Genome-wide identification and characterization of exapted transposable elements in the large genome of sunflower (Helianthus annuus L.).

Transposable elements (TEs) are an important source of genome variability, playing many roles in the evolution of eukaryotic species. Besides well-known phenomena, TEs may undergo the exaptation process and generate the so-called exapted transposable element genes (ETEs). Here we present a genome-wide survey of ETEs in the large genome of sunflower (Helianthus annuus L.), in which the massive amount of TEs, provides a significant source for exaptation. A library of sunflower TEs was used to build TE-specific Hidden Markov Model profiles, to search for all available sunflower gene products. In doing so, 20 016 putative ETEs were identified and further investigated for the characteristics that distinguish TEs from genes, leading to the validation of 3530 ETEs. The analysis of ETEs transcription patterns under different stress conditions showed a differential regulation triggered by treatments mimicking biotic and abiotic stress; furthermore, the distribution of functional domains of differentially regulated ETEs revealed a relevant presence of domains involved in many aspects of cellular functions. A comparative genomic investigation was performed including species representative of Asterids and appropriate outgroups: the bulk of ETEs that resulted were specific to the sunflower, while few ETEs presented orthologues in the genome of all analyzed species, making the hypothesis of a conserved function. This study highlights the crucial role played by exaptation, actively contributing to species evolution.

Transcriptomic Analyses Reveal Insights into the Shared Regulatory Network of Phenolic Compounds and Steviol Glycosides in Stevia rebaudiana.

Stevia rebaudiana (Bertoni) is a highly valuable crop for the steviol glycoside content in its leaves, which are no-calorie sweeteners hundreds of times more potent than sucrose. The presence of health-promoting phenolic compounds, particularly flavonoids, in the leaf of S. rebaudiana adds further nutritional value to this crop. Although all these secondary metabolites are highly desirable in S. rebaudiana leaves, the genes regulating the biosynthesis of phenolic compounds and the shared gene network between the regulation of biosynthesis of steviol glycosides and phenolic compounds still need to be investigated in this species. To identify putative candidate genes involved in the synergistic regulation of steviol glycosides and phenolic compounds, four genotypes with different contents of these compounds were selected for a pairwise comparison RNA-seq analysis, yielding 1136 differentially expressed genes. Genes that highly correlate with both steviol glycosides and phenolic compound accumulation in the four genotypes of S. rebaudiana were identified using the weighted gene co-expression network analysis. The presence of UDP-glycosyltransferases 76G1, 76H1, 85C1, and 91A1, and several genes associated with the phenylpropanoid pathway, including peroxidase, caffeoyl-CoA O-methyltransferase, and malonyl-coenzyme A:anthocyanin 3-O-glucoside-6″-O-malonyltransferase, along with 21 transcription factors like SCL3, WRK11, and MYB111, implied an extensive and synergistic regulatory network involved in enhancing the production of such compounds in S. rebaudiana leaves. In conclusion, this work identified a variety of putative candidate genes involved in the biosynthesis and regulation of particular steviol glycosides and phenolic compounds that will be useful in gene editing strategies for increasing and steering the production of such compounds in S. rebaudiana as well as in other species.

Characterisation of LTR-Retrotransposons of Stevia rebaudiana and Their Use for the Analysis of Genetic Variability.

Stevia rebaudiana is one of the most important crops belonging to the Asteraceae family. Stevia is cultivated all over the world as it represents a valid natural alternative to artificial sweeteners thanks to its leaves, which produce steviol glycosides that have high sweetening power and reduced caloric value. In this work, the stevia genome sequence was used to isolate and characterise full-length long-terminal repeat retrotransposons (LTR-REs), which account for more than half of the genome. The Gypsy retrotransposons were twice as abundant as the Copia ones. A disproportionate abundance of elements belonging to the Chromovirus/Tekay lineage was observed among the Gypsy elements. Only the SIRE and Angela lineages represented significant portions of the genome among the Copia elements. The dynamics with which LTR-REs colonised the stevia genome were also estimated; all isolated full-length elements turned out to be relatively young, with a proliferation peak around 1-2 million years ago. However, a different analysis conducted by comparing sequences encoding retrotranscriptase showed the occurrence of an older period in which there was a lot of LTR-RE proliferation. Finally, a group of isolated full-length elements belonging to the lineage Angela was used to analyse the genetic variability in 25 accessions of S. rebaudiana using the Inter-Retrotransposon Amplified Polymorphism (IRAP) protocol. The obtained fingerprints highlighted a high degree of genetic variability and were used to study the genomic structures of the different accessions. It was hypothesised that there are four ancestral subpopulations at the root of the analysed accessions, which all turned out to be admixed. Overall, these data may be useful for genome sequence annotations and for evaluating genetic variability in this species, which may be useful in stevia breeding.

Epigenetic patterns within the haplotype phased fig (Ficus carica L.) genome.

Due to DNA heterozygosity and repeat content, assembly of non-model plant genomes is challenging. Herein, we report a high-quality genome reference of one of the oldest known domesticated species, fig (Ficus carica L.), using Pacific Biosciences single-molecule, real-time sequencing. The fig genome is ~333 Mbp in size, of which 80% has been anchored to 13 chromosomes. Genome-wide analysis of N6 -methyladenine and N4 -methylcytosine revealed high methylation levels in both genes and transposable elements, and a prevalence of methylated over non-methylated genes. Furthermore, the characterization of N6 -methyladenine sites led to the identification of ANHGA, a species-specific motif, which is prevalent for both genes and transposable elements. Finally, exploiting the contiguity of the 13 pseudomolecules, we identified 13 putative centromeric regions. The high-quality reference genome and the characterization of methylation profiles, provides an important resource for both fig breeding and for fundamental research into the relationship between epigenetic changes and phenotype, using fig as a model species.

LTR-retrotransposon dynamics in common fig (Ficus carica L.) genome.

BACKGROUND: Long Terminal Repeat retrotransposons (LTR-REs) are repetitive DNA sequences that constitute a large part of the genome. The improvement of sequencing technologies and sequence assembling strategies has achieved genome sequences with much greater reliability than those of the past, especially in relation to repetitive DNA sequences. RESULTS: In this study, we analysed the genome of Ficus carica L., obtained using third generation sequencing technologies and recently released, to characterise the complete complement of full-length LTR-REs to study their dynamics during fig genome evolution. A total of 1867 full-length elements were identified. Those belonging to the Gypsy superfamily were the most abundant; among these, the Chromovirus/Tekay lineage was the most represented. For the Copia superfamily, Ale was the most abundant lineage. Measuring the estimated insertion time of each element showed that, on average, Ivana and Chromovirus/Tekay were the youngest lineages of Copia and Gypsy superfamilies, respectively. Most elements were inactive in transcription, both constitutively and in leaves of plants exposed to an abiotic stress, except for some elements, mostly belonging to the Copia/Ale lineage. A relationship between the inactivity of an element and inactivity of genes lying in close proximity to it was established. CONCLUSIONS: The data reported in this study provide one of the first sets of information on the genomic dynamics related to LTR-REs in a plant species with highly reliable genome sequence. Fig LTR-REs are highly heterogeneous in abundance and estimated insertion time, and only a few elements are transcriptionally active. In general, the data suggested a direct relationship between estimated insertion time and abundance of an element and an inverse relationship between insertion time (or abundance) and transcription, at least for Copia LTR-REs.

Interspecific hybridisation and LTR-retrotransposon mobilisation-related structural variation in plants: A case study.

The dynamics of long-terminal-repeat retrotransposons in two poplar species (Populus deltoides and P. nigra) and in an interspecific hybrid, recently synthesized, were investigated by analyzing the genomic abundance and transcription levels of a collection of 828 full-length retroelements identified in the genome sequence of P. trichocarpa, all occurring also in the genomes of P. deltoides and P. nigra. Overall, genomic abundance and transcription levels of many retrotransposons in the hybrid resulted higher or lower than expected by calculating the mean of the parental values. A bioinformatics procedure was established to ascertain the occurrence of the same retrotransposon loci in the three genotypes. The results indicated that retrotransposon abundance variations between the hybrid and the mean value of the parents were due to i) co-segregation of retrotransposon high- or low-abundant haplotypes; ii) new retroelement insertions; iii) retrotransposon loss. Concerning retrotransposon expression, this was generally low, with only 14/828 elements over- or under-expressed in the hybrid than expected by calculating the mean of the parents. It is concluded that interspecific hybridisation between the two poplar species determine quantitative variation and differential expression of some retrotransposons, with possible consequences for the genetic differentiation of the hybrid.

A computational genome-wide analysis of long terminal repeats retrotransposon expression in sunflower roots (Helianthus annuus L.).

Long terminal repeats (LTR) retrotransposons have a major role in determining genome size, structure and function, thanks to their ability to transpose. We performed a meta-analysis of LTR-retrotransposon expression in roots of sunflower plantlets treated with different plant hormones, chemicals and NaCl. By using Illumina cDNA libraries, available from public repositories, we measured the number of reads matching the retrotranscriptase domains isolated from a whole genome library of retrotransposons. LTR-retrotransposons resulted in general barely expressed, except for 4 elements, all belonging to the AleII lineage, which showed high transcription levels in roots of both control and treated plants. The expression of retrotransposons in treated plants was slightly higher than in the control. Transcribed elements belonged to specific chromosomal loci and were not abundant in the genome. A few elements resulted differentially expressed depending on the treatment. Results suggest that, although most retrotransposons are not expressed, the transcription of such elements is related to their abundance, to their position in the chromosome and to their lineage.

Gene expression in Rhizoglomus irregulare at two different time points of mycorrhiza establishment in Helianthus annuus roots, as revealed by RNA-seq analysis.

Arbuscular mycorrhizal fungi (AMF) play a fundamental role in plant growth and nutrition in natural and agricultural ecosystems. Despite the importance of such symbionts, the different developmental changes occurring during the AMF life cycle have not been fully elucidated at the molecular level. Here, the RNA-seq approach was used to investigate Rhizoglomus irregulare specific and common transcripts at two different time points of mycorrhizal establishment in Helianthus annuus in vivo. Four days after inoculation, transcripts related to cellular remodeling (actin and tubulin), cellular signaling (calmodulin, serine/threonine protein kinase, 14-3-3 protein, and calcium transporting ATPase), lipid metabolism (fatty acid desaturation, steroid hormone, and glycerophospholipid biosynthesis), and biosynthetic processes were detected. In addition to such transcripts, 16 days after inoculation, expressed genes linked to binding and catalytic activities; ion (K+, Ca2+, Fe2+, Zn2+, Mn2+, Pi, ammonia), sugar, and lipid transport; and those involved in vacuolar polyphosphate accumulation were found. Knowledge of transcriptomic changes required for symbiosis establishment and performance is of great importance to understand the functional role of AMF symbionts in food crop nutrition and health, and in plant diversity in natural ecosystems.

On the Trail of Tetu1: Genome-Wide Discovery of CACTA Transposable Elements in Sunflower Genome.

Much has been said about sunflower (Helianthus annuus L.) retrotransposons, representing the majority of the sunflower's repetitive component. By contrast, class II transposons remained poorly described within this species, as they present low sequence conservation and are mostly lacking coding domains, making the identification and characterization of these transposable elements difficult. The transposable element Tetu1, is a non-autonomous CACTA-like element that has been detected in the coding region of a CYCLOIDEA (CYC) gene of a sunflower mutant, tubular ray flower (turf). Based on our knowledge of Tetu1, the publicly available genome of sunflower was fully scanned. A combination of bioinformatics analyses led to the discovery of 707 putative CACTA sequences: 84 elements with complete ends and 623 truncated elements. A detailed characterization of the identified elements allowed further classification into three subgroups of 347 elements on the base of their terminal repeat sequences. Only 39 encode a protein similar to known transposases (TPase), with 10 TPase sequences showing signals of activation. Finally, an analysis of the proximity of CACTA transposons to sunflower genes showed that the majority of CACTA elements are close to the nearest gene, whereas a relevant fraction resides within gene-encoding sequences, likely interfering with sunflower genome functionality and organization.

How Quercus ilex L. saplings face combined salt and ozone stress: a transcriptome analysis.

BACKGROUND: Similar to other urban trees, holm oaks (Quercus ilex L.) provide a physiological, ecological and social service in the urban environment, since they remove atmospheric pollution. However, the urban environment has several abiotic factors that negatively influence plant life, which are further exacerbated due to climate change, especially in the Mediterranean area. Among these abiotic factors, increased uptake of Na + and Cl - usually occurs in trees in the urban ecosystem; moreover, an excess of the tropospheric ozone concentration in Mediterranean cities further affects plant growth and survival. Here, we produced and annotated a de novo leaf transcriptome of Q. ilex as well as transcripts over- or under-expressed after a single episode of O3 (80 nl l-1, 5 h), a salt treatment (150 mM for 15 days) or a combination of these treatments, mimicking a situation that plants commonly face, especially in urban environments. RESULTS: Salinity dramatically changed the profile of expressed transcripts, while the short O3 pulse had less effect on the transcript profile. However, the short O3 pulse had a very strong effect in inducing over- or under-expression of some genes in plants coping with soil salinity. Many differentially regulated genes were related to stress sensing and signalling, cell wall remodelling, ROS sensing and scavenging, photosynthesis and to sugar and lipid metabolism. Most differentially expressed transcripts revealed here are in accordance with a previous report on Q. ilex at the physiological and biochemical levels, even though the expression profiles were overall more striking than those found at the biochemical and physiological levels. CONCLUSIONS: We produced for the first time a reference transcriptome for Q. ilex, and performed gene expression analysis for this species when subjected to salt, ozone and a combination of the two. The comparison of gene expression between the combined salt + ozone treatment and salt or ozone alone showed that even though many differentially expressed genes overlap all treatments, combined stress triggered a unique response in terms of gene expression modification. The obtained results represent a useful tool for studies aiming to investigate the effects of environmental stresses in urban-adapted tree species.