Nicotiana Debneyi Research Articles

The complete chloroplast genome sequence of Nicotiana debneyi (Solanaceae)

In present study, we report the complete chloroplast genome of Nicotiana debneyi, a species endemic to eastern coast of Australia. The total genome size of N.debneyi is 156,073 bp in length, containing a large single-copy region of 86,672 bp, a small single-copy region of 18,581 bp, and two inverted repeat regions of 25,410 bp. The all GC content of N.debneyi chloroplast genome is 38.4%. It encodes a total of 129 unique genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes, of which seven tRNA, four rRNA and seven protein-coding genes are duplicated in the IR. Sixteen genes contain a single intron, and only two genes have two introns. Phylogenetic analysis results strongly supported that N.debneyi was closely related to Nicotiana sylvestris and Nicotiana tabacum.

Development of user-friendly markers for disease resistance to black root rot of tobacco through genotyping by sequencing

Black root rot (BRR), a disease caused by the hemibiotrophic fungus Thielaviopsis basicola, seriously compromises yield and leaf quality in tobacco (Nicotiana tabacum). Full resistance to black root rot, conferred by the resistance to BRR 1 (RBRR1) locus from Nicotiana debneyi Domin, was transferred to a burley tobacco cultivar through interspecific hybridization. Some undesirable traits potentially caused by linkage drag restrict wider application of RBRR1 in flue-cured tobacco. Therefore, user-friendly molecular markers are needed to assist selection for resistance to black root rot and to break the unfavorable linkage. Genotyping by sequencing (GBS) is a rapid and robust approach for reduced representation sequencing of multiplexed genomic DNA samples that combines genome-wide molecular marker discovery with genotyping. In the present study, we used GBS to identify single-nucleotide polymorphisms (SNPs) linked to the RBRR1 locus, and PCR-based assays for detection of these SNPs were also developed. Sequence analysis of the SNP markers suggested that RBRR1 is located on chromosome 17, providing a basis for map-based cloning of this valuable gene. Co-dominant CAPS markers that co-segregate with the disease-resistant phenotype offer user-friendly tools for tobacco breeding and variety improvement. Furthermore, tested with diverse N. tabacum germplasm, SS192650 displayed 100% selection accuracy for resistance to BRR, suggesting that this marker can be used in diverse tobacco populations.

A new virus, classifiable in the family Tombusviridae, found infecting Solanum tuberosum in the UK.

A novel virus was discovered in a freeze-dried collection held at SASA, UK, originating from potato (Solanum tuberosum) cv. Nadine. The complete sequence of the viral RNA was determined to be 3674 nucleotides in length encoding five predicted proteins. Based on the deduced genome organization and phylogenetic analysis, this virus represents a putative new member of the genus Alphanecrovirus, family Tombusviridae, most closely related to isolates of Olive mild mosaic virus. The virus was easily transmitted to indicator plants with symptoms that were slower to develop and less severe than those of related viruses. To distinguish this virus, the clearest symptom differences occurred with Nicotiana debneyi, Chenopodium amaranticolor and Ch. quinoa. The virus was detected with antisera to the related viruses tobacco necrosis virus A and tobacco necrosis virus D. The close association to the tobacco necrosis viruses would suggest this virus is not a new introduction to potato but in the past has been misidentified as one of these viruses. The virus isolate has been named potato necrosis virus.

Incidence and characterization of Potato virus V infections in Iran.

A survey was carried out to determine the extent of Potato virus V (PVV) infection, together with other potyviruses, in Iran in both commercial and local potato varieties. We found a low incidence of PVV in commercial varieties compared to a local potato cultivar Zardi, in which we noted a PVV infection up to ~32.9%. We determined the genomic sequence 9,812 nucleotides of one isolate (KER.LAL.P) from cultivar Agria and the 3'-terminal sequence including coat protein (CP) gene of four additional isolates from cultivar Zardi. The Iranian isolate PVV KER.LAL.P was found to share 91% sequence identity with the Scottish isolate DV-42 (AJ243766) of PVV. The CP gene sequences of the PVV isolates from Iran shared 96.5 to 99.3% pairwise nucleotide identity and they shared <97.5% pairwise identity with the CP sequences of all other PVV isolates available in public databases. Our host studies indicated that the Iranian PVV isolate had a narrower host range and infected Nicotiana debneyi and N. glutinosa test plants. Within the commercial varieties of potato in Iran, we noted a significant amount of mixed potyvirus infection. This study is the first report of occurrence and complete genome of PVV in Iran.

Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR

The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in Nicotiana debneyi plants grown in putative viruliferious soils from 20/26 locations. The results were confirmed by back inoculation of sap from both roots and leaves of bait plant on indicator hosts (N. debneyi, Nicotiana benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26 locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566bp were amplified from coat protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait plant bioassay, infectivity assay, ELISA and RT-PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea and tubers of 10 potato cultivars commercially grown in the region.

Nicotiana debneyi has a single dominant gene causing hybrid lethality in crosses with N. tabacum

To elucidate the genetic mechanism of hybrid lethality observed in hybrids between cultivated tobacco, Nicotiana tabacum, and wild tobacco species in the section Suaveolentes, genetic analyses were conducted through the triple cross of the hybrids of wild species, including N. benthamiana and N. fragrans, and N. tabacum. N. benthamiana and N. fragrans produced only viable hybrids after crossing with N. tabacum. Subsequently, N. benthamiana and N. fragrans were crossed with N. africana, N. debneyi, and/or N. suaveolens, which produced inviable hybrids after crossing with N. tabacum. Hybrids of the wild species were obtained from four of the six cross combinations. Only when hybrid plants of N. debneyi × N. fragrans, whose hybridity was confirmed by morphological characteristics, random amplified polymorphic DNA analysis, and chromosome observation, were crossed with N. tabacum, triple hybrids were obtained and segregated 1:1 (lethal:viable). Based on these results, a single dominant gene, designated Hybrid Lethality A1 (HLA1), in N. debneyi was found to control hybrid lethality by the interaction with gene(s) on the Q chromosome in N. tabacum. This represents the first report to identify a causal gene for hybrid lethality in the genus Nicotiana.

Sources of resistance against the Pepper yellow mosaic virus in chili pepper

The Pepper yellow mosaic virus (PepYMV) naturally infects chili and sweet pepper, as well as tomato plants in Brazil, leading to severe losses. This work reports the reaction to the PepYMV of 127 Capsicum spp. accessions, aiming at identifying resistance sources useful in breeding programs. The experiment was carried out in a completely randomized design, with eight replications, in greenhouse conditions. Plants were protected with an insect-proof screen to avoid virus dissemination by aphids. Leaves of Nicotiana debneyi infected with the PepYMV were used as the inoculum source. Plants were inoculated with three to four fully expanded leaves. A second inoculation was done 48 hours later to avoid escapes. Only the youngest fully expanded leaf was inoculated. Two plants were inoculated only with buffer, as negative control. Symptoms were visually scored using a rating scale ranging from 1 (assymptomatic plants) to 5 (severe mosaic and leaf area reduction). Nine accessions were found to be resistant based on visual evaluation. Their resistance was confirmed by ELISA. Two resistance accessions belong to the species C. baccatum var. pendulum, while the seven other were C. chinense. No resistant accessions were identified in C. annuum var. annuum, C. annuum var. glabriusculum, and C. frutescens.

Possible involvement of genes on the Q chromosome of Nicotiana tabacum in expression of hybrid lethality and programmed cell death during interspecific hybridization to Nicotiana debneyi

Hybrid seedlings from the cross between Nicotiana tabacum, an allotetraploid composed of S and T subgenomes, and N. debneyi die at the cotyledonary stage. This lethality involves programmed cell death (PCD). We carried out reciprocal crosses between the two progenitors of N. tabacum, N. sylvestris and N. tomentosiformis, and N. debneyi to reveal whether only the S subgenome in N. tabacum is related to hybrid lethality. Hybrid seedlings from reciprocal crosses between N. sylvestris and N. debneyi showed lethal characteristics identical to those from the cross between N. tabacum and N. debneyi. Conversely, hybrid seedlings from reciprocal crosses between N. tomentosiformis and N. debneyi were viable. Furthermore, hallmarks of PCD were observed in hybrid seedlings from the cross N. debneyi x N. sylvestris, but not in hybrid seedlings from the cross N. debneyi x N. tomentosiformis. We also carried out crosses between monosomic lines of N. tabacum lacking the Q chromosome and N. debneyi. Using Q-chromosome-specific DNA markers, hybrid seedlings were divided into two groups, hybrids possessing the Q chromosome and hybrids lacking the Q chromosome. Hybrids possessing the Q chromosome died with characteristics of PCD. However, hybrids lacking the Q chromosome were viable and PCD did not occur. From these results, we concluded that the Q chromosome belonging to the S subgenome of N. tabacum encodes gene(s) leading to hybrid lethality in the cross N. tabacum x N. debneyi.

IDENTIFICATION OF TOMATO MOSAIC VIRUS INFECTION IN JASMINE

Virus-like symptoms were recently observed on leaves of landscape and nursery downy and star jasmine (Jasminum multiflorum) and wax jasmine (J. gracile) in southeast Florida. Foliar symptoms included mottling, chlorotic ring spots and chlorotic line patterns. An agent was mechanically transmitted with difficulty from symptomatic leaves of downy jasmine to Nicotiana debneyi and N. tabacum 'Xanthi' and subsequently from these hosts to Chenopodium quinoa and other herbaceous test plants. Virions were isolated from N. tabacum 'Xanthi.' Rod-shaped particles (297 x 18 nm) similar to tobamoviruses were observed in partially purified virus preparations, and in leaf dips from symptomatic star jasmine and indicator plants. Extraction of viral-associated double-stranded (ds) RNA revealed a profile consistent with that of a tobamovirus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the virus contained one polypeptide with an approximate molecular weight (Mr) of 18 kDa. The virus reacted specifically with IgG for Tobacco mosaic virus (TMV) and related tobamoviruses [including Tomato mosaic virus (ToMV)] in double antibody sandwich, enzyme-linked immunosorbent assay. No reaction was observed with TMV specific IgG. Reverse transcription-polymerase chain reaction with total RNA isolated from symptomatic jasmine leaves and infected N. tabacum 'Xanthi' using ToMV coat protein specific primers amplified the expected product from downy and star (but not wax) jasmine and N. tabacum 'Xanthi.' The nucleotide and amino acid sequence of the products were 100% identical to the corresponding fragment of a Brazilian isolate of ToMV from Impatiens. To our knowledge, this the first report of ToMV in jasmine in the USA.

Resistance to Tobacco Black Shank in Nicotiana Species

Black shank [caused by Phytophthora parasitica (Dast.) var. nicotianae (B. de Haan) Tucker] is one of the most devastating diseases in tobacco (Nicotiana tabacum L.). Phytophthora parasitica var. nicotianae race 1 is the most damaging race of this fungus. The objective of this study was to identify the Nicotiana species that are resistant to P. parasitica var. nicotianae race 1 as part of the effort to develop Nicotiana‐based plant varieties for applications in the production of plant‐made pharmaceuticals (PMPs). The roots of approximately 26‐d‐old growth room–grown plants were inoculated with zoospores of an isolate of P. parasitica var. nicotianae race 1 by a dipping procedure. The percentage of plants that were free of symptoms 10 to 14 d after inoculation was taken as a measure of resistance to the fungus. We evaluated a total of 97 Nicotiana accessions from 37 species from the USDA collection and other sources. Among the 97 accessions, three from N. debneyi Domin, two from N. repanda Willd. ex Lehm., and one each from N. megalosiphon Van Heurck &amp; Mull. Arg., N. plumbaginifolia Viv., N. suaveolens Lehm., and N. sylvestris Speg. &amp; Comes were found to be resistant. Their resistances were further confirmed after they were evaluated for an additional three times. These nine accessions were then challenged by another isolate of race 1. All accessions except for the N. sylvestris accession were found to be resistant. Nicotiana debneyi, N. megalosiphon, and N. suaveolens were not previously reported to be resistant to P. parasitica var. nicotianae race 1. Our study indicated that the undomesticated Nicotiana species constitute a rich genetic source for resistance to the tobacco disease black shank.

Development of SCAR markers linked to three disease resistances based on AFLP within Nicotiana tabacum L

Amplified fragment length polymorphism (AFLP) was conducted on a set of 92 Nicotiana tabacum L. accessions from diverse types (flue-cured, dark air-cured, burley, oriental, and cigar wrapper) and breeding origins to identify markers associated with disease resistances. Eleven primer combinations were required to identify 33 polymorphic fragments. This allowed the identification of 92% of these accessions, and yielded sufficient information for building a neighbor joining tree. Clusters of accessions with common traits or breeding origins were observed. An important part of this polymorphism could be related to interspecific introgressions from other Nicotiana species, performed during the breeding history of N. tabacum to confer resistance to pathogens. Seven fragments were associated with three different resistances: two for the blue-mold (Peronospora tabacina Adam) resistance derived from Nicotiana debneyi Domin, two for the Va gene (Potato Virus Y susceptibility), and three for the black root rot (Chalara elegans) resistance of N. debneyi origin. Some of these markers were converted into sequence characterized amplified region markers, and validated on recombinant inbred lines or doubled-haploid lines.

Nicotiana megalosiphon, a Highly Susceptible, New, and Useful Host for Potato Virus A.

Potato virus A (PVA; genus Potyvirus, family Potyviridae) occurs wherever potatoes are grown and may reduce tuber yields as much as 40%. Its host range consists of six experimental hosts (Lycopersicon pimpinellifolium (Jusl.) P. Mill., Nicandra physalodes (L.) Gaertn., Nicotiana tabacum L., Solanum demissum Lindl., S. demissum × S. tuberosum [2], and Nicotiana debneyi Domin.) and two natural hosts (S. tuberosum L. and S. betaceae (Cav.) Sendt.) (2). Aphids transmit PVA in a stylet-born manner. Its difficult mechanical transmission, caused by a low virus concentration in potato and other hosts (1), has constrained pathological research on the virus. In routine studies to identify virus isolates from the field, we discovered that N. megalosiphon Van Heurck & Mull. Agr. is a superior host of PVA that markedly facilitated diagnosis, selection for resistance to PVA, and other research applications. The efficiency of mechanical transmission of PVA to potato (5 duplicated assays and 10 plants per assay) ranged from 0 to 10% with PVA-infected potato as the virus source, 0 to 30% with Nicandra physalodes, 10 to 30% with N. tabacum cv. Samsun, and 20 to 80% with N. megalosiphon as the source of virus. The efficiency of mechanical transmission to four systemic hosts of PVA with potato (cv. Russet Burbank) as the source of virus (5 duplicated assays and 10 plants per assay) ranged from 0 to 20% to potato, 0 to 30% to Nicandra physalodes, 10 to 40% to N. tabacum cv. Samsun, and 80 to 100% to N. megalosiphon. The superiority of N. megalosiphon as a host and source of PVA was associated with a high virus concentration in tissues. Infected potato leaves yielded 0.32 to 0.54 mg of virus per kg of infected leaves, Nicandra physalodes yielded 0.37 to 0.66 mg per kg, N. tabacum cv. Samsun yielded 0.78 to 1.22 mg per kg, and N. megalosiphon yielded 5.16 to 9.39 mg per kg of infected leaves in five different purification experiments. These yields are based on the amount of virus isolated in sucrose gradients subjected to rate-zonal centrifugation as the last step in purification (3). The virus antigen concentrations of the original PVA-infected tissues measured using quantitative enzyme-linked immunosorbent assay ranked virus concentrations in the same relative order as purification but were nearly 2 times higher than were the purification yields. Similarly, local lesion assays on S. demissum A leaves (4) ranked infectious virus concentrations in the same order as did purification. Efficiency of aphid transmission from the four hosts was not assayed. Infected N. megalosiphon plants survived and served as sources of PVA for at least 1 year in a greenhouse. N. megalosiphon is an important new host of PVA because it facilitates the routine transmission of the virus and other manipulations essential for efficient research on control of the virus disease.

Black and Hairy Nightshade, the First Weedy Hosts of Potato virus A in the United States

Potato virus A (PVA; genus Potyvirus, family Potyviridae) occurs wherever potatoes are grown and may reduce tuber yields as much as 40%. Its host range consists of six experimental hosts (Lycopersicon pimpinellifolium (Jusl.) P. Mill., Nicandra physalodes (L.) Gaertn., Nicotiana tabacum L., Solanum demissum Lindl., S. demissum × S. tuberosum, and Nicotiana debneyi Domin.) and two natural hosts (S. tuberosum L. and S. betaceae (Cav.) Sendt.) (1). N. physalodes and Nicotiana tabacum sometimes grow in nature in the eastern and southern United States (2) but have not been reported to be PVA sources in nature. PVA is known to survive winter in temperate climates only in tubers used for potato propagation and potato volunteers. We detected PVA in all six Black Nightshade (S. nigrum L.) and all six Hairy Nightshade (S. sarrachoides Sendt.) weeds expressing mild mosaic symptoms and growing in PVA-infected potato fields near Prosser, WA in September of 2002. PVA was not detected in any of the 24 Cutleaf Nightshade (S. trifolium) plants selected from the same fields. The virus was detected using double antibody sandwich enzyme-linked immunosorbent assay with a test kit obtained from Agdia, Inc. (Elkhart, IN) and by reverse transcription-polymerase chain reaction using primers designated 3 (gta-ctg-aac-tgg-aaa-agt-act) and 4 (ccc-tga-cag-ttg-aaa-cat-aag) (3). One positive control for these assays was a PVA virus isolate from the "Shultz Collection" provided by Robert Goth (USDA, ARS, Beltsville, MD) in 1986. The Agdia, Inc. test kit also provided a positive control. We also transmitted PVA from one of the Black Nightshade and one of the Hairy Nightshade weeds to greenhouse-produced Nicotiana megalosiphon Van Heurck & Mull. Agr., Nicotiana tabacum cv. Samsum NN, S. tuberosum cv. Russet Burbank, S. sarrachoides, and S. nigrum plants by Myzus persicae Sulzer in a stylet-borne manner. The virus was transmitted from the greenhouse test plants back to S. sarrachoides and S. nigrum plants where it produced mild mosaic symptoms identical to virus from the original sources. PVA was not transmitted from weedy sources to Datura tatula L., Physalis floridana Rydb., or S. trifolium. M. persicae readily colonized S. sarrachoides, S. nigrum, and S. trifolium in the field and greenhouse. Since black and hairy nightshade are frequently found in potato fields (4) and our findings show these weeds to be hosts of PVA, their role in the epidemiology of this disease should be more fully evaluated.

Detection and Confirmation of Potato mop-top virus in Potatoes Produced in the United States and Canada.

Potato mop-top virus (PMTV) was detected in potatoes grown in the United States and Canada during surveillance testing by a reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene in RNA3. Out of 3,221 lots of seed and ware potatoes that were tested, 4.3% were positive for PMTV. The reliability of the survey results was confirmed by reextraction of selected samples and additional RT-PCR tests using two primer sets targeting gene segments in RNA2 and RNA3. Amplicons generated from RNA2 and RNA3 were identified by analysis of fragment length polymorphisms after digestion with BamHI and HindIII, respectively. PMTV was further identified by enzyme-linked immunosorbent assay, bioassay on Nicotiana debneyi, and transmission electron microscopy. Sequencing of a portion of the coat protein gene revealed near 100% identity among isolates from the United States and Canada and >97% homology of the North American isolates with European isolates.

Effect of the Nicotiana debneyi black root rot resistance gene on the yield and quality characteristics of flue-cured tobacco in Ontario

The effects of Nicotiana debneyi-derived resistance to black root rot disease were evaluated for yield, agronomic and quality traits by comparing the near isogenic cultivars AC Gayed (resistant) and Delgold (susceptible). Over 7 yr of trials the possession of resistance led to yields and economic returns that averaged 6 and 7% lower, respectively, than for the susceptible line. Key words: Flue-cured tobacco, Nicotiana tabaccum, Black Root Rot, Chalara elegans, Nicotiana debneyi, yield, quality

A new host plant species for the cassava biotype of Bemisia tabaci (Gennadius) (Hom., Aleyrodidae)

Abstract: The cassava biotype of Bemisia tabaci (Gennadius) is known to colonize cassava (Manihot esculenta Crantz) whereas the sweet potato biotype colonizes various plant and weed species but not cassava. Both biotypes have Solanum melongena L. and Solanum aethiopicum L. as common host plant species. This study shows for the first time that both biotypes can successfully feed, survive and reproduce on a new host plant species, Nicotiana debneyi Domin. These findings have important implications for better understanding the disease epidemics, related to whitefly transmissible geminiviruses.

Temperature-dependent Apoptosis Detected in Hybrids between Nicotiana debneyi and N. tabacum Expressing Lethality

Interspecific hybrids of Nicotiana debneyi × N. tabacum L. show hybrid lethality, which is one of the mechanisms for reproductive isolation. Apoptotic changes were detected in the cells of hybrid seedlings expressing lethality at 28°C, but not at high temperature (34°C) indicating lethality was suppressed. When hybrid seedlings cultured at 34°C were transferred to 28°C, lethal symptoms appeared. Chromatin condensation and nuclear fragmentation were observed in leaf protoplasts isolated from hybrid seedlings expressing lethality. Agarose gel analysis of DNA extracted from leaves of hybrid seedlings revealed a specific ladder pattern, suggesting nucleosomal fragmentation. Nuclear fragmentation was correlated with lethal symptoms in hybrid seedlings, as confirmed by flow cytometry. These results were observed in hybrid seedlings transferred to 28°C from 34°C but not in hybrid seedlings maintained at 34°C. This phenomenon suggests that apoptosis in hybrid seedlings from the cross N. debneyi×N. tabacum is temperature dependent.

The roles of Ri rol and Ng rol genes in hairy root induction in Nicotiana debneyi

The function of Ri rol genes in TL-DNA of the Ri plasmid of Agrobacterium rhizogenes has been previously studied in Nicotiana tabacum and Daucus carota, but it was reported that these plants have a TL-DNA-similar sequence in their genome. We investigated the function of Ri rol genes in N. debneyi by infection with A. tumefaciens harboring these genes, because the genome of N. debneyi does not contain a TL-DNA-similar sequence. The single gene Ri rolB induced adventitious roots in N. debneyi. Introduction of a DNA fragment that contained Ri rolB, Ri rolC, RiORF13 and RiORF14 resulted in more intense and earlier root formation than that of Ri rolB. Ng rol genes (Ng rolB, Ng rolC, NgORF13, and NgORF14) in the genome of Nicotiana glauca that are similar in sequence to Ri rol genes were also examined. In contrast with Ri rol genes, Ng rol genes did not induce adventitious roots on leaf segments of N. debneyi. Further infection analysis revealed that one of the reasons for this diversity of their functions might be the difference in the rolB region between the sequence of bacteria and plants. The difference in function between the genes of plants and bacteria is analyzed and the molecular evolution of Ng rol genes is discussed.

Isolation and characterization of Tnd-1, a retrotransposon marker linked to black root rot resistance in tobacco

In Nicotiana debneyi, resistance to a wide range of black root rot (Chalara elegans) isolates is conferred by a single dominant gene. This gene has been transferred to cultivated tobacco (Nicotiana tabacum) and was recently discovered to be linked in coupling to a 1050-bp random amplified polymorphic DNA (RAPD) marker generated with the UBC 418 primer. We have cloned and sequenced the UBC4181050 marker and found it to be part of a retrotransposon. This retrotransposon is a remnant of the N. debneyi genome and is the first to be isolated from this species. Transposon N. debneyi (Tnd)-1 is present in the tobacco genome as two directly repeated copies, and in multiple copies in the donor species N. debneyi and in a number of related Nicotiana species. The retrotransposon appears to have been introduced into the Nicotiana genome after the development of the Suavolentes progenitors. The gene associated with black root rot resistance co-segregates with the retrotransposon in tobacco and is thought to be contained within the introgressed fragment marked by Tnd-1. The retrotransposon will therefore be a useful species-specific landmark that can be used for future cloning of the resistance gene.

Three novel Nicotiana debneyi specific repetitive DNA elements derived from a RAPD marker

The RAPD marker UBC4181050, tightly linked in coupling with the Nicotiana debneyi gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris), has been cloned and sequenced. The terminal 10 bases of this RAPD marker exactly match the sequence of the primer UBC418. UBC4181050 was restricted into 6 subfragments of 75, 91, 110, 174, 274, and 336 bp (the accession numbers for these 6 subfragments in the GenBank Data Base are U84217, U84218, U84219, U84220, U84221, and U84222, respectively) by combined restriction with HindIII and RsaI. The entire UBC4181050 marker and its 6 subfragments were used as probes in RFLP analyses. The RFLP analyses were performed on 'Delgold' tobacco (Nicotiana tabacum L.), the tobacco plants (2n = 48) recovered from the somatic hybrids between 'Delgold' and N. debneyi, the 'Delgold' tobacco translocation line carrying the N. debneyi gene for resistance to black root rot, the 'Delgold' tobacco addition lines carrying various N. debneyi chromosomes, and an additional 27 species in the genus Nicotiana. The analyses indicated that UBC4181050 is composed of at least 6 different repetitive DNA elements that are independently interspersed in N. debneyi genomes. Three of them (subfragments of 91, 274, and 336 bp) are N. debneyi specific. The N. debneyi specific repetitive DNA elements are distributed primarily within the taxonomic section (Suaveolentes) to which N. debneyi belongs.Key words: RAPD, repetitive DNA, Nicotiana debneyi, species-specific.