Agronomic Performance of Tobacco Mosaic Virus ...

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Apr 20, 2010 - Current U.S. burley and flue-cured tobacco cul- tivars carry the introgressed resistance gene on chromosome H of the. N. tabacum genome ...
RESEARCH

Agronomic Performance of Tobacco Mosaic Virus-Resistant Tobacco Lines and Hybrids Possessing the Resistance Gene N Introgressed on Different Chromosomes Ramsey S. Lewis* and Cara Rose ABSTRACT Resistance to tobacco mosaic virus (TMV) is conferred by the single dominant gene, N, in Nicotiana glutinosa L. This gene has been transferred to cultivated tobacco (N. tabacum L.) via interspecific hybridization and backcrossing. Current TMV-resistant (TMVR) cultivars carry N introgressed on chromosome H of the N. tabacum genome. Undesirable linkage drag effects have caused associations with reduced yields and/or quality in flue-cured tobacco, however. Other germplasm lines possess the gene transferred onto an alternative chromosome. The objective of this research was to compare the agronomic performance of nearly isogenic lines (NILs) and hybrids possessing N on different chromosomes and originating from four N donor lines. Regardless of the source of the gene, Nn heterozygotes were intermediate in value for yield, cash return, and cured leaf chemistry relative to nn and NN homozygotes. Lines and hybrids carrying N transferred from Xanthi nc produced the highest yields, whereas those possessing N introduced from TI 1473 exhibited the lowest yields. Overall, materials possessing N on chromosome H were not found to be significantly different for yield, grade index, value per hundred weight (US$ cwt–1), or cash return from those carrying the resistance gene on the alternative chromosome. Breeding strategies designed to reduce the amount of N. glutinosa chromatin linked to N are needed to develop TMVR flue-cured tobacco cultivars that do not exhibit an accompanying yield penalty.

R.S. Lewis and C. Rose, Campus Box 7620, Crop Science Dep., North Carolina State Univ., Raleigh, NC 27695. Received 21 Oct. 2009. *Corresponding author ([email protected]). Abbreviations: AFLP, amplified fragment length polymorphism; NIL, nearly isogenic line; TMV, tobacco mosaic virus; TMV R, TMVresistant; TMVS, TMV-susceptible; US$ cwt–1 or $ cwt–1, value per hundred weight.

T

obacco mosaic virus (TMV) is one of the most important pathogens affecting tobacco (Nicotiana tabacum L.) production 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 utilization of the single gene, N, derived from the diploid wild relative N. glutinosa L. (2n = 24) that confers resistance to TMV via a hypersensitive response. Several researchers independently worked to transfer N from N. glutinosa to allotetraploid cultivated tobacco (Holmes, 1938; Ternovsky, 1941, 1945; Goodspeed, 1942; Kostoff and Georgieva, 1944; Gerstel, 1945; Kostoff, 1948; Valleau, 1952; Oka, 1961), and multiple independent introgression lines were probably produced (Lewis et al., 2005). Current U.S. burley and flue-cured tobacco cultivars carry the introgressed resistance gene on chromosome H of the N. tabacum genome (Lewis et al., 2005). An array of other lines carry N on an alternative chromosome of the N. tabacum genome (Chaplin and Gooding, 1969; Gwynn, 1977; Beekwilder, 1999; Bagley, 2002; Lewis et al., 2005). Molecular marker data of Lewis et al. (2005) provided evidence of appreciable differences in sizes of the introgressed alien chromosome segments in TMV-resistant (TMVR) germplasm.

Published in Crop Sci. 50:1339–1347 (2010). doi: 10.2135/cropsci2009.10.0615 Published online 20 Apr. 2010. © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

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Materials possessing disease resistance genes transferred using interspecific hybridization and backcrossing often exhibit associations between the presence of the resistance gene and characteristics such as reduced yields or quality. This might be a result of pleiotropic effects of the resistance gene per se or because of linkage drag effects caused by the presence of deleterious genes of alien origin linked to the gene of interest (Legg et al., 1981; Zeven et al., 1983; Friebe et al., 1996; Brown, 2002). Suppressed recombination within introgressed chromatin (Paterson et al., 1990; Messeguer et al., 1991; Causse et al., 1994; Ganal and Tanksley, 1996; Liharska et al., 1996) can make it difficult to alleviate linkage drag effects through backcrossing (Stam and Zeven, 1981; Young and Tanksley, 1989). Reductions in yield and quality have been associated with conventionally introgressed N in flue-cured tobacco (Chaplin et al., 1966; Chaplin and Mann, 1978), which has impeded development of commercially viable TMV R cultivars of this market class. N was the first plant virus resistance gene to be cloned (Whitham et al., 1994), however, and this permitted introduction of the resistance gene into tobacco without any flanking alien N. glutinosa genes using plant transformation (Lewis et al., 2007). Results indicated that reduced yields associated with N in flue-cured tobacco were attributable to linkage drag rather than pleiotropic effects per se. In principle, introduction of N into elite cultivars via transformation offers the opportunity to bypass potential for undesirable linkage drag effects (Lewis et al., 2007). Current international objection by manufacturers and leaf dealers to genetically engineered tobacco, however, prohibits the use of this approach for development of commercial tobacco cultivars. Studies to investigate the possible value of alternative introgression events might therefore be a worthwhile research objective. Compensating alien chromosome segment transfers are generally presumed to be the most desirable type from an agronomic standpoint, and introgression site could therefore affect the practical value of derived materials. Likewise, the size of an alien chromosome segment is often inversely correlated with yield or quality characteristics. The first objective of this investigation was to use the backcross breeding procedure to transfer N from four donor lines to two elite flue-cured tobacco cultivars and one breeding line. The first donor line, NC 1125-2, carries N on chromosome H of the N. tabacum genome and is representative of U.S. TMV R breeding materials. The other three donor lines (Xanthi nc, TI 1473, and TI 1500) possess the gene on a yet-to-be-identified alternative chromosome. The second objective was to evaluate the BC5F3 NN homozygous nearly isogenic lines (NILs) and Nn hybrids for yield and quality through field studies conducted in six environments. Comparisons were made among materials receiving N from different donor lines 1340

and also between groups possessing N in heterozygous or homozygous condition.

MATERIALS AND METHODS Generation of Genetic Materials Two TMV-susceptible (TMVS) commercial flue-cured tobacco cultivars (‘NC 55’ and ‘Speight 168’) and one TMVS flue-cured breeding line (NCTG 61) were selected to be recipients of N via the backcross breeding procedure. Four lines were selected as donors of N: NC 1125-2, TI 1473, TI 1500, and Xanthi nc. Flue-cured tobacco breeding line NC1125-2 is the TMVR parental line of commercial flue-cured tobacco hybrid ‘NC 297’ and possesses N on chromosome H of the N. tabacum genome. This breeding line is representative of U.S. TMVR tobacco germplasm and was generated by transferring N from flue-cured tobacco cultivar ‘Coker 51’ to flue-cured tobacco cultivar ‘K 326’ using eight backcrosses. TI 1473, TI 1500, and Xanthi nc carry N on the same alternative chromosome of the tobacco genome (Lewis et al., 2005). TI 1473 is a primitive tobacco accession donated to the U.S. Nicotiana Germplasm Collection (Lewis and Nicholson, 2007) by the Universidad Central de Venezuela. TI 1500 is a tobacco accession obtained from the Union of Soviet Socialist Republics as Immune 580. Xanthi nc is an Oriental-type tobacco. Based on molecular marker genotyping, TI 1473, TI 1500, and Xanthi nc probably possess larger introgressed N. glutinosa chromosome segments than NC 1125-2. The alien segments in TI 1473 and TI 1500 may be larger than that present in Xanthi nc (Lewis et al., 2005). Tobacco mosaic virus resistance was introduced into NC 55 (fertile version), Speight 168, and NCTG 61 using conventional methods by initially hybridizing each TMVS line with each TMV R donor line. F1 plants were then backcrossed to their respective recurrent parents with selection for TMV resistance at each generation using the method of Rufty et al. (1987). After five backcrosses, TMV R BC5F1 plants were selfpollinated to produce BC5F2 families. Single TMV R BC5F2 plants homozygous for N were then identified for each family through evaluation of testcross progeny. Individual homozygous (NN) BC5F2 plants were both self-pollinated and hybridized with their respective TMVS recurrent parents to produce homozygous (NN) and heterozygous (Nn) nearly isogenic versions of NC 55, Speight 168, and NCTG 61 using each of four N donor lines. A total of 24 seed lots were generated (3 recipient lines × 4 N donor lines × 2 zygosities).

Agronomic Evaluation Field evaluation of the 24 NILs and hybrids outlined above, in addition to their TMVS counterparts, was conducted in a total of six environments: the Central Crops Research Station (Clayton, NC), the Oxford Tobacco Research Station (Oxford, NC), and the Upper Coastal Research Station (Rocky Mount, NC) during 2007; and the Oxford Tobacco Research Station, the Upper Coastal Plain Research Station, and the Cunningham Research Station (Kinston, NC) during 2008. A randomized complete block design with seven replications was used at each location. Each plot consisted of a single row with 22 competitive plants. Interrow spacing was 1.14, 1.16, 1.20, and 1.22 m at Clayton, Rocky Mount, Oxford, and Kinston, respectively. Within-row spacing was 0.56 m at all locations. The end

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plants of each plot served as guard plants and were removed before harvest. Suggested management practices for flue-cured tobacco production were used at all four research stations. Leaves were harvested in four separate harvests (primings) and flue-cured. Each priming was weighed to generate yield data, and official USDA grades were assigned by a former USDA grader. A numerical reflection of cured leaf quality for each plot was generated using the 2008 North Carolina Flue-Cured Tobacco Grade Index (Fisher and Smith, 2008). Value per hundred weight ($ cwt–1) was calculated based on average price paid for standard grades during the 2008 growing season. Plot values for grade index and $ cwt–1 were calculated using a weighted average across all four primings. Fifty-gram cured leaf samples were prepared for each plot by compositing cured leaf from each priming on a weightedmean basis. Oven-dried samples were ground to pass through a 1-mm sieve and analyzed for percent total alkaloids and percent reducing sugars using the method of Davis (1976).

Data Analysis A combined ANOVA for all entries was fi rst performed using PROC GLM of SAS version 9.1 (SAS Institute, Cary, NC) and by applying F-tests as outlined by McIntosh (1983). Each location × year combination was considered as a single environment. Entries were treated as fi xed effects and environments as random effects. Entry-means were produced using the LSMEANS statement and LSDs were calculated according to Steel et al. (1997). An ANOVA was also performed on a reduced data set produced by removing data associated with the three TMVS checks. Here, the data were treated as coming from a three-factor experiment in a randomized complete block design. The three factors were N donor (NC 1125-2, TI 1473, TI 1500, and Xanthi nc), N recipient (NC 55, Speight 168, and NCTG 61), and N zygosity (heterozygous versus homozygous). Analysis of variance and calculation of LSDs were performed according to Carmer et al. (1989). Single degree of freedom group mean comparisons of interest were also made using CONTRAST statements in PROC GLM.

RESULTS Agronomic Evaluation The 24 TMV R NILs and hybrids, along with their three corresponding TMVS counterparts, were evaluated in a

total of six field environments for yield, quality, and cured leaf chemistry. Using an ANOVA on the complete data set, very significant differences among environments were observed for all six of the measured traits (p < 0.0001) (Table 1). Very significant differences were also observed between entries for yield, cash return, percent total alkaloids, and percent reducing sugars (p ≤ 0.001). Significant genotype × environment interactions were observed for all measured traits (p < 0.0001). The TMVS parental line NC 55 was the highest yielding entry and also provided the highest cash return per hectare (Table 2). When averaged across all TMV R genotypes, the presence of N was found to be associated with significant reductions in yield, cash return, and percent reducing sugars relative to the TMVS recurrent parent lines (p ≤ 0.005) (Table 3). Percent total alkaloids was significantly increased (p < 0.0001) in TMV R materials relative to the TMVS recurrent parents. No significant differences were observed for grade index or $ cwt–1 for these comparisons, however. An ANOVA was also performed on a reduced data set generated by eliminating data for the three TMVS checks. This permitted analysis of the experiment as a three-factor experimental design, the three factors being N donor, N zygosity, and N recipient. This analysis revealed highly significant differences (p < 0.0001) between environments for all measured characteristics (Table 4). Significant differences (p < 0.05) were detected between N donors for yield, percent total alkaloids, and percent reducing sugars. Significant differences were also found between the two levels of N zygosity (NN versus Nn) for yield, cash return, percent total alkaloids, and percent reducing sugars. Significant differences between N recipients were only detected for percent total alkaloids (p < 0.001). A number of interaction effects were also found to be statistically significant. N donor × N zygosity interactions were almost entirely of the noncrossover type, and combination means are therefore not presented. N donor × N recipient interactions are illustrated in Fig. 1. As the number of copies of N increased (from nn to Nn to NN), there were gradual and statistically significant

Table 1. Analysis of variance for TMV-resistant backcross-derived entries and corresponding TMV-susceptible recurrent parents evaluated in six North Carolina environments. Source Environment Replication (environment) Entry Entry × environment Pooled error

df

Yield

5 36 26 130 936

kg ha –1 22,855,668§ 1,597,735 603,243§ 158,564§ 84,539

Grade index

Value

11,685§ 105 41 60§ 16

$ cwt–1† 282,962§ 2369 1040 1215§ 362

Mean squares Cash return $ ha –1 506,443,143§ 28,338,909 8,900,886*** 3,735,510§ 1,448,987

Total alkaloids

Reducing sugars

%‡ 19.69§ 2.45 4.05§ 0.27§ 0.13

%† 1336.79§ 63.22 42.84§ 7.60§ 3.49

***Significant at p = 0.001 level. †

$ cwt–1, value per hundred weight.



Expressed as a percentage of dry wt.

§

Significant at p = 0.0001 level.

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Table 2. Entry-means for TMV-resistant backcross derivatives and corresponding TMV-susceptible recurrent parents evaluated in six North Carolina environments. Entries are ranked in order of increasing return ($ ha –1). Measured trait mean Entry Speight 168 NN (TI 1473)§ NC 55 NN (Xanthi nc) NC 55 NN (TI 1500) NCTG 61 NN (NC 1125-2) NC 55 NN (TI 1473) Speight 168 NN (NC 1125-2) NCTG 61 NN (TI 1473) NC 55 Nn (Xanthi nc) Speight 168 NN (TI 1500) Speight 168 NCTG 61 NN (Xanthi nc) Speight 168 Nn (TI 1473) NCTG 61 Nn (TI 1473) Speight 168 Nn (TI 1500) NCTG 61 NN (TI 1500) NCTG 61 Nn (NC 1125-2) Speight 168 Nn (Xanthi nc) Speight 168 NN (Xanthi nc) NC 55 NN (NC 1125-2) NCTG 61 Nn (TI 1500) NC 55 Nn (TI 1473) NCTG 61 NCTG 61 Nn (Xanthi nc) Speight 168 Nn (NC 1125-2) NC 55 Nn (NC 1125-2) NC 55 Nn (TI 1500) NC 55 Overall Mean LSD 0.05 CV %

Yield kg ha –1 2702.2 2912.6 2889.9 2910.5 2928.3 2977.5 2934.4 3045.0 2963.7 3046.9 3034.4 2978.0 3032.1 3097.6 3047.9 3085.9 3090.4 3068.0 3004.6 3103.0 3098.1 3189.7 3145.1 3173.6 3133.0 3110.2 3330.2 3038.2 171.9 13.1



$ cwt–1, value per hundred weight.



Expressed as a percentage of dry wt.

§

Name in parenthesis indicates source of N gene.

Grade index

Value

76.2 73.6 74.8 76.4 76.2 76.8 77.5 75.5 77.6 76.4 76.0 77.3 76.7 75.7 76.7 76.6 76.3 76.9 78.4 76.2 76.4 75.1 76.0 76.1 77.7 77.6 77.6 76.5 3.3 10.1

$ cwt–1† 343.37 330.01 339.58 343.31 345.67 344.94 349.82 340.09 349.50 343.73 342.59 348.50 345.60 340.35 345.26 343.25 343.26 346.19 354.19 342.89 345.42 336.92 343.18 342.37 350.45 351.69 350.72 344.55 15.05 10.12

decreases in yield and percent reducing sugars and also significant increases in percent total alkaloids (Tables 3 and 5; Fig. 2). Average yields of Nn and NN genotypic groups were 97.9 and 241.1 kg ha–1 lower than the mean yield for the TMVS (nn) recurrent parents (3.1 and 7.6% reductions, respectively). This translated into a significantly lower cash return (reduced by $770.11 ha–1, or 7.0%) for the NN group relative to the TMVS recurrent parents (p = 0.0001). Average percent total alkaloids for the Nn and NN groups were 0.19 and 0.33% points higher, respectively, than the mean for the TMVS parents (significantly different at p ≤ 0.0003). Average percent reducing sugars for the Nn and NN groups were 0.55 and 1.54% points lower than the means for the TMVS recurrent parents, respectively (significantly different at p ≤ 0.05). In comparisons between TMV R groups possessing N derived from different donor lines, materials carrying N derived from Xanthi nc were found to produce the 1342

Cash return $ ha –1 9295.84 9646.00 9863.33 10014.46 10181.42 10258.60 10315.69 10375.89 10390.70 10434.30 10449.17 10472.59 10532.46 10532.82 10599.77 10617.53 10659.95 10664.61 10692.04 10693.11 10740.60 10789.93 10822.95 10866.29 10997.56 11039.14 11679.03 10504.66 834.40 18.40

Total alkaloids %‡ 3.13 4.1 3.12 3.38 3.11 2.68 3.44 3.54 3.03 2.72 3.50 2.93 3.27 2.80 3.31 3.20 2.78 2.81 3.03 3.25 3.11 3.02 3.21 2.71 2.97 3.18 2.93 3.12 0.22 16.51

Reducing sugars %‡ 12.85 13.11 12.11 15.33 12.65 15.21 14.23 14.99 13.86 15.32 13.88 14.43 15.40 15.42 14.80 15.37 14.32 13.53 14.61 15.60 13.86 15.80 15.36 14.98 15.04 13.28 15.04 14.46 1.19 19.08

highest yields and highest percent total alkaloids (Table 6). Materials possessing N derived from TI 1473 exhibited the lowest yields and were significantly lower than the other groups for this trait (p < 0.05). N transferred from NC 1125-2 had less of a numerical effect on yield and cash return when in heterozygous condition as compared with N in heterozygous condition when transferred from the other donor lines (Fig. 2). In addition, phenotypic changes for percent total alkaloids and percent reducing sugars were less in materials carrying N introduced from NC 1125-2 as compared with those possessing N transferred from alternative sources (Fig. 2). N donor line NC 1125-2 possesses N on chromosome H, whereas N donor lines TI 1473, TI 1500, and Xanthi nc carry the resistance gene on an alternative chromosome (Lewis et al., 2005). In the current investigation, backcrossderived TMVR materials possessing N on chromosome H were not found to be significantly different from those with

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Table 3. Various comparisons of group means for materials evaluated in six North Carolina environments. Difference† Comparison

Yield

Grade index

kg ha –1 TMV-susceptible checks versus TMV-resistant lines

Value

Cash return

Total alkaloids

Reducing sugars

$ cwt–1‡

$ ha –1





169 (< 0.0001)

–0.09 (0.9000)

–0.86 (0.7954)

520.98 (0.0050)

–0.26 (< 0.0001)

1.04 (0.0001)

TMV-susceptible recipient lines versus Nn heterozygotes

98 (0.0149)

–0.14 (0.8551)

–0.97 (0.7816)

271.85 (0.1603)

–0.19 (0.0003)

0.55 (0.0479)

TMV-susceptible recipient lines versus NN homozygotes

241 (< 0.0001)

–0.043 (0.9556)

–0.75 (0.8300)

770.12 (0.0001)

–0.33 (< 0.0001)

1.54 (< 0.0001)

Nn heterozygotes versus NN homozygotes

143 (< 0.0001)

0.10 (0.8409)

0.22 (0.9212)

498.27 (< 0.0001)

–0.14 (< 0.0001)

0.99 (< 0.0001)

N-gene on chromosome H versus N-gene on alternative chromosome

37 (0.1982)

0.71 (0.2089)

2.36 (0.3530)

170.19 (0.2283)

–0.21 (< 0.0001)

1.00 (< 0.0001)

TI 1473 as N donor versus TI 1500 and Xanthi nc as N donors

–97 (0.0020)

0.62 (0.2976)

3.51 (0.1935)

–221.69 (0.1395)

–0.06 (0.1560)

–0.28 (0.1857)



Difference calculated by subtracting the latter group or entry-mean indicated in the comparison from the former. Number in parenthesis indicates the probability of observing the given difference if the true means were equal. These probabilities were generated using CONTRAST statements in PROC GLM of SAS (SAS Institute, Cary, NC).



$ cwt–1, value per hundred weight.

§

Expressed as a percentage of dry wt.

Table 4. Analysis of variance for TMV-resistant entries evaluated using a three-way factorial in a randomized complete block design. Mean squares Source of variation Environment Replication (environment) N donor N donor × environment N zygosity N zygosity × environment N donor × N zygosity N donor × N zygosity × environment N recipient N recipient × environment N donor × N recipient N donor × N recipient × environment N zygosity × N recipient N zygosity × N recipient × environment N donor × N zygosity × N recipient N donor × N zygosity × N recipient × environment Pooled error

df 5 36 3 15 1 5 3 15 2 10 6 30 2 10 6 30 828

Yield kg ha –1 19823223§ 1478809 621375* 120760 5165750*** 102156 105505 75498 81538 652615§ 409090 174447 71012 83924 112506 93851 83122

Grade index §

10249.8 104.4 74.1 66.0§ 2.4 72.1*** 7.9 24.6 10.2 348.5§ 54.9 44.7 55.2 24.7 40.6* 16.5 16.8

Value

Cash return

Total alkaloids

Reducing sugars

$ cwt–1† 249889§ 2486 1713 1426§ 12 1840§ 233 537 9 6481§ 1523 866 1198 433 923 403 365

$ ha –1 451262895§ 26816915 4868944 2527140* 62565093** 3227345* 872103 1857515 1077721 20621676§ 8664233* 2950082 2908194 1642819 4458293* 1819745 1434518

%‡ 17.068§ 2.345 4.697§ 0.184 4.882** 0.240 0.680*** 0.051 21.557*** 1.383§ 4.45§ 0.258 0.084 0.071 0.778§ 0.110 0.123

%‡ 1175.2§ 57.3 66.9** 8.5** 246.8*** 5.0 23.6** 2.9 139.8 38.2§ 26.0** 5.7 2.1 4.0 3.6 3.7 3.5

*Significant at p = 0.05 level. **Significant at p = 0.01 level. ***Significant at p = 0.001 level. †

$ cwt–1, value per hundred weight.



Expressed as a percentage of dry wt.

§

Significant at p = 0.0001 level.

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Fig. 1. Illustration of N donor × N recipient interactions for (A) cash return, (B) % total alkaloids, and (C) % reducing sugars. Table 5. Means for nn, Nn, and NN genotypic groups evaluated in six North Carolina environments. Measured trait mean Group

Yield

Grade index

Value

Cash return

Total alkaloids

Reducing sugars

TMV susceptible parent (nn) average Heterozygote (Nn) average Homozygote (NN) average

kg ha –1 3188.9 3091.0 2947.8

76.4 76.5 76.4

$ cwt–1† 343.79 344.75 344.54

$ ha –1 10967.75 10695.91 10197.64

%‡ 2.89 3.08 3.22

%‡ 15.39 14.84 13.85



$ cwt–1, value per hundred weight.



Expressed as a percentage of dry wt.

an alternative introgression site for yield, grade index, $ cwt–1, or cash return (Table 3). Derived materials possessing the TMV resistance gene on chromosome H were significantly lower for percent total alkaloids and significantly higher for percent reducing sugars, however (p < 0.0001). Materials carrying N introduced from TI 1473 produced significantly lower yields as compared with those carrying N transferred from either TI 1500 or Xanthi nc (p = 0.002) (Table 3).

DISCUSSION The N gene was initially introduced into cultivated tobacco from N. glutinosa using interspecific hybridization followed by backcrossing (Holmes, 1938; Ternovsky, 1344

1941, 1945; Goodspeed, 1942; Kostoff and Georgieva, 1944; Gerstel, 1945; Kostoff, 1948; Valleau, 1952; Oka, 1961). The negative association between the presence of this resistance gene and reduced yields or quality in flue-cured tobacco is a classic example of linkage drag in plant breeding, however, and few commercially successful TMV R flue-cured tobacco cultivars have ever been released. Although these linkage drag effects can be circumvented via introduction of the cloned resistance gene into elite cultivars through transformation procedures (Lewis et al., 2007), transgenic commercial tobacco cultivars are not currently accepted by the industry. Motivation therefore exists to increase the probability of successfully

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Fig. 2. Group means for backcross-derived materials carrying N at three levels of zygosity (nn, Nn, and NN) for (A) yield, (B) cash return, (C) % total alkaloids, and (D) % reducing sugars. Means are averages over three genetic backgrounds (NC 55, Speight 168, and NCTG 61) for four N donor lines. Table 6. Group means for backcross-derived materials possessing the N-gene derived from four different sources. Measured trait mean N-gene source TI 1473 TI 1500 Xanthi nc NC 1125-2 LSD (0.05) CV% †

$ cwt–1, value per hundred weight.



Expressed as a percentage of dry wt.

Yield kg ha –1 2945.5 3035.4 3049.3 3047.5 66.0 4.7

using materials possessing the resistance gene introgressed into tobacco via conventional methods. In the data reported here, the reduced yields and cash returns observed for backcross-derived TMV R materials are consistent with earlier reports describing this unfavorable association (Chaplin et al., 1966; Chaplin and Mann, 1978; Lewis et al., 2007). Previously reported reductions in quality (Chaplin et al., 1966; Chaplin and Mann, 1978) as reflected by grade index or $ cwt–1 were not observed in the current investigation, however. The superior yield of CROP SCIENCE, VOL. 50, JULY– AUGUST 2010

Grade index

Value

Cash return

Total alkaloids

Reducing sugars

76.7 76.4 75.7 77.0 1.5 4.3

$ cwt–1† 346.40 344.88 340.89 346.42 7.17 4.47

$ ha –1 10256.43 10519.81 10436.43 10574.42 301.80 6.21

%‡ 3.16 3.12 3.32 3.00 0.08 5.55

%‡ 13.90 14.18 14.20 15.09 0.55 8.30

Nn heterozygous over NN homozygotes is also consistent with prior reports (Chaplin et al., 1966) and supports the deployment of TMV R in commercial F1 Nn hybrids rather than NN lines. All current U.S. TMV R flue-cured and burley tobacco cultivars carry the N gene on chromosome H of the N. tabacum genome (Lewis et al., 2005). A series of germplasm lines have been identified that also carry the resistance gene on an alternative yet-to-be-identified chromosome, however (Beekwilder, 1999; Bagley, 2002;

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Lewis et al., 2005). Homeologous chromosome segments in closely related Nicotiana species probably have similar gene contents and can replace and compensate for each other. Compensating chromatin transfers between N. tabacum and alien chromosomes are probably the most agronomically desirable, whereas noncompensating introgressions may cause duplications or deficiencies that can complicate their use in cultivar development. The results of Whitham et al. (1994) indicated that at least a fraction of the introgressed chromosome segment in Samsun NN (carrying N on chromosome H) may have replaced orthologous sequences from the T subgenome of N. tabacum. This subgenome is probably 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 possibility that N. glutinosa chromatin replaced homologous regions of the S genome in TI 1473, TI 1500, or Xanthi nc is currently unknown. Compensating or noncompensating chromatin transfers during multiple, independent introgressions of N could result in end products that are more or less acceptable from an agronomic point of view. The current study was performed to determine whether or not the yield penalty in materials possessing N on chromosome H would be more or less as compared with materials carrying the gene in an alternative genomic position. On average, the two groups of TMV R materials were not found to be statistically different for yield, grade index, $ cwt–1, or cash return. Significant differences were observed between the two groups for the two measured leaf chemistry traits, percent total alkaloids, and percent reducing sugars. These two traits are of less importance from a field production point of view, however. Based on amplified fragment length polymorphism (AFLP) marker genotyping, it was previously reported that there are probably substantial differences in the sizes of the introgressed N. glutinosa chromosome segments in the N donor lines used in the current study (Lewis et al., 2005). Under the assumption of even AFLP marker distribution across introgressed N. glutinosa chromatin, NC 1125-2 probably carries an alien segment approximately one-fi fth the size of that in TI 1473 and TI 1500 and approximately one-fourth the size of that in Xanthi nc. Despite the probable size differences, TI 1500 and Xanthi nc-derived lines were comparable with those derived from NC 1125-2 for yield. The group of genetic materials possessing N transferred from TI 1473 was inferior to those carrying N introduced from NC 1125-2, TI 1500, or Xanthi nc. In conclusion, our data indicate that the introgressed N. glutinosa chromosome segments in TI 1500 and Xanthi nc are similar to the alien segments in currently used 1346

sources of TMV resistance in terms of their negative impact on yield. Lewis et al. (2005) demonstrated that it is possible to obtain recombination within the introgressed N. glutinosa region when in either genomic position. Application of marker-assisted backcrossing may permit selection against undesirable N. glutinosa alleles and may increase the probability of developing commercially acceptable TMV R flue-cured tobacco cultivars (Young and Tanksley, 1989). Acknowledgments This research was supported, in part, by Altria Client Services Inc. and Philip Morris International.

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