BACKGROUND
over the past dozen years, a number of crops, such as soybean, maize, rice, cotton and canola, have been genetically engineered to contain a variety of fitness enhancing transgenes.
some of these transgenes can increase a crop's defenses by conferring resistance to a number of diseases, herbicides, abiotic stresses, and yield reducing herbivores  <cit> .
consequently, there are environmental and regulatory concerns about the adventitious presence of transgenes, especially with regards to hybridization and introgression into weedy relatives  <cit> .
specifically, could the introgression of fitness-enhancing transgenes result in new hard-to-manage, weedy or invasive biotypes possessing competitive advantages such as herbicide resistance, drought and salt tolerance, or pathogen and insect resistance?
if this were to occur, these hybrids could potentially disrupt agricultural and non-agricultural systems  <cit> .
transgene introgression and competition has been studied in many weed × crop systems including brassica rapa  × brassica napus .
halfhill et al.
analyzed the fitness of four b. rapa × transgenic b. napus backcrossed transgenic hybrid lines that originated from a single transgenic event.
they found that the average vegetative growth and nitrogen use efficiency of the transgenic hybrids were lower than the wild-type b. rapa parent indicating that the transgenic brassica hybrids would likely be less fit in an agronomic setting.
they concluded that the observed decrease in fitness could be the result of species or hybridization effects, initial transgene insertion loci , ecological conditions, and/or linkage-derived genetic load  <cit> .
linkage-disequilibrium or genetic load could be one of the main causes of fitness depression witnessed in hybrid and backcrossed generations  <cit> .
until now, the specific effects of linkage-derived genetic load have not been empirically tested under agronomic conditions.
muller  <cit>  first described genetic load as the total amount of deleterious mutations in the genome of an organism.
since then, genetic load has been studied in the contexts of population and conservation genetics, but not in regards to transgenic plants.
here we define genetic load as the unfavorable consequences of hitchhiking crop or domestication alleles  as they become introgressed into a weedy genome  <cit> .
crops have traditionally been bred and selected for domestication traits, such as lack of seed dormancy, reduced seed dispersal, and non-shattering pods, apical dominance, homogenous fruit ripening, reduced competitive ability, and loss of self-incompatibility  <cit> .
in contrast, weeds have been selected for "weediness" traits that are the counterpoint for the crop traits listed above  <cit> .
therefore, it follows that the incorporation of crop alleles into weedy genetic backgrounds would be disadvantageous for weedy recipients  <cit> .
the purpose of this study was two-fold.
the first objective was to test the hypothesis that genetic load could have a negative effect on introgression and transgene persistence.
specifically, that hybrid inferiority in b. rapa × b. napus populations could be caused by the genetic load of crop alleles that are inadvertently transferred with the transgene into the genome of the weed host.
by measuring brassica hybrid and parental productivity , under competitive  and non-competitive field conditions, the first objective was designed to assess the fitness consequences of genetic load in different competitive environments.
these fitness data were then correlated with genetic load, as assessed by number of amplified fragment length polymorphism  markers specific to the crop parent  and to the weed parent , to determine whether weed × crop hybrids with more crop genes were less competitive than hybrids containing fewer crop genes.
our hypothesis was that hybrids possessing more b. napus-specific aflp markers would be less fit under both competitive and non-competitive conditions.
the second objective of this study was to analyze the advantages of utilizing a transgenic-mitigation  strategy  to reduce the number or frequency of transgenic progeny under field conditions.
a number of gene flow prevention models have been proposed in the literature  <cit> .
these include: use of buffer zones or barrier crops to block or hinder pollen flow  <cit> , production of male-sterile crop plants to prevent pollen flow  <cit> , insertion of gene-deletor constructs   <cit> , insertion of transgenes in "safe" integration sites or areas that are less likely to be transferred during homologous recombination, thereby inhibiting the transgene from being transferred to subsequent generations  <cit> , insertion of transgenes into the maternally inherited plastid genomes  <cit> , and tm strategies  <cit> .
of these proposed strategies, tm is a focal point of this study.
our hypotheses was that direct incorporation of engineered genetic load via tm, would be a stronger mitigator of transgene persistence than random transgene-site-mediated genetic load, which can easily undergo trait segregation in surviving offspring.
this is a follow-up field study from greenhouse and shade-house experiments that demonstrated that the above tm strategy was effective in limiting seed production and thereby mitigating transgene flow from b. napus to b. rapa  <cit> .
we chose the bc1/f <dig> generation since it is the first generation towards introgression in which genetic segregation is expected to vary among transgenic events.
transgene mitigation is made possible by fusing or linking agronomically beneficial transgenes  with transgenes that would decrease the fitness of the weed host, hence inhibiting the persistence of the tm construct.
these same weed-mitigating genes are either positive or neutral for the crop  <cit> .
the mitigating gene used in this study was the Δgai  gene.
this gene confers a dwarf phenotype, a principal trait of the green revolution.
incorporation of this gene has been shown to increase crop biomass and seed yield, but render weeds less competitive  <cit> .
the fitness consequences of this engineered genetic load was also estimated in the present study from brassica hybrid and parental seed yield and above-ground dry biomass measures, under competitive  and non-competitive field conditions.
it was hypothesized that tm would be able to eliminate the persistence of transgenes in progeny and derivative populations, or keep them at exceedingly low frequencies if gene flow and subsequent transgene introgression were to occur.
RESULTS
genetic load study
productivity data: no competition
when lacking competition, from wheat or weeds, non-transgenic b. napus out-performed b. rapa and the three hybrid lines .
the gt <dig> hybrid line had significantly  higher seed biomass than b. rapa.
the other hybrid lines, gt <dig> and gt <dig>  were significantly less fit  than b. rapa.
the same pattern was observed for vegetative dry mass.
regardless of treatment or line, there was a strong correlation between biomass and seed yield  .
productivity data: competition with wheat
when the two non-transgenic parental lines and three gt hybrid lines were grown in competition with wheat, b. rapa produced the most, and b. napus and gt <dig>  the fewest seeds .
there were no statistical differences between the gt <dig> and gt <dig> hybrid lines for seed production.
the gt <dig> line produced significantly  fewer seeds than the other hybrid lines and was not significantly different that b. napus.
however, all of the hybrid lines produced significantly  fewer seeds than b. rapa  under competitive conditions.
two of the hybrid lines had lower biomass than b. rapa, whereas the gt <dig> hybrid line produced as much biomass as b. rapa in competition with wheat .
no statistical differences were observed between non-transgenic b. napus and gt <dig> and gt <dig> hybrid lines when they were grown in competition with wheat .
the effect of brassica competition on wheat productivity differed among lines, but overall, no significant difference was observed between wheat growth in the absence of brassica competition vs. the presence of brassica competition under the specific conditions of this experiment .
aflp marker analysis
the five selective primer sets used  <cit>  resulted in a total of  <dig> b. napus-specific markers.
a marker was considered b. napus-specific if it was present in the bulked b. napus sample and absent in the bulked b. rapa sample.
the five selective primer sets also resulted in  <dig> b. rapa-specific markers.
a marker was considered b. rapa-specific if it was present in the bulked b. rapa sample and absent in the bulked b. napus sample.
as the b. rapa-specific markers were absent in the b. napus crop, they can be considered markers of weediness, the more present, the weedier the hybrid or segregant.
the number of b. napus- and b. rapa-specific aflp markers differed among the gt hybrids and treatments   .
in the absence of interspecific competition, gt <dig> hybrids had significantly more b. rapa markers and significantly fewer b. napus markers than the other lines.
however, when gt <dig> hybrids were grown in competition with wheat, gt <dig> had significantly more b. napus markers  and significantly fewer b. rapa markers than the other lines in the competition treatment.
in contrast, more b. rapa markers were measured in the gt <dig> hybrids grown in competition with wheat than in gt <dig> hybrids grown under no competition, while no differences were seen in b. napus markers for either of the treatments.
thus, gt <dig> and gt <dig> and different patterns of response to competition with regards of genomic constitution.
in the gt <dig> line, there were no significant differences between the number or type of marker in either of the growing conditions .
even though significant differences were observed for the markers and lines under different competitive conditions, correlations were only observed between markers and productivity data for the gt <dig> hybrids .
b. napus aflp marker correlation with productivity
our hypothesis that hybrids containing more b. napus crop alleles or genetic load would be less competitive than weedy species can be conditionally rejected, but only in the case where there is no interspecific competition .
there was an overall moderate positive correlation between the number of b. napus-specific aflp markers and seed yield and vegetative biomass under non-competitive conditions: for seed yield vs. marker number   and vegetative dry biomass vs. marker number .
the statistical significance can be accounted for by gt <dig> hybrids .
no correlations were observed for the other events.
when the gt hybrids were grown in competition with wheat, there were no significant correlations between the number of b. napus crop markers and seed yield, vegetative dry biomass or with wheat vegetative dry biomass .
in addition, no correlations between b. napus markers and wheat vegetative dry biomass existed for any of the hybrid lines .
* denotes significant results
the numeric values represent pearson correlation coefficients .
hybrids were grown in the presence and absence of interspecific competition with wheat.
b. rapa aflp marker correlation with productivity
there were no correlations between the number of b. rapa specific aflp markers and the productivity of the gt hybrids grown alone, i.e., in the absence of interspecific competition .
conversely, under competitive conditions , there were strong positive correlations between b. rapa weediness markers and seed yield but only for the gt <dig> hybrids.
in the gt <dig> line, the correlations between b. rapa markers under competitive conditions were slightly higher than those for the b. napus markers under no competition: seed yield vs. marker  .
overall, the correlations between the number of b. rapa markers and seed yield under competitive conditions were significant .
no correlations were observed in the gt <dig> and gt <dig> hybrid lines with respect to the b. rapa markers and seed yield or vegetative dry biomass.
in addition, no correlations between b. rapa markers and wheat vegetative dry biomass existed for any of the hybrid lines .
transgenic-mitigation study
productivity data
for the tm study, we analyzed populations of non-transgenic b. napus and b. rapa, a transgenic homozygous tm  b. napus population and transgenic segregating populations of bc1/f <dig> hybrids .
the gt hybrids used in the tm study were the same lines that were analyzed in the genetic load study, whereas the tm and the tm lines were unique the tm study.
when grown in competition with wheat, the non-transgenic b. napus and b. rapa plants reached heights of 130- <dig> cm at plant maturity  whereas the homozygous tm b. napus plants  were only 40- <dig> cm at plant maturity.
at the time of harvest , the siliques on all of the non-transgenic and hybrid plants were completely mature and ready to shatter, while the siliques on the tm plants  were still green-to-yellow and maturing.
throughout the field season, it was observed that the tm line had delayed emergence, reduced height, and delayed flowering.
however, based on productivity data, when grown alone, the tm populations performed as well as their non-transgenic crop counterpart , indicating that dwarfing could potentially be utilized in true cropping systems without any yield penalties .
these results are congruent with those of al-ahmad et al.
.
when populations of transgenic and non-transgenic segregants of the gt and tm hybrid lines were grown in the absence of competition with wheat, significant  differences in biomass and seed yield were observed , however, all hybrid lines  performed similarly to b. rapa in the absence of competition.
in contrast under competitive conditions, there were no statistical differences observed for vegetative biomass  or seed yield  for any of the hybrid lines, but there was a great deal of endogenous variation among data in the competition experiment.
transgene persistence data
after one field season of natural hybridization among individuals of a hybrid line, the progeny from the bc1/f <dig> hybrid lines was comprised of bc <dig> and f <dig> individuals.
transgenic segregation ratios in the progeny populations differed among the gt and tm lines .
the frequencies of transgenic individuals decreased in all gt and tm hybrid progeny populations after one field season.
the frequencies of transgenic individuals in these populations differed among lines and under interspecific and intraspecific competitive conditions .
the proportion of transgenic individuals in the progeny populations of the gt <dig>  gt <dig>  gt <dig> and tm bc1/f <dig> hybrid lines were the same for both non- and competitive conditions .
however, there was a significant difference observed for the tm line.
the progeny population of the tm line grown in competition with wheat had fewer transgenic individuals than that grown in the absence of interspecific competition .
the green fluorescent marker  was also detected in the parental  progeny lines indicating the occurrence of gene flow among plots .
the f <dig> progeny populations for the bn and br parental lines were not screened on ms medium containing the selecting imazapyr herbicide, hence, the transgene persistence data reported in table  <dig> only applies to gene flow from gt hybrid plots and not tm hybrid or homozygous plots.
the decrease in transgenic individuals was greater for the tm hybrid line than the gt lines.
the initial frequency of transgenic individuals in the tm hybrid line  was low , and after one field season, the frequency of transgenic individuals dropped further  .
percent germination and transgenic frequencies were calculated for the parental  and hybrid bc1/f <dig> populations  pre-season and post-harvest.
transgene persistence data reported here only applies to gene flow from gt hybrid plots and not tm hybrid or homozygous plots.
column ms represents transgenic ratio mean separation obtained by fisher's lsd .
data was rank transformed and sas software was used for data analysis.
discussion
there is growing evidence that many interspecific hybrids between crops and weeds, including transgenic hybrids, have inferior performance compared with their weedy parent , although a few exceptions have been reported  <cit> .
there are many possible explanations for these observations, most of which focus on the fitness penalties associated with hybridization and backcrossing.
a variety of genetic mechanisms can affect hybrid fitness  <cit> .
heterosis or hybrid vigor could be enhanced by the segregation of additive genetic traits or optimal environmental conditions.
in cases where the crop and weedy or wild relative are the same species, there should be fewer genetic barriers to hybrid fitness and transgene persistence  <cit> .
unless the trait or transgene is deleterious, as in the case of tm, hybridization and introgression might easily progress, thereby potentially allowing transgene persistence.
this is far more likely to occur with related weeds existing and competing in the same agro-ecosystem as the crop than with wild species, residing in non-agricultural areas where a transgenic trait may have little or no value.
the performance of new genotypes introduced via transgenesis and subsequent hybridization are likely also subject to genotype × environment  interactions  <cit> .
when distantly related taxa hybridize, recombination between homologous or homoeologous chromosomes can result in irregular chromosome pairing.
the latter can lead to reduced growth and fertility  <cit> , thus decreasing potential for introgression.
in addition, the dilution of weed alleles by crop alleles, i.e., genetic load, might also account for slower introgression regardless of environment  <cit> .
our results showed a positive correlation between genetic load  and plant productivity in only one of our transgenic events, gt <dig>  a result congruent with a prior study  <cit> .
no correlation was observed for the other transgenic events suggesting a strong transgenic event or genotype effect.
the experimental design allowed the detection of a surprising effect--a counter genetic load finding .
in our experiments, the number of b. napus crop-specific aflp markers was associated with an increase in hybrid performance  in the gt <dig> hybrids in the absence of interspecific competition with wheat and weeds.
conversely, in the presence of interspecific competition with wheat, there was a strong correlation between b. rapa weed-specific aflp markers and productivity, again, only for the gt <dig> line.
these results indicate a strong g × e interaction and that inherited crop alleles, introgressed as a result of hybridization, can be preferentially selected to ensure maximum growth potential and productivity under crop-favorable conditions .
conversely, inherited weedy alleles can be preferentially selected under more "weed-like" conditions   <cit> .
we anticipate that this will continue to occur as the lines continue to backcross.
this evidence of local and situational adaptation demonstrated in our study as well as other studies indicates that many alleles are beneficial in some but not all environments  <cit> .
as sessile organisms, plants must be able to maximize resource acquisition; this can be accomplished with adapted genomes.
in agriculture and in our study, environmental genomic adaptation could translate to favoring some crop alleles in crops grown in monocultures where weeds are controlled; weed alleles might be favored in weeds in competitive environments  <cit> .
dilution in either direction could be maladaptive.
environmental adaptation and trait selection are further demonstrated in a case-study performed on recombinant inbred sunflower lines  <cit> .
baack et al.
<cit>  showed that crop traits  were preferentially favored in one site, while weedy traits  were favored at another location  <cit> .
these studies indicate that, with regards to genetic load and trait selection, environmentally-dependent selection must be taken into consideration for every transgene, species, and situation studied  <cit> .
transgenic event effects and genetic load
since transgene insertion is random, the flanking host dna can differ significantly for individual transgenic events; hence the analysis of multiple events in the present study.
zhu et al.
<cit>  examined the transgene segregation ratios for each of our lines using controlled crosses and progeny analysis.
the gt <dig> and gt <dig> lines did not deviate from expected mendelian segregation, indicating homeologous recombination of the transgene locus on an a-genome chromosome , and hence the transgene would have a decreased chance of being lost in subsequent backcrossing as a result of genomic incompatibility.
thus, linkage effects could likely cause decreased introgression and genetic load in the gt <dig> line.
transgenes located on the c-genome of b. napus might be subject to greater genomic incompatibility since b. rapa does not have the c-genome of b. napus.
thus c-genome-localized transgenic events could be an important investigative avenue for decreasing introgression  <cit> , although recent research found that homeologous recombination occurs at the same rate as homologous recombination in the b. rapa × b. napus system  <cit> , suggesting that "safe-integration" sites in b. napus are unlikely  <cit> .
engineered vs. randon transgenic event genetic load
tm b. napus plants containing the dwarfing gene Δgai were previously analyzed under shade-house conditions  <cit> .
homozygous tm dwarf b. napus plants  grown alone  had a much higher seed yield , and double the shoot and root biomass compared to non-transgenic counterparts .
the tm line also produced more leaves than tall non-transgenic plants when grown alone at  <dig> -cm spacing  and 10-cm spacing .
however, when grown in competition with tall non-transgenic cohorts, these same plants were exceedingly unfit  <cit> .
the reproductive fitness of tm plants, when grown in competition, was less than 12%, and the harvest index  was less than 40% of that of the non-transgenic b. napus competitors.
when grown in the absence of competition, the tm line produced the greatest amount of seeds per plant  <cit> .
from these studies, al-ahmad et al.
<cit>  concluded that the Δgai gene greatly enhanced seed and biomass yield in a weed-free transgenic crop.
however, if dwarfed plants emerged as volunteer weeds and competed with non-transgenic cohorts , dwarfed plants would be eliminated from poor competitive ability, especially if selective herbicides were not used.
our field results were congruent with the previous tm research  <cit> .
when the homozygous tm line  was grown under agronomic non-competitive conditions, it had equivalent performance as the non-transgenic b. napus line with regards to seed yield and biomass.
however, under competitive conditions, the tm line produced the least seed and biomass and, hence, performed the poorest of all the lines that were analyzed.
in terms of transgene persistence, the transgenic progeny from the backcrossed tm line approached a 50- to 85-fold reduction, under competitive and non-competitive conditions, compared with the 3-fold reduction in transgene persistence with the non-tm  lines.
it must be noted that the final portion of the transgene persistence studies; i.e., germination of seed collected from field grown plants were carried out under optimal growth chamber conditions.
this environment did not take into consideration the effects of over-wintering survivorship, dormancy issues or seedling dynamics.
consequently, when environmental factors are considered, one could potentially expect a decrease in performance  under realistic field conditions.
hence, ex situ analysis of transgene persistence could appear higher than it actually would be in the field  <cit> .
regardless, these data do indicate that tm constructs were effective in severely limiting the impact of gene flow from transgenic crops to their wild relatives and transgene persistence.
the tm approach could further be enhanced by stacking other weed mitigating transgenes, such as those that prevent seedpods from shattering or those that prevent secondary dormancy.
in addition to making plants shorter the dwarfing gene could also confer pleiotropic effects such as altered flowering traits.
these could conceivably also have g × e interactions and effect fitness  <cit> .
depending on the trait, the pleiotropic effect could increase or decrease fitness.
in this particular instance, dwarfed plants were very late flowering and also had delayed seed maturation and decreased germination.
in this case, the pleiotropic effects would almost certainly decrease introgression.
some researchers point out that dwarfing in tm is a trait that could foreseeably be selected against in the hemizygous state; e.g., in semi-dwarfed plants  <cit> .
consequently, the dwarfing allele might not be expressed as highly in first-generation hybrids because of the presence of the dominant gai allele from the weedy parent genome.
therefore, hybrids containing the tm construct might not have sufficient dwarfing to be selected against in competition, yet harbor the fitness-enhancing gene from the tm construct.
in subsequent generations, the deleterious allele would only be expressed in homozygous individuals, which would strongly reduce its capability to decrease fitness.
moreover, if the hybrids are fertile   <cit> , this strategy would not prevent them acting as a genetic bridge and pollinating the wild parent  <cit> .
reuter et al.
<cit>  challenged the theory that dwarfed volunteers or transgenic escapees would be less competitive in feral environments.
they noted that feral non-transgenic b. napus populations, growing in rural and urban area in northern germany, were, on average, 41% shorter than cultivated non-transgenic b. napus.
they attributed this height difference to phenotypic adaptation to local environments.
reuter et al.
<cit>  concluded that, under certain environmental and ecological conditions, the proposed mitigation approach  could actually increase escape and persistence of transgenic varieties rather than reducing them, but their study did not consider genotype differences, local adaptation, and environmental effects such as differences in nutrient availability between agronomic and feral conditions.
our transgene persistence data indicate that even if the dwarfing trait is in the semi-dominant state, it is still effective in limiting transgene persistence.
our results indicate that when dwarfing is utilized for a containment strategy, the plants are not able to compete as well and hence persist in subsequent generations.
these observations hold true regardless of whether the plants were grown in the presence or absence of interspecific competition.
hence, under the conditions and situations analyzed in this study, hybrids containing tm constructs were not effective weeds.
different species will likely require various mitigators, and possible also for different environments  <cit> .
anti-shattering genes will be appropriate for many row crops that have seed shattering weedy relatives, and anti-secondary dormancy genes would be appropriate where weeds possess seed-bank longevity as part of their survival mechanisms.
anti-bolting genes  would be appropriate mitigators for biennial crops  or storage crops  that have weedy relatives.
sterility genes are appropriate for vegetatively propagated species .
the waxy transgene in maize would be a good mitigator for pharmaceutical genes expressed in maize embryos, as seeds from self or cross pollination would be shrunken, and unable to survive over-winter in most soils  <cit> .
thus, to choose an appropriate mitigator for each crop and environment, the researcher must seek out genes that are positive or neutral for the crop in that environment and would be detrimental to the related weedy or wild species.
CONCLUSIONS
there is a paradox regarding the apparent absence of introgression in species where gene flow is expected on the basis of sexual compatibility data.
it might be that genetic load of endogenous genes on crop chromosomes moderates introgression, and this same effect could be extended and applied to transgene containment.
perhaps we do not observe extensive transgene persistence in weedy relatives, i.e., only one introgressed transgenic weedy b. rapa plant has been observed in the field despite the extensive commercial release of transgenic b. napus  <cit> , because of genetic load conferred by the lack of homeologous recombination or hitchhiking of crop alleles.
if additional genetic load is imposed using tm, it could dramatically decrease introgression and transgene persistence to an even lower level.

 over the past dozen years, a number of crops, such as soybean, maize, rice, cotton and canola, have been genetically engineered to contain a variety of fitness enhancing transgenes. some of these transgenes can increase a crop's defenses by conferring resistance to a number of diseases, herbicides, abiotic stresses, and yield reducing herbivores. consequently, there are environmental and regulatory concerns about the adventitious presence of transgenes, especially with regards to hybridization and introgression into weedy relatives. specifically, could the introgression of fitness-enhancing transgenes result in new hard-to-manage, weedy or invasive biotypes possessing competitive advantages such as herbicide resistance, drought and salt tolerance, or pathogen and insect resistance? if this were to occur, these hybrids could potentially disrupt agricultural and non-agricultural systems. transgene introgression and competition has been studied in many weed × crop systems including brassica rapa × brassica napus. halfhill et al. analyzed the fitness of four b. rapa × transgenic b. napus backcrossed transgenic hybrid lines that originated from a single transgenic event. they found that the average vegetative growth and nitrogen use efficiency of the transgenic hybrids were lower than the wild-type b. rapa parent indicating that the transgenic brassica hybrids would likely be less fit in an agronomic setting. they concluded that the observed decrease in fitness could be the result of species or hybridization effects, initial transgene insertion loci, ecological conditions, and/or linkage-derived genetic load. linkage-disequilibrium or genetic load could be one of the main causes of fitness depression witnessed in hybrid and backcrossed generations. until now, the specific effects of linkage-derived genetic load have not been empirically tested under agronomic conditions. muller first described genetic load as the total amount of deleterious mutations in the genome of an organism. since then, genetic load has been studied in the contexts of population and conservation genetics, but not in regards to transgenic plants. here we define genetic load as the unfavorable consequences of hitchhiking crop or domestication alleles as they become introgressed into a weedy genome. crops have traditionally been bred and selected for domestication traits, such as lack of seed dormancy, reduced seed dispersal, and non-shattering pods, apical dominance, homogenous fruit ripening, reduced competitive ability, and loss of self-incompatibility. in contrast, weeds have been selected for "weediness" traits that are the counterpoint for the crop traits listed above. therefore, it follows that the incorporation of crop alleles into weedy genetic backgrounds would be disadvantageous for weedy recipients. the purpose of this study was two-fold. the first objective was to test the hypothesis that genetic load could have a negative effect on introgression and transgene persistence. specifically, that hybrid inferiority in b. rapa × b. napus populations could be caused by the genetic load of crop alleles that are inadvertently transferred with the transgene into the genome of the weed host. by measuring brassica hybrid and parental productivity, under competitive and non-competitive field conditions, the first objective was designed to assess the fitness consequences of genetic load in different competitive environments. these fitness data were then correlated with genetic load, as assessed by number of amplified fragment length polymorphism markers specific to the crop parent and to the weed parent, to determine whether weed × crop hybrids with more crop genes were less competitive than hybrids containing fewer crop genes. our hypothesis was that hybrids possessing more b. napus-specific aflp markers would be less fit under both competitive and non-competitive conditions. the second objective of this study was to analyze the advantages of utilizing a transgenic-mitigation strategy to reduce the number or frequency of transgenic progeny under field conditions. a number of gene flow prevention models have been proposed in the literature. these include: use of buffer zones or barrier crops to block or hinder pollen flow, production of male-sterile crop plants to prevent pollen flow, insertion of gene-deletor constructs, insertion of transgenes in "safe" integration sites or areas that are less likely to be transferred during homologous recombination, thereby inhibiting the transgene from being transferred to subsequent generations, insertion of transgenes into the maternally inherited plastid genomes, and tm strategies. of these proposed strategies, tm is a focal point of this study. our hypotheses was that direct incorporation of engineered genetic load via tm, would be a stronger mitigator of transgene persistence than random transgene-site-mediated genetic load, which can easily undergo trait segregation in surviving offspring. this is a follow-up field study from greenhouse and shade-house experiments that demonstrated that the above tm strategy was effective in limiting seed production and thereby mitigating transgene flow from b. napus to b. rapa. we chose the bc1/f generation since it is the first generation towards introgression in which genetic segregation is expected to vary among transgenic events. transgene mitigation is made possible by fusing or linking agronomically beneficial transgenes with transgenes that would decrease the fitness of the weed host, hence inhibiting the persistence of the tm construct. these same weed-mitigating genes are either positive or neutral for the crop. the mitigating gene used in this study was the Δgai gene. this gene confers a dwarf phenotype, a principal trait of the green revolution. incorporation of this gene has been shown to increase crop biomass and seed yield, but render weeds less competitive. the fitness consequences of this engineered genetic load was also estimated in the present study from brassica hybrid and parental seed yield and above-ground dry biomass measures, under competitive and non-competitive field conditions. it was hypothesized that tm would be able to eliminate the persistence of transgenes in progeny and derivative populations, or keep them at exceedingly low frequencies if gene flow and subsequent transgene introgression were to occur.  genetic load study productivity data: no competition when lacking competition, from wheat or weeds, non-transgenic b. napus out-performed b. rapa and the three hybrid lines. the gt hybrid line had significantly higher seed biomass than b. rapa. the other hybrid lines, gt and gt were significantly less fit than b. rapa. the same pattern was observed for vegetative dry mass. regardless of treatment or line, there was a strong correlation between biomass and seed yield. productivity data: competition with wheat when the two non-transgenic parental lines and three gt hybrid lines were grown in competition with wheat, b. rapa produced the most, and b. napus and gt the fewest seeds. there were no statistical differences between the gt and gt hybrid lines for seed production. the gt line produced significantly fewer seeds than the other hybrid lines and was not significantly different that b. napus. however, all of the hybrid lines produced significantly fewer seeds than b. rapa under competitive conditions. two of the hybrid lines had lower biomass than b. rapa, whereas the gt hybrid line produced as much biomass as b. rapa in competition with wheat. no statistical differences were observed between non-transgenic b. napus and gt and gt hybrid lines when they were grown in competition with wheat. the effect of brassica competition on wheat productivity differed among lines, but overall, no significant difference was observed between wheat growth in the absence of brassica competition vs. the presence of brassica competition under the specific conditions of this experiment. aflp marker analysis the five selective primer sets used resulted in a total of b. napus-specific markers. a marker was considered b. napus-specific if it was present in the bulked b. napus sample and absent in the bulked b. rapa sample. the five selective primer sets also resulted in b. rapa-specific markers. a marker was considered b. rapa-specific if it was present in the bulked b. rapa sample and absent in the bulked b. napus sample. as the b. rapa-specific markers were absent in the b. napus crop, they can be considered markers of weediness, the more present, the weedier the hybrid or segregant. the number of b. napus- and b. rapa-specific aflp markers differed among the gt hybrids and treatments. in the absence of interspecific competition, gt hybrids had significantly more b. rapa markers and significantly fewer b. napus markers than the other lines. however, when gt hybrids were grown in competition with wheat, gt had significantly more b. napus markers and significantly fewer b. rapa markers than the other lines in the competition treatment. in contrast, more b. rapa markers were measured in the gt hybrids grown in competition with wheat than in gt hybrids grown under no competition, while no differences were seen in b. napus markers for either of the treatments. thus, gt and gt and different patterns of response to competition with regards of genomic constitution. in the gt line, there were no significant differences between the number or type of marker in either of the growing conditions. even though significant differences were observed for the markers and lines under different competitive conditions, correlations were only observed between markers and productivity data for the gt hybrids. b. napus aflp marker correlation with productivity our hypothesis that hybrids containing more b. napus crop alleles or genetic load would be less competitive than weedy species can be conditionally rejected, but only in the case where there is no interspecific competition. there was an overall moderate positive correlation between the number of b. napus-specific aflp markers and seed yield and vegetative biomass under non-competitive conditions: for seed yield vs. marker number and vegetative dry biomass vs. marker number. the statistical significance can be accounted for by gt hybrids. no correlations were observed for the other events. when the gt hybrids were grown in competition with wheat, there were no significant correlations between the number of b. napus crop markers and seed yield, vegetative dry biomass or with wheat vegetative dry biomass. in addition, no correlations between b. napus markers and wheat vegetative dry biomass existed for any of the hybrid lines. * denotes significant results the numeric values represent pearson correlation coefficients. hybrids were grown in the presence and absence of interspecific competition with wheat. b. rapa aflp marker correlation with productivity there were no correlations between the number of b. rapa specific aflp markers and the productivity of the gt hybrids grown alone, i.e., in the absence of interspecific competition. conversely, under competitive conditions, there were strong positive correlations between b. rapa weediness markers and seed yield but only for the gt hybrids. in the gt line, the correlations between b. rapa markers under competitive conditions were slightly higher than those for the b. napus markers under no competition: seed yield vs. marker. overall, the correlations between the number of b. rapa markers and seed yield under competitive conditions were significant. no correlations were observed in the gt and gt hybrid lines with respect to the b. rapa markers and seed yield or vegetative dry biomass. in addition, no correlations between b. rapa markers and wheat vegetative dry biomass existed for any of the hybrid lines. transgenic-mitigation study productivity data for the tm study, we analyzed populations of non-transgenic b. napus and b. rapa, a transgenic homozygous tm b. napus population and transgenic segregating populations of bc1/f hybrids. the gt hybrids used in the tm study were the same lines that were analyzed in the genetic load study, whereas the tm and the tm lines were unique the tm study. when grown in competition with wheat, the non-transgenic b. napus and b. rapa plants reached heights of 130- cm at plant maturity whereas the homozygous tm b. napus plants were only 40- cm at plant maturity. at the time of harvest, the siliques on all of the non-transgenic and hybrid plants were completely mature and ready to shatter, while the siliques on the tm plants were still green-to-yellow and maturing. throughout the field season, it was observed that the tm line had delayed emergence, reduced height, and delayed flowering. however, based on productivity data, when grown alone, the tm populations performed as well as their non-transgenic crop counterpart, indicating that dwarfing could potentially be utilized in true cropping systems without any yield penalties. these results are congruent with those of al-ahmad et al.. when populations of transgenic and non-transgenic segregants of the gt and tm hybrid lines were grown in the absence of competition with wheat, significant differences in biomass and seed yield were observed, however, all hybrid lines performed similarly to b. rapa in the absence of competition. in contrast under competitive conditions, there were no statistical differences observed for vegetative biomass or seed yield for any of the hybrid lines, but there was a great deal of endogenous variation among data in the competition experiment. transgene persistence data after one field season of natural hybridization among individuals of a hybrid line, the progeny from the bc1/f hybrid lines was comprised of bc and f individuals. transgenic segregation ratios in the progeny populations differed among the gt and tm lines. the frequencies of transgenic individuals decreased in all gt and tm hybrid progeny populations after one field season. the frequencies of transgenic individuals in these populations differed among lines and under interspecific and intraspecific competitive conditions. the proportion of transgenic individuals in the progeny populations of the gt gt gt and tm bc1/f hybrid lines were the same for both non- and competitive conditions. however, there was a significant difference observed for the tm line. the progeny population of the tm line grown in competition with wheat had fewer transgenic individuals than that grown in the absence of interspecific competition. the green fluorescent marker was also detected in the parental progeny lines indicating the occurrence of gene flow among plots. the f progeny populations for the bn and br parental lines were not screened on ms medium containing the selecting imazapyr herbicide, hence, the transgene persistence data reported in table only applies to gene flow from gt hybrid plots and not tm hybrid or homozygous plots. the decrease in transgenic individuals was greater for the tm hybrid line than the gt lines. the initial frequency of transgenic individuals in the tm hybrid line was low, and after one field season, the frequency of transgenic individuals dropped further. percent germination and transgenic frequencies were calculated for the parental and hybrid bc1/f populations pre-season and post-harvest. transgene persistence data reported here only applies to gene flow from gt hybrid plots and not tm hybrid or homozygous plots. column ms represents transgenic ratio mean separation obtained by fisher's lsd. data was rank transformed and sas software was used for data analysis. discussion there is growing evidence that many interspecific hybrids between crops and weeds, including transgenic hybrids, have inferior performance compared with their weedy parent, although a few exceptions have been reported. there are many possible explanations for these observations, most of which focus on the fitness penalties associated with hybridization and backcrossing. a variety of genetic mechanisms can affect hybrid fitness. heterosis or hybrid vigor could be enhanced by the segregation of additive genetic traits or optimal environmental conditions. in cases where the crop and weedy or wild relative are the same species, there should be fewer genetic barriers to hybrid fitness and transgene persistence. unless the trait or transgene is deleterious, as in the case of tm, hybridization and introgression might easily progress, thereby potentially allowing transgene persistence. this is far more likely to occur with related weeds existing and competing in the same agro-ecosystem as the crop than with wild species, residing in non-agricultural areas where a transgenic trait may have little or no value. the performance of new genotypes introduced via transgenesis and subsequent hybridization are likely also subject to genotype × environment interactions. when distantly related taxa hybridize, recombination between homologous or homoeologous chromosomes can result in irregular chromosome pairing. the latter can lead to reduced growth and fertility, thus decreasing potential for introgression. in addition, the dilution of weed alleles by crop alleles, i.e., genetic load, might also account for slower introgression regardless of environment. our results showed a positive correlation between genetic load and plant productivity in only one of our transgenic events, gt a result congruent with a prior study. no correlation was observed for the other transgenic events suggesting a strong transgenic event or genotype effect. the experimental design allowed the detection of a surprising effect--a counter genetic load finding. in our experiments, the number of b. napus crop-specific aflp markers was associated with an increase in hybrid performance in the gt hybrids in the absence of interspecific competition with wheat and weeds. conversely, in the presence of interspecific competition with wheat, there was a strong correlation between b. rapa weed-specific aflp markers and productivity, again, only for the gt line. these results indicate a strong g × e interaction and that inherited crop alleles, introgressed as a result of hybridization, can be preferentially selected to ensure maximum growth potential and productivity under crop-favorable conditions. conversely, inherited weedy alleles can be preferentially selected under more "weed-like" conditions. we anticipate that this will continue to occur as the lines continue to backcross. this evidence of local and situational adaptation demonstrated in our study as well as other studies indicates that many alleles are beneficial in some but not all environments. as sessile organisms, plants must be able to maximize resource acquisition; this can be accomplished with adapted genomes. in agriculture and in our study, environmental genomic adaptation could translate to favoring some crop alleles in crops grown in monocultures where weeds are controlled; weed alleles might be favored in weeds in competitive environments. dilution in either direction could be maladaptive. environmental adaptation and trait selection are further demonstrated in a case-study performed on recombinant inbred sunflower lines. baack et al. showed that crop traits were preferentially favored in one site, while weedy traits were favored at another location. these studies indicate that, with regards to genetic load and trait selection, environmentally-dependent selection must be taken into consideration for every transgene, species, and situation studied. transgenic event effects and genetic load since transgene insertion is random, the flanking host dna can differ significantly for individual transgenic events; hence the analysis of multiple events in the present study. zhu et al. examined the transgene segregation ratios for each of our lines using controlled crosses and progeny analysis. the gt and gt lines did not deviate from expected mendelian segregation, indicating homeologous recombination of the transgene locus on an a-genome chromosome, and hence the transgene would have a decreased chance of being lost in subsequent backcrossing as a result of genomic incompatibility. thus, linkage effects could likely cause decreased introgression and genetic load in the gt line. transgenes located on the c-genome of b. napus might be subject to greater genomic incompatibility since b. rapa does not have the c-genome of b. napus. thus c-genome-localized transgenic events could be an important investigative avenue for decreasing introgression, although recent research found that homeologous recombination occurs at the same rate as homologous recombination in the b. rapa × b. napus system, suggesting that "safe-integration" sites in b. napus are unlikely. engineered vs. randon transgenic event genetic load tm b. napus plants containing the dwarfing gene Δgai were previously analyzed under shade-house conditions. homozygous tm dwarf b. napus plants grown alone had a much higher seed yield, and double the shoot and root biomass compared to non-transgenic counterparts. the tm line also produced more leaves than tall non-transgenic plants when grown alone at -cm spacing and 10-cm spacing. however, when grown in competition with tall non-transgenic cohorts, these same plants were exceedingly unfit. the reproductive fitness of tm plants, when grown in competition, was less than 12%, and the harvest index was less than 40% of that of the non-transgenic b. napus competitors. when grown in the absence of competition, the tm line produced the greatest amount of seeds per plant. from these studies, al-ahmad et al. concluded that the Δgai gene greatly enhanced seed and biomass yield in a weed-free transgenic crop. however, if dwarfed plants emerged as volunteer weeds and competed with non-transgenic cohorts, dwarfed plants would be eliminated from poor competitive ability, especially if selective herbicides were not used. our field results were congruent with the previous tm research. when the homozygous tm line was grown under agronomic non-competitive conditions, it had equivalent performance as the non-transgenic b. napus line with regards to seed yield and biomass. however, under competitive conditions, the tm line produced the least seed and biomass and, hence, performed the poorest of all the lines that were analyzed. in terms of transgene persistence, the transgenic progeny from the backcrossed tm line approached a 50- to 85-fold reduction, under competitive and non-competitive conditions, compared with the 3-fold reduction in transgene persistence with the non-tm lines. it must be noted that the final portion of the transgene persistence studies; i.e., germination of seed collected from field grown plants were carried out under optimal growth chamber conditions. this environment did not take into consideration the effects of over-wintering survivorship, dormancy issues or seedling dynamics. consequently, when environmental factors are considered, one could potentially expect a decrease in performance under realistic field conditions. hence, ex situ analysis of transgene persistence could appear higher than it actually would be in the field. regardless, these data do indicate that tm constructs were effective in severely limiting the impact of gene flow from transgenic crops to their wild relatives and transgene persistence. the tm approach could further be enhanced by stacking other weed mitigating transgenes, such as those that prevent seedpods from shattering or those that prevent secondary dormancy. in addition to making plants shorter the dwarfing gene could also confer pleiotropic effects such as altered flowering traits. these could conceivably also have g × e interactions and effect fitness. depending on the trait, the pleiotropic effect could increase or decrease fitness. in this particular instance, dwarfed plants were very late flowering and also had delayed seed maturation and decreased germination. in this case, the pleiotropic effects would almost certainly decrease introgression. some researchers point out that dwarfing in tm is a trait that could foreseeably be selected against in the hemizygous state; e.g., in semi-dwarfed plants. consequently, the dwarfing allele might not be expressed as highly in first-generation hybrids because of the presence of the dominant gai allele from the weedy parent genome. therefore, hybrids containing the tm construct might not have sufficient dwarfing to be selected against in competition, yet harbor the fitness-enhancing gene from the tm construct. in subsequent generations, the deleterious allele would only be expressed in homozygous individuals, which would strongly reduce its capability to decrease fitness. moreover, if the hybrids are fertile, this strategy would not prevent them acting as a genetic bridge and pollinating the wild parent. reuter et al. challenged the theory that dwarfed volunteers or transgenic escapees would be less competitive in feral environments. they noted that feral non-transgenic b. napus populations, growing in rural and urban area in northern germany, were, on average, 41% shorter than cultivated non-transgenic b. napus. they attributed this height difference to phenotypic adaptation to local environments. reuter et al. concluded that, under certain environmental and ecological conditions, the proposed mitigation approach could actually increase escape and persistence of transgenic varieties rather than reducing them, but their study did not consider genotype differences, local adaptation, and environmental effects such as differences in nutrient availability between agronomic and feral conditions. our transgene persistence data indicate that even if the dwarfing trait is in the semi-dominant state, it is still effective in limiting transgene persistence. our results indicate that when dwarfing is utilized for a containment strategy, the plants are not able to compete as well and hence persist in subsequent generations. these observations hold true regardless of whether the plants were grown in the presence or absence of interspecific competition. hence, under the conditions and situations analyzed in this study, hybrids containing tm constructs were not effective weeds. different species will likely require various mitigators, and possible also for different environments. anti-shattering genes will be appropriate for many row crops that have seed shattering weedy relatives, and anti-secondary dormancy genes would be appropriate where weeds possess seed-bank longevity as part of their survival mechanisms. anti-bolting genes would be appropriate mitigators for biennial crops or storage crops that have weedy relatives. sterility genes are appropriate for vegetatively propagated species. the waxy transgene in maize would be a good mitigator for pharmaceutical genes expressed in maize embryos, as seeds from self or cross pollination would be shrunken, and unable to survive over-winter in most soils. thus, to choose an appropriate mitigator for each crop and environment, the researcher must seek out genes that are positive or neutral for the crop in that environment and would be detrimental to the related weedy or wild species.  there is a paradox regarding the apparent absence of introgression in species where gene flow is expected on the basis of sexual compatibility data. it might be that genetic load of endogenous genes on crop chromosomes moderates introgression, and this same effect could be extended and applied to transgene containment. perhaps we do not observe extensive transgene persistence in weedy relatives, i.e., only one introgressed transgenic weedy b. rapa plant has been observed in the field despite the extensive commercial release of transgenic b. napus, because of genetic load conferred by the lack of homeologous recombination or hitchhiking of crop alleles. if additional genetic load is imposed using tm, it could dramatically decrease introgression and transgene persistence to an even lower level. 
in the absence of interspecific competition, transgenic weed × crop hybrids benefited from having more crop-specific alleles. tm strategies are proposed to decrease transgene persistence if gene flow and subsequent transgene introgression to a wild host were to occur. however under interspecific competition with wheat or more weed-like conditions, there was a positive correlation between the number of b. rapa weed-specific aflp markers and seed yield, although no such correlation was detected for vegetative biomass. the effects of directly incorporated genetic load were analyzed through transgene persistence data. however, when genetic load was directly incorporated into transgenic events, e.g., using a tm construct, the number of transgenic hybrids and persistence in weedy genomic backgrounds was significantly decreased. there was a positive correlation between performance and number of b. napus crop-specific aflp markers. one theoretical explanation for the relatively poor performance of brassica rapa × brassica napus transgenic hybrids suggests that hybridization imparts a negative genetic load. consequently, in hybrids genetic load could overshadow any benefits of fitness enhancing transgenes and become the limiting factor in transgenic hybrid persistence. two types of genetic load were analyzed in this study: random/linkage-derived genetic load, and directly incorporated genetic load using a transgenic mitigation strategy. in order to measure the effects of random genetic load, hybrid productivity was correlated with crop- and weed-specific aflp genomic markers. when genetic load was directly incorporated into the hybrid genome, by inserting a fitness-mitigating dwarfing gene that that is beneficial for crops but deleterious for weeds, there was a dramatic decrease in the number of transgenic hybrid progeny persisting in the population. the effects of genetic load of crop and in some situations, weed alleles might be beneficial under certain environmental conditions. this portion of the study was designed to answer whether or not weed × transgenic crop hybrids possessing more crop genes were less competitive than hybrids containing fewer crop genes.
 one theoretical explanation for the relatively poor performance of brassica rapa × brassica napus transgenic hybrids suggests that hybridization imparts a negative genetic load. consequently, in hybrids genetic load could overshadow any benefits of fitness enhancing transgenes and become the limiting factor in transgenic hybrid persistence. two types of genetic load were analyzed in this study: random/linkage-derived genetic load, and directly incorporated genetic load using a transgenic mitigation strategy. in order to measure the effects of random genetic load, hybrid productivity was correlated with crop- and weed-specific aflp genomic markers. this portion of the study was designed to answer whether or not weed × transgenic crop hybrids possessing more crop genes were less competitive than hybrids containing fewer crop genes. the effects of directly incorporated genetic load were analyzed through transgene persistence data. tm strategies are proposed to decrease transgene persistence if gene flow and subsequent transgene introgression to a wild host were to occur.  in the absence of interspecific competition, transgenic weed × crop hybrids benefited from having more crop-specific alleles. there was a positive correlation between performance and number of b. napus crop-specific aflp markers. however under interspecific competition with wheat or more weed-like conditions, there was a positive correlation between the number of b. rapa weed-specific aflp markers and seed yield, although no such correlation was detected for vegetative biomass. when genetic load was directly incorporated into the hybrid genome, by inserting a fitness-mitigating dwarfing gene that that is beneficial for crops but deleterious for weeds, there was a dramatic decrease in the number of transgenic hybrid progeny persisting in the population.  the effects of genetic load of crop and in some situations, weed alleles might be beneficial under certain environmental conditions. however, when genetic load was directly incorporated into transgenic events, e.g., using a tm construct, the number of transgenic hybrids and persistence in weedy genomic backgrounds was significantly decreased. 