Evolutionary divergence of duplicated genomes in newly described allotetraploid cottons.

Allotetraploid cotton (Gossypium) species represents a model system for the study of plant polyploidy, molecular evolution, and domestication. Here, chromosome-scale genome sequences were obtained and assembled for two recently described wild species of tetraploid cotton, Gossypium ekmanianum [(AD)6, Ge] and Gossypium stephensii [(AD)7, Gs], and one early form of domesticated Gossypium hirsutum, race punctatum [(AD)1, Ghp]. Based on phylogenomic analysis, we provide a dated whole-genome level perspective for the evolution of the tetraploid Gossypium clade and resolved the evolutionary relationships of Gs, Ge, and domesticated G. hirsutum. We describe genomic structural variation that arose during Gossypium evolution and describe its correlates-including phenotypic differentiation, genetic isolation, and genetic convergence-that contributed to cotton biodiversity and cotton domestication. Presence/absence variation is prominent in causing cotton genomic structural variations. A presence/absence variation-derived gene encoding a phosphopeptide-binding protein is implicated in increasing fiber length during cotton domestication. The relatively unimproved Ghp offers the potential for gene discovery related to adaptation to environmental challenges. Expanded gene families enoyl-CoA δ isomerase 3 and RAP2-7 may have contributed to abiotic stress tolerance, possibly by targeting plant hormone-associated biochemical pathways. Our results generate a genomic context for a better understanding of cotton evolution and for agriculture.

Ultrasensitive and fast detection of SARS-CoV-2 using RT-LAMP without pH-dependent dye.

This study investigates the performance of reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the colorimetric detection of SARS-CoV-2 using fluorometric dye, namely, calcein. The detection limit (LoD) with the N-ID1 primer set resulted in superior performance, corresponding to ~ 2 copies/reaction or ~ 0.1 copies/µL of the RNA sample. The color development can be observed by the naked eye, using an ultraviolet (UV) transilluminator or a hand-UV light without the requirement of expensive devices. The average time-to-reaction (TTR) value was 26.2 min in high-copy number samples, while it was about 50 min in rRT-PCR. A mobile application was proposed to quantify the positive and negative results based on the three-color spaces (RGB, Lab, and HSB). Compared to rRT-PCR (n = 67), this assay allows fast and sensitive visual detection of SARS-CoV-2, with high sensitivity (90.9%), selectivity (100%), and accuracy (94.03%). Besides, the assay was sensitive regardless of variants. Since this assay uses a fluorescent dye for visual observation, it can be easily adapted in RT-LAMP assays with high sensitivity. Thus, it can be utilized in low-source centers and field testing such as conferences, sports meetings, refugee camps, companies, and schools.

High-quality chromosome-level genome assembly and multi-omics analysis of rosemary (Salvia rosmarinus) reveals new insights into the environmental and genome adaptation.

High-quality genome of rosemary (Salvia rosmarinus) represents a valuable resource and tool for understanding genome evolution and environmental adaptation as well as its genetic improvement. However, the existing rosemary genome did not provide insights into the relationship between antioxidant components and environmental adaptability. In this study, by employing Nanopore sequencing and Hi-C technologies, a total of 1.17 Gb (97.96%) genome sequences were mapped to 12 chromosomes with 46 121 protein-coding genes and 1265 non-coding RNA genes. Comparative genome analysis reveals that rosemary had a closely genetic relationship with Salvia splendens and Salvia miltiorrhiza, and it diverged from them approximately 33.7 million years ago (MYA), and one whole-genome duplication occurred around 28.3 MYA in rosemary genome. Among all identified rosemary genes, 1918 gene families were expanded, 35 of which are involved in the biosynthesis of antioxidant components. These expanded gene families enhance the ability of rosemary adaptation to adverse environments. Multi-omics (integrated transcriptome and metabolome) analysis showed the tissue-specific distribution of antioxidant components related to environmental adaptation. During the drought, heat and salt stress treatments, 36 genes in the biosynthesis pathways of carnosic acid, rosmarinic acid and flavonoids were up-regulated, illustrating the important role of these antioxidant components in responding to abiotic stresses by adjusting ROS homeostasis. Moreover, cooperating with the photosynthesis, substance and energy metabolism, protein and ion balance, the collaborative system maintained cell stability and improved the ability of rosemary against harsh environment. This study provides a genomic data platform for gene discovery and precision breeding in rosemary. Our results also provide new insights into the adaptive evolution of rosemary and the contribution of antioxidant components in resistance to harsh environments.

Genome-wide exploration of oil biosynthesis genes in cultivated olive tree varieties (Olea europaea): insights into regulation of oil biosynthesis.

Genome-wide oil biosynthesis was explored by de novo sequencing two cultivated olive tree (Olea europaea) varieties (cv. Ayvalik and Picual). This is the first report of the former variety sequencing. As outgroups, raw reads of cv. Leccino and scaffold-level assembly of cv. Farga were also retrieved. Each of these four cultivars was chromosome-scale assembled into 23 pseudochromosomes, with 1.31 Gbp (Farga), 0.93 Gbp (Ayvalik), 0.7 Gbp (Picual), and 0.54 Gbp (Leccino) in size. Ab initio gene finding was performed on these assemblies, using wild olive tree (oleaster)-trained parameters. High numbers of gene models were predicted and anchored to the pseudochromosomes: 69,028 (Ayvalik), 55,073 (Picual), 63,785 (Farga), and 40,449 (Leccino). Using previously reported oil biosynthesis genes from wild olive tree genome project, the following homologous sequences were identified: 1,355 (Ayvalik), 1,269 (Farga), 812 (Leccino), and 774 (Picual). Of these, 358 sequences were commonly shared by all cultivars. Besides, some sequences were cultivar unique: Ayvalik (126), Farga (118), Leccino (46), and Picual (52). These putative sequences were assigned to various GO terms, ranging from lipid metabolism to stress tolerance, from signal transactions to development, and to many others, implicating that oil biosynthesis is synergistically regulated with involvement of various other pathways.

Global gene expression profiling in congenital diaphragmatic hernia (CDH) patients.

Congenital diaphragmatic hernia (CDH) is an anomaly characterized by a defect in the diaphragm, leading to the passage of intra-abdominal organs into the thoracic cavity. Herein, the presented work analyzes the global gene expression profiles in nine CDH and one healthy newborn. All of the patients had left posterolateral (Bochdalek) diaphragmatic hernia, operated via an abdominal approach, and stomach and bowels in the thorax cavity. Some patients also had additional anomalies. A total of 560 differentially regulated genes were measured. Among them, 11 genes showed significant changes in expression associated with lung tissue, vascular structure development, and vitamin A metabolism, which are typical ontologies related to CDH etiology. Among them, SLC25A24 and RAB3IL1 are involved in angiogenesis, HIF1A and FOXC2-AS1 are related with the alveolus, MAGI2-AS3 is associated with the diaphragm, LHX4 and DHH are linked with the lung, and BRINP1, FZD9, WNT4, and BLOC1S1-RDH5 are involved in retinol. Besides, the expression levels of some previously claimed genes with CDH etiology also showed diverse expression patterns in different patients. All these indicated that CDH is a complex, multigenic anomaly, requiring holistic approaches for its elucidation.

Colorimetric and fluorometric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for diagnosis of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since it infected humans almost 3 years ago. Improvements of current assays and the development of new rapid tests or to diagnose SARS-CoV-2 are urgent. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and propitious assay, allowing to detect both colorimetric and/or fluorometric nucleic acid amplifications. This study describes the analytical and clinical evaluation of RT-LAMP assay for detection of SARS-CoV-2, by designing LAMP primers targeting N (nucleocapsid phosphoprotein), RdRp (polyprotein), S (surface glycoprotein), and E (envelope protein) genes. The assay's performance was compared with the gold standard RT-PCR, yielding 94.6% sensitivity and 92.9% specificity. Among the tested primer sets, the ones for S and N genes had the highest analytical sensitivity, showing results in about 20 min. The colorimetric and fluorometric comparisons revealed that the latter is faster than the former. The limit of detection (LoD) of RT-LAMP reaction in both assays is 50 copies/µl of the reaction mixture. However, the simple eye-observation advantage of the colorimetric assay (with a color change from yellow to red) serves a promising on-site point-of-care testing method anywhere, including, for instance, laboratory and in-house applications.

Comparative transcriptome analysis of resistant and cultivated tomato lines in response to Clavibacter michiganensis subsp. michiganensis.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive bacterium causing destructive bacterial wilt and canker disease in tomato. Herein, a comparative transcriptome analysis was performed on Cmm-resistant and -susceptible tomato lines. Tomato seedlings were inoculated with Cmm and harvested for transcriptome analysis after 4 and 8 day time-points. Twenty-four transcriptome libraries were profiled by RNA sequencing approach. Total of 545 million clean reads was generated. 1642 and 2715 differentially expressed genes (DEG) were identified in susceptible lines within 4 and 8 days after inoculation (DAI), respectively. In resistant lines, 1731 and 1281 DEGs were found following 4 and 8 DAI, respectively. Gene Ontology analysis resulted in a higher number of genes involved in biological processes and molecular functions in susceptible lines. On the other hand, such biological processes, "defense response", and "response to stress" were distinctly indicated in resistant lines which were not found in susceptible ones upon inoculation, according to the gene set enrichment analyses. Upon Cmm-inoculation, several defense responsive genes were found to be differentially expressed. Of which 26 genes were in the resistant line and three were in the susceptible line. This study helps to understand the transcriptome response of Cmm-resistant and -susceptible tomato lines. The results provide comprehensive data for molecular breeding studies, for the purpose to control of the pathogen in tomato.

Barley long non-coding RNAs (lncRNA) responsive to excess boron.

Long non-coding RNA (lncRNA) has a misleading name, since although they do not encode proteins, they may encode small peptides. Such transcripts are emerging as regulatory molecules. With the advent of next-generation sequencing technologies and novel bioinformatics tools, a tremendous amount of lncRNAs have been identified in several plant species. Recent reports demonstrated roles of plant lncRNAs such as development and environmental response. Here, we reported a genome-wide discovery of ~8000 barley lncRNAs and measured their expression pattern upon excessive boron (B) treatment. According to the tissue-based comparison, leaves have a greater number of B-responsive differentially expressed lncRNAs than the root. Functional annotation of the coding transcripts, which were co-expressed with lncRNAs, revealed that molecular function of the ion transport, establishment of localization, and response to stimulus significantly enriched only in the leaf. On the other hand, 32 barley endogenous target mimics (eTM) as lncRNAs, which potentially decoy the transcriptional suppression activity of 18 miRNAs, were obtained. Also, six lncRNAs, differentially expressed upon B-treatment, were selected and quantitatively analyzed in both B-sensitive and B-tolerant cultivars treated by excess B-level. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed the B-responsive expressional changes obtained by RNA sequencing. Notably, some lncRNAs (i.e., TCONS_00045190 and TCONS_00056415) over-expressed only in B-tolerant cultivar upon excess B treatment. Presented data including identification, expression measurement, and functional characterization of barley lncRNAs suggest that B-stress response might also be regulated by lncRNA expression, via cooperative interaction of miRNA-eTM-coding target transcript modules.

Genome of wild olive and the evolution of oil biosynthesis.

Here we present the genome sequence and annotation of the wild olive tree (Olea europaea var. sylvestris), called oleaster, which is considered an ancestor of cultivated olive trees. More than 50,000 protein-coding genes were predicted, a majority of which could be anchored to 23 pseudochromosomes obtained through a newly constructed genetic map. The oleaster genome contains signatures of two Oleaceae lineage-specific paleopolyploidy events, dated at ∼28 and ∼59 Mya. These events contributed to the expansion and neofunctionalization of genes and gene families that play important roles in oil biosynthesis. The functional divergence of oil biosynthesis pathway genes, such as FAD2, SACPD, EAR, and ACPTE, following duplication, has been responsible for the differential accumulation of oleic and linoleic acids produced in olive compared with sesame, a closely related oil crop. Duplicated oleaster FAD2 genes are regulated by an siRNA derived from a transposable element-rich region, leading to suppressed levels of FAD2 gene expression. Additionally, neofunctionalization of members of the SACPD gene family has led to increased expression of SACPD2, 3, 5, and 7, consequently resulting in an increased desaturation of steric acid. Taken together, decreased FAD2 expression and increased SACPD expression likely explain the accumulation of exceptionally high levels of oleic acid in olive. The oleaster genome thus provides important insights into the evolution of oil biosynthesis and will be a valuable resource for oil crop genomics.

Correction to: Two novel methylesterases from Olea europaea contribute to the catabolism of oleoside-type secoiridoid esters.

Page 5, paragraph 3, line 14, GenBank Accession Number which should read MK234850 instead of MK23485.

Two novel methylesterases from Olea europaea contribute to the catabolism of oleoside-type secoiridoid esters.

MAIN CONCLUSION: Two newly identified phytohormone cleaving esterases from Olea europaea are responsible for the glucosidase-initiated activation of the specialized metabolites ligstroside and oleuropein. Biosynthetic routes leading to the formation of plant natural products are tightly orchestrated enzymatic sequences usually involving numerous specialized catalysts. After their accumulation in plant cells and tissues, otherwise non-reactive compounds can be enzymatically activated, e.g., in response to environmental threats, like pathogen attack. In olive (Olea europaea), secoiridoid-derived phenolics, such as oleuropein or ligstroside, can be converted by glucosidases and as yet unidentified esterases to oleoside aldehydes. These are not only involved in pathogen defense, but also bear considerable promise as pharmaceuticals or neutraceuticals. Making use of the available olive genomic data, we have identified four novel methylesterases that showed significant homology to the polyneuridine aldehyde esterase (PNAE) from Rauvolfia serpentina, an enzyme acting on a distantly related metabolite group (monoterpenoid indole alkaloids, MIAs) also featuring a secoiridoid structural component. The four olive enzymes belong to the α/ß-hydrolase fold family and showed variable in vitro activity against methyl esters of selected plant hormones, namely jasmonic acid (MeJA), indole acetic acid (MeIAA), as well as salicylic acid (MeSA). None of the identified catalysts were directly active against the olive metabolites oleuropein, ligstroside, or oleoside 11-methyl ester. When employed in a sequential reaction with an appropriate glucosidase, however, two were capable of hydrolyzing these specialized compounds yielding reactive dialdehydes. This suggests that the esterases play a pivotal role in the activation of the olive secoiridoid polyphenols. Finally, we show that several of the investigated methylesterases exhibit a concomitant in vitro transesterification capacity-a novel feature, yielding ethyl esters of jasmonic acid (JA) or indole-3-acetic acid (IAA).

CRISPR/Cas9: An RNA-guided highly precise synthetic tool for plant genome editing.

CRISPR/Cas9 is a newly developed and naturally occurred genome editing tool, which is originally used by bacteria for immune defence. In the past years, it has been quickly employed and modified to precisely edit genome sequences in both plants and animals. Compared with the well-developed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 has lots of advantages, including easier to design and implement, higher targeting efficiency, and less expensive. Thus, it is becoming one of the most powerful tools for knockout of an individual gene as well as insertion of one gene and/or control of gene transcription. Studies have shown that CRISPR/Cas9 is a great tool to edit many genes in a variety of plant species, including the model plant species as well as agriculturally important crops, such as cotton, maize, wheat, and rice. CRISPR/Cas9-based genome editing can be used for plant functional studies and plant improvement to yield, quality, and tolerance to environmental stress.

miRNA-based drought regulation in wheat.

MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. Drought is a common environmental stress influencing crop growth and development. To date, it has been reported that a number of plant miRNA are involved in drought stress response. In this study, we comparatively investigated drought stress-responsive miRNAs in the root and leaf of bread wheat (Triticum aestivum cv. Sivas 111/33) by miRNA microarray screening. miRNA microarray analysis showed that 285 miRNAs (207 upregulated and 78 downregulated) and 244 miRNAs (115 upregulated and 129 downregulated) were differentially expressed in leaf and root tissues, respectively. Among the differentially expressed miRNAs, 23 miRNAs were only expressed in the leaf and 26 miRNAs were only expressed in the root of wheat growth under drought stress. Upon drought treatment, expression of miR159, miR160, miR166, miR169, miR172, miR395, miR396, miR408, miR472, miR477, miR482, miR1858, miR2118, and miR5049 were found to be significantly differentiated in bread wheat. The regulatory network analysis showed that miR395 has connections with a number of target transcripts, and miR159 and miR319 share a number of target genes. Drought-tolerant and drought-sensitive wheat cultivars showed altered expression pattern upon drought stress in terms of investigated miRNA and their target transcript expression level.

Genome-wide characterization and expression analysis of common bean bHLH transcription factors in response to excess salt concentration.

Members of basic helix-loop-helix (bHLH) gene family found in all eukaryotes play crucial roles in response to stress. Though, most eukaryotes carry the proteins of this family, biological functions of the most bHLH family members are not deeply evaluated in plants. In this study, we conducted a comprehensive genome-wide analysis of bHLH transcription factors in salt tolerant common bean. We identified 155 bHLH protein-encoding genes (PvbHLH) by using in silico comparative genomics tools. Based on the phylogenetic tree, PvbHLH genes were classified into 8 main groups with 21 subfamilies. Exon-intron analysis indicated that proteins belonging to same main groups exhibited a closely related gene structure. While, the PvbHLH gene family has been mainly expanded through segmental duplications, a total of 11 tandem duplication were detected. Genome-wide expression analysis of bHLH genes showed that 63 PvbHLH genes were differentially expressed in at least one tissue. Three of them displayed higher expression values in both leaf and root tissues. The in silico micro-RNA target transcript analyses revealed that totally 100 PvHLH genes targeted by 86 plant miRNAs. The most abundant transcripts, which were targeted by all 18 plant miRNA, were belonging to PvHLH-22 and PvHLH-44 genes. The expression of 16 PvbHLH genes in the root and leaf tissues of salt-stressed common bean was evaluated using qRT-PCR. Among them, two of PvbHLHs, PvbHLH-54, PvbHLH-148, were found to be up-regulated in both tissues in correlation with RNA-seq measurements. The results of this study could help improve understanding of biological functions of common bean bHLH family under salt stress. Additionally, it may provide basic resources for analyzing bHLH protein function for improving economic, agronomic and ecological benefit in common bean and other species.

Isolation, molecular characterization and functional analysis of OeMT2, an olive metallothionein with a bioremediation potential.

Metallothioneins are essential in plants for metal detoxification in addition to their other roles in plant life cycle. This study reports the characterization of an olive (Olea europaea L. cv. Ayvalik) metallothionein with respect to molecular and functional properties. A cDNA encoding a type 2 metallothionein from olive was isolated from a leaf cDNA library, characterized and named OeMT2 after its molecular and functional properties. OeMT2 was expressed in Escherichia coli, and a single protein band was confirmed by protein gel blot analysis. Metal tolerance ability of bacterial cells expressing OeMT2 was determined against 0.2 mM CdCl2, 0.4 mM CdCl2 and 1 mM CuSO4 in the growth medium. Metal ion contents of bacterial cells expressing OeMT2 were measured by ICP. Metal tolerance assays and ICP measurements suggested that OeMT2 effectively binds Cu and Cd. Molecular analysis of OeMT2 revealed two introns, three exons, a short 3' UTR and a long 5' UTR. Comparing the genomic sequences from 14 olive cultivars revealed OeMT2 had both intron and exon polymorphisms dividing the cultivars into three groups. Real-time PCR analysis demonstrated that OeMT2 expresses more or less the same amounts in all tissues of the olive tree examined. The genomic copy number of OeMT2 was also determined employing real-time PCR which suggested a single copy gene in the olive genome while three other MT2 members were determined from the draft olive genome sequences of Ayvalik cultivar and that of wild olive. This is the first report on molecular and functional characterization of an olive metallothionein and shows that OeMT2 expressed in E. coli has the capability of effectively binding toxic heavy metals. This may suggest that OeMT2 plays an important role in metal homeostasis in addition to a good potential for environmental and industrial usage.

Regulation of the alkaloid biosynthesis by miRNA in opium poppy.

Opium poppy (Papaver somniferum) is an important medicinal plant producing benzylisoquinoline alkaloids (BIA). MicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) of approximately 21 nucleotides. They are noncoding, but regulate gene expression in eukaryotes. Although many studies have been conducted on the identification and functions of plant miRNA, scarce researches on miRNA regulation of alkaloid biosynthesis have been reported. In this study, a total of 316 conserved and 11 novel miRNAs were identified in opium poppy using second-generation sequencing and direct cloning. Tissue-specific regulation of miRNA expression was comparatively analysed by miRNA microarray assays. A total of 232 miRNAs were found to be differentially expressed among four tissues. Likewise, 1469 target transcripts were detected using in silico and experimental approaches. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates carbohydrate metabolism and genetic-information processing. Additionally, miRNA target transcripts were mostly involved in response to stress against various factors and secondary-metabolite biosynthesis processes. Target transcript identification analyses revealed that some of the miRNAs might be involved in BIA biosynthesis, such as pso-miR13, pso-miR2161 and pso-miR408. Additionally, three putatively mature miRNA sequences were predicted to be targeting BIA-biosynthesis genes.

Transcriptome-wide identification of bread wheat WRKY transcription factors in response to drought stress.

The WRKY superfamily of transcription factors was shown to be involved in biotic and abiotic stress responses in plants such as wheat (Triticum aestivum L.), one of the major crops largely cultivated and consumed all over the world. Drought is an important abiotic stress resulting in a considerable amount of loss in agronomical yield. Therefore, identification of drought responsive WRKY members in wheat has a profound significance. Here, a total of 160 TaWRKY proteins were characterized according to sequence similarity, motif varieties, and their phylogenetic relationships. The conserved sequences of the TaWRKYs were aligned and classified into three main groups and five subgroups. A novel motif in wheat, WRKYGQR, was identified. To putatively determine the drought responsive TaWRKY members, publicly available RNA-Seq data were analyzed for the first time in this study. Through in silico searches, 35 transcripts were detected having an identity to ten known TaWRKY genes. Furthermore, relative expression levels of TaWRKY16/TaWRKY16-A, TaWRKY17, TaWRKY19-C, TaWRKY24, TaWRKY59, TaWRKY61, and TaWRKY82 were measured in root and leaf tissues of drought-tolerant Sivas 111/33 and susceptible Atay 85 cultivars. All of the quantified TaWRKY transcripts were found to be up-regulated in root tissue of Sivas 111/33. Differential expression of TaWRKY16, TaWRKY24, TaWRKY59, TaWRKY61 and TaWRKY82 genes was discovered for the first time upon drought stress in wheat. These comprehensive analyses bestow a better understanding about the WRKY TFs in bread wheat under water deficit, and increased number of drought responsive WRKYs would contribute to the molecular breeding of tolerant wheat cultivars.

Genome-wide fungal stress responsive miRNA expression in wheat.

MicroRNAs (miRNAs) are small non-coding class of RNAs. They were identified in many plants with their diverse regulatory roles in several cellular and metabolic processes. A number of miRNAs were involved in biotic and abiotic stress responses. Here, fungal stress responsive wheat miRNAs were analyzed by using miRNA-microarray strategy. Two different fungi (Fusarium culmorum and Bipolaris sorokiniana) were inoculated on resistant and sensitive wheat cultivars. A total of 87 differentially regulated miRNAs were detected in the 8 × 15 K array including all of the available plant miRNAs. Using bioinformatics tools, the target transcripts of responsive miRNAs were predicted, and related biological processes and mechanisms were assessed. A number of the miRNAs such as miR2592s, miR869.1, miR169b were highly differentially regulated showing more than 200-fold change upon fungal-inoculation. Some of the miRNAs were identified as fungal-inoculation responsive for the first time. The analyses showed that some of the differentially regulated miRNAs targeted resistance-related genes such as LRR, glucuronosyl transferase, peroxidase and Pto kinase. The comparison of the two miRNA-microarray analyses indicated that fungal-responsive wheat miRNAs were differentially regulated in pathogen- and cultivar-specific manners.

Isolation and expression analysis of cDNAs that are associated with alternate bearing in Olea europaea L. cv. Ayvalik.

BACKGROUND: 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 (when fruits start to appear) and in November (harvest time) were constructed. Randomly selected 100 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. RESULTS: Quantitative real-time RT-PCR (qRT-PCR) analyses of the cDNA libraries confirmed cDNA molecules that are associated with different developmental stages (e. g. "on year" leaves in July, "off year" leaves in July, leaves in November) 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 500 screened cDNAs, 48 cDNA elements were obtained after eliminating ribosomal RNA sequences. These independent transcripts were analyzed using BLAST searches (cutoff E-value of 1.0E-5) against the KEGG and GenBank nucleotide databases and 37 putative transcripts corresponding to known gene functions were annotated with gene names and Gene Ontology (GO) terms. Transcripts in the biological process were found to be related with metabolic process (27%), cellular process (23%), response to stimulus (17%), localization process (8.5%), multicellular organismal process (6.25%), developmental process (6.25%) and reproduction (4.2%). CONCLUSIONS: A putative P450 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.