The nature and organization of satellite DNAs in Petunia hybrida, related, and ancestral genomes

IntroductionThe garden petunia, Petunia hybrida (Solanaceae) is a fertile, diploid, annual hybrid species (2n=14) originating from P. axillaris and P. inflata 200 years ago. To understand the recent evolution of the P. hybrida genome, we examined tandemly repeated or satellite sequences using bioinformatic and molecular cytogenetic analysis.MethodsRaw reads from available genomic assemblies and survey sequences of P. axillaris N (PaxiN), P. inflata S6, (PinfS6), P. hybrida (PhybR27) and the here sequenced P. parodii S7 (PparS7) were used for graph and k-mer based cluster analysis of TAREAN and RepeatExplorer. Analysis of repeat specific monomer lengths and sequence heterogeneity of the major tandem repeat families with more than 0.01% genome proportion were complemented by fluorescent in situ hybridization (FISH) using consensus sequences as probes to chromosomes of all four species.ResultsSeven repeat families, PSAT1, PSAT3, PSAT4, PSAT5 PSAT6, PSAT7 and PSAT8, shared high consensus sequence similarity and organisation between the four genomes. Additionally, many degenerate copies were present. FISH in P. hybrida and in the three wild petunias confirmed the bioinformatics data and gave corresponding signals on all or some chromosomes. PSAT1 is located at the ends of all chromosomes except the 45S rDNA bearing short arms of chromosomes II and III, and we classify it as a telomere associated sequence (TAS). It is the most abundant satellite repeat with over 300,000 copies, 0.2% of the genomes. PSAT3 and the variant PSAT7 are located adjacent to the centromere or mid-arm of one to three chromosome pairs. PSAT5 has a strong signal at the end of the short arm of chromosome III in P. axillaris and P.inflata, while in P. hybrida additional interstitial sites were present. PSAT6 is located at the centromeres of chromosomes II and III. PSAT4 and PSAT8 were found with only short arrays.DiscussionThese results demonstrate that (i) repeat families occupy distinct niches within chromosomes, (ii) they differ in the copy number, cluster organization and homogenization events, and that (iii) the recent genome hybridization in breeding P. hybrida preserved the chromosomal position of repeats but affected the copy number of repetitive DNA

Participation of Multifunctional RNA in Replication, Recombination and Regulation of Endogenous Plant Pararetroviruses (EPRVs)

Pararetroviruses, taxon Caulimoviridae, are typical of retroelements with reverse transcriptase and share a common origin with retroviruses and LTR retrotransposons, presumably dating back 1.6 billion years and illustrating the transition from an RNA to a DNA world. After transcription of the viral genome in the host nucleus, viral DNA synthesis occurs in the cytoplasm on the generated terminally redundant RNA including inter- and intra-molecule recombination steps rather than relying on nuclear DNA replication. RNA recombination events between an ancestral genomic retroelement with exogenous RNA viruses were seminal in pararetrovirus evolution resulting in horizontal transmission and episomal replication. Instead of active integration, pararetroviruses use the host DNA repair machinery to prevail in genomes of angiosperms, gymnosperms and ferns. Pararetrovirus integration – leading to Endogenous ParaRetroViruses, EPRVs – by illegitimate recombination can happen if their sequences instead of homologous host genomic sequences on the sister chromatid (during mitosis) or homologous chromosome (during meiosis) are used as template. Multiple layers of RNA interference exist regulating episomal and chromosomal forms of the pararetrovirus. Pararetroviruses have evolved suppressors against this plant defense in the arms race during co-evolution which can result in deregulation of plant genes. Small RNAs serve as signaling molecules for Transcriptional and Post-Transcriptional Gene Silencing (TGS, PTGS) pathways. Different populations of small RNAs comprising 21–24 nt and 18–30 nt in length have been reported for Citrus, Fritillaria, Musa, Petunia, Solanum and Beta. Recombination and RNA interference are driving forces for evolution and regulation of EPRVs

Virus integration and tandem repeats in the genomes of Petunia

The integration of endogenous pararetroviruses (EPRVs) and tandemly repeated sequences were examined in whole genome raw reads, and two genome assemblies, in diploid Petunia species including hybrid-derivatives and their ancestors, using bioinformatics, molecular biology, cytogenetics and microscopy. Three types of EPRV clusters (petuvirus-, florendovirus- and caulimovirus-like sequences) were found. Chromosomal signals of PVCV (Petunia vein clearing virus) were seen by in situ hybridization in all Petunia species. Fragmented parts of four novel florendovirus-like sequences were found and the complete sequence was reconstructed, adding petunia to the 27 known host species. Chromosome III of P. axillaris and P. hybrida Rdc showed strong pericentromeric signal of PVCV and Florendovirus suggesting both EPRVs have similar positions, integration patterns and endogenization events (unlike P. integrifolia subsp inflata and P. axillaris subsp parodii). The caulimovirus-like sequence cluster was less abundant in genomes, with four novel members. RNA analysis from infected and healthy petunia samples revealed expression of endogenous PVCV and Caulimovirus sequences, unlike Florendovirus (not detected in RNA). The episomal form of vertically transmitted PVCV was integrated near the telomere of heterologous chromosomes. Transmission electron microscopy (TEM) showed differences in number and size of PVCV particles and inclusion bodies for both chlorotic spots and vein clearing symptoms, the latter correlated with PVCV particles in cytoplasm from vascular bundle cells. In plants with chlorotic symptoms, infected cells contained virions in parenchyma cells, while scattered virions were seen in chlorotic spots in P. hybrida W138 after heat induction of symptoms. Eight unique types of tandem repeat clusters were analysed within Petunia raw reads with variable genome proportions and different loci on mitotic chromosomes. Three were useful markers for chromosome identification. Taken together, the work shows the contribution of repetitive DNA to diversity and variation within petunia genomes, and has consequences for evolution, and both resistance and spread of some viruses