5 research outputs found
In silico identification and characterization of a diverse subset of conserved microRNAs in bioenergy crop Arundo donax L.
MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the post-transcriptional regulation of gene expression in plants. Arundo donax L. is a perennial C-3 grass considered one of the most promising bioenergy crops. Despite its relevance, many fundamental aspects of its biology still remain to be elucidated. In the present study we carried out the first in silico mining and tissue-specific characterization of microRNAs and their putative targets in A. donax. We identified a total of 141 miRNAs belonging to 14 families along with the corresponding primary miRNAs, precursor miRNAs and a total of 462 high-confidence predicted targets and novel target sites were validated by 5'-race. Gene Ontology functional annotation showed that miRNA targets are constituted mainly by transcription factors, but three of the newly validated targets are enzymes involved in novel functions like RNA editing, acyl lipid metabolism and post-Golgi trafficking. Folding variability of pre-miRNA loops and phylogenetic analyses indicate variable selective pressure acting on the different miRNA families. The set of miRNAs identified in this study will pave the road to further miRNA research in Arundo donax and contribute towards a better understanding of miRNA-mediated gene regulatory processes in other bioenergy crops.Peer reviewe
Phylogenomic proof of Recurrent Demipolyploidization and Evolutionary Stalling of the “Triploid Bridge” in Arundo (Poaceae)
Polyploidization is a frequent phenomenon in plants, which entails the increase from one generation to the next by multiples of the haploid number of chromosomes. While tetraploidization is arguably the most common and stable outcome of polyploidization, over evolutionary time triploids often constitute only a transient phase, or a “triploid bridge”, between diploid and tetraploid levels. In this study, we reconstructed in a robust phylogenomic and statistical framework the evolutionary history of polyploidization in Arundo, a small genus from the Poaceae family with promising biomass, bioenergy and phytoremediation species. Through the obtainment of 10 novel leaf transcriptomes for Arundo and outgroup species, our results prove that recurrent demiduplication has likely been a major driver of evolution in this species-poor genus. Molecular dating further demonstrates that the species originating by demiduplication stalled in the “triploid bridge” for evolutionary times in the order of millions of years without undergoing tetratploidization. Nevertheless, we found signatures of molecular evolution highlighting some of the processes that accompanied the genus radiation. Our results clarify the complex nature of Arundo evolution and are valuable for future gene functional validation as well as reverse and comparative genomics efforts in the Arundo genus and other Arundinoideae
Phylogenomic proof of Recurrent Demipolyploidization and Evolutionary Stalling of the “Triploid Bridge” in Arundo (Poaceae)
Polyploidization is a frequent phenomenon in plants, which entails the increase from one generation to the next by multiples of the haploid number of chromosomes. While tetraploidization is arguably the most common and stable outcome of polyploidization, over evolutionary time triploids often constitute only a transient phase, or a “triploid bridge”, between diploid and tetraploid levels. In this study, we reconstructed in a robust phylogenomic and statistical framework the evolutionary history of polyploidization in Arundo, a small genus from the Poaceae family with promising biomass, bioenergy and phytoremediation species. Through the obtainment of 10 novel leaf transcriptomes for Arundo and outgroup species, our results prove that recurrent demiduplication has likely been a major driver of evolution in this species-poor genus. Molecular dating further demonstrates that the species originating by demiduplication stalled in the “triploid bridge” for evolutionary times in the order of millions of years without undergoing tetratploidization. Nevertheless, we found signatures of molecular evolution highlighting some of the processes that accompanied the genus radiation. Our results clarify the complex nature of Arundo evolution and are valuable for future gene functional validation as well as reverse and comparative genomics efforts in the Arundo genus and other Arundinoideae
Utility of RNA-Seq and bioinformatics for gene discovery and evolutionary analyses in the Arundinoideae (Poaceae)
Perennial grass species have been used as renewable resource to produce biofuel, and especially the Arundinoideae, one subfamily of Poaceae perennial grasses has attracted great research attention. Arundo donax L. (also known as the giant reed) is a perennial C3 grass with fast growth. It is considered as the next generation most promising bioenergy and phytoremediation crop in the Mediterranea area. Despite its importance and value, many fundamental aspects of its biology and the precise phylogenetic relationships with respect to other species of the Arundo genus still remain to be fully elucidated.
In this PhD thesis I first applied, based on the reference transcriptome of Arundo donax, a computational step-by-step workflow for identifying a total of 141 miRNAs belonging to 14 families and a total of 462 high-confidence predicted targets in A. donax. Among the different miRNA families identified, MIR444 family was commonly expressed in four tissues (bud, culm, root and leaf) in A. donax, indicating it may be related to the high resistance to viruses of these species.
Secondly, a total of 235 miRNAs belonging to 37 miRNA families and a total of 175 high-confidence putative targets were identified by using computational approaches in de novo assembly of several Arundo species leaf transcriptomes. Conserved miRNAs tend to regulate homologous targets at conserved target sites in different species.
The in depth analysis of the leaf transcriptomes for all taxa of the Arundo genus and closely related outgroups yielded a total of 1,016,877 unigenes with average length ranging from 741 to 1065 bp. Phylogenomic reconstruction based on 150 one-to-one orthologous groups (OGs) showed that A. formosana was sister to the other members of the Arundo genus. The probabilistic models suggested that the ancestral haploid chromosome number of Arundo was thirty-six and revealed that demi-duplication was responsible for the evolutionary increase in chromosome numbers throughout the Arundo genus radiation. In addition, evolutionary analyses identified some genes under positive selection, suggesting their potential for future gene functional validation and improvement of the biomass species A. donax.
Lastly, in silico identification and comparative analysis of lignin and cellulose biosynthesis gene families across the Arundinoideae (Poaceae) was carried out. A total of 741 CesA/Csl protein sequences and 1118 lignin biosynthesis proteins were identified from the de novo assemblies of Arundinoideae leaf transcriptomes. Phylogenetic analysis of CesA/Csl proteins showed that CesA/Csl genes classified into 8 clades. CSLA and CSLC subfamily is an independent lineage to other CesA/Csl genes family, indicating that they probably originated from a separate duplication event. Lignin biosynthetic gene were highly divergent between eudicots and monocots, indicating that these genes might have experienced expansion after species differentiation. Further, these gene families divided into different groups based on reference species, namely rice, Arabidopsis and Amborella, indicating that diverse functions might exist in these gene family. The cellulose and lignin biosynthesis genes identified in this study will be helpful for establishing mutagenesis-based reverse genetics and functional genomics approaches in A.donax.
In summary, leveraging on the availability of the leaf transcriptomes for all taxa of the Arundo genus and closely related outgroups, and reference transcriptomes of Arundo donax, gene discovery and evolutionary analyses were carried out in this study. These results pave the road to further elucidate the biology and evolution of Arundo donax and other Arundo species. The dissection of the patterns of evolution in Arundo genus will support ongoing efforts to establish reverse genetics and functional genomics approaches in A. donax, thus contributing to provide promising candidate genes for the improvement of this biomass species.Le Arundinoideae, una sottofamiglia di erbe perenni di Poaceae, ha attirato grande attenzione come risorsa rinnovabile per la produzione di biocarburanti. Arundo donax L. (canna gigante) è un erba perenne di tipo C3 a crescita rapida. È considerata la specie più promettente per produzione di bioenergia e fitodepurazione nella zona mediterranea. Nonostante la sua importanza e valore, molti aspetti fondamentali della sua biologia e le precise relazioni filogenetiche rispetto ad altre specie del genere Arundo restano ancora da chiarire.
In questa tesi di dottorato ho dapprima applicato una pipeline computazionale per identificare un totale di 141 miRNA appartenenti a 14 famiglie e un totale di 462 geni target in A. donax. Tra le diverse famiglie di miRNA identificate, la famiglia MIR444 è comunemente espressa in quattro tessuti (gemma, fusto, radice e foglia) in A. donax, indicando che potrebbe essere correlata all'elevata resistenza ai virus di queste specie.
In secondo luogo, un totale di 235 miRNA appartenenti a 37 famiglie di miRNA e un totale di 175 target putativi sono stati identificati utilizzando approcci computazionali tramite l'assemblaggio de novo di diversi trascrittomi fogliari di specie del genere Arundo. I miRNA conservati tendono a regolare obiettivi omologhi presso siti bersaglio conservati in diverse specie.
L'analisi dei transcrittomi di foglia per tutti i taxa del genere Arundo e outgroup strettamente correlati ha prodotto un totale di 1.016.877 unigenes con una lunghezza media di 741-1065 bp. La ricostruzione filogenomica basata su 150 gruppi ortologhi uno-a-uno (OG) ha dimostrato che A. formosana è la specie sorella degli altri membri del genere Arundo. I modelli probabilistici suggeriscono che il numero aploide di cromosomi ancestrale di Arundo è di 36 e rivela che la semi-duplicazione è stata responsabile dell'aumento evolutivo dei numeri cromosomici in tutto il genere Arundo. Inoltre, le analisi evolutive hanno identificato alcuni geni soggetti a selezione positiva, suggerendo il loro potenziale per il miglioramento della specie da biomassa A. donax.
Infine, è stata effettuata l'identificazione in silico e l'analisi comparativa delle famiglie di geni di biosintesi della lignina e della cellulosa nelle Arundinoideae (Poaceae). Un totale di 741 sequenze di proteine CesA / Csl e 1118 proteine di biosintesi della lignina sono state identificate dagli assemblaggi de novo dei trascrittomi di foglie di Arundinoideae. L'analisi filogenetica delle proteine CesA / Csl ha dimostrato che i geni CesA / Csl sono classificati in 8 cladi. Le sottofamiglie CSLA e CSLC sono una linea evolutiva indipendente rispetto ad altre famiglie di geni CesA / Csl, indicando che probabilmente hanno avuto origine da un evento di duplicazione separato. I geni biosintetici della lignina sono altamente divergenti tra eudicotiledoni e monocotiledoni, indicando che questi geni potrebbero essere andati incontro ad espansione dopo la differenziazione delle specieInoltre, queste famiglie di geni si dividono in diversi gruppi basati sulle specie di riferimento, indicando che potrebbero esistere diverse funzioni in questa famiglia di geni. I geni identificati in questo studio saranno utili per stabilire approcci di genomica funzionale e di genetica inversa basati sulla mutagenesi in A.donax.
In sintesi, facendo leva sulla disponibilità dei trascrittomi fogliari per tutti i taxa del genere Arundo, in questo studio sono stati condotti sia la scoperta genica che l’analisi evolutiva in Arundinoidee. Questi risultati aprono la strada per chiarire ulteriormente la biologia e l'evoluzione di Arundo donax e di altre specie di Arundo. La dissezione dei modelli di evoluzione nel genere Arundo sosterrà gli sforzi in corso per metter a punto gli approcci di genetica inversa e genomica funzionale in A. donax, contribuendo così a fornire promettenti geni candidati per il miglioramento di questa specie da biomassa
Phylogenomic proof of Recurrent Demipolyploidization and Evolutionary Stalling of the “Triploid Bridge” in Arundo (Poaceae)
Polyploidization is a frequent phenomenon in plants, which entails the increase from one generation to the next by multiples of the haploid number of chromosomes. While tetraploidization is arguably the most common and stable outcome of polyploidization, over evolutionary time triploids often constitute only a transient phase, or a “triploid bridge”, between diploid and tetraploid levels. In this study, we reconstructed in a robust phylogenomic and statistical framework the evolutionary history of polyploidization in Arundo, a small genus from the Poaceae family with promising biomass, bioenergy and phytoremediation species. Through the obtainment of 10 novel leaf transcriptomes for Arundo and outgroup species, our results prove that recurrent demiduplication has likely been a major driver of evolution in this species-poor genus. Molecular dating further demonstrates that the species originating by demiduplication stalled in the “triploid bridge” for evolutionary times in the order of millions of years without undergoing tetratploidization. Nevertheless, we found signatures of molecular evolution highlighting some of the processes that accompanied the genus radiation. Our results clarify the complex nature of Arundo evolution and are valuable for future gene functional validation as well as reverse and comparative genomics efforts in the Arundo genus and other Arundinoideae