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PLANT GENETIC RESOURCES

Identification of Germplasm of Possible Value for Confronting an Unfavorable Inverse Genetic Correlation in Tobacco

Campus Box 7620, Crop Science Dep., North Carolina State Univ., Raleigh, NC 27695

^* Corresponding author (ramsey_lewis{at}ncsu.edu)

	ABSTRACT

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To be commercially viable in the USA, a flue-cured tobacco (Nicotiana tabacum L.) cultivar must be high-yielding and also meet minimum requirements for percent total alkaloids (PTA). The negative correlation between yield and PTA complicates development of higher-yielding cultivars with acceptable leaf chemistry, however. Identification and use of germplasm possessing alternative alleles positively affecting PTA could be an important component of strategies to develop commercially acceptable, higher-yielding cultivars. Choice of donor germplasm should be done carefully, however, because yield modulates the phenotypic expression of PTA. Consequently, comparison of materials for genetic potential to accumulate alkaloids might best be done at common levels of yield. This investigation used manual control of leaf number to manipulate yield of fifteen diverse tobacco genotypes grown in a split-plot design in two North Carolina environments. Within genotypes, the relationships between PTA and yield were found to be negative. Through statistical analyses, germplasm accessions TI 464 and TI 959 were found to exhibit the highest levels of PTA at given levels of yield in both environments. Data on N-partitioning indicated that these genotypes may have increased genetic potential for utilizing accumulated N for alkaloid synthesis. Transfer of alleles from these genotypes to elite germplasm pools may facilitate development of higher-yielding cultivars with acceptable PTA levels.

Abbreviations: N, nitrogen • N-ACC, nitrogen accumulation efficiency • N-UPT, nitrogen uptake efficiency • PTA, percent total alkaloids • PTN, percent total nitrogen • RMSP, Regional Minimum Standards Program • TAP, total alkaloid production

	INTRODUCTION

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CORRELATIONS between quantitative characters in crop plants often serve as obstacles to simultaneous improvement of two or more traits. For example, the negative correlation between grain yield and grain protein content complicates the development of high-yield, high-protein wheat (Triticum aestivum L.) and soybean [Glycine max (L.) Merr.] cultivars (Wehrmann et al., 1987; Delzer et al., 1995; Wilcox and Cavins, 1995; Feil, 1997; Cober and Voldeng, 2000). A similar problem exists in flue-cured tobacco breeding. To be commercially viable, a new cultivar of flue-cured tobacco must be high-yielding and also satisfy PTA requirements as outlined by the United States Regional Minimum Standards Program (RMSP). This program dictates that, before commercial release, an experimental cultivar exhibit PTA levels within a specific window (typically 2.5–3.5%) defined by two long-term check cultivars (Bowman, 1996). An inverse genetic correlation exists between yield and PTA in tobacco, however (Matzinger et al., 1960, 1972, 1989; Matzinger and Mann, 1964; Legg et al., 1965; Matzinger and Wernsman, 1968). This unfavorable relationship has made it difficult to develop higher-yielding flue-cured tobacco cultivars that continue to satisfy the chemical requirements of the RMSP.

Breeding strategies that would facilitate development of higher-yielding flue-cured tobacco cultivars with acceptable PTA levels are desired. Lerner (1958) indicated that genetic correlations might originate from genetic linkages and/or pleiotropy and suggested that gains in correlated characters might be made through strategies designed to break unfavorable linkages or to replace alleles with pleiotropic effects with alleles that are without, or have a reduced influence, on the correlated trait. Consequently, germplasm enhancement approaches have been investigated for confronting undesirable correlations in several crop species (McNeal et al., 1978; Wehrmann et al., 1987; Wilcox and Cavins, 1995).

A large degree of phenotypic variation for PTA has been reported for tobacco accessions maintained by the United States Nicotiana Germplasm Collection (Sisson and Saunders, 1982). One might attempt to transfer alleles positively affecting PTA from exotic donor materials to elite, high-yielding flue-cured tobacco lines that exhibit lower-than-acceptable levels of PTA. Selection of potential donor germplasm should be done carefully, however, if much of the phenotypic variation for PTA is caused by genetic differences for yield. Nicotine normally comprises greater than 90% of the total alkaloids in flue-cured tobacco (Bush et al., 1993; Bush and Crowe, 1989). This alkaloid is synthesized in the roots (Dawson, 1942) and subsequently translocated to the leaves in processes that are stimulated by herbivore damage (Baldwin, 1989, 1999) and removal of the apical inflorescence (Collins and Hawks, 1993). Some researchers have suggested the involvement of a dilution effect in the relationship between PTA and yield in tobacco, where nicotine becomes diluted by incremental increases in above-ground biomass (Matzinger and Mann, 1964; Wolf and Bates, 1964). Consequently, some tobacco accessions may exhibit high PTA because of the fact that they possess genes contributing to low yielding ability.

Kramer (1979) suggested that, in wheat, if cultivar differences for grain protein content were due primarily to differences in harvest index, that cultivars could only be compared sensibly for protein content at some common level of harvest index. He suggested that if the relationship between grain protein content and harvest index could be known for multiple genotypes, one could make predictions about the protein content of the genotypes at a predetermined harvest index. To select tobacco germplasm with increased genetic potential for alkaloid accumulation, it might be advisable to make PTA comparisons among possible donor materials at some common level of yield in a shared environment. The primary objectives of this research were to (i) determine the genotypic response for PTA for 15 diverse tobacco genotypes when yield was manipulated via decapitation at three target leaf numbers and (ii) to use this information in analyses to compare these genotypes for PTA at common levels of yield. Genotypes possessing higher PTA at given levels of yield might have an increased probability of possessing alleles positively affecting alkaloid accumulation that are not currently present in elite flue-cured germplasm pools.

Finally, genotypes exhibiting increased PTA may do so for a number of reasons, including enhanced absorption of soil N or alternative pathways for N partitioning. Secondary objectives were to investigate possible relationships between increased alkaloid accumulation and several nitrogen absorption and use indices. The ultimate goals of this research were to identify genetic materials that may be of value to breeding programs designed to develop higher-yielding flue-cured tobacco cultivars with commercially acceptable cured leaf chemistry.

	MATERIALS AND METHODS

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Genetic Material
Fifteen tobacco genotypes were selected for this investigation (Table 1). Cultivars Hicks, K326, and NC 95 were selected as being representative of the U.S. flue-cured germplasm base. The Canadian cultivar Delgold was selected because it has N. rustica L. (a high alkaloid Nicotiana species) in its pedigree and was derived from a breeding program emphasizing selection for increased PTA (Pandeya and White, 1984). TI 501, TI 216, TI 959, and TI 464 are accessions from the United States Nicotiana Germplasm Collection that were selected for high PTA per se on the basis of data from a one-replicate field test reported by Sisson and Saunders (1982). TI 281, TI 648, TI 835, TI 859, TI 934, and TI 973 are germplasm accessions selected on the basis of high best-possible estimates of total alkaloid production (TAP, kg ha^–1) calculated using PTA and green leaf yield data publicly available from the website of the Germplasm Resources Information Network (http://www.ars-grin.gov). It should be noted that these estimates were weak because the PTA and yield data were not collected from the same field experiments. The accessions selected for high PTA and TAP were not necessarily the highest for their respective categories because some selection for plant type was conducted so that the selected accessions could be grown efficiently under a conventional tobacco production regime. ‘Silver Dollar’ is an old-line U.S. flue-cured tobacco cultivar that was selected for high TAP on the basis of published data (Chaplin et al., 1962).

Plants were decapitated at the target leaf number when the leaf immediately above the target leaf number was approximately 11 cm in length. Control of axillary bud growth was achieved by pouring flumetralin [N-(2-chloro-6-fluorobenzyl)-N-ethyl-,,-trifluoro-2,6-dinitro-p-toluidine] at the recommended rate down the stalk at the time of decapitation, followed by hand removal, as necessary. Fertilization regimes were based on recommended rates for standard flue-cured tobacco production. On the basis of soil tests, plots were fertilized approximately 2 d after transplanting with sidedress applications of 560 kg ha^–1 8–8-24 (N-P-K) in 2003 and 336 kg ha^–1 8–8-24 (N-P-K) in 2004. Approximately 2 to 3 wk after the initial fertilizer applications, supplemental sidedress applications of 30% liquid urea ammonium nitrate (UAN) were made (93.5 L ha^–1 and 130.9 L ha^–1 in 2003 and 2004, respectively).

Leaves were harvested in four separate harvests (primings) according to the rate of leaf ripening for the standard U.S. cultivars. Approximately one-fourth of the leaves were harvested for each plot at each priming. Harvested leaves were flue-cured using a regime appropriate for the leaves from the standard cultivars. Fifty-gram cured leaf samples were prepared for each plot by compositing cured leaf from each priming on a weighted-mean basis. Oven-dried samples were ground to pass through a 1-mm sieve and analyzed for PTA by the method of Davis (1976) and percent total N (PTN) by the procedure of Nelson and Sommers (1973).

The relationships between PTA and several N absorption and use indexes were also of interest. Nitrogen-accumulation efficiency (N-ACC) and N-uptake efficiency (N-UPT) were calculated for each genotype averaged over whole-plots and locations according to Sisson et al. (1991) by the following formulae:

In addition, the ratio of alkaloids to total N in the cured leaf on a w/w basis (LALN) was calculated as:

Also, the ratio of alkaloids in the cured leaf to fertilizer-N on a w/w basis (LANF) was computed using the formula:

It should be noted that N-ACC, N-UPT, and LALN are indices based on total N in the leaves only and not total N for all plant parts. For this paper, we have made the assumption that tissue N concentrations of stalk, midribs, and roots are proportional to leaf N concentrations among the genotypes that were tested. Moll et al. (1982) suggested that estimates of N accumulation in leaves can provide insight into variation for N uptake and use.

Statistical Analyses
An analysis of variance (ANOVA) appropriate for a split-plot design (McIntosh, 1983) was conducted using PROC MIXED of SAS, ver. 9.1 (Littell et al., 1996). Whole-plots and subplots were considered as fixed factors and environments were considered as random. The ANOVA was used to test the main effects and their interactions on the measured variables yield, PTA, TAP, PTN, N-ACC, N-UPT, LALN, and LANF. Mean separations for whole-plots and subplots were conducted using LSD tests at the 0.05 and 0.01 probability levels (Steel et al., 1997).

The intragenotypic relationships between yield and PTA were investigated through visual inspection of plotted data points and also by using PROC REG of SAS to determine the degree of association between the two measured traits through calculation of simple linear regression and correlation coefficients. Confidence intervals (95%) were computed for each regression coefficient to test for significant differences among genotypes for the degree of association between yield and PTA. These analyses were conducted independently for each year to allow for comparisons across the two environments.

To compare genotypes for PTA at common levels of yield, genotype means for PTA were adjusted to both 1500 and 1800 kg ha^–1 of yield for each of the two environments independently using PROC MIXED as outlined by Littell et al. (1996) under the assumption of uncommon slopes describing the relationship between yield and PTA for the different genotypes. These two values of yield were selected to maximize the number of pairwise comparisons that could be made between genotypes without extrapolation beyond the ranges of yield observed in the 2 yr. This analysis included the continuous variable yield as an explanatory variable in the model statement of PROC MIXED to adjust genotype values for PTA based on the measured values of yield for each genotype. The LSMEANS statement with the AT option (SAS Online Documentation found at http://v8doc.sas. com//sashtml/; verified 29 March 2006) was used to estimate PTA for each genotype at the chosen yield levels for each environment. The PDIFF option within the LSMEANS statement was used to report p-values for all possible pairwise comparisons among genotypes at the two levels of yield. To provide genotype comparisons in a more reader-friendly format, conservative LSD values for each environment x yield level combination were calculated on the basis of PDIFF output. LSD values were computed using the largest standard error value from all possible pairwise comparisons from each combination.

Finally, the relationship between yield and TAP for the 15 different genotypes was also investigated through examination of plotted data points for these two traits for both environments and also via the use of PROC REG of SAS to calculate regression and correlation coefficients.

	RESULTS

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Analysis of Variance
Analysis of variance of the split plot design over environments revealed highly significant differences among genotypes for all measured traits (Table 2). The whole-plot effect (target leaf number) was statistically significant at the P = 0.05 level for PTA and LALN. A significant genotype x environment interaction was observed for PTA. Spearman's rank correlation analysis (Steel et al., 1997) was performed to assess the nature of this interaction. The correlation coefficient was found to be highly significant (r_s = 0.95, P < 0.0001), suggesting that the interaction was largely due to differences in scale among the environments. Significant whole-plot x environment interactions were detected for yield and PTN. Graphical representations of this data (not shown) indicated that these interactions were of the noncrossover type. Significant target leaf number x genotype interactions were detected for yield, TAP, LALN, and LANF. Graphical representations (not shown) indicated that some genotypes responded at different rates (and not necessarily in the same direction) for these traits as leaf number increased.

Comparison of Genotypes for PTA at Chosen Levels of Yield
When computed over all genotypes and both environments, a strong negative relationship was observed between yield and PTA (b = –0.00158, R² = 0.521, P < 0.0001). Similar relationships were observed when regression statistics were calculated for the two environments independently (b = –0.00155, R² = 0.523, P < 0.0001, for the 2003 environment; b = –0.00163, R² = 0.550, P < 0.0001, for the 2004 environment). It was important to know whether the change in PTA per unit change in yield was similar for each genotype. Hence, the relationships between PTA and yield were determined for each genotype in each environment. Regression coefficients were negative in value for all genotypes in both environments, and were significantly different from zero at the 0.05 level for all genotypes except TI 216, TI 281, TI 464, TI 648, and TI 859 in 2003 (Table 5). For the 2004 environment, only the regression coefficients for TI 835, TI 959, TI 973, Hicks, NC 95, and Silver Dollar were significantly different from zero (Table 5). Regression coefficients were smallest for TI 281 and TI 216 for the 2003 environment, and smallest for K326 and TI 501 for the 2004 environment. On the basis of calculated 95% confidence intervals using the standard errors for b in Table 5, the regression coefficient for TI 835 was found to be significantly greater in magnitude than the coefficients for Delgold, Hicks, K326, NC 95, Silver Dollar, TI 216, and TI 281 in both the 2003 and 2004 environments. The regression coefficient for TI 973 in 2003 was also found to be significantly greater in magnitude that those calculated for NC 95 and TI 281. The regression coefficient for TI 835 in 2004 was also found to be significantly greater in magnitude than b calculated for TI 934. No other significant differences among computed values for b were found.

Relationship between Yield and Total Alkaloid Production
If a dilution effect were solely responsible for the negative intragenotypic relationship between yield and PTA, one would expect total alkaloid production (TAP, kg ha^–1) to remain relatively constant over all levels of yield. When computed for all genotypes over both years, a positive relationship was found between the two traits (b = 0.00901, R² = 0.138, P < 0.0001). Positive relationships were also found when each environment was considered separately (b = 0.01011, R² = 0.191, P < 0.0001, for the 2003 environment; b = 0.00734, R² = 0.089, P = 0.0005, for the 2004 environment). Regression coefficients reflecting the relationship between yield and TAP were positive in value in almost all cases but usually not significantly different from zero (Table 5). The regression coefficient for TI 835 was negative in value for both years, and the regression coefficient for TI 973 was negative in value for the 2004 environment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The negative genetic correlation between yield and PTA in tobacco has served as a barrier to the development of higher-yielding cultivars possessing leaf chemistry that continues to satisfy the requirements of the RMSP. This barrier must not be absolute in view of the fact that improved genetics contributed to the development of commercially acceptable higher-yielding cultivars from the period from 1964 to 1981 (Bowman et al., 1984). In the last 20 yr, however, it has been very difficult develop higher-yielding flue-cured tobacco cultivars that continue to satisfy the PTA requirements of the RSMP. The extremely narrow germplasm base that exists in most flue-cured tobacco breeding programs (Murphy et al., 1987) may be exacerbating the problem.

It is typically not difficult to develop breeding lines with increased yield potential. These materials typically exhibit unacceptable PTA levels, however. Under the current PTA requirements of the RMSP, any yield increases must be accompanied by simultaneous increases in total alkaloid production (kg ha^–1). Identification of germplasm possessing alleles positively affecting alkaloid accumulation that are not currently present in the elite flue-cured tobacco germplasm base may increase the potential for developing higher-yielding cultivars with acceptable cured leaf chemistry. Since yield modulates the phenotypic expression of PTA (Chaplin, 1963; Chaplin et al., 1964), this research was conducted with the goal of comparing a set of diverse genotypes for PTA at common levels of yield. Genotypes with higher levels of PTA at given levels of yield might have a higher probability of possessing alleles of value for dealing with this problem.

Manual control of leaf number was useful for generating wide variation for yield for the 15 genotypes and allowed for the application of regression techniques for PTA comparisons at common levels of yield. The inverse intragenotypic relationships that were observed between yield and PTA are consistent with data from previous experiments in which nonflowering cultivars were decapitated at different leaf numbers (Chaplin, 1963; Chaplin et al., 1964). These findings are also in line with the belief that alkaloid accumulation after decapitation is affected largely by dry matter accumulation during the same period (Bush and Saunders, 1977). Germplasm accessions TI 464 and TI 959 were found to have the highest levels of PTA at chosen levels of yield. These genotypes would be considered as "exotic" germplasm for flue-cured tobacco breeding, however, and long-term effort might be required for their utilization. Desirable leaf quality in tobacco appears to be controlled by a complex network of genes, and there seems to be little tolerance for high proportions of exotic germplasm (Wernsman and Rufty, 1987). In the more near term, the Canadian flue-cured tobacco cultivar Delgold might be a valuable source of alleles to permit further increases in yield while keeping PTA at acceptable levels. Delgold has N. rustica, a high-alkaloid species, in its pedigree (Pandeya and White, 1984), but it is currently unknown if any N. rustica germplasm is actually present in the genome of this cultivar.

Consideration might also be given to selection of potential donor germplasm on the basis of weak observed associations between yield and PTA. Among the germplasm accessions examined in the current study, the lowest negative regression coefficients for this relationship were observed for accessions TI 216, TI 281, and TI 501. These materials would also be considered exotic germplasm, however, and long-term effort would likely be required for their use.

The genetic–physiological reason for the apparent increased capacity to accumulate alkaloids in certain genotypes is not clear. Several alkaloids are among the most N-intensive secondary metabolites in plants. Nicotine contains the same proportion of N as protein (17%), and it has been estimated that 3.62 g of glucose are required to synthesize 1 g of nicotine (Gershenzon, 1994). In addition to genetic variation for yield, genotypic differences for alkaloid accumulation in leaves might be attributed to several factors, including (i) differential N-absorption capacity per unit of biomass or (ii) variability for partitioning of N metabolism into alkaloid accumulation. Collection of data for PTN allowed for examination of the possible influence of these factors on alkaloid accumulation in the 15 genotypes that were evaluated. TI 464 and TI 959 had the highest PTN levels but ranked poorly for N-ACC and N-UPT because of their relatively low cured leaf yields. On the other hand, Delgold exhibited the lowest levels of PTN but the highest levels of N-ACC and N-UPT because of its high yield. The LALN index may be considered a measure of how efficiently the plant utilized acquired N for alkaloid production. The observation that TI 464 and TI 959 exhibited the highest values for this index may point to the possibility that these accessions have a greater genetic potential for partitioning N into alkaloid accumulation rather than other N-containing products such as nitrates, amines, peptides, and proteins. These genotypes were intermediate in value for LANF. Delgold, on the other hand, ranked the highest for this index and may have an increased potential for accumulating fertilizer N into alkaloids in the leaves. Incorporation of either of these characteristics into elite, high-yielding flue-cured tobacco germplasm may aid in the development of higher-yielding cultivars possessing acceptable leaf chemistry. It should be noted that these indices are based on total N in the leaves and not total N throughout the entire plant. Previous research has demonstrated that N is not distributed uniformly throughout parts of the tobacco plant (Williamson and Johnson, 1981). For this paper, we have made the assumption that tissue N concentrations of stalk, midribs, and roots are proportional to leaf N concentration among cultivars. Since genotypic differences for N partitioning are known to occur in plants (Hay et al., 1953; Chevalier and Schrader, 1977), these results should be interpreted with caution. Moll et al. (1982) suggested, however, that estimates of N-accumulation in leaves can provide insight into genotypic variation in response to N supply.

Additional characteristics that might contribute to differences in observed levels of alkaloid accumulation might include (i) differences in root mass or number of root tips where synthesis is believed to occur, (ii) differences in synthetic capability of alkaloid-producing cells, (iii) differences in photosynthetic capacity of above-ground plant parts which might drive N uptake and alkaloid synthesis, (iv) variations in the efficiency of translocation of nicotine from the roots to leaves, or (v) differences in alkaloid turnover in the leaves. The impact of some of these factors might be investigated in future experiments involving reciprocal grafting techniques involving TI 464 and TI 959 rootstocks/scions and commercial flue-cured tobacco cultivar rootstocks/scions.

Previous researchers have suggested the involvement of a dilution effect in the relationship between yield and PTA (Matzinger and Mann, 1964; Wolf and Bates, 1964). The comparison of genotypes evaluated here at different yield levels does suggest that, for a fixed genotype, such a dilution relationship does occur. The tendency for TAP to increase as yield increased suggests that the relationship between yield and PTA is not simply one of dilution, however. These observations suggest a role for factors or processes operating in the leaves in alkaloid synthesis. This should not be unexpected, however, in view of the fact that photosynthate is necessary for nitrogen uptake and metabolism. Seltmann et al. (1962) and Hall (1975) also reported that processes operating in both the shoot and roots probably affect alkaloid accumulation.

This investigation adds to the body of knowledge regarding the relationship between alkaloid accumulation and yield in N. tabacum and also to the data of Sisson and Saunders (1982) on genetic diversity for alkaloid accumulation in this species. We conclude the TI 464, TI 959, and Delgold may have a greater probability of possessing alleles of value for achieving the goal of increasing tobacco cultivar yields while maintaining commercially acceptable PTA levels. Germplasm accessions TI 216, TI 281, and TI 501 might also be considered because of the weak observed associations between yield and PTA for these genotypes. This information may be valuable to tobacco breeders worldwide interested in this problem.

	ACKNOWLEDGMENTS

 
The author would like to express his appreciation to personnel at the Upper Coastal Plain Research Station and the North Carolina State University Tobacco Analytical Services Lab for their assistance in this research. The author also is grateful to Philip Morris USA for financial support of the NCSU tobacco breeding program. Appreciation is also extended to two anonymous reviewers for their helpful suggestions on a previous version of this manuscript.

Received for publication February 20, 2006.

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