Host resistance is an important strategy for managing Globodera tabacum subsp. solanacearum and G. tabacum subsp. tabacum, important nematode pests of flue-cured tobacco (Nicotiana tabacum) in Virginia, and cigar wrapper tobacco (N. tabacum) in Connecticut and Massachusetts, respectively. Field research from 1992 to 2005 evaluated reproduction of G. tabacum subsp. solanacearum on genotypes with and without a chromosome segment from N. plumbaginifolia containing a gene (Php) that conferred resistance to race 0 of Phytophthora nicotianae (causal agent of tobacco black shank). Ratios of G. tabacum subsp. solanacearum eggs/500 cm3 soil at the end versus the beginning of experiments (Pf/Pi) were significantly lower in cultivars and breeding lines possessing the Php-containing chromosome segment from N. plumbaginifolia compared with genotypes without the segment. Numbers of vermiform G. tabacum subsp. solanacearum juveniles in roots were similar among genotypes but numbers of swollen and pyriform nematodes were significantly lower for the known G. tabacum subsp. solanacearum resistant cv. NC 567 and in genotypes possessing the Php gene compared with genotypes and cultivars without the gene. In a 2003 greenhouse test, the percentage of plants with visible G. tabacum subsp. tabacum cysts was also significantly lower for parental and progeny genotypes homozygous and heterozygous, respectively, for Php compared with similar lines without the gene. These results indicate a close linkage or association between a likely single, dominant gene (Php) for resistance to P. nicotianae and suppressed reproduction by G. tabacum subsp. solanacearum and G. tabacum subsp. tabacum. Further research to accurately elucidate the relationships among these genes could lead to significant improvements in tobacco disease control.

Resistance to tobacco mosaic virus (TMV) is controlled by the single dominant gene N in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) by interspecific hybridization and backcrossing, but has historically been associated with reduced yields and/or quality in flue-cured tobacco breeding materials. Past researchers have suggested the role of pleiotropy and/or linkage drag effects in this unfavorable relationship. Introduction of the cloned N gene into a TMV-susceptible tobacco genotype (cultivar 'K326') via plant transformation permitted investigation of the relative importance of these possibilities. On average, yield and cash return ($ ha(-1)) of 14 transgenic NN lines of K326 were significantly higher relative to an isoline of K326 carrying N introduced via interspecific hybridization and backcrossing. The negative effects of tissue culture-induced genetic variation confounded comparisons with the TMV-susceptible cultivar, K326, however. Backcrossing the original transgenic lines to non-tissue cultured K326 removed many of these unfavorable effects, and significantly improved their performance for yield and cash return. Comparisons of the 14 corresponding transgenic NN backcross-derived lines with K326 indicated that linkage drag is the main factor contributing to reduced yields in TMV-resistant flue-cured tobacco germplasm. On average, these transgenic lines outyielded the conventionally-developed TMV-resistant K326 isoline by 427 kg ha(-1) (P < 0.05) and generated $1,365 ha(-1) more (P < 0.05). Although transgenic tobacco cultivars are currently not commercially acceptable, breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N may increase the likelihood of developing high-yielding TMV-resistant flue-cured tobacco cultivars.

Flue-cured tobacco (Nicotiana tabacum) cultivar Coker 371-Gold (C 371-G) possesses a dominant gene, Ph, that confers high resistance to black shank disease, caused by race 0 of the soil-borne pathogen Phytophthora parasitica var. nicotianae. The origin of this gene is unknown. Breeding lines homozygous for the Ph gene were hybridized with NC 1071 and L8, flue-cured and burley genotypes known to possess qualitative resistance genes from Nicotiana plumbaginifolia and N. longiflora, respectively. The F1 hybrids were out-crossed to susceptible testers and the progenies evaluated in field black shank nurseries and in greenhouse disease tests with P. parasitica var. nicotianae race 0. Results showed that Ph was allelic to Php from N. plumbaginifolia in NC 1071. Testcross populations of hybrids between burley lines homozygous for Ph and L8, possessing Phl from N. longiflora, showed that Ph and Phl integrated into the same tobacco chromosome during interspecific transfer. Nevertheless, the two loci were estimated to be 3 cM apart. Random amplified polymorphic DNA (RAPD) analyses of the testcross progenies confirmed that recombination between the two loci was occurring. Forty-eight RAPD markers linked to Ph in doubled haploid lines were used in cluster analyses with multiple accessions of N. longiflora and N. plumbaginifolia, breeding lines L8, NC 1071, and DH92-2770-40, and cultivars K 326, Hicks, and C 371-G. A cladogram or region tree confirmed the data obtained from field and greenhouse trials, that Ph, transferred from C 371-G to DH92-2770-40, and Php in NC 1071 were allelic and originated from N. plumbaginifolia.

Bulked segregant (BSA) and random amplified polymorphic DNA (RAPD) analyses were used to identify markers linked to the dominant black shank resistance gene, Ph, from flue-cured tobacco (Nicotiana tabacum) cv. Coker 371-Gold. Sixty RAPD markers, 54 in coupling and 6 in repulsion phase linkage to Ph, were identified in a K 326-derived BC1F1 (K 326-BC1F1) doubled haploid (DH) population. Thirty RAPD markers, 26 in coupling and 4 in repulsion phase linkage to Ph, were used to screen 149 K 326-BC2F1 haploid plants. Complete linkage between the 26 coupling phase markers and Ph was confirmed by screening 149 K 326-BC2F1 DH lines produced from the haploid plants in black shank nurseries. RAPD markers OPZ-5770 in coupling and OPZ-7370 in repulsion phase linkage were used to select plants homozygous for the Ph gene for further backcrossing to the widely grown flue-cured cultivar K 326. Black shank disease nursery evaluation of 11 K 326-BC4S1 lines and their testcross hybrids to a susceptible tester confirmed linkage between Ph and OPZ-5770. The results demonstrated the efficiency of marker-assisted selection for Ph using a RAPD marker linked in coupling and repulsion. Complete linkage between 26 RAPD markers and the Ph gene was confirmed in the K 326-BC5 generation, and RAPD phenotypes were stable across generations and ploidy levels. These RAPD markers are useful in marker-assisted selection for Ph, an important black shank resistance gene in tobacco.

Gene cloning and transformation can be used to circumvent linkage drag effects that can plague conventional interspecific gene transfers. These techniques can also be used to create desirable genetic linkages. Use of Nicotiana glutinosa L. N‐gene mediated TMV (tobacco mosaic virus) resistance in flue‐cured tobacco, N tabacum L., has been limited due to linkage drag effects. Transformation was used to introduce the cloned N‐gene into NC152, a chromosome addition line possessing a chromosome pair from N africana. This chromosome has been proposed to be used as a “designer chromosome” into which numerous transgenes could be inserted to form a desirable linkage package. The system could be used to shuttle a large number of transgenes from genotype to genotype. One hundred thirty‐six primary transformants possessing the N transgene were produced and hybridized with TMV‐susceptible ‘Petite Havana.’ These may serve as valuable TMV‐resistant breeding materials. For each independent transformant, BC1F1 families which segregated for TMV resistance and the addition chromosome were generated. Data from cosegregation, transmission, and molecular analyses were used to conclude that one transformant possessed an insertion of the N‐gene in the addition chromosome. By inserting N in the chromosome, we initiated construction of a disease resistance package by linking the TMV resistance gene with a potyvirus resistance gene(s) native to the chromosome. Occasional loss of the transgene, however, may be evidence of previously undetected interchromosomal recombination, and may have implications for use of this system in cultivar development.

Random amplified polymorphic DNA (RAPD) analysis was conducted to map the Rk gene in tobacco which conditions resistance to races 1 and 3 of the root-knot nematode, Meloidogyne incognita. Resistant burley tobacco genotype NC 528, containing the Rk gene, and the susceptible cultivar Ky 14 were screened with 1,500 random decamers. A low rate of genetic polymor-phism (<10%) was detected among these lines. Two populations (F1 and F3) of maternally de-rived doubled haploid (MDH) lines of burley tobacco, developed from the cross NC 528 × Ky 14, were used to map the Rk gene. NC 528, Ky 14, three Rk-resistant (Rk-R) DNA bulks, andthree Rk-susceptible (Rk-S) bulks generated from F1-derived MDH individuals were screenedwith the primers that amplified bands polymorphic between Rk-R and Rk-S lines. A total of 67 F1MDH lines and 59 F3MDH lines were screened with the primers that amplified bands polymorphic between Rk-R bulks and Rk-S bulks to confirm linkage between candidate markers and the Rk gene. Sixteen RAPD markers were positioned at six loci in a map 24.1 centimorgans long. Six RAPD markers, including one identified in the F3MDH population, were mapped at the Rk locus.

Tobacco lines expressing transgenes that encode tobacco etch virus (TEV) coat protein (CP) mRNA with or without nonsense codons give rise to TEV-resistant tissues that have reduced levels of TEV CP mRNA while maintaining high levels of transgene transcriptional activity. Two phenotypes for virus resistance in the lines containing the transgene have been described: immune (no virus infection) and recovery (initial systemic symptoms followed by gradual recovery over several weeks). Here, we show that at early times in development, immune lines are susceptible to TEV infection and accumulate full-length CP mRNA. Therefore, immune lines also exhibit meiotic resetting, as is seen in the recovery lines, providing molecular evidence for a common mechanism of gene silencing and virus resistance in both cases. We also investigated the characteristics of two sets of low molecular weight RNAs that appear only in silenced tissue. One set has nearly intact 5[prime] ends, lacks poly(A) tails, and is associated with polyribosomes; the second set contains the 3[prime] end of the mRNA. Treating silenced leaf tissue with cycloheximide resulted in decreased levels of full-length mRNA and an increase in the levels of the low molecular weight RNAs, supporting a cytoplasmic decay mechanism that does not require ongoing translation. Surprisingly, mRNA from the transgene containing nonsense codons was associated with more ribosomes than expected, possibly resulting from translation from a start codon downstream of the introduced translational stop codons. We present a hypothesis for transgene/viral RNA degradation in which RNA degradation occurs in the cytoplasm while in association with polyribosomes.

Black shank, caused by Phytophthora parasitica var. nicotianae, is a widespread and severe disease of tobacco throughout the southeastern United States. Partial resistance derived from the cigar tobacco cultivar Florida 301 has been the primary means of reducing losses to the disease for many years. The recently released tobacco cultivar, Coker 371-Gold (C 371-G), was found to provide an additional source of resistance to P. parasitica var. nicotianae. Although the resistance in C 371-G is being used widely by breeders, the origin and inheritance of this resistance mechanism was unknown. Two populations of doubled haploid lines derived from C 371-G were used to determine that C 371-G possesses a single, dominant gene designated Ph, which confers a very high level of resistance to race 0 of P. parasitica var. nicotianae. A greenhouse inoculation procedure was developed that provided an efficient means of screening for the presence of this resistance gene prior to selection in the field, and confirmed that Ph provides complete resistance to race 0 but no resistance to race 1 of P. parasitica var. nicotianae. Because Florida 301 resistance is effective against both races of the pathogen that occur in the major tobacco growing areas of the United States, combination of these two sources of resistance should provide enhanced protection of new tobacco cultivars to P. parasitica var. nicotianae.

RNA-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. This study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (TEV). Transgenic tobacco line 2RC-6.13, expressing an untranslatable mRNA containing the TEV coat protein open reading frame, had three distinct transgene integration events that segregated as two linkage groups. A genetic series of plants that contained zero, one, two, or all three transgene inserts in both homozygous and heterozygous conditions was produced and examined. Genetic and biochemical data suggested that RNA-mediated virus resistance is a multigenic trait in line 2RC-6.13; three or more transgenes were necessary to establish the highly resistant state. One or two transgene copies resulted in an inducible form of resistance (i.e., recovery). Transcription rates and steady state RNA levels of the transgene-derived transcript present in different members of the genetic series supported a post-transcriptional RNA degradation process as the underlying mechanism for transgene transcript reduction and virus resistance. This degradation process appeared to initiate via cleavage of specific sites within the target RNA sequence, as determined by RNA get blot and primer extension analyses of transgene-derived mRNA from various transgenic plant lines.

Transgenic burley (cultivars KY14, NC3433-33, and TN90) and flue-cured (cultivar K326) tobacco with resistance to four necrotic isolates of potato virus Y (PVY) were constructed. These tobacco cultivars were transformed with a chimeric gene designed to express the coat protein (CP) from the necrotic Chilean isolate of PVY CP expression among R(0) plants was undetectable by indirect enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis. Transgenic R(0) plants that harbored from one to five neomycin phosphotransferase II (NPT II) transgene loci were identified. Although the NPT II and the PVY CP chimeric genes were linked within a common T-DNA, no correlation was found between the number of NPT II and CP transgenes. Inoculation of 41 independent transgenic KY14 R(0) plants with PVY-Chilean identified eight resistant plants, while inoculation of 30 transgenic K326 Ro plants identified six resistant plants. Inoculation of 17 transgenic NC3433-33 R(0) plants with PVY-Chilean identified six resistant plants, while inoculation of 50 transgenic TN90 R(0) plants with PVY isolate VAM B resulted in nine resistant plants. Progeny derived from PVY-inoculated but symptomless Ro plants were also resistant to the Chilean, Europe H, M(s)N(r), N-Canada, and VAM B isolates of PVY, demonstrating sexual transmission of the transgenes that condition resistance. We discuss the ability of the PVY-Chilean CP gene to protect transgenic tobacco K326, KY14, NC3433-33, and TN90 from infection by four necrotic isolates of PVY. The flue-cured tobacco cultivar K326, which carries a gene simultaneously conferring resistance against root-knot nematode and susceptibility to PVY strain M(S)N(R), is protected against this strain when transformed with the PVY-Chilean CP gene.

Haploid leaf tissue of tobacco cultivars K326 and K149 was transformed with several transgenes containing cDNA of the potato virus Y (PVY) coat protein (CP) open reading frame (ORF). The various transgenes containing the PVY CP ORF sequence produced (1) the expected mRNA and CP product, (2) an mRNA rendered untranslatable by introduction of a stop codon immediately after the initiation codon, or (3) an antisense RNA that was untranslatable as a result of the incorrect orientation of the PVY CP ORF behind the transcriptional promoter. Homozygous doubled haploid (DH) (diploid) plants were generated, and selfed progeny from these plants were examined. Resistance was virus specific, functioning only against PVY. An inverse correlation between transgene-derived PVY transcript steady state levels and resistance was generally noted with lines expressing the untranslatable sense version of the PVY CP ORF. A collection of DH lines, derived from a single transformation event of a common haploid plant and isogenic for the PVY transgenes expressing untranslatable sense RNA, displayed different levels of PVY resistance. Lines with actively transcribed, methylated transgene sequences had low steady state levels of transgene transcript and a virus-resistant phenotype. These results are discussed within the context of sense suppression in plants.

Black shank, caused by Phytophthora parasitica Dast var. nicotianae [Breda de Haan] Tucker, is a common disease of tobacco (Nicotinna tabacum L.). Selection for genetic resistance to black shank within an F1‐derived haploid population is more efficient than selection within an F2 diploid population. Our objectives were to identify black shank resistance in three segregating haploid tobacco populations and to verify that disease resistance assessed on haploids corresponded to that of doubled haploids (DHs). Population 1 was derived from ‘McNair 944’ × ‘ZA 692’, Populations 2 from ‘Coker 371‐Gold’ (C 371‐G) × ‘K 326’, and Population 3 from ‘VA 509’ × breeding line MDH 410. Haploids were transplanted into a disease nursery and disease incidence was recorded weekly throughout the growing season. We observed segregation for resistance in all three haploid populations. Rescue and chromosome doubling was successful for 85% of selected haploids. In the flue‐cured populations (1 and 2), all but one selected haploid was resistant as a DH. Transgressive segregation for resistance was identified in Population 1. The resistance of a population produced by Intel‐mating Population 1 DHs was equal to that of McNair 944, the resistant parent. Resistance of Population 2 DHs derived from selected haploids was equal to that of C 371‐G, the resistant parent. Selected DHs from Population 2 were significantly more resistant than both K 326 and an unselected DH population derived from the same cross. In Population 3, the hurley population, differences between parents and among DHs were difficult to detect and should be assessed in multiple environments.

The tobacco (Nicotiana tabacum L.) breeding line NC 152 is a doubled haploid that possesses an addition chromosome from N. africana [Merxm. and Buttler]. A gene on this chromosome confers potyvirus resistance (Poty(R)). Our objective was to use the addition chromosome as a base on which to construct a designer chromosome containing a foreign gene linkage package. A mutant dhfr gene conferring resistance to methotrexate (Mtx) was inserted into NC 152-haploid (n = 25) leaf tissue via Agrobacterium tumefaciens-mediated transformation. After chromosome doubling, 135 NC 152dhfr transformants (2n = 50) were pollinated with the potyvirus-susceptible (Poty(S)) cultivar 'McNair 944' (2n = 48). Linkage analysis was performed in the BC1 generation. Two transformants, NC 152dhfr-996 and NC 152dhfr-1517 exhibited complete linkage between Mtx resistance (Mtx(R)) and Poty(R). Segregants from these two transformants which were Mtx(R) and Poty(R) possessed 49 chromosomes, while Mtx sensitive (Mtx(S)) and Poty(S) progeny possessed 48 chromosomes. Eighty percent of the NC 152dhfr transformants transmitted the dhfr gene as one locus. Other foreign genes can be directed to the addition chromosome through transformation followed by selection for single loci with linkage to Poty(R) or Mtx(R). The integrity of both the foreign-gene linkage package and the rest of the genome will be maintained because recombination between the N. africiana and the N. tabacum chromosomes has not been observed.

Atypical quadrivalents, resulting from pairing between homologous amplified chromosome segments, have been observed in hybrids between low‐yielding, second‐cycle doubled haploid (DH) lines of tobacco (Nicotiana tabacum L.) and their source cultivars. The first objective of this study was to determine if homologous amplified chromosome segments are present in other DHs, such as those that have been through only one cycle of anther culture and those that are only moderately depressed in yield. The second objective was to trace the development of amplified chromosomal regions through consecutive cycles of anther culture. Chromosome pairing was studied in DH × cultivar hybrids involving high‐ and low‐yielding first‐cycle DHs and second‐cycle DHs derived from the high‐yielding first‐cycle DHs. There appeared to be no correlation between multivalent formation and yield. The one DH that was found to contain homologous amplified chromosome segments was a low‐yielding first‐cycle DH, NC95 DHL. NC95 DHL was then crossed to two second‐cycle DHs derived from it that had previously been determined to have homologous amplified chromosome segments. Cytological analysis of these DH × DH hybrids indicated that the amplified chromosomes in NC95 DHL passed unchanged to one of its second‐cycle derivatives. In contrast, a number of modifications occurred in another of its derivatives during the second cycle of anther culture.

Complementary DNA libraries representing the capsid protein cistron of the potato virus Y (PVY) isolate 'Chilean', 'Hungarian', MsNr, NsNr, O, and 'Potato US' were synthesized and used as template for polymerase chain reaction (PCR) amplification. An AUG codon for initiating a discrete capsid protein (CP) open reading frame was embedded upstream of the first codon of the CP cistrons. PCR-amplified products of the expected size of 0.8 kilo bases were cloned into the transcription vector pBS(+). The fidelity of each PCR-amplified PVY CP cistron was tested by transcribing recombinant plasmids in vitro and translating the transcripts in two cell free translation systems. Translation analysis of in vitro transcribed PVY CP cistrons consistently yielded a polypeptide co-migrating with authentic CP that was immunoprecipitated by anti PVY 'Chilean' antibodies. The nucleotide sequence of each capsid protein gene was determined by dideoxy sequence analysis. Each capsid protein gene was determined to be 801 nucleotides in length, encoding a deduced protein of 267 amino acids with calculated M(r) ranging from 29,799 to 29,980. The nucleic acid sequence similarity between the six isolates ranged between 89 to 97% and the amino acid similarity between 91 to 99%. The high level of amino acid sequence similarity confirms the classification of these viruses as isolates of PVY.

Atypical quadrivalents, which appear to be the result of pairing between amplified homologous segments in nonhomologous chromosomes, have been found in tobacco (Nicotiana tabacum L.) doubled haploid (DH) × cultivar hybrids. The purpose of this study was to determine if the same amplified chromosomes are involved in these quadrivalents in different DH lines. Chromosome pairing was studied in hybrids between four DHs, of which two were derived from ‘NC95’ and the other two from ‘C139’. Results of this study indicate that all four of the DHs contain two homologous amplified chromosomes regions. The chromosomal locations of both of these amplified segments in three of the DHs appear to be the same. One of the amplified segments in the fourth DH is located on the same and the other on a different chromosome than in the other three DHs. The difference in location of one of the amplified segments in this fourth DH could be the result of a translocation or the amplification of nontandem multigene families. That similar genetic sequences have been amplified in all four DHs studied suggests there may be some specificity to the DNA amplification that occurs as a result of anther culture in tobacco.

Amplication of DNA has been reported for another culture‐derived doubled haploid (DH) lines of tobacco (Nicotiana tabacum L.). The purpose of this study was to evaluate the cytological changes associated with this DNA amplification. Crosses were made between three second‐cycle DHs and their source cultivars, NC95 and C139. Examination of chromosome pairing in pollen mother cells (PMCs) revealed that two of the hybrids produced a single quadrivalent at metaphase I, whereas the other hybrid formed two quadrivalents. In all of the hybrids, quadrivalents either were in a ring, or consisted of two ring bivalents connected by a single strand of chromatin. This latter form, which was termed a side‐arm quadrivalent, appears to represent pairing between amplified homologous chromosome regions in nonhomologous chromosomes. These findings suggest that at least some of the DNA that is amplified in tobacco DHs is present in large blocks, and occurs in identical or similar sequences located on different chromosome pairs. Observations of chromosome and spindle behavior in somatic anaphases and meiotic anaphase I and tetrad cells revealed the presence of a low frequency of spindle errors in the hybrids. while the genotypic effectofr the frequency of abnormal mitotic anaphases was not significant, a highly significant genotypic effect was found fot the frequency of abnormal anaphase I cells. All hybrids had significantly more anaphase I errors than did their parental lines. These spindle errors may be caused by the amplification of specific sequences within the DH lines or by the differences between the parental lines in amount of DNA.

Potato virus Y (PVY), susceptible tobacco (Nicotiana tabacum L.) cultivar, McNair 944, was subjected to in vitro anther culture to determine if genetic variability for virus resistance could be induced among resulting haploids. Five hundred and forty-five haploids were produced and inoculated with a highly necrotic strain (NN) of PVY. One haploid plant survived, even though it was infected with the virus. Selfed progenies of a chromosome-doubled plant of this variant, designated NC 602, proved to be highly resistant to the necrotic effects of the virus. An investigation into the genetic nature of this variant showed the resistance mechanism to be controlled by a single gene exhibiting incomplete dominance. Cytoplasmic and maternal effects were not involved in the disease resistance reaction. The variant was challenged with ten additional strains of PVY from an international collection, and it proved to be resistant to three (VAM-B, MM, and Spanish) strains. NC 602 was evaluated for five agronomic traits and concentrations of total alkaloids as nicotine and reducing sugars in cured leaf. The gametoclonal variant differed from McNair 944 only for cured leaf yield, where an 18.4% reduction was measured.

A previously identified gametoclonal tobacco variant, NC 602, exhibits resistance to certain potato virus Y (PVY) strains. NC 602 was subjected to a second cycle of anther culture and colchicine treatment. The resulting doubled haploid lines were evaluated for back mutation to susceptibility and any additional mutation that might provide resistance to tobacco etch virus (TEV). The PVY resistance was stable in anther culture, and although no TEV resistance was found, three lines were identified with delayed symptom onset. NC 602 was crossed to breeding line NC 744, which contained the PVY resistance gene found in the Virgin A Mutante (VAM) (Tobacco Introduction line 1406). Maternal doubled haploid lines of this cross were screened for both resistance genes by using an array of PVY strains to distinguish genotypes. Results demonstrated that the two genes could be combined into a single genome with improved levels of resistance, thus indicating different loci for each resistance. NC 602 was also crossed to a root-knot nematode (Rk) resistant cultivar (Coker 209) in an effort to combine PVY and Rk resistance for protection against the MN strain of PVY in Rk resistant varieties. Plants were identified at the haploid level, which contained both PVY(NN) and Rk resistance, but when these plants were doubled and challenged with the PVY(MN) strain, all lines resistant to Rk showed susceptibility to the virus.