Tommaso Giordani

Genetic variability in sunflower (Helianthus annuus L.) and in the Helianthus genus as assessed by retrotransposon-based molecular markers

The inter-retrotransposon amplified polymorphism (IRAP) protocol was applied for the first time within the genus Helianthus to assess intraspecific variability based on retrotransposon sequences among 36 wild accessions and 26 cultivars of Helianthus annuus L., and interspecific variability among 39 species of Helianthus. Two groups of LTRs, one belonging to a Copia-like retroelement and the other to a putative retrotransposon of unknown nature (SURE) have been isolated, sequenced and primers were designed to obtain IRAP fingerprints. The number of polymorphic bands in H. annuus wild accessions is as high as in Helianthus species. If we assume that a polymorphic band can be related to a retrotransposon insertion, this result suggests that retrotransposon activity continued after Helianthus speciation. Calculation of similarity indices from binary matrices (Shannon’s and Jaccard’s indices) show that variability is reduced among domesticated H. annuus. On the contrary, similarity indices among Helianthus species were as large as those observed among wild H. annuus accessions, probably related to their scattered geographic distribution. Principal component analysis of IRAP fingerprints allows the distinction between perennial and annual Helianthus species especially when the SURE element is concerned.

Expression of a dehydrin gene during embryo development and drought stress in ABA-deficient mutants of sunflower (Helianthus annuus L.)

The synthesis of a particular class of proteins, the dehydrins, is a common response to drought in plants. Dehydrins are known to be synthesized by the cell in response to abscisic acid, which represents a link between environment and nuclear activity, though dehydrin genes may be expressed even constitutively. We have investigated the relationship between abscisic acid (ABA) and accumulation of a dehydrin mRNA in sunflower, in which a dehydrin cDNA (HaDhn1a) was isolated. In particular, we studied changes in the steady-state level of dehydrin transcripts in two mutants for ABA synthesis and accumulation: nd-1 (an albino, non-dormant and lethal mutant with a very low ABA content and no ABA accumulation in response to stress) and w-1 (a wilty mutant, with reduced ABA accumulation) during embryo and plantlet development and drought stress. Differences between genotypes were observed through embryogenesis: w-1 shows a lower content of dehydrin transcripts in the early stages compared to control plants, indicating that ABA affects dehydrin mRNA accumulation; however, dehydrin transcripts level appears independent of ABA content in late embryogenesis. Also during drought stress in w-1 adult leaves, ABA is not quantitatively related to the steady-state level of the HaDhn1a transcripts. Finally, data on nd-1 mutant show a high level of dehydrin transcripts after drought stress in plantlet cotyledons and leaflets. These results indicate the existence of two regulation pathways of HaDhn1a transcripts accumulation, an ABA-dependent and an ABA-independent one, which may have cumulative effects.

The repetitive component of the sunflower genome as shown by different procedures for assembling next generation sequencing reads

BackgroundNext generation sequencing provides a powerful tool to study genome structure in species whose genomes are far from being completely sequenced. In this work we describe and compare different computational approaches to evaluate the repetitive component of the genome of sunflower, by using medium/low coverage Illumina or 454 libraries.ResultsBy varying sequencing technology (Illumina or 454), coverage (0.55 x-1.25 x), assemblers and assembly procedures, six different genomic databases were produced. The annotation of these databases showed that they were composed of different proportions of repetitive DNA families. The final assembly of the sequences belonging to the six databases produced a whole genome set of 283,800 contigs. The redundancy of each contig was estimated by mapping the whole genome set with a large Illumina read set and measuring the number of matched Illumina reads. The repetitive component amounted to 81% of the sunflower genome, that is composed mainly of numerous families of Gypsy and Copia retrotransposons. Also many families of non autonomous retrotransposons and DNA transposons (especially of the Helitron superfamily) were identified.ConclusionsThe results substantially matched those previously obtained by using a Sanger-sequenced shotgun library and a standard 454 whole-genome-shotgun approach, indicating the reliability of the proposed procedures also for other species. The repetitive sequences were collected to produce a database, SUNREP, that will be useful for the annotation of the sunflower genome sequence and for studying the genome evolution in dicotyledons.

Retrotransposon-based molecular markers for grapevine species and cultivars identification.

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.

Copia and Gypsy retrotransposons activity in sunflower (Helianthus annuus L.)

BackgroundRetrotransposons are heterogeneous sequences, widespread in eukaryotic genomes, which refer to the so-called mobile DNA. They resemble retroviruses, both in their structure and for their ability to transpose within the host genome, of which they make up a considerable portion. Copia- and Gypsy-like retrotransposons are the two main classes of retroelements shown to be ubiquitous in plant genomes. Ideally, the retrotransposons life cycle results in the synthesis of a messenger RNA and then self-encoded proteins to process retrotransposon mRNA in double stranded extra-chromosomal cDNA copies which may integrate in new chromosomal locations.ResultsThe RT-PCR and IRAP protocol were applied to detect the presence of Copia and Gypsy retrotransposon transcripts and of new events of integration in unstressed plants of a sunflower (Helianthus annuus L.) selfed line. Results show that in sunflower retrotransposons transcription occurs in all analyzed organs (embryos, leaves, roots, and flowers). In one out of sixty-four individuals analyzed, retrotransposons transcription resulted in the integration of a new element into the genome.ConclusionThese results indicate that the retrotransposon life cycle is firmly controlled at a post transcriptional level. A possible silencing mechanism is discussed.

The Peculiar Landscape of Repetitive Sequences in the Olive (Olea europaea L.) Genome

Analyzing genome structure in different species allows to gain an insight into the evolution of plant genome size. Olive (Olea europaea L.) has a medium-sized haploid genome of 1.4 Gb, whose structure is largely uncharacterized, despite the growing importance of this tree as oil crop. Next-generation sequencing technologies and different computational procedures have been used to study the composition of the olive genome and its repetitive fraction. A total of 2.03 and 2.3 genome equivalents of Illumina and 454 reads from genomic DNA, respectively, were assembled following different procedures, which produced more than 200,000 differently redundant contigs, with mean length higher than 1,000 nt. Mapping Illumina reads onto the assembled sequences was used to estimate their redundancy. The genome data set was subdivided into highly and medium redundant and nonredundant contigs. By combining identification and mapping of repeated sequences, it was established that tandem repeats represent a very large portion of the olive genome (∼31% of the whole genome), consisting of six main families of different length, two of which were first discovered in these experiments. The other large redundant class in the olive genome is represented by transposable elements (especially long terminal repeat-retrotransposons). On the whole, the results of our analyses show the peculiar landscape of the olive genome, related to the massive amplification of tandem repeats, more than that reported for any other sequenced plant genome.

A computational study of the dynamics of LTR retrotransposons in the Populus trichocarpa genome

Retrotransposons are an ubiquitous component of plant genomes, especially abundant in species with large genomes. Populus trichocarpa has a relatively small genome, which was entirely sequenced; however, studies focused on poplar retrotransposons dynamics are rare. With the aim to study the retrotransposon component of the poplar genome, we have scanned the complete genome sequence searching full-length long-terminal repeat (LTR) retrotransposons, i.e., characterised by two long terminal repeats at the 5′ and 3′ ends. A computational approach based on detection of conserved structural features, on building multiple alignments, and on similarity searches was used to identify 1,479 putative full-length LTR retrotransposons. Ty1-copia elements were more numerous than Ty3-gypsy. However, many LTR retroelements were not assigned to any superfamily because lacking of diagnostic features and non-autonomous. LTR retrotransposon remnants were by far more numerous than full-length elements, indicating that during the evolution of poplar, large amplification of these elements was followed by DNA loss. Within superfamilies, Ty3-gypsy families are made of more members than Ty1-copia ones. Retrotransposition occurred with increasing frequency following the separation of Populus sections, with different waves of retrotransposition activity between Ty3-gypsy and Ty1-copia elements. Recently inserted elements appear more frequently expressed than older ones. Finally, different levels of activity of retrotransposons were observed according to their position and their density in the linkage groups. On the whole, the results support the view of retrotransposons as a community of different organisms in the genome, whose activity (both retrotransposition and DNA loss) has heavily impacted and probably continues to impact poplar genome structure and size.

Genomic alterations in the interspecific hybrid Helianthus annuus×Helianthus tuberosus

Abstract The genome of a Helianthus annuus (2n=34) ×Helianthus tuberosus (2n=102) hybrid was studied at cytological, biochemical and molecular levels and compared to those of the parental species. Cytophotometric analyses showed that the hybrid has a 4C DNA content higher than expected and with a larger variability than in the parents. This high variability is probably not related to chromosome-number variations since the hybrid always had 2n=68 chromosomes. Moreover, hybrid interphase nuclei showed lower heterochromatin condensation than the parental ones. Thermal denaturation of genomic DNAs indicated that quantitative variation of some DNA families occurred in the hybrids compared to parents. Finally, molecular analyses of DNAs restricted with different enzymes, after Southern blotting and hybridization with HR probes, showed restriction patterns in the hybrid different from those observed in parents. These results indicate that interspecific hybridization between H. annuus and H. tuberosus may determine quantitative variation of some DNA families and differential DNA methylations that probably modify the nuclear structure. These phenomena are probable responses to a “genomic shock” following the interspecific cross.

Sequence variability of a dehydrin gene within Helianthus annuus

Abstract.Dehydrins are proteins produced during the late stages of plant embryo development and following any environmental stimulus involving dehydration. In order to investigate the variability of a dehydrin-encoding gene (Dhn1) in cultivated and wild sunflower (Helianthus annuus) genotypes, near-complete alleles were isolated by the polymerase chain reaction and sequenced. All of the isolated sequences were found to contain the typical dehydrin domains, and interrupted by an intron. The number of nucleotide substitutions and indels per site was calculated. With respect to the overall sequence, variation in both the coding and noncoding [intron and 3′-UTR (untranslated region)] sequences was much larger among wild accessions than among cultivars. No variation was observed in 3′-UTRs from cultivated sunflowers. Different coding regions showed a different numbers of synonymous and nonsynonymous substitutions. The Y and K domains were the most conserved in both wild and cultivated genotypes. Sequence analysis of the deduced dehydrin proteins showed that nucleotide substitutions in wild accessions should also determine large biochemical differences at the protein level. All of the isolated alleles were however functional, at least at the transcription level. To our knowledge these are the first data on intraspecific genetic variability of such a stress response gene. The low variability of dehydrin genes from cultivated sunflower is discussed in relation to the origin of sunflower cultivars. The possibility of rescuing general genetic variability through crosses to wild accessions of H. annuus rather than using wild Helianthus species is also discussed.

LTR retrotransposon dynamics in the evolution of the olive (Olea europaea) genome.

Improved knowledge of genome composition, especially of its repetitive component, generates important information for both theoretical and applied research. The olive repetitive component is made up of two main classes of sequences: tandem repeats and retrotransposons (REs). In this study, we provide characterization of a sample of 254 unique full-length long terminal repeat (LTR) REs. In the sample, Ty1-Copia elements were more numerous than Ty3-Gypsy elements. Mapping a large set of Illumina whole-genome shotgun reads onto the identified retroelement set revealed that Gypsy elements are more redundant than Copia elements. The insertion time of intact retroelements was estimated based on sister LTR’s divergence. Although some elements inserted relatively recently, the mean insertion age of the isolated retroelements is around 18 million yrs. Gypsy and Copia retroelements showed different waves of transposition, with Gypsy elements especially active between 10 and 25 million yrs ago and nearly inactive in the last 7 million yrs. The occurrence of numerous solo-LTRs related to isolated full-length retroelements was ascertained for two Gypsy elements and one Copia element. Overall, the results reported in this study show that RE activity (both retrotransposition and DNA loss) has impacted the olive genome structure in more ancient times than in other angiosperms.