Nicotine represents the predominant alkaloid in cultivated tobacco. In burley varieties, during senescence and curing of leaves, nicotine can be converted to nornicotine, which is highly undesirable because of its relationship with the tobacco specific nitrosamines formation. Thus, an alternative for producing varieties with low or null nornicotine content would be to select plants or progenies for traits related to nicotine conversion. Therefore, understanding genetic control and inheritance underlying this process is important to chooseappropriate strategies in breeding programs. Two contrasting inbred lines for nicotine conversion to nornicotine, TN 90 and BY 37, have been crossed to obtain the segregating generations (F<sub>2</sub> and BCs). Genetic parameters of mean and variance, as well as the average degree of dominance and heritability were estimated. It has been found that nicotine conversion has predominantly additive effects, and in addition, narrow sense heritabilities at the individual level were higher than 65%. Both are desirable conditions for conducting a selection program of burley tobacco aiming at development of inbred lines associating yield and other traits with low or null nicotine conversion.
One of the major alkaloids produced by tobacco (Nicotianatabacum L.) is nicotine and represents 90% - 97% of the total alkaloid content [
For that reason and considering that tobacco is consumed primarily in the form of cigarettes, one of the goals of a tobacco plant breeder is to develop very productive varieties keeping nornicotine levels to a minimum [
Conversion occurs mainly during senescence and curing [
Several studies aiming to determine the genetic mechanism underlying this process have been conducted. The first studies have shown that a single dominant gene has controlled the difference between high and low norni- cotine varieties [
Many technologies are being studied in an attempt to reduce nornicotine produced in tobacco leaf. RNA in- terference (RNAi) has been used to induce post-transcriptional gene silencing on genes controlling nornicotine synthesis [
An alternative strategy would be to select plants and progenies in a conventional manner with the same effect, without using genetic engineering. Therefore, understanding the genetic control and inheritance underlying the nicotine conversion in superior varieties is important for selecting efficient methods and strategies for increasing yield while minimizing nornicotine content. This was the aim of the present study. Thus, we show the predomi- nant effect in genetic control of nicotine conversion and alternatives that may lead to a higher gain in decreasing nornicotine in tobacco leaves.
All plant materials used in this study were provided by Souza Cruz SA. Two contrasting burley inbred lines, TN 90 (“non-converter”) and BY 37 (“converter”), were crossed to obtain the F1 generation. F1 plants were crossed with both parents and self-pollinated to generate backcrosses (BCs) and the F2 population, respectively (
Field evaluation was carried out in Rio Negro, PR, Brazil in the 2013/2014 crop season. A randomized com- plete block design with two replications was used. Different numbers of plants were evaluated per generation: 40 from TN 90, 40 from BY 37, 200 from the F2 generation, 200 from BC-TN 90, and 200 from BC-BY 37. They were randomized into the two replications in experimental units consisting of a single 10-plant row. Data was collected at the individual level.
Quantitative determinations of nicotine and nornicotine in cured leaf samples were carried out for each plant in the plot. The analysis was done at the Product Center Laboratory of Souza Cruzlocated in Porto Alegre, RS, Brazil. The samples were extracted and the quantification of the nicotine and nornicotine was performed by gas chromatography. The results were reported as μg/g of leaf tissue for nicotine and nornicotine content, and as percentages for conversion. The percentage of nicotine conversion was calculated as:
Data was subjected to analysis of variance for randomized complete block design using R software [
Genetic components of mean were estimated using generation means (parents, F2 and BCs), through the approach proposed by Rowe and Alexander [
Genetic components of variance were estimated by iterative weighted least square [
timated using the following expression:
Pearson phenotypic correlation was estimated between the total nicotine content before leaf curing, and the nornicotine after curing using plants of the F2 generation by:
typic covariance between the traits and Vx and Vy are the phenotypic variances of each trait independently. The t
test was applied to rP to check whether the estimate is not equal to zero.
The means of each trait for the generations are shown in
Nicotine (μg/g) | Nornicotine (μg/g) | Conversion (%) | ||||
---|---|---|---|---|---|---|
Generation | Mean | Variance | Mean | Variance | Mean | Variance |
TN 90 | 12,599 | 3,570,178 | 397 | 45,556 | 3.14 | 3.88 |
BY 37 | 202 | 168,382 | 8674 | 1,304,658 | 98.06 | 15.08 |
Parents average | 6401 | 4535 | 50.60 | |||
F2 | 6295 | 20,506,162 | 5272 | 13,325,445 | 48.36 | 1108.81 |
BC-TN 90 | 10,411 | 11,542,958 | 1642 | 5,857,273 | 15.15 | 523.55 |
BC-BY 37 | 1697 | 5,433,430 | 8753 | 5,630,094 | 84.53 | 421.90 |
parents proved to be contrasting for all traits, which is vital for this kind of study. The means of the F2 with re- spect to all the characters studied were approximately equal to the means of the parents. The mean values of BCs tend to the recurrent parent, as expected.
Estimates of genetic parameters are shown in
The estimates of environmental (VE), additive (VA) and dominance (VD) variance, as well as the narrow sense of heritability (h2) are shown in
Nicotine (μg/g) | Nornicotine (μg/g) | Conversion (%) | ||||
---|---|---|---|---|---|---|
Parameters | Estimate | SD1 | Estimate | SD | Estimate | SD |
Mean | 6893.20 | ±138.76 | 4878.57 | ±86.78 | 50.91 | ±0.34 |
a | −6739.84 | ±135.65 | 4506.62 | ±85.99 | 47.98 | ±0.34 |
d | −2229.98 | ±349.34 | 746.38 | ±282.49 | −0.45 | ±2.12 |
add | −0.32 | 0.15 | −0.01 | |||
R2 | 98.11 | 95.60 | 93.25 |
1Standard deviation.
Nicotine (μg/g) | Nornicotine (μg/g) | Conversion (%) | ||||
---|---|---|---|---|---|---|
Parameters | Estimate | SD1 | Estimate | SD | Estimate | SD |
VE | 175,932 | ±38,587 | 47,090 | ±10,445 | 4.57 | ±0.86 |
VA | 13,734,820 | ±2,585,295 | 8,664,871 | ±1,685,781 | 726.95 | ±140.14 |
VD | 6,595,410 | ±802,806 | 4,613,485 | ±539,551 | 377.28 | ±44.48 |
h2 | 0.67 | 0.65 | 0.66 | |||
R2 (%) | 94.25 | 94.10 | 96.72 |
1Standard deviation.
and VD were different from zero. The estimated VA and VD components were consistent with a and d described before. Heritability (h2) represents another parameter that provides information about the genetic control of the trait and allows us to infer whether the trait can be easily selected. In the present study, narrow sense heritability at plant level was relatively high for all characters (>65%). In this case, h2 only represents additive genetic va- riance, which is associated with the breeding value an individual can transmit to its progeny.
Quantification of nicotine and nornicotine content, as well as the conversion rate, were performed with high precision, as can be confirmed by the high experimental accuracies (>92%, data not shown) and the low envi- ronmental variances related to the genetic variance components (
Understanding the genetic determination of traits helps the breeder in formulating breeding techniques for combining desirable characters that are dispersed in two or more genotypes into one. Identification of the sources contributing to genetic variation and the type of gene actions involved will assist in the selection of the most ap- propriate breeding strategy.
If we consider one gene with two alleles, B and b, we have one of three possible genotypes in the F2 genera- tion or any other segregating generation: BB, Bb and bb. The phenotypic expression of each genotype is deter- mined as the departure from the mid-point (m) between two homozygous parents (BB and bb). Thus, parameter aB measures the departure of each homozygote from m, and dB measures the departure of the heterozygote from m (
The model can also be extended to include non-allelic (epistatic) interaction components [
cient can be used to choose the most adequate model. Since the estimates of this coefficient were high for all cases (>94%), the epistatic effects should be minimum or null. Thus, adequacy of the simple model, i.e., main effects alone, was satisfactory.
The estimates obtained showed predominance of additive effects, evidenced by add. Nevertheless, d is not negligible. In early literature, the information found is that trait is controlled by one gene with dominance for the allele controlling nicotine conversion [
VA and VD estimates were consistent with a and d, as already stated. According to Bernardo [
In the literature, estimates of mean and variance components were not found for the traits evaluated in this study. There are reports referring to the number of genes controlling the phenotypic expression. Moreover, in a study performed involving crosses between N. tabacum and related amphidiploids two genes controlling nico- tine conversion were found [
For that reason, the selection of plants with low or null nicotine conversion is likely the best strategy for achieving success in breeding programs. Additionally, the narrow sense heritability estimate at the individual level for nicotine conversion was relatively high (76%), which indicates a favorable condition for selection in early generations when practicable. Even so, selection should be associated with a recurrent program because it is not possible to reach the desired nornicotine content after just one selection cycle. One more point to take into considerations is the low correlation estimate (rP) between the total nicotine content before leaf curing and the nornicotine content after curing (−0.09; p-value = 0.22). Hence, selecting for nicotine content does not imply in any consequences for nicotine conversion. In other words, the breeder can select “non-converters” plants with low nicotine content.
In production of foundation seeds, for example, a screening is carried out on all plants to eliminate individuals that exhibit nicotine conversion higher than 3% [
“Although this screening procedure leads to significant reductions in nornicotine (and NNN) in comparison to tobacco populations that have not been screened, this process can never be perfect, since high nornicotine producing converter plants can spontaneously arise with each generation.”
Therefore, genetic control of this trait is not as simple as has been stated by many authors. These unexpected converter plants could be arising from changes in the epigenetic state [
Since evaluation of nicotine and nornicotine is expensive and laborious, the use of molecular markers linked to one of the major genes controlling the trait could be an alternative for screening the progenies or plants in early generation before being subjected to field evaluation. Markers can be selected in a study like this through the use of a segregating population, but the sequences for primers can also already be found in scientific articles published on the issue [
Nicotine conversion had predominantly additive effects. Narrow sense heritabilities at the individual level were higher than 65%. Both of these desirable conditions for conducting a selection program for burley tobacco aim- ing at the development of inbred lines associating yield and other traits with low or null nicotine conversion.
This is an academic study supported by Souza Cruz SA. We also thank the Coordenação de AperfeiçoamentoProfissional do Ensino Superior (CAPES) for the Ph.D. scholarship and the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) for the grant.