A distinct endogenous pararetrovirus family in Nicotiana tomentosiformis, a diploid progenitor of polyploid tobacco

A distinct endogenous pararetrovirus (EPRV) family corresponding to a previously unknown virus has been identified in the genome of Nicotiana tomentosiformis, a diploid ancestor of allotetraploid tobacco (Nicotiana tabacum). The putative virus giving rise to N. tomentosiformis EPRVs (NtoEPRVs) is most similar to tobacco vein clearing virus, an episomal form of a normally silent EPRV family in Nicotiana glutinosa; it is also related to a putative virus giving rise to the NsEPRV family in Nicotiana sylvestris (the second diploid progenitor of tobacco) and in the N. sylvestris fraction of the tobacco genome. The copy number of NtoEPRVs is significantly higher in N. tomentosiformis than in tobacco. This suggests that after the polyploidization event, many copies were lost from the polyploid genome or were accumulated specifically in the diploid genome. By contrast, the copy number of NsEPRVs has remained constant in N. sylvestris and tobacco, indicating that changes have occurred preferentially in the NtoEPRV family during evolution of the three Nicotiana species. NtoEPRVs are often flanked by Gypsy retrotransposon-containing plant DNA. Although the mechanisms of NtoEPRV integration, accumulation, and/or elimination are unknown, these processes are possibly linked to retrotransposon activity.     

Next generation sequencing reveals genome downsizing in allotetraploid Nicotiana tabacum, predominantly through the elimination of paternally derived repetitive DNAs

We used next generation sequencing to characterize and compare the genomes of the recently derived allotetraploid, Nicotiana tabacum (<200,000 years old), with its diploid progenitors, Nicotiana sylvestris (maternal, S-genome donor), and Nicotiana tomentosiformis (paternal, T-genome donor). Analysis of 14,634 repetitive DNA sequences in the genomes of the progenitor species and N. tabacum reveal all major types of retroelements found in angiosperms (genome proportions range between 17-22.5% and 2.3-3.5% for Ty3-gypsy elements and Ty1-copia elements, respectively). The diploid N. sylvestris genome exhibits evidence of recent bursts of sequence amplification and/or homogenization, whereas the genome of N. tomentosiformis lacks this signature and has considerably fewer homogenous repeats. In the derived allotetraploid N. tabacum, there is evidence of genome downsizing and sequences loss across most repeat types. This is particularly evident amongst the Ty3-gypsy retroelements in which all families identified are underrepresented in N. tabacum, as is 35S ribosomal DNA. Analysis of all repetitive DNA sequences indicates the T-genome of N. tabacum has experienced greater sequence loss than the S-genome, revealing preferential loss of paternally derived repetitive DNAs at a genome-wide level. Thus, the three genomes of N. sylvestris, N. tomentosiformis, and N. tabacum have experienced different evolutionary trajectories, with genomes that are dynamic, stable, and downsized, respectively.     

Distribution of the Tnt1 retrotransposon family in the amphidiploid tobacco (Nicotiana tabacum) and its wild Nicotiana relatives

Transposable elements can generate considerable genetic diversity. Here we examine the distribution of the Tnt1 retrotransposon family in representative species of the genus Nicotiana . We show that multiple Tnt1 insertions are found in all Nicotiana species. However, Tnt1 insertions are too polymorphic to reveal species relationships. This indicates that Tnt1 has amplified rapidly and independently after Nicotiana speciation. We compare patterns of Tnt1 insertion in allotetraploid tobacco ( N. tabacum ) with those in the diploid species that are most closely related to the progenitors of tobacco, N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). We found no evidence for Tnt1 insertion sites of N. otophora origin in tobacco. Nicotiana sylvestris has a higher Tnt1 content than N. tomentosiformis and the elements are distributed more uniformly across the genome. This is reflected in tobacco where there is a higher Tnt1 content in S-genome chromosomes. However, the total Tnt1 content of tobacco is not the sum of the two modern-day parental species. We also observed tobacco-specific Tnt1 insertions and an absence of tobacco Tnt1 insertion sites in the diploid relatives. These data indicate Tnt1 evolution subsequent to allopolyploidy. We explore the possibility that fast evolution of Tnt1 is associated with 'genomic-shock' arising out of interspecific hybridization and allopolyploidy.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 639–649.     

Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum.

Origin and rearrangement of ribosomal DNA repeats in natural allotetraploid Nicotiana tabacum are described. Comparative sequence analysis of the intergenic spacer (IGS) regions of Nicotiana tomentosiformis (the paternal diploid progenitor) and Nicotiana sylvestris (the maternal diploid progenitor) showed species-specific molecular features. These markers allowed us to trace the molecular evolution of parental rDNA in the allopolyploid genome of N. tabacum; at least the majority of tobacco rDNA repeats originated from N. tomentosiformis, which endured reconstruction of subrepeated regions in the IGS. We infer that after hybridization of the parental diploid species, rDNA with a longer IGS, donated by N. tomentosiformis, dominated over the rDNA with a shorter IGS from N. sylvestris; the latter was then eliminated from the allopolyploid genome. Thus, repeated sequences in allopolyploid genomes are targets for molecular rearrangement, demonstrating the dynamic nature of allopolyploid genomes.     

The origin of tobacco's T genome is traced to a particular lineage within Nicotiana tomentosiformis (Solanaceae)

Nicotiana tabacum (tobacco) is a natural allotetraploid. The maternal genome donor is not controversial and is probably derived from an ancestor of N. sylvestris. The paternal, T-genome donor has been less clear, with N. tomentosiformis, N. otophora, or an introgression hybrid proposed. Here we provide evidence that the T genome of N. tabacum is derived from a particular lineage of N. tomentosiformis. We show that the repetitive sequences of geminiviral origin, GRD53 and GRD3, are present in the genomes of N. tabacum cultivars, a tobacco cell suspension culture TBY-2, and N. tomentosiformis ac. NIC 479/84. Surprisingly, they are not present in another three varieties of N. tomentosiformis. A detailed cytogenetic analysis also revealed that N. tomentosiformis ac. NIC 479/84 most closely resembles the N. tabacum T genome in the location of other tandem repetitive sequences. Thus, tobacco formed after divergence within N. tomentosiformis, and the spectrum of potential donors of the paternal genome can be narrowed to a genotype of N. tomentosiformis characterized by the presence of GRD53 and GRD3 repeats. It is clear that future paternity studies in tobacco should use N. tomentosiformis ac. NIC 479/84 rather than any other accession.     

Concerted evolution of 18–5.8–26S rDNA repeats in Nicotiana allotetraploids

We review and extend data showing concerted evolution of parental 18–5.8–26S nuclear ribosomal DNA (18–26S rDNA) gene families in three natural Nicotiana allotetraploids ( N. tabacum , N. rustica and N. arentsii , each 2 n  = 4 x  = 48) and one synthetic N. tabacum line (Th37, ♀ N. sylvestris (2 n  = 24) × ♂ N. tomentosiformis (2 n  = 24)). The origin of the gene families was analysed by sequence polymorphisms in the intergenic spacer (IGS) region and the number of chromosomal loci by fluorescence in situ hybridization (FISH). FISH revealed that the number and locations of 18–26S rDNA in the natural allopolyploids was the sum of those found in the diploid progenitors. However, the rDNA restriction patterns showed polymorphisms in the IGS that were not additive, suggesting that parental rDNA clusters were partially ( N. tabacum, N. rustica ) or completely ( N. arentsii ) overwritten by hybrid-specific units. Thus the Nicotiana allotetraploids show evidence of concerted evolution, including both intralocus and interlocus gene conversion. A feral N. tabacum collected in Bolivia had a higher proportion of unconverted parental rDNA units than cultivated tobacco varieties, suggesting either that rDNA homogenization is accelerated by inbreeding or multiple origins of tobacco. There is no evidence for the elimination of N. sylvestris- derived rDNA units in the synthetic Th37 tobacco line as occurred in natural tobacco, although several novel rDNA unit variants were found in most but not all the hybrid plants. Factors that may control the occurrence and extent of rDNA homogenization are discussed for allopolyploids in Nicotiana and other taxa.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 615–625.     

Origin of Nicotiana tabacum detected by primary structure of fraction I protein.

Nicotiana tabacum is believed to have arisen after hybridization of Nicotiana sylvestris with a species in the Tomentosae section of the genus Nicotiana. Recent biochemical experiments have confirmed the conclusions from previous cytogenetic studies that N. sylvestris was the maternal parent and have indicated that Nicotiana tomentosiformis was the paternal parent. However, these studies did not take into account the possibility that a new species of Nicotiana, called K-12, discovered in South America in 1968, could also have been one of the parents. Fraction I proteins were purified from N. tabacum and its putative progenitors, and separated into large and small subunits. Chymotryptic peptides of each subunit were analyzed by ion exchange column chromatography with a gradient elution system. Among 38 resolved peaks of the large subunits, 2 peaks were found to be different among the putative species. Since only N. sylvestris showed an identical chromatogram with N. tabacum, N. sylvestris was concluded to be the maternal progenitor, as the genetic information for the large subunit of Fraction I protein was known to be inherited by the cytoplasmic mode. On the other hand, the small subunit of Fraction I protein is inherited by the Mendelian mode and therefore N. tabacum, an allopolyploid, could be expected to contain two types of small subunits, one derived from N. sylvestris and the other from a paternal progenitor. N. sylvestris lacks two of the 25 chymotryptic peptides of the small subunit of N. tabacum. Among 3 putative paternal progenitors, these two peaks appeared only in N. tomentosiformis, but not in Nicotiana otophora or K-12. Thus, N. tomentosiformis was concluded to be a paternal progenitor of N. tabacum. The conclusion was verified by comparing chymotryptic peptides of small subunits from three amphidiploids of N. sylvestris crossed with N. tomentosiformis, N. sylvestris crossed with N. otophora snd N. sylvestris crossed with K-12. The analytical results showed that only the progeny of N. sylvestris crossed with N. tomentosiformis contained the same small subunits as N. tabacium.     

AFLP analysis of genetic polymorphism and evolutionary relationships among cultivated and wild Nicotiana species.

Amplified fragment length polymorphism (AFLP) analysis was used to determine the degree of intra- and inter-specific genetic variation in the genus Nicotiana. Forty-six lines of cultivated tobacco (Nicotiana tabacum L.) and seven wild Nicotiana species, including N. sylvestris, N. tomentosiformis, N. otophora, N. glutinosa, N. suaveolens, N. rustica, and N. longiflora, were analyzed, using at least eight different oligonucleotide primer combinations capable of detecting a minimum of 50 polymorphic bands per primer pair. The amount of genetic polymorphism present among cultivated tobacco lines (N. tabacum) was limited, as evidenced by the high degree of similarity in the AFLP profiles of cultivars collected worldwide. Six major clusters were found within cultivated tobacco that were primarily based upon geographic origin and manufacturing quality traits. A greater amount of genetic polymorphism exists among wild species of Nicotiana than among cultivated forms. Pairwise comparisons of the AFLP profiles of wild and cultivated Nicotiana species show that polymorphic bands present in N. tabacum can be found in at least one of three proposed wild progenitor species (i.e., N. sylvestris, N. tomentosiformis, and N. otophora). This observation provides additional support for these species contributing to the origin of N. tabacum.     

Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species

Nicotiana tabacum (tobacco) is an allotetraploid derived from ancestors of the modern diploids, N. sylvestris and N. tomentosiformis. We identified and characterized two distinct families of 5S ribosomal DNA (rDNA) in N. tabacum; one family had an average 431 bp unit length and the other a 646 bp unit length. In the diploid species, N. sylvestris and N. tomentosiformis, the 5S rDNA unit lengths are 431 bp and 644 bp respectively. The non-coding spacer sequence of the short unit in tobacco had high sequence homology to the spacer of N. sylvestris5S rDNA, while the longer spacer of tobacco had high homology with the 5S spacer of N. tomentosiformis. This suggests that the two 5S families in tobacco have their origin in the diploid ancestors. The longer spacer sequence had a GC rich sub-region (called the T-genome sub-region) that was absent in the short spacer. Pulsed field gel analysis and fluorescent in situ hybridization to tobacco metaphase chromosomes showed that the two families of 5S rDNA units are spatially separate at two chromosomal loci, on chromosomes S8 (short family) and T8 (long family). The repeat copy number at each chromosomal locus showed heterogeneity between different tobacco cultivars, with a tendency for a decrease in the copy number of one family to be compensated by an increase in the copy number of the second family. Sequence analysis reveals there is as much diversity in 5S family units within the diploid species as there is within the T and S-genome 5S family units respectively, suggesting 5S diversification within each family had occurred before tobacco speciation. There is no evidence of interlocus homogenization of the two 5S families in tobacco. This is therefore substantially different to 18-26S rDNA where interlocus gene conversion has substantially influenced most sequences of S and T genome origin; possible reasons are discussed.     

Use of transferable Nicotiana tabacum L. microsatellite markers for investigating genetic diversity in the genus Nicotiana.

The recent development of microsatellite markers for tobacco, Nicotiana tabacum L., may be valuable for genetic studies within the genus Nicotiana. The first objective was to evaluate transferability of 100 N. tabacum microsatellite primer combinations to 5 diploid species closely related to tobacco. The number of primer combinations that amplified scorable bands in these species ranged from 42 to 56. Additional objectives were to assess levels of genetic diversity amongst available accessions of diploid relatives closely related to tobacco (species of sections Sylvestres and Tomentosae), and to evaluate the efficacy of microsatellite markers for establishing species relationships in comparison with existing phylogenetic reconstructions. A subset of 46 primer combinations was therefore used to genotype 3 synthetic tobaccos and an expanded collection of 51 Nicotiana accessions representing 15 species. The average genetic similarity for 7 diverse accessions of tobacco was greater than the average similarity for N. otophora accessions, but lower than the average genetic similarities for N. sylvestris, N. tomentosa, N. kawakamii, and N. tomentosiformis accessions. A microsatellite-based phylogenetic tree was largely congruent with taxonomic representations based on morphological, cytological, and molecular observations. Results will be useful for selection of parents for creation of diploid mapping populations and for germplasm introgression activities.     

Preferential elimination of repeated DNA sequences from the paternal, Nicotiana tomentosiformis genome donor of a synthetic, allotetraploid tobacco

Nicotiana tabacum (tobacco, 2n = 4x = 48) is a natural allotetraploid combining two ancestral genomes closely related to modern Nicotiana sylvestris and Nicotiana tomentosiformis. Here we examine the immediate consequences of allopolyploidy on genome evolution using 20 S4-generation plants derived from a single synthetic, S0 plant made by Burk in 1973 (Th37). Using molecular and cytogenetic methods we analysed 14 middle and highly repetitive sequences that together total approximately 4% of the genome. Two repeats related to endogenous geminiviruses (GRD5) and pararetroviruses (NtoEPRV), and two classes of satellite repeats (NTRS, A1/A2) were partially or completely eliminated at variable frequency (25-60%). These sequences are all from the N. tomentosiformis parent. Genomic in situ hybridization revealed additivity in chromosome numbers in two plants (2n = 48), while a third was aneuploid for an N. tomentosiformis-origin chromosome (2n = 49). Two plants had homozygous translocations between chromosomes of the S- and T-genomes. * The data demonstrate that genetic changes in synthetic tobacco were fast, targeted to the paternal N. tomentosiformis-donated genome, and some of the changes showed concordance with changes that presumably occurred during evolution of natural tobacco.     

Molecular evolutionary analysis of the psbP gene family of the photosystem II oxygen-evolving complex in Nicotiana.

Photosystem II psbP protein of the oxygen-evolving complex is involved in the photosynthetic oxygen evolution in plants. Four psbP polypeptides were detected in Nicotiana tabacum on a two-dimensional gel by immunostaining the proteins with antiserum against the pea psbP Comparison of the protein patterns of psbP from N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, indicated that each of the ancestral parents has contributed a pair of psbP proteins. This was supported by Southern hybridization results, which suggested that psbP in Nicotiana is encoded by a gene family consisting of four members in N. tabacum and two members each in N. glauca, N. langsdorffii, N. sylvestris, and N. tomentosiformis. A scheme of molecular evolution of the psbP genes in Nicotiana is also proposed.     

Effects of mutagens on androgenesis in some species of the genusNicotiana L.

Chosen species and cultivars(Nicotiana sanderae, N. glutinosa, N. otophora, N. paniculata, N. sylvestris, N. velutina, N. tabacum cv. Samsun, N. tabacum cv. Samsun — a dihaploid line, and an amphidiploid N.sylvestris x N tomentosiformis) were treated with adequate concentration of n-buthylmethane sulphonate. The anthers of M₁ plants were cultured on the medium of NITSCH 1969 plus 0.1 mg IAA/1. Special attention was paid to quantitative characteristics of androgenesis. Androgenesis occurred more often in the treated variants. The conclusions have been confirmed of a heterosis effect of adequate chemomutagen concentration at the diploid level (an increase in the number of androgenic anthers in the treated variants, and a decrease in the mean number of haploids per androgenic anther) in the broad range of the material. We have recorded a high androgenic ability in the amphidiploidN. sylvestris x N. tomentosiformis, and androgenesis inN. velutina for the first time.     

Repetitive DNA variation and pivotal-differential evolution of wild wheats.

Several polyploid species in the genus Triticum contain a U genome derived from the diploid T. umbellulatum. In these species, the U genome is considered to be unmodified from the diploid based on chromosome pairing analysis, and it is referred to as pivotal. The additional genome(s) are considered to be modified, and they are thus referred to as differential genomes. The M genome derived from the diploid T. comosum is found in many U genome polyploids. In this study, we cloned three repetitive DNA sequences found primarily in the U genome and two repetitive DNA sequences found primarily in the M genome. We used these to monitor variation for these sequences in a large set of species containing U and M genomes. Investigation of sympatric and allopatric accessions of polyploid species did not show repetitive DNA similarities among sympatric species. This result does not support the idea that the polyploid species are continually exchanging genetic information through introgression. However, it is also possible that repetitive DNA is not a suitable means of addressing the question of introgression. The U genomes of both diploid and polyploid U genome species were similar regarding hybridization patterns observed with U genome probes. Much more variation was found both among diploid T. comosum accessions and polyploids containing M genomes. The observed variation supports the cytogenetic evidence that the M genome is more variable than the U genome. It also raises the possibility that the differential nature of the M genome may be due to variation within the diploid T. comosum, as well as among polyploid M genome species and accessions.