other library .
the remaining cdnas in the nnf library were detected only once .
the cold stress-induced protein / dehydrin homolog  along with the other putative dehydrin  and the putative metallothionein  were the most abundant cdnas in both of the november libraries  from the leaves .
after ariadne - like protein homologs, the second most abundant  cdnas in fruit flesh library  were homologs of udp-glucose 4-epimerase , acyl-coa binding protein  and triosephosphate isomerase .
qrt-pcr revealed all the cdnas  isolated from fruit flesh library were specific to fruits.
a cdna  that has a weak similarity to predicted nuclear-pore anchor protein  was also fruit specific .
bioinformatics
among the olive ests analyzed,  <dig> sequences displayed significant blastx matches within the genes registered in the ncbi database.
in order to predict the reliability assessment of the ests or alignment quality, sequence similarity  and e-value distribution graphs  were generated from sequences based on the blastx results.
the species distribution of olive ests based on blastx hits had the highest sequence homology to vitis vinifera , and followed by populus trichocarpa , arabidopsis thaliana  and oryza sativa   .
the functional annotation and categorization of each olive est based on gene ontology  terms were analyzed using the blast2go suite.
for each transcript, a set of go term information including; accession, annotation term and basic definition is shown in table .
in addition, these transcripts representing genes with known function were categorized by biological process, cellular component and molecular function according to the ontological definitions of the go terms.
the transcripts in the biological process  category were related to metabolic process , cellular process , response to stimulus , cellular localization , multicellular organismal process , developmental process , reproduction , multi - organism process , biological regulation , cell wall organization or biogenesis  and cellular component biogenesis .
in the cellular component category , most of the go terms were mainly related to cellular  and organelle  components such as cell periphery and intracellular organelle parts, followed by macromolecular complex , extracellular region  and membrane enclosed lumen .
as for the molecular function  category, most abundant go terms were involved in binding  such as nucleic acid and transition metal ion binding, catalytic activity , electron carrier activity , transporter  and structural molecule  activity as well as enzyme regulator  activity .
using kaas, each olive est was assigned with a kegg orthology  number with the sbh  assignment method and the numbers subsequently were mapped to one of the kegg's reference metabolic pathways.
consequently, a total of  <dig> main metabolic pathways were generated through the use of kaas pathway mapping and the sequences largely correlated with essential metabolisms of galactose metabolism  , amino sugar and nucleotide sugar metabolism , photosynthesis , other glycan degradation , monoterpenoid biosynthesis  and followed by spliceosome , ribosome  and circadian rhythm - plant  , e-value distribution , species distribution  and top-hit species distribution  graphs generated after olive sequences were processed.
*see tables 1– <dig> for the accession numbers of these sequences.
according the table, there is a correlation between the biosynthesis  and ribosomal activity in fruited and non- fruited leaves
discussion
the approach to isolate differentially expressed genes
to isolate differentially expressed cdnas, it is essential to start with total rna molecules extracted from tissues of identical conditions.
with this respect, the trees we selected were genetically identical and grew virtually in the same micro-environment.
the same constitutive cdna  was detected in four  of the five libraries  and was confirmed with qrt-pcr while homologs of cdnas reported to express at certain stress conditions such as cold stress induced protein / dehydrin  and temperature induced lipocalin  or at certain metabolic processes such as acyl-coa binding protein  and triosephosphate isomerase  were detected at the expected libraries .
at least  <dig> cdnas from each library  were confirmed by qrt-pcr to be specific to the tissues  that they were initially detected through sequencing of the plasmid inserts from arbitrarily selected colonies.
hence, the approach  we used to detect differentially expressed cdnas in the libraries, has proven to be reasonable.
furthermore, more than half of the cdnas of each library did not match olive records in nucleotide  and est  databases of ncbi which contains cdnas derived from leaves, fruits and flowers .
overall cdna profile of the libraries
in july, “on year” leaves  and in “off year” leaves , homologs of cytochrome p <dig> monooxygenases  appeared to be dominating cdnas .
embryo defective binding / small gtpase protein homolog  appeared to be strictly specific to jf.
interestingly, jf <dig> was detected more in “on year” leaves and fruits but less in “off year” leaves  suggesting it might have a role in “on year” but more in july than that of november.
on the contrary, the putative metallothionein  appeared to be associated with “on year” leaves but more in november.
one  of the two unknown cdnas isolated from july “off year” leaves were found to express in all the tissues studied except fruits while the other  was specific to july leaves only.
in november, “on year” leaves , a homolog of emb lyase  was the most abundant cdna , yet it was detected in this library only.
based on the ncbi record , this putative lyase is predicted to function in breaking of c-c, c-o and c-n bonds during embryo development.
detecting this cdna in maturing fruit bearing leaves makes meaningful sense as for developing embryo , nutrients from the leaves  should be supplied  <cit> .
most other cdnas of november “on year” leaves  also appeared to be associated with cold stress and embryo development which were the specific conditions for nf library: nf <dig> is a homolog of dehydrin  that has been reported to function in low temperatures and seed development  <cit> .
nf <dig> is similar to metallothionein  and has been reported to function in senescence  <cit> .
likewise nf <dig> homolog jerf <dig>  has been reported to involve in gene expression at cold  <cit> , and nf <dig> homolog glycosyl hydrolase  is associated with biotic / abiotic stress, lignification and cell wall reconstruction  <cit> .
the cdna that is homolog of both ariadne  and a zinc finger family protein  was the most abundant in both november, “off year” leaves  and in fruit flesh  libraries.
qrt-pcr results revealed  <dig> fold to  <dig> fold more expression of this cdna in fruits than in other libraries but did not confirm as one of the most abundant cdnas in fruits nor in “off year” leaves.
combined with ubiquitin association, these results suggest ariadne homolog in olive is most probably a constitutively expressed cdna.
nnf <dig> / nnf <dig> and nnf <dig> are homologs of a splicing factor subunit  and a translation initiation factor , respectively, and they both were detected at very low level  in all libraries .
given the fact that these two trees are genetically identical and grow virtually in the same micro-environment, overall results present cdnas differentially expressed in leaves, “on year” leaves, and in “off year” leaves.
constitutively expressed genes, most of which have not been detected in olive before, and several unknown cdnas and / or genes are also reported.
it should be kept in mind that alternate bearing is a result of complicated biotic and abiotic processes including environmental factors, physiological responses of the trees in the form of activation and repression of endogenous metabolic pathways  <cit> , which in turn are also based on the genetic background of the tree.
large phenotypic variation has been observed, including year-to-year variation of a single genotype, as well as variation among and within  cultivars under the same environment.
hence it is not possible to clearly enlighten the genetic players of alternate bearing in a single cdna screening or even a complete transcriptome analysis.
multiple approaches involving several years follow up of the selected trees / cultivars are needed to identify certain or key genetic players of alternate bearing in olive.
there are no comprehensive reports on the genetic basis of alternate bearing in olive, however, and hence these results constitute important information for one of the first steps of a genetic dissection of olive periodicity which causes significant economic loss for olive growers.
through exploring these cdnas further, it is possible to isolate genes that are key regulator of fruit formation and / or periodicity in olive.
bioinformatic analyses
through the bioinformatic analyses it was possible to extract further additional information about the cdnas as well as about olive in general.
blastx analysis revealed olive has a surprisingly high  similarity to grapevine , although these two plants are not even the same order  in systematics.
the second most similar plant to olive is populus  with a much lower  similarity.
this suggests the cdnas captured are directly or indirectly associated with the pathways of fruit formation and / or production that are in turn related to periodicity.
go terms categorization grouped the cdnas into common processes, localizations and functions such as metabolic process, cellular localization and binding, respectively, which reflect a general profile of typical cell while differentially expressed cdnas were also significantly represented such as 17% of the cdnas in the “response to stimulus” category, and 25% of the cdnas in the “nucleic acid and transition metal ion binding, catalytic activity” category.
the metabolic pathways generated through the use of kaas pathway mapping were largely correlated with essential metabolisms such as galactose, amino sugar, nucleotide sugar metabolisms and photosynthesis confirming the constitutive status of the majority of the cdnas obtained.
CONCLUSIONS
in summary, we have isolated and analyzed cdnas that are associated with alternate bearing in olive.
a p <dig> monooxigenase homolog expressed more in the “on year” than that of “off year” leaves in july.
two putative dehydrins expressed significantly more in “on year” leaves than that of “off year” leaves in november.
homologs of triose phosphate isomerise, udp - glucose epimerase, acyl - coa binding protein, and a putative nuclear core anchor protein appeared fruit specific, while a homolog of an embryo binding protein / small gtpase regulator was detected in “on year” leaves only.
an unknown cdna was specific to leaves in july.
kegg pathway analyses of the sequences correlated with essential metabolisms such as galactose metabolism, amino sugar and nucleotide sugar metabolisms and photosynthesis.
detailed analysis of the results presents candidate cdnas that can be used to dissect further the genetic basis of fruit production and / or alternate bearing.

 olive has long been among important topics of agricultural research due to its well - known nutritional and health value. therefore, numerous studies on physiological phytochemical, molecular systematic and molecular genetics / genomics aspects of olive have been reported. further genetic studies involving molecular mechanism of fruit set, fruit development, fruit detachment and alternate bearing in olive have not been widely reported though there are reports on various aspects of alternate bearing such as endogenous and environmental factors. while the idea of generating a genetically modified olive tree has not been welcome, it is possible to explore molecular mechanisms of the common problems of olive through molecular genetics approaches. identifying transcription factors specific for important genes of fruit senescence only, for instance, can lead to subsequent steps of controlling these molecules for potentially getting a relatively more uniform harvest. one of the first steps to achieve such long term aims is constructing and characterizing cdna libraries to identify genes that are specific to certain tissues and / or developmental stages. furthermore, since the olive genome has not yet been sequenced, olive molecular genetics studies mostly depend on cdna libraries to identify novel genes, or genes associated with certain processes such as fruit development and senescence. although numerous cdna libraries of various plant tissues and organs under very specific conditions such as phosphorus stressed roots, root hairs, glucose stressed root tips, nodules, ripe fruit detachment tissues and leaves are available, olive cdna libraries have largely been restricted to fruit libraries. hence, reports such as comparison of olive genes expressed in leaves of “on year” and that of “off year”, or comparison of olive genes expressed in fruited leaves and non-fruited leaves are rare. we have recently reported micro rnas and global transcripts associated with alternate bearing in olive. in this study, olive leaves of trees in “on year” and in “off year” in july and in november were harvested into liquid nitrogen and used to construct cdna libraries to identify cdnas specific for each time and condition. additionally, a fruit cdna library was also constructed to further confirm the specificity of the cdnas obtained from each library. analyses of the results revealed cdnas specific for each library, and hence, a number of candidate cdnas associated with alternate bearing were identified. additionally, bioinformatics tools such as detailed blast searches, genonthology and kegg analyzes on the obtained sequences were applied to extract further information about these cdna molecules.  selection of the reference gene using the appropriate reference gene in quantitative real-time pcr to normalize the initial total rna template amounts is one of the most important factors affecting the reliability of the qrt-pcr results. that is why commonly used reference genes should be tested first in the organism in use to accurately pick the one that has no variation based on changes because of diverse factors. since reference genes in olive have not widely been reported for qrt-pcr, expression levels of seven commonly used reference genes in various plants were determined via qrt-pcr and gapdh was decided to be an appropriate reference gene to use with olive. brief overview of the cdnas obtained from the libraries genbank homologous records for each insert sequence obtained through blastn search at ncbi, revealed 11% - 16% protein coding gene homologs and 3% unknown cdnas while the remaining 84% to 89% constituted non-coding rna molecules including rrnas and trnas. all the cdna sequences had similarity to cdna records of other plants previously registered in the genbank databases while jnf jnf and jnf had no similarity for any records in any available database. in each library, more than half of the cdnas were new for olive genome. * cdnas that are new for olive. ** obtained from genbank for the sequences generated through this study. * cdnas that are new for olive. ** obtained from genbank for the sequences generated through this study. * cdnas that are new for olive. ** obtained from genbank for the sequences generated through this study. * cdnas that are new for olive. ** obtained from genbank for the sequences generated through this study. * cdnas that are new for olive. ** obtained from genbank for the sequences generated through this study. cdna contents and qrt-pcr validation of the individual libraries in july, “on year” leaves, 4% of the protein coding gene homologs was detected as p monooxygenases while homologs of menthone:neomenthol reductase a transcription factor and a protease were represented by 2%. the remaining five cdnas were represented with 1%. qrt-pcr confirmed the abundance of p monooxygenases mentioned in jf library, and revealed embryo defective binding / small gtpase regulator to be specific to “on year” leaves in july. in july “off year” leaves, the predominating cdnas were again homologs of p monooxygenase that was also confirmed by qrt-pcr. the second most abundant cdnas were homologs of ethylene responsive protein, wound stress protein, a gene complex of multiple constitutive proteins and intergenic spacers and ribosomal protein l. only in this library were detected cdnas that were completely novel to any available nucleotide database. qrt-pcr revealed wound stress protein, one of the abundant cdnas in jnf library, as a specific cdna to “off year” leaves in july. although the abundance of the cdnas detected in november, “on year” leaves were the least among all libraries generated, nf, nf and a cdna from jnf library appeared to express at very high levels. homologs of cryptochrome mrna, dnaj heat shock family protein, and a calmodulin binding protein were detected as 2% but their abundance was not confirmed by qrt-pcr. a cdna that is homologous to both ariadne and a zinc finger family protein was the most abundant cdna of the remaining two libraries. it was detected at a rate of 3% in each library. qrt-pcr analysis revealed this cdna’s expression as fold to fold more in f library than in any other library. the remaining cdnas in the nnf library were detected only once. the cold stress-induced protein / dehydrin homolog along with the other putative dehydrin and the putative metallothionein were the most abundant cdnas in both of the november libraries from the leaves. after ariadne - like protein homologs, the second most abundant cdnas in fruit flesh library were homologs of udp-glucose 4-epimerase, acyl-coa binding protein and triosephosphate isomerase. qrt-pcr revealed all the cdnas isolated from fruit flesh library were specific to fruits. a cdna that has a weak similarity to predicted nuclear-pore anchor protein was also fruit specific. bioinformatics among the olive ests analyzed, sequences displayed significant blastx matches within the genes registered in the ncbi database. in order to predict the reliability assessment of the ests or alignment quality, sequence similarity and e-value distribution graphs were generated from sequences based on the blastx results. the species distribution of olive ests based on blastx hits had the highest sequence homology to vitis vinifera, and followed by populus trichocarpa, arabidopsis thaliana and oryza sativa. the functional annotation and categorization of each olive est based on gene ontology terms were analyzed using the blast2go suite. for each transcript, a set of go term information including; accession, annotation term and basic definition is shown in table. in addition, these transcripts representing genes with known function were categorized by biological process, cellular component and molecular function according to the ontological definitions of the go terms. the transcripts in the biological process category were related to metabolic process, cellular process, response to stimulus, cellular localization, multicellular organismal process, developmental process, reproduction, multi - organism process, biological regulation, cell wall organization or biogenesis and cellular component biogenesis. in the cellular component category, most of the go terms were mainly related to cellular and organelle components such as cell periphery and intracellular organelle parts, followed by macromolecular complex, extracellular region and membrane enclosed lumen. as for the molecular function category, most abundant go terms were involved in binding such as nucleic acid and transition metal ion binding, catalytic activity, electron carrier activity, transporter and structural molecule activity as well as enzyme regulator activity. using kaas, each olive est was assigned with a kegg orthology number with the sbh assignment method and the numbers subsequently were mapped to one of the kegg's reference metabolic pathways. consequently, a total of main metabolic pathways were generated through the use of kaas pathway mapping and the sequences largely correlated with essential metabolisms of galactose metabolism, amino sugar and nucleotide sugar metabolism, photosynthesis, other glycan degradation, monoterpenoid biosynthesis and followed by spliceosome, ribosome and circadian rhythm - plant, e-value distribution, species distribution and top-hit species distribution graphs generated after olive sequences were processed. *see tables 1– for the accession numbers of these sequences. according the table, there is a correlation between the biosynthesis and ribosomal activity in fruited and non- fruited leaves discussion the approach to isolate differentially expressed genes to isolate differentially expressed cdnas, it is essential to start with total rna molecules extracted from tissues of identical conditions. with this respect, the trees we selected were genetically identical and grew virtually in the same micro-environment. the same constitutive cdna was detected in four of the five libraries and was confirmed with qrt-pcr while homologs of cdnas reported to express at certain stress conditions such as cold stress induced protein / dehydrin and temperature induced lipocalin or at certain metabolic processes such as acyl-coa binding protein and triosephosphate isomerase were detected at the expected libraries. at least cdnas from each library were confirmed by qrt-pcr to be specific to the tissues that they were initially detected through sequencing of the plasmid inserts from arbitrarily selected colonies. hence, the approach we used to detect differentially expressed cdnas in the libraries, has proven to be reasonable. furthermore, more than half of the cdnas of each library did not match olive records in nucleotide and est databases of ncbi which contains cdnas derived from leaves, fruits and flowers. overall cdna profile of the libraries in july, “on year” leaves and in “off year” leaves, homologs of cytochrome p monooxygenases appeared to be dominating cdnas. embryo defective binding / small gtpase protein homolog appeared to be strictly specific to jf. interestingly, jf was detected more in “on year” leaves and fruits but less in “off year” leaves suggesting it might have a role in “on year” but more in july than that of november. on the contrary, the putative metallothionein appeared to be associated with “on year” leaves but more in november. one of the two unknown cdnas isolated from july “off year” leaves were found to express in all the tissues studied except fruits while the other was specific to july leaves only. in november, “on year” leaves, a homolog of emb lyase was the most abundant cdna, yet it was detected in this library only. based on the ncbi record, this putative lyase is predicted to function in breaking of c-c, c-o and c-n bonds during embryo development. detecting this cdna in maturing fruit bearing leaves makes meaningful sense as for developing embryo, nutrients from the leaves should be supplied. most other cdnas of november “on year” leaves also appeared to be associated with cold stress and embryo development which were the specific conditions for nf library: nf is a homolog of dehydrin that has been reported to function in low temperatures and seed development. nf is similar to metallothionein and has been reported to function in senescence. likewise nf homolog jerf has been reported to involve in gene expression at cold, and nf homolog glycosyl hydrolase is associated with biotic / abiotic stress, lignification and cell wall reconstruction. the cdna that is homolog of both ariadne and a zinc finger family protein was the most abundant in both november, “off year” leaves and in fruit flesh libraries. qrt-pcr results revealed fold to fold more expression of this cdna in fruits than in other libraries but did not confirm as one of the most abundant cdnas in fruits nor in “off year” leaves. combined with ubiquitin association, these results suggest ariadne homolog in olive is most probably a constitutively expressed cdna. nnf / nnf and nnf are homologs of a splicing factor subunit and a translation initiation factor, respectively, and they both were detected at very low level in all libraries. given the fact that these two trees are genetically identical and grow virtually in the same micro-environment, overall results present cdnas differentially expressed in leaves, “on year” leaves, and in “off year” leaves. constitutively expressed genes, most of which have not been detected in olive before, and several unknown cdnas and / or genes are also reported. it should be kept in mind that alternate bearing is a result of complicated biotic and abiotic processes including environmental factors, physiological responses of the trees in the form of activation and repression of endogenous metabolic pathways, which in turn are also based on the genetic background of the tree. large phenotypic variation has been observed, including year-to-year variation of a single genotype, as well as variation among and within cultivars under the same environment. hence it is not possible to clearly enlighten the genetic players of alternate bearing in a single cdna screening or even a complete transcriptome analysis. multiple approaches involving several years follow up of the selected trees / cultivars are needed to identify certain or key genetic players of alternate bearing in olive. there are no comprehensive reports on the genetic basis of alternate bearing in olive, however, and hence these results constitute important information for one of the first steps of a genetic dissection of olive periodicity which causes significant economic loss for olive growers. through exploring these cdnas further, it is possible to isolate genes that are key regulator of fruit formation and / or periodicity in olive. bioinformatic analyses through the bioinformatic analyses it was possible to extract further additional information about the cdnas as well as about olive in general. blastx analysis revealed olive has a surprisingly high similarity to grapevine, although these two plants are not even the same order in systematics. the second most similar plant to olive is populus with a much lower similarity. this suggests the cdnas captured are directly or indirectly associated with the pathways of fruit formation and / or production that are in turn related to periodicity. go terms categorization grouped the cdnas into common processes, localizations and functions such as metabolic process, cellular localization and binding, respectively, which reflect a general profile of typical cell while differentially expressed cdnas were also significantly represented such as 17% of the cdnas in the “response to stimulus” category, and 25% of the cdnas in the “nucleic acid and transition metal ion binding, catalytic activity” category. the metabolic pathways generated through the use of kaas pathway mapping were largely correlated with essential metabolisms such as galactose, amino sugar, nucleotide sugar metabolisms and photosynthesis confirming the constitutive status of the majority of the cdnas obtained.  in summary, we have isolated and analyzed cdnas that are associated with alternate bearing in olive. a p monooxigenase homolog expressed more in the “on year” than that of “off year” leaves in july. two putative dehydrins expressed significantly more in “on year” leaves than that of “off year” leaves in november. homologs of triose phosphate isomerise, udp - glucose epimerase, acyl - coa binding protein, and a putative nuclear core anchor protein appeared fruit specific, while a homolog of an embryo binding protein / small gtpase regulator was detected in “on year” leaves only. an unknown cdna was specific to leaves in july. kegg pathway analyses of the sequences correlated with essential metabolisms such as galactose metabolism, amino sugar and nucleotide sugar metabolisms and photosynthesis. detailed analysis of the results presents candidate cdnas that can be used to dissect further the genetic basis of fruit production and / or alternate bearing. 
transcripts in the biological process were found to be related with metabolic process, cellular process, response to stimulus, localization process, multicellular organismal process, developmental process and reproduction. detailed analysis of the results presents candidate cdnas that can be used to dissect further the genetic basis of fruit production and / or alternate bearing which causes significant economical loss for olive growers. olive cdna libraries to isolate candidate genes that can help enlightening the molecular mechanism of periodicity and / or fruit production were constructed and analyzed. quantitative real-time rt-pcr analyses of the cdna libraries confirmed cdna molecules that are associated with different developmental stages and fruits. comparison of the detected cdnas to the current est database of genbank along with other non - redundant databases of ncbi revealed homologs of previously described genes along with several unknown cdnas. one of the two unknown cdnas was specific to leaves in july while the other was detected in all of the libraries except fruits. for this purpose, cdna libraries from the leaves of trees in “on year” and in “off year” in july and in november were constructed. kegg pathway analyses for the obtained sequences correlated with essential metabolisms such as galactose metabolism, amino sugar and nucleotide sugar metabolisms and photosynthesis. these independent transcripts were analyzed using blast searches against the kegg and genbank nucleotide databases and putative transcripts corresponding to known gene functions were annotated with gene names and gene ontology terms. homologs of udp – glucose epimerase, acyl - coa binding protein, triose phosphate isomerase and a putative nuclear core anchor protein were significant in fruits only, while a homolog of an embryo binding protein / small gtpase regulator was detected in “on year” leaves only. two putative dehydrins expressed significantly more in “on year” leaves than that of “off year” leaves in november. hence, a number of candidate cdnas associated with “on year” and “off year” were isolated.
 olive cdna libraries to isolate candidate genes that can help enlightening the molecular mechanism of periodicity and / or fruit production were constructed and analyzed. for this purpose, cdna libraries from the leaves of trees in “on year” and in “off year” in july and in november were constructed. randomly selected positive clones from each library were analyzed with respect to sequence and size. a fruit-flesh cdna library was also constructed and characterized to confirm the reliability of each library’s temporal and spatial properties.  quantitative real-time rt-pcr analyses of the cdna libraries confirmed cdna molecules that are associated with different developmental stages and fruits. hence, a number of candidate cdnas associated with “on year” and “off year” were isolated. comparison of the detected cdnas to the current est database of genbank along with other non - redundant databases of ncbi revealed homologs of previously described genes along with several unknown cdnas. of around screened cdnas, cdna elements were obtained after eliminating ribosomal rna sequences. these independent transcripts were analyzed using blast searches against the kegg and genbank nucleotide databases and putative transcripts corresponding to known gene functions were annotated with gene names and gene ontology terms. transcripts in the biological process were found to be related with metabolic process, cellular process, response to stimulus, localization process, multicellular organismal process, developmental process and reproduction.  a putative p monooxigenase expressed fivefold more in the “on year” than that of “off year” leaves in july. two putative dehydrins expressed significantly more in “on year” leaves than that of “off year” leaves in november. homologs of udp – glucose epimerase, acyl - coa binding protein, triose phosphate isomerase and a putative nuclear core anchor protein were significant in fruits only, while a homolog of an embryo binding protein / small gtpase regulator was detected in “on year” leaves only. one of the two unknown cdnas was specific to leaves in july while the other was detected in all of the libraries except fruits. kegg pathway analyses for the obtained sequences correlated with essential metabolisms such as galactose metabolism, amino sugar and nucleotide sugar metabolisms and photosynthesis. detailed analysis of the results presents candidate cdnas that can be used to dissect further the genetic basis of fruit production and / or alternate bearing which causes significant economical loss for olive growers. oliveolea europaea lperiodicity associated genesgene onthologykegg 