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Molecular and genetic characterization of Nicotiana glutinosa L. chromosome segments in tobacco mosaic virus-resistant tobacco accessions.

TOBACCO MOSAIC VIRUS is one of the most important pathogens of tobacco worldwide. Development of cultivars possessing genetic resistance offers the best opportunity for reducing economic loss from this pathogen. Although several mechanisms of resistance have been evaluated (Valleau, 1952; Cameron, 1958; Holmes, 1960; Thomas and Fulton, 1968), most breeding efforts have focused on the utilization of the single gene N because of its effectiveness, simple inheritance and ease at which resistant genotypes can be identified (Wernsman, 1992). The N gene confers resistance via a hypersensitive response (HR) and was initially transferred to N. tabacum in the development of the line 'Holmes Samsoun' by substituting a N. glutinosa chromosome carrying the resistance gene for chromosome H of the N. tabacum genome (Holmes, 1938; Gerstel, 1945). Independent introgressions of the N gene may also have been achieved by other workers, although information on the derivation and existence of this material is fragmentary (Ternovsky, 1941,1945; Goodspeed, 1942; Kostoff and Georgieva, 1944; Kostoff, 1948; Valleau, 1952; Oka, 1961).

It was later demonstrated that recombination could occur between chromosome

H and the N-carrying chromosome in Holmes Samsoun (Gerstel and Burk, 1960), and this line is believed to have been the ultimate source of TMV resistance used in all tobacco breeding in the USA (Valleau, 1952; Wernsman, 1992). High yielding cultivars of burley tobacco with N-mediated resistance have been developed and accepted by growers. TMV-resistant flue-cured cultivars have also been developed. These have not been widely grown, however, because of decreased yield and quality associated with the resistance. Available data suggest that these negative associations are due to linkage drag effects rather than pleiotropy (Chaplin et al., 1966; Chaplin and Mann, 1978; Linger et al., 2000). The N gene has been cloned (Whitham et al., 1994) and, in principle, introduction of the cloned N-gene sequence into tobacco via transformation offers opportunity for bypassing linkage drag effects (Linger et al., 2000). Strong international objection to genetically engineered tobacco, however, and costs associated with intellectual property rights issues provide incentives to explore alternative means of developing TMV-resistant flue-cured tobacco cultivars. Chaplin and Mann (1978) recommended that either additional backcrossing be performed or new substitutional events be explored. Unfavorable linkages surrounding a gene of interest can be difficult to break because of the low probability of selecting for crossover events within small regions (Young and Tanksley, 1989). The problem can be exacerbated in derivatives from interspecific crosses (Ganal and Tanksley, 1996). Young and Tanksley (1989) concluded that conventional backcrossing is generally ineffective in decreasing linkage drag effects and recommended the use of marker-assisted backcrossing for selecting for reduced introgressed segment sizes.

Several tobacco accessions from the U.S. Nicotiana germplasm collection (Sisson, 1992) have been identified that exhibit a HR type of resistance to TMV (Chaplin and Gooding, 1969; Gwynn, 1977). Previous research has demonstrated that a TMV resistance gene may be present on more than one chromosome of the N. tabacum genome (Beekwilder, 1999; Bagley, 2002). The first objective of this work was to determine the relative chromosome positions of TMV resistance loci in a set of 12 TMV-resistant (TM[V.sup.R]) tobacco accessions and to determine the identity of these resistance genes. The second objective was to identify a large set of AFLP markers specific to the N. glutinosa chromosome present in the chromosome substitution line Holmes Samsoun. These markers were subsequently used to characterize the 12 accessions and to determine the relative propensity for crossing over within the introgressed segments when in different positions within the N. tabacum genome. These steps were taken as a preliminary effort to increase the probability of developing commercially acceptable TMV-resistant flue-cured tobacco cultivars. Molecular marker-assisted selection of genotypes possessing minimal amounts of deleterious alien chromatin in genomic positions with less of an unfavorable effect on yield and/or quality could increase the potential for Success.

MATERIALS AND METHODS

Relative Positions and Identification of TMV-Resistance Genes within the Tobacco Genome

Nine tobacco accessions identified as possessing a HR type of TMV resistance by Gwynn (1977), plus three additional TM[V.sup.R] accessions from the U.S. tobacco germplasm collection (Table 1), were tested to determine if they possessed a TMV resistance gene that segregated independently of the N gene in line 'NC1125-2.' NC1125-2 is the TM[V.sup.R] parental line of commercial flue-cured tobacco hybrid 'NC297' and is representative of U.S. TM[V.sup.R] tobacco germplasm. Each of the 12 accessions was hybridized with NC1125-2 and [F.sub.1] individuals were subsequently crossed as females to TMV-susceptible (TM[V.sup.S]) cultivar 'K326' to produce 12 testcross families. Approximately 200 plants from each family were inoculated with a common strain of TMV maintained by the N.C. State University tobacco breeding program. Inoculum was prepared according to Rufty et al. (1987) and was applied to two leaves per plant at the two-leaf stage (approximately 30 d old) with cotton tipped applicators. Inoculated plants were maintained in a growth room at a temperature of 24 to 26[degrees]C. Plants whose leaves exhibited the localized lesions of a hypersensitive response 4- to 5-d postinoculation were classified as TMVR. Leaves that did not produce a hypersensitive response were classified as coming from TM[V.sup.S] plants. Chi-square tests (Steel et al., 1997) were used to determine if observed ratios of TM[V.sup.R] to TM[V.sup.S] plants conformed to that expected if there were two independently segregating TM[V.sup.R] loci present in the testcross families.

Single plants representing each of the 12 TM[V.sup.R] tobacco accessions were tested by means of the polymerase chain reaction (PCR) to determine if a sequence corresponding to the cloned N-gene sequence derived from N. glutinosa was present. The TM[V.sup.S] lines 'Samsun', 'Connecticut Broadleaf', 'Red Russian', and K326 were also tested for comparison. Two sets of primer pairs were designed to amplify portions of the published N-gene sequence (Whitham et al., 1994). Primer pair E1 + E2 (5'-ACCAGAATGATATGTTCCAC-3' and 5'-GGACTCAACGTTAATTCTCTG-3') was expected to amplify a 545-bp product, while primer pair N1 + N2 (5'CGTCGACACATTATGCCATC-3' and 5'-GAGGGGTCTTACCCCATTGT-3') was expected to amplify a 359-bp product. DNA was isolated according to Johnson et al. (1995), except that a BIO 101 FastPrep machine (BIO 101, Inc., Vista, CA) was used for tissue grinding. Amplifications were performed in a 96-well PTC 100 thermal cycler (MJ Research, Watertown, MA) and reaction mixes for PCR assays were constructed according to Taq DNA polymerase manufacturer's recommendations (Boehringer Mannheim, Indianapolis, IN). Reaction parameters were 94[degrees]C for 1 min., 55[degrees]C for 1 min, and 72[degrees]C for 1 min for 25 cycles, using 250 ng of genomic DNA and 250 ng of each of the above primers in 100-[micro]L reaction volumes. Reaction products were electrophoresed in 1.5% (w/v) agarose gels containing 0.15 [micro]g m[L.sup.-1] ethidium bromide. Gels were run for 4 h at 60 V and were visualized with a UV transilluminator. Band sizes were determined by RFLPscan Version 2.1 Gel Analysis Software (Scanalytics, Billerica, MA).

On the basis of data from experiments described above, seven selected accessions (TI 1407, TI 1473, TI 1492, TI 1500, TI 1501, Xanthi nc, and Xanthi NN) were crossed in diallel fashion (excluding reciprocals) to determine the number of N. tabacum chromosomes that carried a HR type of TMV-resistance gene. Individual [F.sub.1] plants from each cross were then crossed as females to TM[V.sup.S] cultivar K326 and approximately 200 testcross progeny from each cross were inoculated with TMV by the procedure described above. Chi-square tests were used to determine if the observed data conformed to that expected if there were two independently segregating TM[V.sup.R] loci present in these progenies.

The initial transfer of the N gene in the development of Holmes Samsoun reportedly occurred by replacing chromosome H of the N. tabacum genome with the N-carrying chromosome from N. glutinosa (Gerstel, 1945; Gerstel and Burk, 1960). Identification of N. tabacum individuals monosomic for chromosome H is relatively simple, and monosomic analysis (Clausen and Cameron, 1944) was used to determine which accessions, if any, actually possessed the N gene on chromosome H. On the basis of data from the diallel crossing scheme which indicated the total number of N. tabacum chromosomes carrying a HR type of TMV-resistance gene, two accessions (TI 1500 and Xanthi nc) plus Holmes Samsoun and line NC1125-2 were selected. These were crossed as males to a genetic stock of the line Red Russian that was monosomic for chromosome H (Red Russian Haplo H). A single monosomic individual (2n = 47) was then isolated from each cross and was subsequently crossed as a female to TM[V.sup.S] cultivar K326. Approximately 200 plants from each resulting family were then inoculated with TMV by procedures described above. Red Russian genotypes monosomic for chromosome H transmit the unpaired chromosome through the egg at a frequency of only 0.296 (Clausen and Cameron, 1944). If the resistance gene was present on chromosome H, the ratio of TM[V.sup.R]:TM[V.sup.S] plants was predicted to differ significantly from a 1:1 ratio. Chi-square tests were used to determine if segregation ratios differed from those which were expected on the basis of normal distribution of a paired chromosome to female gametes (Gerstel, 1945). For comparison, the four lines that were crossed with Red Russian Haplo H were also crossed with the disomic version of Red Russian. The [F.sub.1] individuals were crossed to K326 and approximately 200 progeny from each cross were inoculated with TMV. Chi-square tests were used to determine if segregation for TMV resistance differed from 1:1 ratios.

Identification of AFLP Markers Associated with the N. glutinosa Donor Chromosome

A total of 160 AFLP primer combinations (LI-COR, Lincoln, Nebraska) were screened for their ability to reveal polymorphism between DNA from Holmes Samsoun and a bulk of DNA from TM[V.sup.S] lines Red Russian, Connecticut Broadleaf, and Samsun (lines involved in the pedigree of Holmes Samsoun). DNA was isolated as described above. AFLP reactions were performed according to Myburg and Remington (2000) and gels were run on a LI-COR model 4200 automated sequencer. Gels were scored initially by the software package AFLP-Quantar 1.0 (Keygene Products B.V., Wageningen, the Netherlands) and scoring was subsequently verified visually.

Markers present for Holmes Samsoun but absent for the TM[V.sup.S] bulk were tentatively assigned to the N. glutinosa donor chromosome. Assignment of a subset of these markers to this chromosome was confirmed by means of progeny derived by pollinating a plant monosomic for the N-carrying chromosome of Holmes Samsoun (derived by crossing Red Russian Haplo H with Holmes Samsoun) with pollen from TM[V.sup.S] cultivar K326. Twenty-two progeny from this cross were inoculated with TMV and genotyped with the subset of AFLP markers tentatively assigned to the N. glutinosa chromosome. Gerstel (1945) found the N-carrying chromosome of Holmes Samsoun, when in the monosomic condition, to be transmitted through the egg only 19.4% of the time. Cosegregation and transmission analyses were used to confirm assignment of the selected subset of polymorphic AFLP markers to the N. glutinosa chromosome. The presence or absence of each of these markers was also tested on DNA extracted from a representative accession of N. glutinosa (PI 241768). AFLP marker names were designated according to the primers used to amplify the DNA, followed by the band size in base pairs. E primers refer to those

corresponding to EcoRI restriction sites, while M primers refer to those corresponding to MseI restriction sites.

Genotyping of TM[V.sup.R] Lines and Estimation of Relative Degrees of Recombination

The 12 TMV[V.sup.R] accessions, three TM[V.sup.R] cultivars-breeding lines ('Burley 21', 'Coker 176', and NC1125-2), and two TM[V.sup.S] cultivars (K326 and 'TN 86') were genotyped with the subset of AFLP markers found to be specific to the N. glutinosa chromosome in Holmes Samsoun to estimate the relative amounts of N. glutinosa chromatin present.

On the basis of information regarding relative genomic positions of introgression events, accessions TI 1462, TI 1500, and Samsun NN were crossed to TM[V.sup.S] cultivar K326. [F.sub.1] hybrids were backcrossed to K326 to produce B[C.sub.1][F.sub.1] families representing each of the three accessions. Ninety-four individuals from each family were phenotyped for TMV-resistance and genotyped with those AFLP markers present in the TM[V.SUP.R] parents. Gels were scored as described above. Chi-square tests (Steel et al., 1997) were applied to each marker to test for segregation distortion and estimation of recombination fractions and linkage map construction were performed by MAPMAKER/EXP 3.0 (Lander et al., 1987). Linkage was determined on the basis of a minimum logarithm of odds (LOD) score of 3.0 and a linkage threshold of 40 cM by the Group command. The best linkage order was established by the Compare command. Map distances (centimorgan, cM) were esti mated from recombination fractions and the Kosambi mapping function (Kosambi, 1944).

RESULTS

Relative Positions and Identification of TMV-Resistance Genes within the Tobacco Genome

Segregation of TMV resistance in testcross families involving NC1125-2 and the 12 accessions from the U.S. Nicotiana germplasm collection allowed placement of the accessions into one of two groups (Table 2). No segregation of resistance was observed for the testcross progeny involving the five accessions placed in Group 1: TI 1459, TI 1462, TI 1463, TI 1504, and Samsun NN. Seven accessions contained a TMV resistance factor that segregated independently of the resistance gene in NC1125-2 and were placed into Group 2 (TI 1407, TI 1473, TI 1492, TI 1500, TI 1501, Xanthi nc, and Xanthi NN). Portions of these results agreed with data previously reported by Beekwilder (1999) and Bagley (2002). Accessions placed into Group 2 were crossed in diallel fashion (excluding reciprocals) and F1 individuals were then crossed as females to TM[V.sup.S] cultivar K326. No segregation for resistance was observed in any of these testcross families (data not shown), indicating that the same chromosome carried resistance in each of the accessions in Group 2. When PCR was performed with two sets of primer pairs designed to amplify specific portions of the cloned N gene, strong amplification products of predicted sizes were produced for each of the 12 TM[V.sup.R] accessions (Fig. 1). These bands were identical in size to that produced for NC1125-2 and were absent for four TM[V.sup.S] tobacco lines (K326, Red Russian, Connecticut Broadleaf, and Samsun) also included in the PCR experiment.

[FIGURE 1 OMITTED]

Collectively, the results described above indicated that the N gene (and not a previously undocumented resistance gene) is present on two chromosomes of the tobacco genome. Monosomic analysis was performed to determine which group of TM[V.sup.S] tobacco lines, if any, actually possess the N gene on chromosome H. Line NC1125-2 was chosen to represent Group 1, and significant deviation from a 1:1 ratio of TM[V.sup.R]:TM[V.sup.S] individuals was observed for progeny segregating for chromosome H donated by this line (Table 3). The percentage of TM[V.sup.R] individuals among the progeny (27.3%) was very close to the previously reported ovular transmission rate of 29.6% for the H univalent (Clausen and Cameron, 1944). Segregation of resistance in progeny segregating for chromosome H donated by accessions representing Group 2 (TI 1500 and Xanthi nc) did not deviate significantly from a 1:1 ratio (Table 3). Aberrant segregation of TMV resistance in progeny derived from crosses with the chromosome substitution line, Holmes Samsoun, agreed with previously reported observations (Gerstel, 1945).

Identification of AFLP Markers Associated with the N. glutinosa Donor Chromosome

Screening of 160 AFLP primer combinations on DNA from Holmes Samsoun, and a bulk of DNA from three TM[V.sup.S] genotypes involved in the development of Holmes Samsoun, revealed 266 bands that were tentatively assigned as AFLP markers specific to the N. glutinosa donor chromosome. A set of 177 of these markers was selected for confirmation of this assignment using a monosomic approach. Gerstel (1945) found the N-carrying chromosome of Holmes Samsoun to be transmitted through the egg only 19.4% of the time. Of the 177 markers that were genotyped on 22 progeny derived from a cross between a plant monosomic for this chromosome and K326, 168 were found to cosegregate as a group with the N gene in a highly distorted fashion (3 TM[V.SUP.R]:19 TM[V.SUP.S]; significant deviation from 1:1 at the 0.01 level of significance). Of these 168 markers, 164 (97.6%) were also present in N. glutinosa accession TW60 (PI 241768).

Genotyping TM[V.SUP.R] Accessions

The 12 accessions, Holmes Samsoun, and several cultivars selected to represent U.S. burley and flue-cured tobacco cultivars were genotyped with the 168 markers described above. Variability was present among the accessions for the number of N. glutinosa AFLP markers that were present (Fig. 2). All accessions possessed a small fraction of the markers that were present in the line Holmes Samsoun. Accessions carrying the N gene on chromosome H (Group 1) possessed fewer N. glutinosa markers than accessions carrying the N gene on the alternative chromosome (Group 2). None of the accessions possessed fewer N. glutinosa markers than TM[V.SUP.R] flue-cured cultivar Coker 176, which possessed only three of the 168 AFLP markers. None of the AFLP markers were present in TM[V.sup.S] cultivars TN 86 or K326.

[FIGURE 2 OMITTED]

Estimation of the Relative Degrees of Recombination

TI 1462 and Samsun NN were chosen as nonrecurrent parents for the generation of two B[C.sub.1][F.sub.1] families to examine the propensity for recombination of the introgressed N. glutinosa chromosome segment with chromosome H of the N. tabacum genome. A B[C.sub.1][F.sub.1] family was also created involving TI 1500 to investigate the relative potential for recombination of the alien chromosome segment with the alternative chromosome of the N. tabacum genome. Ninety-four individuals from each family were phenotyped for TMV resistance and genotyped with those AFLP markers present in the TM[V.SUP.R] parents. Crossing over within the N. glutinosa chromosome segments was observed for each of the three B[C.sub.1][F.sub.1] families (Fig. 3). The linkage map corresponding to the alien segment present in TI 1462 was 1.1 cM in length and included three AFLP markers in addition to the N gene. A linkage group of 4.6 cM and consisting of nine AFLP markers and the N gene was constructed for the N. glutinosa segment present in Samsun NN. Two AFLP markers (M6 and M13) exhibited segregation distortion in the TI 1500/K326//K326 B[C.sub.1][F.sub.1] population and were not included in the linkage group analysis. After removal of these two markers, AFLP marker M17 was found to be unlinked to the remaining markers on the basis of a minimum LOD score of 3.0 and a linkage threshold of 40 cM. The remaining 13 markers were placed into a linkage group of 6.9 cM to represent recombination of N. glutinosa chromatin with the alternative chromosome. The genetic mechanism behind the distorted segregation for markers M6 and M13 is not known, but such deviation is not entirely unexpected for markers associated with introgressed alien chromatin. A possible explanation for independent segregation between the N gene and AFLP marker M17 is that TI 1500 does not actually possess the true N. glutinosa M17 AFLP marker. Rather, TI 1500 may possess an AFLP marker of N. tabacum origin that is identical in size to the M17 N. glutinosa marker present in Holmes Samsoun and that segregates independently of the N-carrying chromosome in this accession.

[FIGURE 3 OMITTED]

No cases were observed in any of the three B[C.sub.1][F.sub.1] families in which the N gene was separated from all of the linked AFLP markers. Very low frequency of recombination was observed between the N gene and closely linked markers M1, M3, and M9 when the N. glutinosa segment was present on chromosome H. No recombination between the N gene and AFLP markers M1, M2, M3, M4, M5, M10, M12, M14, and M16 was observed when the N. glutinosa alien segment was present on the alternative N. tabacum chromosome. Because of a general lack of recombination between most AFLP markers, it was not possible to thoroughly compare co-linearity of the markers in the different B[C.sub.1][F.sub.1] populations. For markers for which recombination was observed, there were no obvious disagreements between the different linkage groups with respect to marker alignment.

DISCUSSION

This investigation demonstrates that the TMV resistance gene N is present on N. tabacum chromosome H in parental line NC 1125-2 and accessions TI 1459, TI 1462, TI 1463, TI 1504, and Samsun NN. The research also shows that the N gene per se, and not a previously uncharacterized TMV resistance gene, is present on an alternative chromosome in accessions placed into a second group: TI 1407, TI 1473, TI 1492, TI 1500, TI 1501, Xanthi nc, and Xanthi NN. The recipient chromosome of the N gene in accessions placed into Group 2 is not currently known. Further investigation using remaining monosomic stocks, in conjunction with DNA marker genotyping, might reveal the location of the alternate introgression event.

The derivation of the accessions evaluated in this research and their possible relationship to Holmes Samsoun is not known. Given the observed variability among the accessions for genomic position of the N gene and accompanying alien fragment lengths, it seems probable that a number of these accessions were derived from independent introgression events. Workers independent of Holmes (1938) described efforts to transfer TMV resistance from N. glutinosa to N. tabacum (Ternovsky, 1941, 1945; Goodspeed, 1942; Kostoff and Georgieva, 1944; Kostoff, 1948; Valleau, 1952; Oka, 1961). Descriptions of this work are fragmentary, but it seems likely that some of the accessions used in our study may be directly related to these efforts. One cannot rule out the possibility, however, that the 12 accessions were derived from multiple independent occasions in which the N-carrying chromosome of Holmes Samsoun recombined with a chromosome of the N. tabacum genome. This chromosome fails to pair normally in [F.sub.1] hybrids with TM[V.sup.S] lines (Gerstel, 1943), which theoretically increases the potential for physical association with other chromosomes of the N. tabacum genome.

Valleau (1952) developed the first commercial TM[V.SUP.R] tobacco cultivars and the origin of resistance in almost all U.S. tobacco cultivars can be traced to flue-cured tobacco cultivars Vamorr 48 and Vamorr 50 (Henderson, 1949); burley tobacco cultivars Kentucky 56, Burley 1, and Burley 21 (Heggestad, 1966); or dark-fired cured cultivar Ky 151 (Valleau, 1949). Although published information is limited, Holmes Samsoun is believed to have been the source of resistance in these early cultivars, and it is reasonable to assume that all U.S. breeding material possesses the N gene on chromosome H. Although TM[V.sup.R] burley tobacco cultivars have been highly successful with U.S. growers, TM[V.SUP.R] flue-cured cultivars have not been widely adopted by U.S. growers because of associations of resistance with reduced yields and/or quality (Chaplin et al., 1966; Chaplin and Mann, 1978). In principle, introduction of the N gene into elite cultivars via transformation should offer the opportunity to bypass the potential for undesirable linkage drag effects (Linger et al., 2000). Current objection to genetically engineered tobacco by manufacturers and leaf dealers prohibits the use of this approach for development of commercial tobacco cultivars, however. Investigating the possible value of alternative introgression events may be a worthwhile research objective. The effects of N. glutinosa introgression on yield and quality in flue-cured breeding material derived from accessions in Group 2 are not currently known. Our study demonstrated that the introgressed alien chromosome segments in this group are likely much larger than those present in accessions possessing the N gene on chromosome H. Chromosome position effects may affect the practical value of this material, however. Field studies designed to compare backcross-derived lines possessing the N gene on different chromosomes are currently being performed to examine the possibility of position effects on yield and quality of TM[V.SUP.R] flue-cured tobacco.

In the work reported here, there were no cases in which all N. glutinosa AFLP markers had been separated from the N gene. Also, no accessions were identified that possessed fewer N. glutinosa AFLP markers than the small number found to be present in TM[V.SUP.R] flue-cured tobacco cultivar Coker 176. Nevertheless, evidence of recombination within the introgressed segments in either genomic position may indicate the potential for incorporating the use of AFLP markers identified in this research into a breeding program that has TMV resistance as an objective. Young and Tanksley (1989) demonstrated the utility of marker-assisted backcrossing to reduce the potential for linkage drag associated with an interspecifically derived disease resistance gene in cultivated tomato. The potential for eliminating such effects in N. tabacum likely decreases as the taxonomic distance between the donor species and the probable progenitor species of N. tabacum increases.

The results of Whitham et al. (1994) indicated that at least a fraction of the introgressed chromosome segment in Samsun NN (carrying the N gene on chromosome H) may have replaced orthologous sequences from the T subgenome of N. tabacum. This subgenome is likely derived from a species from Section Tomentosae (probably N. tomentosiformis Goodsp., N. otophora Griseb., or an introgressive hybrid between the two species) (Kenton et al., 1993; Kitamura et al., 2001; Lim et al., 2000). The S subgenome was contributed by N. sylvestris Speg. of Section Sylvestres (Bland et al., 1985; Knapp et al., 2004). Goodspeed (1954) placed N. glutinosa into Section Tomentosae along with likely donors of the T subgenome of N. tabacum. A more recent classification places N. glutinosa into Section Undulatae (Knapp et al., 2004). If one prefers the classification of Goodspeed (1954), one might expect the frequency of recombination within the introgressed N. glutinosa segment to be greater in accessions possessing the resistance gene on chromosome H. If one favors the classification of Knapp et al. (2004), one might expect little recombination within the alien chromatin when in either genomic position. In our investigation, the frequency of crossover events was low when in either chromosomal position. Nevertheless, it appeared that chances for recombination around markers most closely linked to the N gene would be greater in accessions possessing the resistance gene on chromosome H. Hundreds or thousands of individuals may need to be genotyped to achieve ultimate success by a molecular marker-assisted backcrossing approach to eliminate linkage drag effects, however. Further investigation will be required to determine whether the alternative introgression events or molecular breeding approaches will increase the potential for developing commercially acceptable TMV-resistant cultivars.

ACKNOWLEDGMENTS

The authors would like to thank the North Carolina Tobacco Research Commission and Philip Morris USA for their financial support of this research. The authors are also appreciative of three anonymous reviewers for their constructive comments that improved this paper.

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R. S. Lewis, * S. R. Milla, and J. S. Levin

Dep. of Crop Science, North Carolina State Univ., Campus Box 7620, Raleigh, NC 27695. Received 5 Feb. 2005. * Corresponding author (ramsey_lewis@ncsu.edu).

Published in Crop Sci. 45:2355-2362 (2005). Plant Genetic Resources doi:10.2135/cropsci2005.0121

Abbreviations: AFLP, amplified fragment length polymorphism; HR, hypersensitive response; TMV, tobacco mosaic virus.
Table 1. Designations and origins of TMV-resistant accessions.

            Plant introduction     Year      Donating
Accession         number         submitted    country

TI 1407         PI 293914          1963       Bulgaria
TI 1459         PI 304898          1965       Germany
TI 1462         PI 304901          1965       Germany
TI 1463         PI 304902          1965       Germany
TI 1473         PI 301017          1964       Venezuela
TI 1492         PI 321709          1967       Bulgaria
TI 1500         PI 329206          1968       Russia
TI 1501         PI 329207          1968       Russia
TI 1504         PI 338510          1968       Taiwan
Xanthi nc       PI 552488          1976       USA
Xanthi NN       PI 552484          1975       USA
Samsun NN       PI 552486          1975       USA

Table 2. Segregation of TMV resistance in testcross progeny derived
by crossing [F.sub.1] hybrids between accessions and NC1125-2 with
K326.

                          Resistance to TMV in progeny

                 Number of            Number of
Accession    plants TM[V.sup.R]   plants TM[V.sup.S]   [chi square]

TI 1407             163                   51               0.16
TI 1459             213                    0              71.00 *
TI 1462             216                    0              72.00 *
TI 1463             216                    0              72.00 *
TI 1473             162                   50               0.23
TI 1492             168                   48               0.89
TI 1500             155                   53               0.03
TI 1501             150                   62               2.04
TI 1504             216                    0              72.00 *
Xanthi nc           161                   54               0.00
Xanthi NN           152                   63               2.12
Samsun NN           214                    0              71.33 *

* Significantly different from the expected 3:1 ratio at the
0.05 probability level.

Table 3. Segregation of TMV resistance in testcross progeny derived
from crosses of Red Russian and Red Russian Haplo H withselected
TMV-resistant tobacco lines.

                                      Resistance to TMV in progeny

                                       Number of     Number of
                                        plants         plants
Pedigree                              TM[V.sup.R]   TM[V.sup.S]

Red Russian/Holmes Samsoun//K326           78           138
Red Russian Haplo H/Holmes
  Samsoun//K326                            25           191
Red RussianfFI 1500//K326                  98           106
Red Russian Haplo H/TI 1500//K326         108           108
Red Russian/Xanthi net/K326               108           108
Red Russian Haplo H/Xanthi nd/K326        104           112
Red Russian/NC1125-2//K326                112           104
Red Russian Haplo H/NC1125-21/K326         59           157

Pedigree                              [chi square]

Red Russian/Holmes Samsoun//K326         16.67 *
Red Russian Haplo H/Holmes
  Samsoun//K326                         127.57 *
Red RussianfFI 1500//K326                 0.31
Red Russian Haplo H/TI 1500//K326         0.00
Red Russian/Xanthi net/K326               0.00
Red Russian Haplo H/Xanthi nd/K326        0.27
Red Russian/NC1125-2//K326                0.27
Red Russian Haplo H/NC1125-21/K326       44.46 *

* Indicates significant deviation (0.05 level of probability) from
the 1:1 ratio of TMV-resistant to TMV-susceptible plants that was
expected if the N gene was present on a paired chromosome with
normal distribution to female gametes.
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Author:Lewis, R.S.; Milla, S.R.; Levin, J.S.
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Date:Nov 1, 2005
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