Temporal dynamics in the evolution of the sunflower genome as revealed by sequencing and annotation of three large genomic regions.

Improved knowledge of genome composition, especially of its repetitive component, generates important informations in both theoretical and applied research. In this study, we provide the first insight into the local organization of the sunflower genome by sequencing and annotating 349,380 bp from 3 BAC clones, each including one single-copy gene. These analyses resulted in the identification of 11 putative gene sequences, 18 full-length LTR retrotransposons, 6 incomplete LTR retrotransposons, 2 non-autonomous LTR-retroelements (LINEs), 2 putative DNA transposons fragments and one putative helitron. Among LTR-retrotransposons, non-autonomous elements (the so-called LARDs), which do not carry any protein-encoding sequence, were discovered for the first time in the sunflower. The insertion time of intact retroelements was measured, based on sister LTRs divergence. All isolated elements were inserted relatively recently, especially those belonging to the Gypsy superfamily. Retrotransposon families related to those identified in the BAC clones are present also in other species of Helianthus, both annual and perennial, and even in other Asteraceae. In one of the three BAC clones, we found five copies of a lipid transfer protein (LTP) encoding gene within less than 100,000 bp, four of which are potentially functional. Two of these are interrupted by LTR retrotransposons, in the intron and in the coding sequence, respectively. The divergence between sister LTRs of the retrotransposons inserted within the genes indicates that LTP gene duplication started earlier than 1.749 MYRS ago. On the whole, the results reported in this study confirm that the sunflower is an excellent system to study transposons dynamics and evolution.

An analysis of sequence variability in eight genes putatively involved in drought response in sunflower (Helianthus annuus L.).

With the aim to study variability in genes involved in ecological adaptations, we have analysed sequence polymorphisms of eight unique genes putatively involved in drought response by isolation and analysis of allelic sequences in eight inbred lines of sunflower of different origin and phenotypic characters and showing different drought response in terms of leaf relative water content (RWC). First, gene sequences were amplified by PCR on genomic DNA from a highly inbred line and their products were directly sequenced. In the absence of single nucleotide polymorphisms, the gene was considered as unique. Then, the same PCR reaction was performed on genomic DNAs of eight inbred lines to isolate allelic variants to be compared. The eight selected genes encode a dehydrin, a heat shock protein, a non-specific lipid transfer protein, a z-carotene desaturase, a drought-responsive-element-binding protein, a NAC-domain transcription regulator, an auxin-binding protein, and an ABA responsive-C5 protein. Nucleotide diversity per synonymous and non-synonymous sites was calculated for each gene sequence. The π (a)/π (s) ratio range was usually very low, indicating strong purifying selection, though with locus-to-locus differences. As far as non-coding regions, the intron showed a larger variability than the other regions only in the case of the dehydrin gene. In the other genes tested, in which one or more introns occur, variability in the introns was similar or even lower than in the other regions. On the contrary, 3'-UTRs were usually more variable than the coding regions. Linkage disequilibrium in the selected genes decayed on average within 1,000 bp, with large variation among genes. A pairwise comparison between genetic distances calculated on the eight genes and the difference in RWC showed a significant correlation in the first phases of drought stress. The results are discussed in relation to the function of analysed genes, i.e. involved in gene regulation and signal transduction, or encoding enzymes and defence proteins.

HACRE1, a recently inserted copia-like retrotransposon of sunflower (Helianthus annuus L.).

In this paper we report on the isolation and characterization, for the first time, of a complete 6511 bp retrotransposon of sunflower. Considering its protein domain order and sequence similarity to other copia elements of dicotyledons, this retrotransposon was assigned to the copia retrotransposon superfamily and named HACRE1 (Helianthus annuus copia-like retroelement 1). HACRE1 carries 5' and 3' long terminal repeats (LTRs) flanking an internal region of 4661 bp. The LTRs are identical in their sequence except for two deletions of 7 and 5 nucleotides in the 5' LTR. Based on the sequence identity of the LTRs, HACRE1 was estimated to have inserted within the last approximately 84 000 years. The isolated sequence contains a complete open reading frame with only one complete reading frame. The absence of nonsense mutations agrees with the very high sequence identity between LTRs, confirming that HACRE1 insertion is recent. The haploid genome of sunflower (inbred line HCM) contains about 160 copies of HACRE1. This retrotransposon is expressed in leaflets from 7-day-old plantlets under different light conditions, probably in relation to the occurrence of many putative light-related regulatory cis-elements in the LTRs. However, sequenced cDNAs show less variability than HACRE1 genomic sequences, indicating that only a subset of this family is expressed under these conditions.

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.

Phylogenetic relationships between annual and perennial species of Helianthus: evolution of a tandem repeated DNA sequence and cytological hybridization experiments.

The amplification and chromosomal localization of tandem repeated DNA sequences from Helianthus annuus (clone HAG004N15) and the physical organization of ribosomal DNA were studied in annual and perennial species of Helianthus. HAG004N15-related sequences, which did not show amplification in other Asteraceae except for Viguiera multiflora, were redundant in all the Helianthus species tested, but their frequency was significantly higher in perennials than in annuals. These sequences were located at the ends and intercalary regions of all chromosome pairs of annual species. A similar pattern was found in the perennials, but a metacentric pair in their complement was not labelled. Ribosomal cistrons were carried on two chromosome pairs in perennials and on three pairs in annuals except for H. annuus, where rDNA loci were on four pairs. No difference was observed between cultivated H. annuus and its wild accessions in the hybridization pattern of the HAG004N15 and ribosomal probes. These findings support the hypothesis that the separation between annual and perennial Helianthus species occurred through interspecific hybridization involving at least one different parent. However, GISH in H. annuus using genomic DNA from the perennial Helianthus giganteus as blocking DNA failed to reveal different genomic assets in annual and perennial species.

Characterization of the chromosome complement of Helianthus annuus by in situ hybridization of a tandemly repeated DNA sequence.

A tandemly repeated sequence isolated from a clone (HAG004N15) of a nebulized genomic DNA library of sunflower (Helianthus annuus L., 2n = 34) was characterized and used to study the chromosome complement of sunflower. HAG004N15 repeat units (368 bp in length) were found to be highly methylated, and their copy number per haploid (1C) genome was estimated to be 7800. After in situ hybridization of HAG004N15 repeats onto chromosome spreads, signals were observed at the end of both chromosome arms in 4 pairs and at the end of only one arm in 8 other pairs. Signals were also observed at the intercalary (mostly subtelomeric) regions in all pairs, in both arms in 8 pairs, and in only one arm in the other 9 pairs. The short arm of 1 pair was labelled entirely. The chromosomal location of ribosomal DNA was also studied by hybridizing the wheat ribosomal probe pTa71. Four chromosome pairs contained ribosomal cistrons at the end of their shorter arm, but a satellite was seen in only 3 pairs. These hybridization patterns were the same in the 3 sunflower lines studied (HA89, RA20031, and HOR). The chromosomal localization of HAG004N15-related sequences allowed all of the chromosome pairs to be distinguished from each other, in spite of small size and similar morphology.

Distribution of Ty3-gypsy- and Ty1-copia-like DNA sequences in the genus Helianthus and other Asteraceae.

Two repeated DNA sequences, pHaS13 and pHaS211, which revealed similarity to the int gene of Ty3-gypsy retrotransposons and the RNAse-H gene of Ty1-copia retroelements, respectively, were surveyed in Asteraceae species and within the genus Helianthus. Southern analysis of the genome of selected Asteraceae that belong to different tribes showed that pHaS13- and pHaS211-related subfamilies of gypsy- and copia-like retroelements are highly redundant only in Helianthus and, to a lesser extent, in Tithonia, a Helianthus strict relative. However, under low stringency posthybridization washes, bands were observed in almost all the other Asteraceae tested when pHaS13 was used as a probe, and in several species when pHaS211 was hybridized. FISH analysis of pHaS13 or pHaS211 probes was performed in species in which labelling was observed in Southern hybridizations carried out under high stringency conditions (Helianthus annuus, Tithonia rotundifolia, Ageratum spp., Leontopodium spp., Senecio vulgaris for pHaS13, and H. annuus, Tithonia rotundifolia, and S. vulgaris for pHaS211). Scattered labelling was observed over all metaphase chromosomes, indicating a large dispersal of both Ty3-gypsy- and Ty1-copia-like retroelements. However, preferential localization of Ty3-gypsy-like sequences at centromeric chromosome regions was observed in all of the species studies but one, even in species in which pHaS13-related elements are poorly represented. Ty1-copia-like sequences showed preferential localization at the chromosome ends only in H. annuus. To study the evolution of gypsy- and copia-like retrotransposons in Helianthus, cladograms were built based on the Southern blot hybridization patterns of pHaS13 or pHaS211 sequences to DNA digests of several species of this genus. Both cladograms agree in splitting the genomes studied into annuals and perennials. Differences that occurred within the clades of perennial and annual species between gypsy- and copia-like retroelements indicated that these retrotransposons were differentially active during Helianthus speciation, suggesting that the evolution of the 2 retroelement families was, within limits, independent.

Analysis of a dehydrin encoding gene and its phylogenetic utility in Helianthus.

Dehydrins are ubiquitous plant proteins, synthesized in late stages of plant embryo development and following any environmental stress involving dehydration. With the aim to study the evolution of such a stress-responsive gene within Helianthus and to test the possibility of using this gene for phylogenetic studies, fragments of the same dehydrin gene were isolated by PCR and sequenced in 16 wild Helianthus species or subspecies. All isolated sequences included the typical dehydrin domains (Y, S and K), a portion of 3'-UTR and an intron, inserted in the same position within the S domain-encoding region. The number of nucleotide substitutions (both synonymous and nonsynonymous) was calculated keeping separate the different gene regions, and differences occur even among coding domains, indicating that evolutionary constraints act differently on each region. The occurrence of indels and/or insertions was also observed. At the deduced protein level, the calculation of isoelectric point, molecular weight and the percentage of alpha-helix showed a diversification of biochemical properties of this protein between annual and perennial Helianthus species. Phylogenetic trees were built by the maximum-likelihood, maximum-parsimony, and neighbor-joining methods. In all cases the same topology was observed; perennial and annual species form a supported clade, and H. annuus was separated from the other annuals and from perennials. These data support the use of this stress-responsive gene to study the phylogeny of Helianthus.

Sequence variability of a dehydrin gene within Helianthus annuus.

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.

Genome plasticity in Festuca arundinacea: direct response to temperature changes by redundancy modulation of interspersed DNA repeats.

The response of the genome of Festuca arundinacea seedlings to changes in the temperature at which they were grown was investigated. Fifteen repeated sequences in the nuclear DNA were isolated and hybridized to the genomic DNA of seedlings grown at 10 degrees C or 30 degrees C. The redundancies of sequences recognized by four probes ( FaA5, FaH8, FaH13 and FaH14), were found to differ significantly in the two DNAs. DNA sequences recognized by FaH8, FaH13 and FaH14 were more represented in the genome of the 30 degrees C-raised seedlings than in the genome of the 10 degrees C-raised seedlings (76.5 x 10(3), 1.9 x 10(3), and 111.8 x 10(3) copies per haploid, 1C genome vs 62.7 x 10(3), 1.3 x 10(3), and 80.8 x 10(3) copies, respectively). In contrast, FaA5-related sequences were more represented in the genome of seedlings grown at the lower temperature (15.5 x 10(3) vs 10.2 x 10(3) copies, respectively). Southern-blot hybridization of these repeats to digested genomic DNA produced patterns which indicated that the probe sequences were part of longer repeated sequences having a limited degree of structural heterogeneity. These patterns were partly different when the probes were hybridized to the DNA from seedlings grown at 10 degrees C or 30 degrees C. In situ hybridization showed that the DNA sequences recognized by each probe were scattered along the length of all the chromosomes, with preferential location of FaA5- and FaH13-related sequences at given, mainly centromeric, regions of certain chromosomes. These findings suggest that redundancy modulations of interspersed repeated sequences allow direct responses of the genome of F. arundinacea to changes in environmental temperature.

Characterization and expression of DNA sequences encoding putative type-II metallothioneins in the seagrass Posidonia oceanica.

Posidonia oceanica is a marine phanerogam, largely widespread in the Mediterranean sea, representing an important food substrate for many marine organisms. A progressive reduction of P. oceanica meadows has been reported, due to anthropogenic coastal activity. Studying mechanisms by which this species responds to environmental stresses, three DNA sequences putatively encoding metallothioneins (MTs) have been isolated, by PCR. Two sequences, Pomt2a (accession no. AJ249603) and Pomt2b (accession no. AJ249602), show high similarities with genes encoding type-II MTs and are interrupted by two and one intron, respectively. The third sequence, Pomt2c (accession no. AJ249604), is supposed to be a pseudogene, originated by retrotranscription of the Pomt2b mRNA. These sequences belong to a multigene family with at least five members. Northern hybridizations indicated that MT transcripts accumulation is constitutive and seasonally regulated. MT encoding RNAs increase after rhyzome harvesting and (at a lesser extent) after 15 d of cultivation in an aquarium. As for animal MTs, transcripts accumulation is observed also after exposure to trace metals such as copper and cadmium. In the case of copper, the effect depends on concentration. Finally, taking into consideration the great interest in studying the biogeochemical cycle of mercury in the Mediterranean basin and since P. oceanica is commonly considered a bioindicator of this metal, the effect of mercury treatments on the accumulation of MT transcripts has been analyzed: in only a few experiments a small increase in the level of transcripts was recorded, suggesting that MTs are not key elements in the mercury accumulation by this species.

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 (HaDhnla) 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.

Variation of repetitive DNA sequences in progenies of regenerated plants of Pisum sativum.

The frequency variation of highly repeated (HR) DNA sequences was studied in plants regenerated through in vitro culture of macerated vegetative apices of Pisum sativum and their progenies. Feulgen cytophotometry showed that regenerated plants (R1) have 6-7% more DNA per nucleus than control plants; this difference is maintained in the subsequent generation (R2). Slot-blot analyses using isolated highly repeated sequences as probe indicated that an increased frequency of these sequences occurs in regenerated plants and their progenies. These results were confirmed by a series of experiments: (a) metaphase chromosomes were longer in R2 than in control plants; (b) cytophotometric analyses of chromatin structure after Feulgen-staining showed that condensed chromatin is more represented in R2 than in control plants; and (c) thermal denaturation of isolated HR sequences indicated that a new HR family appears in R1 and is maintained in R2 plants. These results suggest that, in this species, the DNA extrasynthesized during in vitro culture is, at least in part, integrated in the chromosomes and transmitted to the progeny.

Nuclear DNA changes within Helianthus annuus L.: variations in the amount and methylation of repetitive DNA within homozygous progenies.

Complex alterations in the redundancy and methylation of repeated DNA sequences were shown to differentiate the nuclear genome of individuals belonging to single progenies of homozygous plants of the sunflower. DNA was extracted from seedlings obtained from seeds collected at the periphery of flowering heads (P DNA) or from seedlings obtained from seeds collected in their middle (M DNA). Three fractions of repeated sequences were isolated from genomic DNA: a highly repetitive fraction (HR), which reassociates within an equivalent Cot of about 2 × 10(-1), and two medium repetitive fractions (MR1 and MR2) having Cot ranges of about 2 × 10(-1)-2 and 2-10(2), respectively. Denaturation kinetics allowed different sequence families to be recognized within each fraction of repetitive DNA, and showed significant differences in sequence redundancy to occur between P and M DNA, particularly as far as the MR2 fraction is concerned. Most DNA sequence families are more represented in P DNA than in M DNA. However, the redundancy of certain sequences is greater in the latter than in the former. Each repetitive DNA fraction was hybridized to Southern blots of genomic P or M DNA which was digested to completion by three pairs of isoschizomeric restriction endonucleases which are either insensitive or sensitive to the methylation of a cytosine in the recognition site. The results obtained showed that the repetitive DNA of H. annuus is highly methylated. Clear-cut differences in the degree of methylation of P and M DNA were found, and these differences were particularly apparent in the MR2 fraction. It is suggested that alterations in the redundancy of given DNA sequences and changes in their methylation patterns are complementary ways to produce continuous genotypic variability within the species which can be exploited in environmental adaptation.

Heterochromatin and repetitive DNA frequency variation in regenerated plants of Helianthus annuus L.

Plant regeneration from cotyledons of seeds of a single progeny of a pure line of Helianthus annuus was studied in respect of the nuclear DNA contents of control and regenerated plants. Control plants were divided into two groups: those developed from seeds at the periphery of the inflorescence (showing a high basic 4C DNA content) and those from seeds developed in the middle of the inflorescence (showing a low basic 4C DNA content). It was observed that plants from peripheral seeds have a higher morphogenetic potential than those from central seeds. Cytophotometric analyses indicated that plants regenerated from cotyledons of both peripheral and central seeds show the same basic 4C DNA amount, which is higher that that observed in vivo in peripheral seeds. Molecular analysis by slot blotting and hybridization with different DNA families showed that the difference in nuclear DNA content between plants from peripheral and central seeds in vivo are mainly related to differences in the frequency of highly repeated, "slow" medium repeated (MR2), and ribosomal DNA families; by contrast, the increase in DNA amount in regenerated plants is mainly due to "fast" medium repeated sequences (MR1). Moreover, the frequency of kinetically isolated "unique" sequences was higher in peripheral seeds than in central ones and still higher in regenerated plants. Optical-density measurements of interphase nuclei showed an increase of heterochromatin in regenerated plants, suggesting that, whatever DNA is amplified in these plants, it remains condensed and probably inactive.