Thailand’s northeast (NE) region is an area of high-quality cultivated rice (Oryza sativa L.) production. However, an outbreak of weedy rice has recently spread throughout the region. Weedy rice is phenotypically and morphologically similar to cultivated rice, making identification difficult. The prospective management of weedy rice in the future will involve the study of its genetic diversity and population structure in this region. This study assesses the genetic diversity and population structure of 380 weedy rice samples in the northeast of Thailand through simple sequence repeat (SSR) markers. Thirty-one SSR markers generated 213 alleles with an average of 6.87 per locus and an overall genetic diversity of 0.723. Based on its geographic origin, weedy rice in the Southern NE are showed greater genetic diversity than that in the Central NE and Northern NE areas. The outcrossing rate in all regions was relatively high, with the highest being in the Southern NE at 9.769%. According to genetic distance analysis, the clustering of weedy rice samples in northeast Thailand was not associated with the geographic region. Neighbor-joining and principal coordinate analysis revealed that the 380 weedy rice samples fell into two major clusters. Cluster I contained three weedy rice samples and four wild. In Cluster II, 377 weedy rice samples were closely related to the four cultivated rice cultivars as well as brownbeard rice (Oryza rufipogon Griffiths) wild species. The results suggest that weedy rice in northeast Thailand may have originated as a cross between cultivated and wild rice, as seen in the closely related species, O. rufipogon. Overall, the findings of this study demonstrate the high genetic diversity of weedy rice in this region. Notably, some samples adapted, performing more like cultivated rice, which may be problematic for the future production of high-quality rice in this region. The prevention of weedy rice should, therefore, be given greater consideration in future studies.

Although sorghum [Sorghum bicolor (L.) Moench ssp. bicolor] is the fifth most important grain crop in terms of global production, no commercial hybrids carry genetically engineered (GE) traits for resistance to insect pests or herbicides due to regulatory concerns about gene flow to weedy relatives. However, non-GE herbicide resistance currently is being developed in grain sorghum and will likely transfer to related weeds. Monitoring the impact of this new nuclear technology on the evolution and invasiveness of related weeds requires a baseline understanding of the population biology of grain sorghum genes once they transfer to in situ weed populations. We previously characterized the rate of gene flow from grain sorghum to shattercane [Sorghum bicolor (L.) Moench nothosubsp. drummondii (Steud.) de Wet ex. Davidse], a conspecific weed relatively common in North America; as well as the ecological fitness of an F1 population when S. bicolor nothosubsp. drummondii was the maternal parent. Here we report the ecological fitness of a S. bicolor nothosubsp. drummondii × S. bicolor ssp. bicolor F2 population relative to its crop and weed parents. Parental and F2 populations were grown in two Nebraska environments in 2012 and 2013. Traits evaluated included overwinter survival, field emergence, biomass production and partitioning at anthesis, total seed production, and 100-seed weight. Results indicated that F2 traits were generally intermediate between the parents, but more similar to S. bicolor nothosubsp. drummondii than to grain sorghum. The one exception was overwinter survival, which was nearly 0% for both the F2 and the grain sorghum parent in these northern environments. Thus, the frequency of crop alleles stably introgressed into S. bicolor nothosubsp. drummondii populations appears to primarily depend on overwinter survival of the F2 and which selective pressures are imposed upon it by the cropping system. These data provide needed baseline information about the environmental fate of nuclear genetic technologies deployed in this important global crop.

Two distinctive leaf forms have been described in the morningglory species Ipomoea hederacea (L.) Jacq. These are identified as ivyleaf morningglory [I. hederacea (L.) Jacq. # IPOHE] and entireleaf morningglory [I. hederacea (L.) Jacq. var. integriuscula Gray # IPOHG]. Controlled matings were made between pure breeding forms of plants with these two leaf types. The first generation progeny (F1) were all ivyleaf and the second generation (F2) was segregated in a 3:1 ratio (ivyleaf to entireleaf). The data fit a single gene model for determination of leaf shape, with ivyleaf being the dominant allele. Despite the dominance of the ivyleaf trait, the abundance of the entireleaf phenotype in the Mississippi Delta was higher than that of ivyleaf. Ipomoea hederacea is a facultative self-pollinator and other annual morningglory species found in the same area are also self-pollinated.

Gene flow among herbicide-resistant (HR) canola varieties can lead to the development of multiple HR canola plants, creating volunteer canola management challenges for producers. In western Canada, escaped populations of HR canola are ubiquitous outside of cultivated fields, yet the extent of gene flow resulting in herbicide resistance trait stacking in individuals within these populations remains unknown. The objectives of this study were to document the presence of single and multiple herbicide resistance traits and assess the extent of gene flow within escaped canola populations. Seed was collected from 16 escaped canola populations along the verges of fields and roadways in four agricultural regions in southern Manitoba from 2004 to 2006. Glyphosate resistance was found in 14 (88%) of these populations, glufosinate resistance in 13 (81%) populations, and imidazolinone resistance in five (31%) populations. Multiple herbicide resistance was observed at levels consistent with previously published canola outcrossing rates in 10 (62%) of the tested populations. In 2005 and 2006, maternal plants from two escaped populations were tested using trait indicator test strips for glyphosate and glufosinate resistance to confirm outcrossing events. In 2005, two of 13 tested maternal plants with single herbicide resistance traits produced progeny with both glyphosate and glufosinate resistance. In 2006, of 21 tested plants, 10 single HR maternal plants produced multiple HR progeny, and five nonresistant maternal plants produced resistant offspring. This is the first report indicating that intraspecific gene flow results in stacking of herbicide resistance traits in individuals within escaped canola populations, confirming that multiple HR canola volunteers are not confined to agricultural fields. Results of this study suggest that escaped populations of crop plants can contribute to the spread of genetically engineered novel traits, which has important implications for containment, especially for highly controversial pharmaceutical and industrial traits in crop plants.

Red rice is a major weed of rice in the southern U.S. and can intercross with rice. Knowledge of the plant phenotypes from such crosses would be valuable for identification and management of these plants. Male-sterile long-grain tropical japonicas ‘Kaybonnet-1789’ and ‘Cypress-1819’ were crossed with two awned and two awnless U.S. red rice types. F1 plants produced pubescent leaves, red pericarp, and medium-grain seeds. Crosses involving awned LA3 and TX4 red rice produced F1 plants with reddish-purple basal leaf sheaths and usually flowered within the same time periods as the parents, whereas those involving awnless StgS red rice had green basal leaf sheaths, flowered much later than either parent, and produced awnless F1 and F2 offspring. Crosses involving awned red rice produced F1 plants with long awns and F2 plants with awns ranging in length from zero to that of red rice parents. F1 plants were taller than either parent and produced intermediate culm angles similar to red rice, whereas F2 plants had culms ranging from erect (like rice) to more open than red rice. Thus, true F1 hybrids from crosses between pure breeding (homozygous) rice and red rice can be positively identified by a combination of traits including pubescent leaves, medium-grain seeds with red pericarps, open plant types, and heights greater than the red rice parent. F1 hybrids may be awned or awnless, have purple or green stems, or have normal or delayed heading. F2 plants have a broad combination of phenotypic traits found in both parents and F1 hybrids.

Weedy red rice is a major weed pest of rice in the southern United States. Outcrossing between red rice and commercial tropical japonica rice cultivars has resulted in new weed biotypes that further hinder the effectiveness of weed management. In recent years, indica rice has been used increasingly as a germplasm source for breeding and for reduced-input systems in the United States, but little is known about its outcrossing potential with U.S. weedy red rice biotypes. In a 2-yr study, simple sequence repeat marker analysis was used to show that blackhull (BH) red rice (PI 653424) outcrossing to four, late-maturing indica cultivars averaged 0.0086% and ranged from 0.002% for ‘TeQing’ to 0.0173% for ‘4484’ (PI 615022). Rates of outcrossing to a tropical japonica cultivar standard, ‘Kaybonnet’ (0.032%), were substantially greater than for the indica cultivars. These differences in outcrossing were due largely to synchronization of flowering times between rice and red rice, with Kaybonnet and TeQing exhibiting the greatest and least synchronization, respectively. Outcrossing rates also may have been affected by rice–red rice flower density differences within the rice plots. Outcrossing from cultivated rice to the red rice (as pollen recipient), which was taller than all rice cultivars, was undetectable in these studies, and environmental conditions (e.g., temperature, humidity, solar radiation, and rainfall) were not strongly correlated with the outcrossing rates observed. Grain yields of the original BH red rice line were greatest in the Kaybonnet plots, demonstrating that the indica cultivars were superior competitors against this weed. Collectively, these results suggest that red rice biotypes that flower synchronously with rice cultivars are a potential source of pollen for outcrossing and gene flow in rice fields in the southern United States.

Two separate field experiments were conducted to quantify the degree of plant-to-plant outcrossing and pollen-mediated gene flow (PMGF) in wild oat. The purpose of the study was to determine the extent to which pollen movement could contribute to the spread of herbicide resistance in this species. In both experiments, an acetyl-CoA carboxylase inhibitor–resistant (R) wild oat genotype (UM1) was used as the pollen donor and a susceptible (S) genotype (UM5) was used as the pollen receptor. Hybrid progeny resulting from a cross between UM1 and UM5 were identified using the herbicide resistance trait as a marker. In the plant-to-plant outcrossing experiment, single UM5 plants were closely surrounded by 20 homozygous R UM1 plants in hills. By screening seed from the S parent for resistance, outcrossing was determined to range from 0 to 12.3%, with a mean of 5.2% over 10 hills. In the PMGF experiment, single homozygous R UM1 plants were surrounded by UM5 plants arranged in a hexagonal pattern at low and high densities (total of 19 and 37 wild oat plants m−2), growing within spring wheat and flax crops. In the wheat crop, mean wild oat outcrossing was 0.08 and 0.05% at low and high densities, respectively. In the less competitive flax, corresponding outcrossing values were 0.07 and 0.16% at low and high densities, respectively. Distance from the pollen source was a significant factor only for the high-density planting arrangement in flax. Up to 77 R hybrid seeds were recovered from 6 m2 in the PMGF experiment, indicating that PMGF contributes to the evolution of resistance in wild oat populations. However, the contribution of pollen movement to resistance evolution and the spread of resistance in wild oat populations would be relatively small when compared with R seed production and dispersal from a resistant plant.

EDITOR'S NOTE: This manuscript was reviewed by six colleagues whose recommendations varied widely. Lack of repetition was a major concern. The authors address the problem in the last paragraph of the results section. Factors favoring publication included the worldwide importance of wild oats, the minimal data on gene flow in the species, and the fact that the results are consistent with those of other studies cited in this manuscript. The points raised by reviewers who did not favor publication, especially the role of the environment in pollen production and viability, are acknowledged.

R. L. Zimdahl, Editor

Experiments were conducted to determine the inheritance of resistance in crosses between imazethapyr-resistant rice and red rice. Past experiments on red rice control, using the Clearfield rice technology, resulted in outcrossing between Clearfield rice and Stuttgart strawhull red rice. The F2 generation of these spontaneous crosses were characterized with respect to inheritance of imazethapyr resistance, leaf color and leaf pubescence, and seed shattering, pubescence, color, and size. Agronomic traits of hybrids were also observed in relation to their parents. To determine the segregation of resistance among F2 phenotypes, the response of three- to four-leaf plants to imazethapyr was scored 3 wk after application as resistant (R, no imazethapyr symptoms), susceptible (S, death of plants), or intermediate (I, stunted plants). R, I, and S phenotypes segregated in a 1:2:1 ratio in the F2 generation. Two- or three-gene inheritance was documented for leaf and seed characteristics. A wide range in onset of flowering (70 to 130 d after planting) was observed in F2 families, although 6% of the plants did not flower during the growing season. F2 plants were taller and had more tillers than any of their parents. Resistance to imazethapyr is associated with a single, incompletely dominant allele.

The transfer of herbicide resistance genes from crops to related species is one of the greatest risks of growing herbicide-resistant crops. The recent introductions of imidazolinone-resistant wheat in the Great Plains and Pacific Northwest regions of the United States and research on transgenic glyphosate-resistant wheat have raised concerns about the transfer of herbicide resistance from wheat to jointed goatgrass via introgressive hybridization. Field experiments were conducted from 2000 to 2003 at three locations in Washington and Idaho to determine the frequency and distance that imidazolinone-resistant wheat can pollinate jointed goatgrass and produce resistant F1 hybrids. Each experiment was designed as a Nelder wheel with 16 equally spaced rays extending away from a central pollen source of ‘Fidel-FS4’ imidazolinone-resistant wheat. Each ray was 46 m long and contained three rows of jointed goatgrass. Spikelets were collected at maturity at 1.8-m intervals along each ray and subjected to an imazamox screening test. The majority of all jointed goatgrass seeds tested were not resistant to imazamox; however, 5 and 15 resistant hybrids were found at the Pullman, WA, and Lewiston, ID, locations, respectively. The resistant plants were identified at a maximum distance of 40.2 m from the pollen source. The overall frequency of imazamox-resistant hybrids was similar to the predicted frequency of naturally occurring acetolactate synthase resistance in weeds; however, traits with a lower frequency of spontaneous mutations may have a relatively greater risk for gene escape via introgressive hybridization.

Red rice is a major weed in rice production in the southern United States. Red rice and rice intercross because they are the same species. Our objectives were to determine the genetic diversity represented by accessions of red rice and to identify DNA markers that might be useful in identifying hybrids between red and cultivated rice. Red rice accessions were collected from Arkansas and other rice-producing states. Seventy-nine red rice accessions, 10 known or putative hybrid derivatives of red rice and cultivated rice (RC hybrids), and seven rice cultivars were analyzed using microsatellite DNA markers developed for cultivated rice. Microsatellite markers differentiated awned and awnless red rice accessions, six of the seven rice cultivars, and all 10 RC hybrids tested. Thus, these markers were useful in identifying red rice types and RC hybrids.

Red rice has long been a troublesome, conspecific weed of cultivated rice. Rice varieties carrying certain herbicide-resistant traits acquired through genetic modification (herbicide-resistant varieties) now offer new options for red rice control. In concert with this innovation is the risk of gene flow, which can result in the transfer of that specific herbicide resistance to red rice and thus render this weed control measure ineffective. Gene flow in concept is simple, however, the parameters that determine the establishment of a new trait in a weed population are complex. Cross-pollination to make hybrid seed and the subsequent fate of those hybrid families in the general weed population are some of the biological factors that influence gene flow between red rice and cultivated rice. Natural outcrossing among rice plants is generally low. Most of the pollen dispersal studies published to date indicated that rice × rice outcrossing rates were less than 1.0%. Numerous reports summarized in this study suggest that outcrossing rates between rice and red rice can be highly variable but usually are similar to or lower than this level. However, once hybrids form, they may introgress into a red rice population within only a few generations. If hybrid seed families are to persist and establish herbicide-resistant red rice populations, they must successfully compete in the crop–weed complex. The ability to survive a herbicide applied to a herbicide-resistant rice variety would be a strong selective advantage for these hybrid families. Thus, the well-established principles of weed resistance management appear to be relevant for herbicide-resistant crop systems and should be used in combination with practices to minimize coincident flowering to mitigate the potential impact of gene flow from herbicide-resistant rice into red rice. For the rice–red rice crop–weed complex, there are both biological factors and agricultural practices that can work together to preserve these new weed control options.

Herbicides that target the enzyme acetolactate synthase (ALS) are among the most widely used in the world. Unfortunately, these herbicides are also notorious for their ability to select resistant (R) weed populations. Now, there are more weed species that are resistant to ALS-inhibiting herbicides than to any other herbicide group. In most cases, resistance to ALS-inhibiting herbicides is caused by an altered ALS enzyme. The frequent occurrence of weed populations resistant to ALS inhibitors can be attributed to the widespread usage of these herbicides, how they have been used, the strong selection pressure they exert, and the resistance mechanism. In several cropping systems, ALS-inhibiting herbicides were used repeatedly as the primary mechanism of weed control. These herbicides exert strong selection pressure because of their high activity on sensitive biotypes at the rates used and because of their soil residual activity. Several point mutations within the gene encoding ALS can result in a herbicide-resistant ALS. From investigations of numerous R weed biotypes, five conserved amino acids have been identified in ALS that, on substitution, can confer resistance to ALS inhibitors. Substitutions of at least 12 additional ALS amino acids can also confer herbicide resistance in plants and other organisms but, to date, have not been found in R weed populations. Mutations in ALS conferring herbicide resistance are at least partially dominant, and because the gene is nuclear inherited, it is transmitted by both seed and pollen. Furthermore, in many cases there is apparently a negligible fitness cost of the resistance gene in the absence of herbicide selection. Although resistance to ALS-inhibiting herbicides has been a bane to weed management, it has spurred many advances within and beyond the weed science discipline. As examples, resistance to ALS-inhibiting herbicides has been exploited in the development of herbicide-resistant crops, studies of weed population dynamics, and in developing protocols for targeted gene modification. Resistance to ALS-inhibiting herbicides has greatly affected weed science by influencing how we view the sustainability of our weed management practices, what we consider when developing and marketing new herbicides, and how we train new weed scientists.

The transfer of the imazethapyr-resistant gene from Clearfield™ (CL) rice to red rice is an ecological risk. Flowering synchronization and genetic compatibility between cultivated rice and red rice could influence gene transfer. We examined the (1) variability in maximum outcrossing rate between 12 red rice biotypes and ‘CL161’ rice during their peak flowering overlap in the field and (2) genetic compatibility of red rice biotypes with CL161 rice. Experiments were conducted at Stuttgart, AR, and Fayetteville, AR, from 2005 to 2007. To evaluate the flowering synchrony of red rice and CL161 rice as well as its impact on outcrossing rate, field experiments were conducted at four planting times from early April to late May. The red rice biotypes were planted in the middle row of nine-row CL161 plots and flowering was monitored. Outcrosses were evaluated in subsequent years by herbicide response and simple-sequence-repeat marker assays. To determine compatibility, manual crosses were performed between 12 red rice biotypes and CL161 rice in the greenhouse. The flowering duration of all red rice types ranged from 5 to 16 d after the onset of flowering in contrast to 6 d in CL161 rice. Ten of the twelve types of red rice had ≥ 70% overlap in flowering time with CL161 rice in at least one planting date. The maximum field outcrossing rate between red rice biotypes and CL161 ranged from 0.03 to 0.25%. The field outcrossing rate between red rice biotypes differed (P < 0.01), but flowering synchronization was not directly related to outcrossing rate. Manual crosses resulted in seed sets of 49 to 94%. The majority of red rice biotypes had similar compatibility with CL161 rice. Thus, other factors must contribute to hybridization rates in the field. Follow-up experiments should investigate other plant factors and environmental influence on hybridization rate.

Reports of resistance to grass-selective herbicides, including johnsongrass (Sorghum halepense) resistant to fluazifop-P, have become quite common. Experiments were conducted to determine if fluazifop-P resistance could be transferred from johnsongrass to crop sorghum (Sorghum bicolor) and to identify the heritability of resistance in the progeny. A population of male sterile, fluazifop-P–sensitive sorghum, AKS-82, was interplanted with fluazifop-P–resistant johnsongrass and progeny treated with 0.105 kg ai/ha fluazifop-P postemergence (POST). Surviving seedlings were backcrossed to fluazifop-P–resistant johnsongrass. Viable plants from this successful cross were then testcrossed to another sensitive sorghum, TX622. These testcross progeny were then screened for herbicide resistance by again applying fluazifop-P POST at 0.105 kg /ha. Chi-square analysis revealed that resistance to fluazifop-P was inherited by a single, dominant gene. Natural hybridization of johnsongrass and sorghum is possible, and transfer of herbicide resistance between species can occur.

A study was carried out between 2008 and 2011 to investigate the potential risks of gene flow and its consequences in the crop–wild–weed S. bicolor complex in Ethiopia to aid efforts to conserve genetic diversity. Morphological measurements and genomic DNA samples were taken in situ from 30 wild and eight cultivated populations representing a total of 760 samples from five regions. Genetic diversity, gene flow, population structure and outcrossing rates of wild populations were computed using phenotypic measurements and/or polymorphic simple sequence repeat (SSR) markers. Moreover, morphological analyses of fitness of crop–wild hybrids were studied. High diversity was observed among the wild/weedy sorghum populations for phenotypic traits and SSRs. SSR diversity was high in both wild and cultivated populations, but the magnitude was greater in the former. Gene flow between the wild and the cultivated sorghum was observed to be higher than that within either pool. Wild sorghums exhibited variation in the multilocus outcrossing rate (range = 0.31–0.65) and fitness was not compromised in most wild × crop hybrids. The study indicated that crop-to-wild gene flow is possible in Ethiopia. Thus, genes from transgenic sorghum are expected to enter into the wild and non-transgenic cultivated populations and may spread and persist, if transgenic sorghum is deployed in Ethiopia and in other countries of Africa, which may pose risk of introduction of unwanted effects, which in turn may lead to loss of genetic diversity.

Outcrossing to wild or unrelated strains has been proposed as a counter to the negative effects of adaptation by domesticated animals to captive-breeding environments. However, for mass-reared insects intended for sterile insect technique release, outcrossing may reduce productivity in mass-rearing facilities. In this study, we characterized adaptation to mass-rearing in the Queensland fruit fly Bactrocera tryoni (Froggatt). The features of domesticated B. tryoni were similar to those of other domesticated tephritids: shortened preoviposition period, higher egg production and increased early survival. We also examined the practical use of outcrossing to improve mass-reared strains. The B. tryoni mass-reared strain was crossed with independent strains that had already been domesticated for a varying number of generations. Preoviposition period, egg production, survival and stress tolerance were measured in the resulting hybridized strains. The short preoviposition period of the mass-reared strain was retained in outcrossed flies, indicating a genetically dominant trait. Egg production in outcrossed flies exceeded the mid-parent value, indicating some hybrid vigour for this trait. If both parents exhibited high egg production, the egg production of outcrossed strains was as high as that of the mass-reared strain. The high early survival of domesticated tephritids was reduced in outcrossed females, but not in outcrossed males. Preliminary thermal stress tests revealed that outbred flies were more stress resistant than the mass-reared strain. Therefore, the anticipated loss of productivity in outcrossed B. tryoni mass-reared strains is avoidable by choosing suitable parental strains and the outcrossed flies should have improved field performance. Using already domesticated strains should also facilitate the annual renewal of hybrid strains.

Selfing has been considered the most common mode of reproduction in Echinococcus flatworms. However, population genetic studies on the asexual larval stage involving nuclear co-dominant markers have not always revealed significant heterozygote deficiencies – the expected outcome of a regularly and highly inbred population. In this study, we analysed the genetic structure of Echinococcus granulosus sensu lato populations from Southern Brazil during their adult (sexual) stage using 1 mitochondrial and 1 nuclear marker (cox 1 and mdh, respectively). We show that parasite genetic differentiation is largest among definitive hosts (domestic dogs) from different farms, suggesting that transmission is mostly maintained within a farm. Moreover, we show that heterozygote deficiencies are not significant, and we suggest that outbreeding is the most common mode of reproduction of the parasite in that region.

Field experiments were conducted in Chile and western Canada to measure short-distance (0 to 100 m) outcrossing from transgenic safflower (Carthamus tinctorius L.) intended for plant molecular farming to non-transgenic commodity safflower of the same variety. The transgenic safflower used as the pollen source was transformed with a construct for seed-specific expression of a high-value protein and constitutive expression of a gene conferring resistance to the broad-spectrum herbicide glufosinate. Progeny of non-transgenic plants grown in plots adjacent to the transgenic pollen source were screened for glufosinate resistance to measure outcrossing frequency. Outcrossing frequency differed among locations: values closest to the transgenic pollen source (0 to 3 m) ranged from 0.48 to 1.67% and rapidly declined to between 0.0024 to 0.03% at distances of 50 to 100 m. At each location, outcrossing frequency was spatially heterogeneous, indicating insects or wind moved pollen asymmetrically. A power analysis assuming a binomial distribution and a range of alpha values (type 1 error) was conducted to estimate an upper and lower confidence interval for the probable transgenic seed frequency in each sample. This facilitated interpretation when large numbers of seeds were screened from the outcrossing experiments and no transgenic seeds were found. This study should aid regulators and the plant molecular farming industry in developing confinement strategies to mitigate pollen mediated gene flow from transgenic to non-transgenic safflower.

Outcrossing of non-Bt cotton (Gossypium hirsutum (L.)) in refuges by transgenic Bt cultivars could reduce the efficacy of refuges for delaying resistance in seed-feeding pests. Based on reports that outcrossing decreased as distance from Bt cotton increased in small-scale studies, we hypothesized that increasing refuge width or distance from Bt fields would reduce outcrossing. In a large-scale study in Arizona, we quantified Bt seed in refuges of experimental and commercial fields, comparing outcrossing between in-field (narrow) and external (wide) refuges and among rows of refuges at various distances from Bt fields. Some refuges, including those in tightly controlled experimental plots, contained up to 8% adventitious Bt plants. Some, but not all, Bt plants likely resulted from Bt seed in the non-Bt seed bags. We did not detect a difference in outcrossing between in-field and external refuges. However, statistical power was low because outcrossing was low (< 0.4% of seeds) in both treatments. Higher outcrossing levels (≤ 4.6% of seeds) were observed in the studies measuring outcrossing at various distances from Bt fields, yet outcrossing did not decrease as the distance from Bt fields increased. We hypothesize that Bt plants in refuges cross-pollinated surrounding non-Bt plants, overshadowing the expected association between distance from Bt fields and outcrossing.

Development of transgenic triticale as a platform for novel bio-industrial products is predicated on an environmental biosafety assessment that quantifies the potential risks associated with its release. Pollen-mediated gene flow to related species and conventional triticale varieties is one pathway for transgene movement. A tier 1 quantification of triticale hybridization was conducted by emasculating and hand pollinating flowers under greenhouse conditions. Approximately 2000 manual pollinations were conducted for each cross and its reciprocal between two triticale genotypes: a modern triticale cultivar (AC Alta) and primary triticale (89TT108), and common wheat, durum wheat and rye. The frequency of outcrossing, hybrid seed appearance and weight, and F1 emergence and fertility were recorded. Outcrossing, F1 emergence and fertility rates were high from crosses between triticale genotypes. Outcrossing in inter-specific crosses was influenced by the species, and the genotype and gender of the triticale parent. In crosses to common and durum wheat where triticale was the male parent, outcrossing was ≥73.0% and ≥69.5%, respectively, but ≤23.9% and ≤3.0% when triticale was the female parent. Overall, outcrossing with rye was lower than with common and durum wheat. F1 hybrid emergence was greater when triticale was the female parent. With the exception of a single seed, all wheat-triticale F1 hybrid seeds were non-viable when triticale was the male parent in the cross. Only seven durum wheat-triticale F1 hybrids emerged from 163 seeds sown, and all were produced with triticale 89TT108 as female parent. With rye, 8 F1 hybrids emerged from 38 seeds sown, and all were produced from crosses to AC Alta; five with AC Alta as the female parent and three as the male. Interspecific F1 hybrids were self-sterile, with the exception of those produced in crosses between common wheat and triticale where triticale was the female parent. Tier 2 hybridization quantification will be conducted under field conditions.