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RAPD and SCAR markers linked to an introgressed gene conditioning resistance to Peronospora tabacina D.B. Adam. in tobacco.

BLUE MOLD is one of the most important foliar diseases of tobacco (Lucas, 1980). The disease is capable of infecting tobacco of all types in many tobacco growing regions of the world. The pathogen can attack plants throughout the growing season (including transplant production) and can spread rapidly under favorable weather conditions (Main, 1991). Depending on environmental conditions, losses from the disease can range from only slight to complete destruction of the crop. Use of fungicides is recommended to reduce potential for economic loss from blue mold (Shoemaker, 2004). The fungicide Ridomil (Syngenta Crop Protection, Greensboro, NC; methyl benzimidazol-2-ylcarbamate) has been used effectively in the past, but resistant isolates have developed in several countries in recent years (Main, 1991; Shoemaker, 2004). Deployment of host-plant resistance is the most economic and environmentally sustainable method for controlling blue mold. Naturally occurring resistance within N. tabacum is generally very low, however (Rufty, 1989). Resistance can be found within several Nicotiana species of Australian origin where blue mold is endemic and has reportedly been introgressed into cultivated tobacco from N. debneyi (Clayton, 1967; Clayton et al., 1967; Lea, 1963), N. goodspeedii Wheeler (Wark, 1963, 1970), and N. velutina Wheeler (Wark, 1963, 1970). Resistance induced by treatment of flue-cured cultivar Virginia Gold with the mutagen triethylene iminotrazine has also been reported (Marani et al., 1972). Few resistant cultivars are available to growers worldwide, however (Shoemaker, 2004).

Several studies have been conducted on the control of genetic resistance derived from different sources, but many discrepancies exist (Rufty, 1989). Although some reports have indicated that introgressed resistance may be controlled by several genetic factors (Clayton, 1968), it seems likely that single genes would be of primary importance in controlling resistance in species-derived lines. This presumption is based on the low probability of transferring several unlinked genes to N. tabacum from wild Nicotiana species not closely related to probable progenitor species N. sylvestris Speg. or N. tomentosiformis Goodsp. Incorporation of blue mold resistance into new cultivars has been complicated by the complex interaction between P. tabacina and the tobacco host plant. Peronospora tabacina is an obligate parasite, and selection for resistance is best performed under natural or induced field epidemics. Disease reactions are highly dependent, however, on factors such as plant age, physiological status of the plant, and environmental conditions. These factors can cause field experiments to be highly variable and unpredictable (Rufty, 1989). Identification of molecular markers linked to genes contributing to blue mold resistance would be valuable for increasing the capacity for eliminating susceptible individuals and/or lines during early stages of a breeding program.

Bulked segregant analysis (Michelmore et al., 1991), simple F tests, and interval mapping (Lander and Botstein, 1989) are useful techniques for identifying molecular markers linked to a specific gene or region of the genome. Although DNA polymorphism as revealed by molecular markers is generally low for N. tabacum, identification of markers linked to resistance genes transferred to tobacco from wild relatives has been successful (Bai et al., 1995; Yi and Rufty, 1998; Yi et al., 1998; Johnson et al., 2002). The first objective of this work was to use bulked segregant analysis, F tests, and interval mapping to identify a set of RAPD markers linked to a gene contributing to blue mold resistance found in flue cured tobacco cultivar Ovens 62. Resistance in this cultivar has been reported to be controlled by a major single gene introgressed from either N. velutina or N. goodspeedii in addition to N. tabacum genes with smaller modifying effects (Powell, 1979; Rufty, 1989; Rufty et al., 1990a).

Transformation of RAPD markers into SCAR markers is usually considered desirable before application in marker assisted breeding due to their relative increased specificity and reproducibility (Paran and Michelmore, 1993). We were able to convert two RAPD markers flanking an introgressed QTL influencing blue mold resistance to SCAR markers on the basis of specific forward and reverse primers of at least 21 base pairs in length. We also determined the presence/absence of identified RAPD markers in a set of 45 cultivars or breeding lines and four Nicotiana species of Australian origin to determine the origin and distribution of the Ovens 62 type of blue mold resistance.

MATERIALS AND METHODS

Population Development

Genetic materials used in this research were generated from an applied breeding program with the objective of developing blue-mold resistant burley tobacco cultivars using flue-cured cultivar Ovens 62 as a donor of genetic resistance (Rufty et al., 1990a). This cultivar was believed to contain an introgressed gene from either N. velutina or N. goodspeedii that has a major effect on blue mold resistance (Powell, 1979; Rufty, 1989). The materials were developed over three cycles of doubled haploid (DH) breeding (Fig. 1). In the first cycle, Ovens 62 was hybridized with blue mold susceptible cultivars Ky 15, Ky 17, and McNair 944 (Rufty, 1985). A series of [F.sub.1]-derived DH lines for each cross were evaluated for blue mold resistance, and DH lines NC-BMR 42 and NC-BMR 90 were selected for release as germplasm lines (Rufty et al., 1990b). In the second cycle, NC-BMR 42 and NC-BMR 90 were crossed with Ky 17 and cultivar Ky 14. Derived DH lines were evaluated for blue mold resistance, and lines NC-BMR 113 and NC-BMR 114 were selected for use in crosses with cultivars Ky 14 and TN 90 to develop a set of third-cycle DH breeding lines.

Bulked Segregant Analysis

Two different DNA bulks were prepared from equal volumes of standardized DNA from seven blue mold susceptible tobacco cultivars and seven lines that exhibited excellent blue mold resistance in field experiments. The susceptible bulk was comprised of DNA from blue mold susceptible cultivars Ky 14, Ky 17, TN 86, TN 90, McNair 944, Speight G-28, and Speight G-70. The resistant bulk was comprised of DNA from Ovens 62 and six DH lines derived from the breeding program outlined above: NC-BMR 42, NC-BMR 90, NC-BMR 113, NC-BMR 114, DH 17, and DH 62. DNA was extracted according to Johnson et al. (1995) and was quantified with a Hoefer TKO 100 fluorometer (Hoefer Scientific, San Francisco, CA).

A total of 1216 random 10-mer primers obtained from Operon Technologies Inc. (Alameda, CA) and the Oligonucleotide Synthesis Laboratory of the University of British Columbia, Canada, were screened for their ability to reveal polymorphism between the two bulks following procedures outlined by Williams et al. (1990). PCR reaction components have been described previously by Johnson et al. (2002). Amplifications were performed in a 96-well PTC 100 thermal cycler (MJ Research, Watertown, MA) using the following reaction conditions: 1 cycle 94[degrees]C/1 min; 16 cycles 92[degrees]C/30 s, 50[degrees]C/30 s, 0.5[degrees]C/cycle, 72[degrees]C/2 min; 21 cycles 92[degrees]C/1 min, 40[degrees]C/1 min, 72[degrees]C/ 2 min; i cycle 72[degrees]C/5 min. Reaction products were subjected to electrophoresis in 1.5% (w/v) agarose gels containing 0.15 [micro]g ethidium bromide per liter. Primers generating marker polymorphisms between the DNA bulks were subsequently tested on individual lines comprising the bulks to tentatively identify those that were linked to blue mold resistance.

Verification of Associations between Markers and Blue Mold Resistance

RAPD markers tentatively identified as being linked to blue mold resistance were subsequently tested to verify their association with resistance. Disease resistance data collected for a total of 122 DH lines derived from three crosses and evaluated in four independent field experiments conducted at Papantla, Mexico, was used. Blue mold is endemic in Mexico, and weather conditions were favorable for disease development in these experiments. Experiments were conducted as randomized complete block designs with the number of replications ranging from to two to four. Experimental units consisted of one-row plots containing at least 5 plants per row. Plant spacing was 46 cm within rows and 122 cm between rows. Seed was sown in September, and plants were transplanted in November of each year. The number of DH lines used for marker-trait associations in each experiment ranged from 17 to 50. The pedigrees, population sizes, and parameters for each of the four field experiments are provided in Table 1. Ovens 62 and Ky 14 were included in each experiment as resistant and susceptible checks, respectively. Disease evaluations based on the quantitative Horsfall-Barrat rating scale (Horsfall and Barratt, 1945) were made approximately three months after transplanting.

Data for each experiment were analyzed by the GLM procedure of SAS (SAS Institute, Inc., Cary, NC), and mean disease ratings were calculated for each experimental entry. DH lines from each of the four experiments were genotyped with the RAPD markers tentatively identified as being linked to blue mold resistance using BSA. Chi-square tests were performed to determine if marker segregation differed significantly from an expected 1:1 ratio. For each of the four field experiments, the GLM procedure of SAS was used to conduct single degree of freedom F-tests for each marker (Liu, 1998) to determine if mean blue mold resistance ratings differed significantly for the two RAPD genotype classes. Significance at the P = 0.05 level was considered evidence of linkage between the marker and a gene involved in blue mold resistance. R2 values were also calculated for each marker locus to determine that marker's contribution to the total variation for blue mold resistance ratings.

Linkage and Interval Mapping

Data from experiment BM1 contained the largest number of lines (50) derived from a single cross and were utilized to generate a linkage map by Mapmaker/EXP 3.0 (Lander et al., 1987) and to localize resistance QTL by interval mapping. Linkage was determined on the basis of a minimum logarithm of odds (LOD) score of 3.0 and a linkage threshold of 0.4 by the Group command. Linkage order was established by first selecting a subset of eight markers on the basis of LOD scores and pairwise linkages and then identifying the best order using the Compare command. Remaining markers were added by the Try command. The final marker order was checked by the Ripple command with the default log-likelihood threshold value of 2.0. Map distances (centimorgan, cM) were estimated from recombination fractions and the Kosambi mapping function (Kosambi, 1944).

Interval mapping was performed by Windows QTL Cartographer (Wang et al., 2001-2004). Log-likelihood (LOD) plots for statistically significant associations between genotype and resistance were generated by calculating LOD scores at 2-cM intervals along the linkage group. The LOD threshold significance level was determined using 1000 permutations of the procedure of Churchill and Doerge (1994). A 1 - LOD empiric QTL confidence interval for the likely position of a QTL was also determined by Windows QTL Cartographer.

Origin and Distribution of Resistance

The RAPD markers were also tested to determine their presence/absence in a set of 45 tobacco cultivars/breeding lines and 4 Nicotiana species of Australian origin (N. velutina, N. goodspeedii, N. debneyi, N. excelsior J.M.Black). These species had all previously been reported as donors of blue mold resistance. Eight of the cultivars/breeding lines reportedly carried blue mold resistance transferred from either N. goodspeedii, N. velutina, or N. debneyi (Rufty, 1989) (Table 2). Two were reported to possess resistance induced by chemical mutation (Marani et al., 1972; Rufty, 1989).

Conversion of RAPD Markers to SCAR Markers

Coupling phase RAPD markers UBC180.328 and OPR06.268 were selected for conversion to SCAR markers on the basis of their significant associations with blue mold resistance and for the ease at which the amplified bands could be purified from agarose gels. RAPD bands were excised from an agarose gel and purified with the Promega Wizard Gel and PCR CleanUp Kit (Promega, Madison, WI). RAPD products were reamplified from the 10-mer primers and cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). RAPD fragments were sequenced and specific forward and reverse primers of at least 21 bp in length were designed on the basis of forward and reverse sequences of the RAPD fragment by the program Primer 3 (Rozen and Skaletsky, 2000).

SCAR markers were then tested on 73 DH lines evaluated in experiments BM1 and BM2 described previously. SCAR amplifications were performed in 20-[micro]L volumes with 150 ng genomic DNA, 200 nM each primer, 200 nM each dNTP, 1 X PCR buffer, 1.5 mM Mg[Cl.sub.2], and 0.5 Units Taq polymerase. Reaction conditions for the UBC180.328 SCAR conversion were 1 cycle 94[degrees]C/2 min; 29 cycles 94[degrees]C/30 s, 60[degrees]C/30 s, 72[degrees]C/ 40 s; 1 cycle 72[degrees]C/7 min. Reaction conditions for the OPR06.268 SCAR conversion were the same except that the annealing temperature was 61[degrees]C.

RESULTS

Bulked Segregant Analysis

Out of 1216 RAPD primers that were screened, 40 primers (3.29%) detected 40 polymorphisms between the resistant and susceptible bulks. When these primers were tested on individuals comprising the two bulks, four primers produced putative repulsion phase markers that were present for all members of the susceptible bulk and absent for all members of the resistant bulk. Seventeen markers were found to be present for all members of the resistant bulk except DH 62 and absent for all members of the susceptible bulk. It was speculated that DH 62 was incorrectly classified as resistant before its inclusion in the resistant bulk, and these 17 markers were tentatively assigned as markers linked in coupling with blue mold resistance. Amplifications of tentative markers were performed twice to determine reproducibility and 15 markers (12 coupling and three repulsion) were chosen for further study (Table 3). Primers with the prefix OP were obtained from Operon Technologies Inc. (Alameda, CA), and primers with the prefix UBC were obtained from the Oligonucleotide Synthesis Laboratory of the University of British Columbia, Canada.

Verification of Associations between Markers and Blue Mold Resistance

Good disease pressure was present for each of the field experiments conducted during 1989, 1992, and 1996, and significant differences were present among entries in each experiment. Variability for disease resistance ratings in the four experiments is indicated by the histograms presented in Fig. 2. All 15 markers were found to segregate in a 1:1 fashion in two of the four sets of DH lines (Table 3). Segregation of one marker (UBC610.528) in experiment BM3 and two markers (UBC610.528 and UBC528.528) in experiment BM4 deviated significantly from a 1:1 ratio. F tests indicated significant marker-resistance associations for all 15 markers in experiments BM1, BM2, and BM4 (Table 3). Significant associations were observed for 14 of 15 markers in experiment BM3. R2 values for markers associated with resistance ranged from 0.118 to 0.412. R2 values were highest for experiment BM2, where the range was from 0.213 to 0.412.

Linkage and Interval Mapping

A population of 50 DH lines derived from the cross NC-BMR 113/Ky 14 was used to establish a single linkage group covering 36.6 cM that included all 15 RAPD markers (Fig. 3). The 12 RAPD markers linked in coupling phase with blue mold resistance were all positioned within a contiguous group of 14.2 cM. The three repulsion phase markers were placed at the end of the linkage group.

The interval mapping approach indicated that all 15 RAPD markers within the linkage group had LOD scores that exceeded the LOD threshold significance level of 1.23 determined by the permutation procedure of Churchill and Doerge (1994) (Fig. 3). All but three markers had LOD scores that exceeded 3.0. A 1 - LOD confidence interval of 8.7 cM was determined that included a LOD peak of 4.12 very near the position of RAPD marker UBC544.499. Nine RAPD markers were included within this interval, and the QTL locus at the given LOD maximum explained 31.7% of the variance in disease ratings.

Conversion of RAPD Markers to SCAR Markers

RAPD amplification products for markers UBC180.328 and OPR06.268 were selected for conversion to SCAR markers. These markers flanked a 6.1-cM region that included the LOD peak generated by interval mapping. The intensity of the amplification products and their good separation from neighboring RAPD bands facilitated their isolation and cloning. The bands were sequenced, and the size of the UBC180.328 RAPD fragment was found to be 279 bp on the basis of sequence data, while the size of the OPR06.268 RAPD fragment was found to be 268 bp. BLAST searchers were performed on these sequences, but no significant similarities were found in the Genbank nucleotide sequence database. Forward primer 5'-CTGAGTTTGGCCGA ATAGCAT-3'and reverse primer 5'-CAAACGTCC TAAATGGGGTATAA-3' were designed to amplify a 251-bp SCAR marker corresponding to RAPD marker UBC180.328. Forward primer 5'-GTCTACGGCAAG GGGAGATATTA-3' and reverse primer 5'-GTCTA CGGCAGCAATCAACATG-3' were designed to amplify a 268-bp SCAR marker corresponding to RAPD marker OPRO6.268. These SCAR markers are referred to as SUBC180.251 and SOPR06.268, respectively, and are shown in Fig. 4. The SCAR markers were present in the blue mold resistant bulk and absent for the susceptible bulk. The SCAR markers were used to Genotype 73 DH lines evaluated in experiments BM1 and BM2 and perfect agreement was observed between the RAPD and SCAR genotypes, thus confirming that the SCAR products were, in fact, derived from the RAPD markers, and verifying their value for marker assisted breeding.

Origin and Distribution of Resistance

We investigated the presence or absence of the 15 RAPD markers in 45 tobacco cultivars or breeding lines. Eight of the cultivars or breeding lines reportedly possessed resistance from either N. goodspeedii, N. velutina, or N. debneyi (Delon, 1992; Rufty, 1989). Each of the 12 coupling phase RAPD markers was found to be present for eight cultivars or breeding lines with resistance reportedly derived from a wild Nicotiana species (Table 2). Each of the repulsion phase markers was absent in these lines. The origin of resistance in lines Sota 6505, ZA 684, and ZA 692 was previously unknown to the authors. These lines possessed 11, 12, and 9 of the coupling phase RAPD markers, respectively. Interestingly, Chemical Mutant and NC11-51 (reportedly possessing mutation induced resistance mechanisms) had RAPD genotypes that were identical to those for lines possessing resistance derived from Ovens 62.

We also screened the species N. goodspeedii, N. velutina, N. debneyi, and N. excelsior for the presence of eight coupling phase RAPD markers and the SCAR markers SUBC180.251 and SOPR06.268. A high amount of polymorphism was observed between the four wild Nicotiana species accessions that represented reported possible donors of resistance. The data indicated that N. debneyi is the most likely donor of resistance in all of the cultivars or breeding lines that were genotyped. Nicotiana debneyi (PI 503320) shared in common with the resistant N. tabacum material almost all of the coupling phase RAPD markers and was the only species that possessed the two SCAR markers (Table 4).

DISCUSSION

This report describes identification of RAPD and SCAR markers linked to blue mold resistance derived from flue cured tobacco cultivar Ovens 62. The identification of 15 markers within a single linkage group significantly associated with resistance supported the hypothesis that a single genetic locus with a large effect was of primary importance in controlling resistance in Ovens 62. The apparent close linkage of several molecular markers with this locus suggests their value for marker assisted selection. The precise origin of blue mold resistance in this cultivar was not entirely clear on the basis of available pedigree information. The cultivar was described as being derived from a cross of N. velutina with N. tabacum var. Praecox with subsequent backcrosses to tobacco cultivars Hicks, Sirogo, and Sirone (Powell, 1979). Both Sirogo and Sirone reportedly possess resistance derived from N. goodspeedii (Rufty, 1989). Our marker data suggest that N. debneyi may be the true donor of resistance in Ovens 62 as well as in Sirone, Sirogo, ZA 684, ZA 692, Sota 6505, and Bel 6-10. It was interesting that all 12 coupling phase RAPD markers linked to a factor controlling blue mold resistance in Ovens 62 were also present in lines with resistance supposedly achieved via induced mutation (Marani et al., 1972). These data suggest that resistance in Chemical Mutant may be due to alien gene introgression rather than induced mutation. Indeed, Marani et al. (1972) found no segregation for blue mold resistance in progeny derived from the cross Bel 61-10/Chemical Mutant, a result suggesting that genetic factors conditioning resistance in these two lines were identical. As noted by Rufty (1989), it is unlikely that a resistance allele derived via mutation breeding would occur at the same genome position as that which possesses a gene introgressed from a wild Nicotiana relative. Marani et al. (1972) noted the possibility that accidental mixing of seed may have occurred during the development of Chemical Mutant.

Because of low levels of marker polymorphism, it has been difficult to find markers linked to disease resistance genes originating from within N. tabacum (Nishi et al., 2003; unpublished observations). The work reported here, however, adds to the number of cases in which molecular markers have been identified for resistance genes introgressed from wild Nicotiana relatives (Bai et al., 1995; Yi and Rufty, 1998; Yi et al., 1998; Johnson et al., 2002; Lewis, 2005). The close linkage of a set of nine RAPD markers and two derived SCAR markers to a major gene influencing blue mold resistance indicates their potential value for marker-based selection. Field evaluation of experimental breeding materials for blue mold resistance can be difficult in many countries because of the stringent environmental requirements required for disease development. The set of RAPD and SCAR markers should be a valuable asset to breeding programs worldwide wishing to incorporate resistance into new cultivars.

Markers have also been identified that are linked to genetic resistance to Tobacco mosaic virus (Whitham et al., 1994), wildfire [caused by Pseudomonas syringae pv. tabaci (Wolf & Foster) Young et al.; Yi et al., 1998], root-knot nematodes (Meloidogyne spp.; Yi and Rufty, 1998), black shank [caused by Phytophthora parasitica Dastur var. nicotianae (Breda de Haan) Tucker; Johnson et al., 2002, and Potato virus Y (Lewis, 2005). Molecular marker technology can be effectively integrated into a tobacco breeding program to allow selection of multiple disease resistance genes with a single DNA extraction. Coupling-phase, dominant markers linked to resistance genes are generally more useful than repulsion phase markers in a breeding program. Doubled haploid breeding methodologies are frequently used in tobacco breeding (Legg and Smeeton, 1999). DNA markers for disease resistance could be used very effectively in a doubled haploid breeding program by allowing identification of haploid individuals possessing desired resistance genes before the chromosome doubling step.

ACKNOWLEDGMENTS

This research was supported in part by the North Carolina Tobacco Research Commission.

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

S.R. Milla, J.S. Levin, and R.S. Lewis, Dep. of Crop Science, North Carolina State Univ., Campus Box 7620, Raleigh, NC 27695-7620. R.C. Rufty, Office of the Graduate School, North Carolina State Univ., Campus Box 71-2, Raleigh, NC 27695. Received 22 Dec. 2004. * Corresponding author (ramsey_lewis@ncsu.edu).
Table 1. Experiments conducted at Papantla, Mexico, to evaluate
experimental doubled haploid (DH) lines for resistance to
blue mold.
                      Number of    Number of
Experiment    Year      Reps       DH Lines     Population pedigree

BM1           1989        2           50         NC-BMR 113/Ky 14
BM2           1992        3           23         NC-BMR 113/Ky 14
BM3           1992        3           32         NC-BMR 114/Ky 14
BM4           1996        4           17         NC-BMR 114/TN 90

Table 2. RAPD genotypes for tobacco breeding lines and cultivars
classified as being resistant or susceptible.

                     Resistance                Reported origin
Cultivar/Line        Classification    of resistance ([double dagger])

Ky 14                susceptible                      -
TN 86                susceptible                      -
TN 90                tolerant                         -
Ky 8959              susceptible                      -
Ky 907               susceptible                      -
Ky 15                susceptible                      -
Ky 17                susceptible                      -
Ky 12                susceptible                      -
Burley 21            susceptible                      -
Barley 37            susceptible                      -
Burley 49            susceptible                      -
Burley 64            susceptible                      -
GR 131               susceptible                      -
Speight G-28         susceptible                      -
Speight G-70         susceptible                      -
Hicks                susceptible                      -
NC 88                susceptible                      -
NC 95                susceptible                      -
McNair 944           susceptible                      -
Havana 307           susceptible                      -
Sota 6505            resistant         N. debneyi
Virginia A Mutant    susceptible                      -
Orgaz C-1            susceptible                      -
NC 744               susceptible                      -
Judy's Pride         susceptible                      -
NC 744               susceptible                      -
L8                   susceptible                      -
VA 509               susceptible                      -
VA 510               susceptible                      -
VA 528               susceptible                      -
Bel 61-10            resistant         N. debneyi
Bel W-3              susceptible                      -
Bel B                susceptible                      -
NC11-51              resistant         chemical mutant
NC-BMR 42            resistant         Ovens 62
NC-BMR 90            resistant         Ovens 62
NC-BMR 113           resistant         Ovens 62
NC-BMR 114           resistant         Ovens 62
Ovens 62             resistant         N. velutina
Chemical Mutant      resistant         induced mutation
Sirone               resistant         N. goodspeedii
Sirogo               resistant         N. Goodspeedii
ZA 684               resistant         unknown to authors
ZA 692               resistant         unknown to authors
Z-3                  susceptible                      -

                                Marker ([dagger])

Cultivar/Line        UBC624.480    UBC610.528    UBC563.566

Ky 14                    -             -             -
TN 86                    -             -             -
TN 90                    -             -             -
Ky 8959                  -             -             -
Ky 907                   -             -             -
Ky 15                    -             -             -
Ky 17                    -             -             -
Ky 12                    -             -             -
Burley 21                -             -             -
Barley 37                -             -             -
Burley 49                -             -             -
Burley 64                -             -             -
GR 131                   -             -             -
Speight G-28             -             -             -
Speight G-70             -             -             -
Hicks                    -             -             -
NC 88                    -             -             -
NC 95                    -             -             -
McNair 944               -             -             -
Havana 307               -             -             -
Sota 6505                -             +             +
Virginia A Mutant        -             -             -
Orgaz C-1                -             -             -
NC 744                   -             -             -
Judy's Pride             -             -             -
NC 744                   -             -             -
L8                       -             -             -
VA 509                   -             -             -
VA 510                   -             -             -
VA 528                   -             -             -
Bel 61-10                +             +             +
Bel W-3                  -             -             -
Bel B                    -             -             -
NC11-51                  +             +             +
NC-BMR 42                +             +             +
NC-BMR 90                +             +             +
NC-BMR 113               +             +             +
NC-BMR 114               +             +             +
Ovens 62                 +             +             +
Chemical Mutant          +             +             +
Sirone                   +             +             +
Sirogo                   +             +             +
ZA 684                   +             +             +
ZA 692                   +             +             +
Z-3                      -             -             -

                                Marker ([dagger])

Cultivar/Line        UBC544.499    UBC534.436    UBC528.528

Ky 14                    -             -             -
TN 86                    -             -             -
TN 90                    -             -             -
Ky 8959                  -             -             -
Ky 907                   -             -             -
Ky 15                    -             -             -
Ky 17                    -             -             -
Ky 12                    -             -             -
Burley 21                -             -             -
Barley 37                -             -             -
Burley 49                -             -             -
Burley 64                -             -             -
GR 131                   -             -             -
Speight G-28             -             -             -
Speight G-70             -             -             -
Hicks                    -             -             -
NC 88                    -             -             -
NC 95                    -             -             -
McNair 944               -             -             -
Havana 307               -             -             -
Sota 6505                +             +             +
Virginia A Mutant        -             -             -
Orgaz C-1                -             -             -
NC 744                   -             -             -
Judy's Pride             -             -             -
NC 744                   -             -             -
L8                       -             -             -
VA 509                   -             -             -
VA 510                   -             -             -
VA 528                   -             -             -
Bel 61-10                +             +             +
Bel W-3                  -             -             -
Bel B                    -             -             -
NC11-51                  +             +             +
NC-BMR 42                +             +             +
NC-BMR 90                +             +             +
NC-BMR 113               +             +             +
NC-BMR 114               +             +             +
Ovens 62                 +             +             +
Chemical Mutant          +             +             +
Sirone                   +             +             +
Sirogo                   +             +             +
ZA 684                   +             +             +
ZA 692                   +             -             +
Z-3                      -             -             -

                                Marker ([dagger])

Cultivar/Line        UBC243.335    UBC240.545    UBC180.328

Ky 14                    +             +             -
TN 86                    +             +             -
TN 90                    +             +             -
Ky 8959                  +             +             -
Ky 907                   +             +             -
Ky 15                    +             +             -
Ky 17                    +             +             -
Ky 12                    +             +             -
Burley 21                +             +             -
Barley 37                +             +             -
Burley 49                +             +             -
Burley 64                +             +             -
GR 131                   +             +             -
Speight G-28             +             +             -
Speight G-70             +             +             -
Hicks                    +             +             -
NC 88                    +             +             -
NC 95                    +             +             -
McNair 944               +             +             -
Havana 307               +             +             -
Sota 6505                -             -             +
Virginia A Mutant        +             +             -
Orgaz C-1                +             +             -
NC 744                   +             +             -
Judy's Pride             +             +             -
NC 744                   +             +             -
L8                       +             +             -
VA 509                   +             +             -
VA 510                   +             +             -
VA 528                   +             +             -
Bel 61-10                -             -             +
Bel W-3                  +             +             -
Bel B                    +             +             -
NC11-51                  -             -             +
NC-BMR 42                -             -             +
NC-BMR 90                -             -             +
NC-BMR 113               -             -             +
NC-BMR 114               -             -             +
Ovens 62                 -             -             +
Chemical Mutant          -             -             +
Sirone                   -             -             +
Sirogo                   -             -             +
ZA 684                   -             -             +
ZA 692                   -             -             +
Z-3                      +             +             -

                                Marker ([dagger])

Cultivar/Line        UBC149.228    UBC024.589    OPAE07.316

Ky 14                    -             -             +
TN 86                    -             -             +
TN 90                    -             -             +
Ky 8959                  -             -             +
Ky 907                   -             -             +
Ky 15                    -             -             +
Ky 17                    -             -             +
Ky 12                    -             -             +
Burley 21                -             -             +
Barley 37                -             -             +
Burley 49                -             -             +
Burley 64                -             -             +
GR 131                   -             -             +
Speight G-28             -             -             +
Speight G-70             -             -             +
Hicks                    -             -             +
NC 88                    -             -             +
NC 95                    -             -             +
McNair 944               -             -             +
Havana 307               -             -             +
Sota 6505                +             +             -
Virginia A Mutant        -             -             +
Orgaz C-1                -             -             +
NC 744                   -             -             +
Judy's Pride             -             -             +
NC 744                   -             -             +
L8                       -             -             +
VA 509                   -             -             +
VA 510                   -             -             +
VA 528                   -             -             +
Bel 61-10                +             +             -
Bel W-3                  -             -             +
Bel B                    -             -             +
NC11-51                  +             +             -
NC-BMR 42                +             +             -
NC-BMR 90                +             +             -
NC-BMR 113               +             +             -
NC-BMR 114               +             +             -
Ovens 62                 +             +             -
Chemical Mutant          +             +             -
Sirone                   +             +             -
Sirogo                   +             +             -
ZA 684                   +             +             -
ZA 692                   -                           -
Z-3                      -             -             +

                                Marker ([dagger])

Cultivar/Line        OPAE02.335    OPR06.268     OPC09.670

Ky 14                    -             -             -
TN 86                    -             -             -
TN 90                    -             -             -
Ky 8959                  -             -             -
Ky 907                   -             -             -
Ky 15                    -             -             -
Ky 17                    -             -             -
Ky 12                    -             -             -
Burley 21                -             -             -
Barley 37                -             -             -
Burley 49                -             -             -
Burley 64                -             -             -
GR 131                   -             -             -
Speight G-28             -             -             -
Speight G-70             -             -             -
Hicks                    -             -             -
NC 88                    -             -             -
NC 95                    -             -             -
McNair 944               -             -             -
Havana 307               -             -             -
Sota 6505                +             +             +
Virginia A Mutant        -             -             -
Orgaz C-1                -             -             -
NC 744                   -             -             -
Judy's Pride             -             -             -
NC 744                   -             -             -
L8                       -             -             +
VA 509                   -             -             -
VA 510                   -             -             -
VA 528                   -             -             -
Bel 61-10                +             +             +
Bel W-3                  -             -             -
Bel B                    -             -             -
NC11-51                  +             +             +
NC-BMR 42                +             +             +
NC-BMR 90                +             +             +
NC-BMR 113               +             +             +
NC-BMR 114               +             +             +
Ovens 62                 +             +             +
Chemical Mutant          +             +             +
Sirone                   +             +             +
Sirogo                   +             +             +
ZA 684                   +             +             +
ZA 692                   +             +             +
Z-3                      -             -             -

([dagger]) Markers UBC243.335, UBC240.545, and OPAE07.316 are markers
linked in repulsion phase with the blue mold resistance gene. The
remaining markers are linked in coupling phase with the resistance
gene. '+' = marker presence; '-' = marker absence.

([double dagger]) Origins of resistance have been discussed primarily
by Rufty (1989). The origin of resistance in GA 955 was listed by Delon
(1992).

Table 3. Chi-Square tests for segregation distortion and F tests for
associations between marker genotype and blue mold resistance for
four experiments conducted at Papantla, Mexico.

                 Primer
                sequence                      Experiment BM1
                              Linkage
RAPD Marker     5' to 3'       phase      [chi square] ([dagger])

UBC624.480     GTGATAAGCC    coupling              0.08
UBC610.528     TTTGCCGCCC    coupling              0.00
UBC563.566     CGCCTCTCCT    coupling              0.00
UBC544.499     TAGAGACTCC    coupling              0.32
UBC534.436     CACCCCCTGC    coupling              0.08
UBC528.528     GGATCTATGC    coupling              0.00
UBC243.335     GGGTGAACCG    repulsion             1.28
UBC240.545     ATGTTCCAGG    repulsion             2.00
UBC180.328     GGGCCACGCT    coupling              0.00
UBC149.228     AGCAGCGTGG    coupling              0.08
UBC024.589     ACAGGGGTGA    coupling              0.00
OPAE07.316     GTGTCAGTGG    repulsion             0.32
OPAE02.335     TCGTTCACCC    coupling              0.08
OPR06.268      GTCTACGGCA    coupling              0.03
OPC09.670      CTCACCGTCC    coupling              0.08

                           Experiment BM1

RAPD Marker    F value ([double dagger])    [R.sup.2]

UBC624.480             21.37 ***              0.190
UBC610.528             20.04 ***              0.181
UBC563.566             20.04 ***              0.181
UBC544.499             25.73 ***              0.219
UBC534.436             16.60 ***              0.156
UBC528.528             20.04 ***              0.181
UBC243.335             20.67 ***              0.185
UBC240.545             19.74 ***              0.178
UBC180.328             20.04 ***              0.181
UBC149.228             16.60 ***              0.156
UBC024.589             20.04 ***              0.181
OPAE07.316             21.37 ***              0.190
OPAE02.335             16.60 ***              0.156
OPR06.268              21.37 ***              0.190
OPC09.670              21.37 ***              0.190

                           Experiment BM2

RAPD Marker    [chi square]     F value     [R.sup.2]

UBC624.480         0.04        42.05 ***      0.412
UBC610.528         0.39        15.58 ***      0.214
UBC563.566         0.39        15.58 ***      0.214
UBC544.499         0.04        42.05 ***      0.412
UBC534.436         0.39        15.58 ***      0.214
UBC528.528         0.04        42.05 ***      0.412
UBC243.335         0.04        42.05 ***      0.412
UBC240.545         0.04        42.05 ***      0.412
UBC180.328         0.04        42.05 ***      0.412
UBC149.228         0.39        15.58 ***      0.214
UBC024.589         0.04        42.05 ***      0.412
OPAE07.316         0.04        42.05 ***      0.412
OPAE02.335         0.39        15.58 ***      0.214
OPR06.268          0.04        42.05 ***      0.412
OPC09.670          0.04        42.05 ***      0.412

                           Experiment BM3

RAPD Marker    [chi square]     F value     [R.sup.2]

UBC624.480        0.50         11.10 **       0.119
UBC610.528        3.90 *        9.67 **       0.118
UBC563.566        2.00         14.24 ***      0.145
UBC544.499        3.13          1.70          0.030
UBC534.436        2.00         14.24 ***      0.145
UBC528.528        2.00         14.24 ***      0.145
UBC243.335        2.00         14.24 ***      0.145
UBC240.545        2.00         14.24 ***      0.145
UBC180.328        2.00         14.24 ***      0.145
UBC149.228        2.00         14.24 ***      0.145
UBC024.589        2.00         14.24 ***      0.145
OPAE07.316        2.00         14.24 ***      0.145
OPAE02.335        2.00         14.24 ***      0.145
OPR06.268         2.00         14.24 ***      0.145
OPC09.670         2.00         14.24 ***      0.145

                           Experiment BM4

RAPD Marker    [chi square]    F value      [R.sup.2]

UBC624.480        1.47          7.01 *        0.206
UBC610.528        4.00 *        4.09 *        0.177
UBC563.566        2.88          6.07 *        0.195
UBC544.499        0.53         11.60 **       0.255
UBC534.436        2.88          6.07 *        0.195
UBC528.528        4.00 *        5.91 *        0.215
UBC243.335        0.53         20.40 ***      0.334
UBC240.545        0.53         20.40 ***      0.334
UBC180.328        2.88          6.07 *        0.195
UBC149.228        2.88          6.07 *        0.195
UBC024.589        0.53         15.89 ***      0.295
OPAE07.316        0.16         16.48 ***      0.301
OPAE02.335        2.88          6.07 *        0.195
OPR06.268         2.88          6.07 *        0.195
OPC09.670         0.53          8.23 **       0.220

* Denotes significance at 0.05 probability level.

** Denotes significance at 0.01 probability level.

*** Denotes significance at 0.001 probability level.

([dagger]) Chi-square tests were used to test for significant
deviations from a 1:1 marker segregation ratio.

([double dagger]) F-tests were applied to each marker to test for
signficant differences for blue mold resistance between the two marker
classes.

Table 4. RAPD and SCAR genotypes for Nicotiana species accessions
respresenting possible donors of resistance.

                                        Marker ([dagger])

Cultivar or line             UBC624.480    UBC563.566    UBC544.499

N. excelsior (PI 224063)          -             -             +
N. velutina (PI 244638)           -             -             +
N. goodspeedii (PI 241012)        -             -             +
N. debneyi (PI 503320)            +             +             +

                                        Marker ([dagger])

Cultivar or line             UBC528.528    UBC149.228    UBC024.589

N. excelsior (PI 224063)          -             -             +
N. velutina (PI 244638)           +             -             +
N. goodspeedii (PI 241012)        -             -             +
N. debneyi (PI 503320)            -             +             +

                                        Marker ([dagger])

Cultivar or line             OPAE02.335     OPC09.670    SUBC180.251

N. excelsior (PI 224063)          +             +             -
N. velutina (PI 244638)           +             +             -
N. goodspeedii (PI 241012)        +             +             -
N. debneyi (PI 503320)            +             +             +

                             Marker ([dagger])

Cultivar or line                UOPR06.268

N. excelsior (PI 224063)             -
N. velutina (PI 244638)              -
N. goodspeedii (PI 241012)           -
N. debneyi (PI 503320)               +

([dagger]) `+' = marker presence; `-' = marker absence.
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Author:Milla, S.R.; Levin, J.S.; Lewis, R.S.; Rufty, R.C.
Publication:Crop Science
Date:Nov 1, 2005
Words:6469
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