Crop-Weed Introgression Plays Critical Roles in Genetic Differentiation and Diversity of Weedy Rice: A Case Study of Human-Influenced Weed Evolution.

As an important driving force, introgression plays an essential role in shaping the evolution of plant species. However, knowledge concerning how introgression affects plant evolution in agroecosystems with strong human influences is still limited. To generate such knowledge, we used InDel (insertion/deletion) molecular fingerprints to determine the level of introgression from japonica rice cultivars into the indica type of weedy rice. We also analyzed the impact of crop-to-weed introgression on the genetic differentiation and diversity of weedy rice, using InDel (insertion/deletion) and SSR (simple sequence repeat) molecular fingerprints. Results based on the STRUCTURE analysis indicated an evident admixture of some weedy rice samples with indica and japonica components, suggesting different levels of introgression from japonica rice cultivars to the indica type of weedy rice. The principal coordinate analyses indicated indica-japonica genetic differentiation among weedy rice samples, which was positively correlated with the introgression of japonica-specific alleles from the rice cultivars. In addition, increased crop-to-weed introgression formed a parabola pattern of dynamic genetic diversity in weedy rice. Our findings based on this case study provide evidence that human activities, such as the frequent change in crop varieties, can strongly influence weed evolution by altering genetic differentiation and genetic diversity through crop-weed introgression in agroecosystems.

Reduced Carbon Dioxide by Overexpressing EPSPS Transgene in Arabidopsis and Rice: Implications in Carbon Neutrality through Genetically Engineered Plants.

With the increasing challenges of climate change caused by global warming, the effective reduction of carbon dioxide (CO2) becomes an urgent environmental issue for the sustainable development of human society. Previous reports indicated increased biomass in genetically engineered (GE) Arabidopsis and rice overexpressing the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, suggesting the possibility of consuming more carbon by GE plants. However, whether overexpressing the EPSPS gene in GE plants consumes more CO2 remains a question. To address this question, we measured expression of the EPSPS gene, intercellular CO2 concentration, photosynthetic ratios, and gene expression (RNA-seq and RT-qPCR) in GE (overexpression) and non-GE (normal expression) Arabidopsis and rice plants. Results showed substantially increased EPSPS expression accompanied with CO2 consumption in the GE Arabidopsis and rice plants. Furthermore, overexpressing the EPSPS gene affected carbon-fixation related biological pathways. We also confirmed significant upregulation of four key carbon-fixation associated genes, in addition to increased photosynthetic ratios, in all GE plants. Our finding of significantly enhanced carbon fixation in GE plants overexpressing the EPSPS transgene provides a novel strategy to reduce global CO2 for carbon neutrality by genetic engineering of plant species, in addition to increased plant production by enhanced photosynthesis.

Reducing Seed Shattering in Weedy Rice by Editing SH4 and qSH1 Genes: Implications in Environmental Biosafety and Weed Control through Transgene Mitigation.

Mitigating the function of acquired transgenes in crop wild/weedy relatives can provide an ideal strategy to reduce the possible undesired environmental impacts of pollen-mediated transgene flow from genetically engineered (GE) crops. To explore a transgene mitigation system in rice, we edited the seed-shattering genes, SH4 and qSH1, using a weedy rice line ("C9") that originally had strong seed shattering. We also analyzed seed size-related traits, the total genomic transcriptomic data, and RT-qPCR expression of the SH4 or qSH1 gene-edited and SH4/qSH1 gene-edited weedy rice lines. Substantially reduced seed shattering was observed in all gene-edited weedy rice lines. The single gene-edited weedy rice lines, either the SH4 or qSH1 gene, did not show a consistent reduction in their seed size-related traits. In addition, reduced seed shattering was closely linked with the weakness and absence of abscission layers and reduced abscisic acid (ABA). Additionally, the genes closely associated with ABA biosynthesis and signaling transduction, as well as cell-wall hydrolysis, were downregulated in all gene-edited weedy rice lines. These findings facilitate our deep insights into the underlying mechanisms of reduced seed shattering in plants in the rice genus Oryza. In addition, such a mitigating technology also has practical applications for reducing the potential adverse environmental impacts caused by transgene flow and for managing the infestation of weedy rice by acquiring the mitigator from GE rice cultivars through natural gene flow.

Sympatric genetic divergence between early- and late-season weedy rice populations.

Sympatric genetic divergence is the most appealing and controversial pattern in the theory of ecological speciation. Examples that support sympatric genetic divergence in plant species are extremely rare. Solid evidence of sympatric genetic divergence will provide deep insights for revealing the underlying mechanisms of ecological speciation. We analysed the total genomic DNA sequences of 120 weedy rice (WR; Oryza sativa f. spontanea) plants, representing three WR population pairs separately from three early- and late-season rice fields, in comparison with those of the co-occurring rice cultivars and other rice materials. We detected substantial genetic divergence within the pairs of the sympatric early- and late-season WR populations, although genetic divergence was unevenly distributed across the genomes. Restricted gene flow was determined between the sympatric WR populations, resulting in their distinct genetic structures. We also detected relatively low genetic diversity that was likely to be associated with stronger selection in early-season WR populations. Our findings provide strong evidence for sympatric genetic divergence between the WR populations in the same fields but in different seasons. We conclude that temporal isolation plays an important role in creating genetic divergence between sympatric populations/species in plants.

Key Roles of De-Domestication and Novel Mutation in Origin and Diversification of Global Weedy Rice.

Agricultural weeds pose great challenges to sustainable crop production, owing to their complex origins and abundant genetic diversity. Weedy rice (WD) infests rice fields worldwide causing tremendous losses of rice yield/quality. To explore WD origins and evolution, we analyzed DNA sequence polymorphisms of the seed shattering genes (sh4 and qsh1) in weedy, wild, and cultivated rice from a worldwide distribution. We also used microsatellite and insertion/deletion molecular fingerprinting to determine their genetic relationship and structure. Results indicate multiple origins of WD with most samples having evolved from their cultivated progenitors and a few samples from wild rice. WD that evolved from de-domestication showed distinct genetic structures associated with indica and japonica rice differentiation. In addition, the weed-unique haplotypes that were only identified in the WD samples suggest their novel mutations. Findings in this study demonstrate the key role of de-domestication in WD origins, in which indica and japonica cultivars stimulated further evolution and divergence of WD in various agroecosystems. Furthermore, novel mutations promote continued evolution and genetic diversity of WD adapting to different environments. Knowledge generated from this study provides deep insights into the origin and evolution of conspecific weeds, in addition to the design of effective measures to control these weeds.

Increased Longevity and Dormancy of Soil-Buried Seeds from Advanced Crop-Wild Rice Hybrids Overexpressing the EPSPS Transgene.

Estimating the fitness effect conferred by a transgene introgressed into populations of wild relative species from a genetically engineered (GE) crop plays an important role in assessing the potential environmental risks caused by transgene flow. Such estimation has essentially focused on the survival and fecundity-related characteristics measured above the ground, but with little attention to the fate of GE seeds shattered in the soil seed banks after maturation. To explore the survival and longevity of GE seeds in soil, we examined the germination behaviors of crop-wild hybrid seeds (F4-F6) from the lineages of a GE herbicide-tolerant rice (Oryzasativa) line that contains an endogenous EPSPS transgene hybridized with two wild O. rufipogon populations after the seeds were buried in soil. The results showed significantly increased germination of the GE crop-wild hybrid seeds after soil burial, compared with that of the non-GE hybrid seeds. Additionally, the proportion of dormant seeds and the content of the growth hormone auxin (indole-3-acetic acid, IAA) in the GE crop-wild hybrid seeds significantly increased. Evidently, the EPSPS transgene enhances the survival and longevity of GE crop-wild rice seeds in the soil seed banks. The enhanced survival and longevity of the GE hybrid seeds is likely associated with the increases in seed dormancy and auxin (IAA) by overexpressing the rice endogenous EPSPS transgene. Thus, the fate of GE seeds in the soil seed banks should be earnestly considered when assessing the environmental risks caused by transgene flow.

Increases in Genetic Diversity of Weedy Rice Associated with Ambient Temperatures and Limited Gene Flow.

Hypotheses regarding the association of increased species or genetic diversity with gradually warmer regions as a global pattern have been proposed, but no direct and solid experimental data are available to approve the association between plant genetic diversity and ambient temperatures. To test the diversity-temperature hypothesis, we studied genetic diversity and genetic differentiation of weedy rice (Oryza sativa f. spontanea) populations occurring naturally in early- and late-season rice fields that share nearly the same ecological conditions but with slightly different temperatures. Data collected from 10-year historical climatic records indicated a ~2 ℃ higher average air temperature in the late rice-cultivation seasons than in the early seasons. Results based on molecular fingerprints of 27 SSR (simple sequence repeat) loci showed a higher level of genetic diversity in the late-season weedy rice populations than in the early-season populations. In addition, a positive correlation was detected between the increased proportion of genetic diversity (ΔHe ) and genetic differentiation among the weedy rice populations, suggesting limited gene flow. Therefore, we conclude from this study that increased genetic diversity in the late-season weedy rice populations is probably caused by the higher ambient temperatures. This finding provides evidence for the possible association between genetic diversity and ambient temperatures.

Overexpressing Exogenous 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Genes Increases Fecundity and Auxin Content of Transgenic Arabidopsis Plants.

Transgenic glyphosate-tolerant plants overproducing EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) may exhibit enhanced fitness in glyphosate-free environments. If so, introgression of transgenes overexpressing EPSPS into wild relative species may lead to increased competitiveness of crop-wild hybrids, resulting in unpredicted environmental impact. Assessing fitness effects of transgenes overexpressing EPSPS in a model plant species can help address this question, while elucidating how overproducing EPSPS affects the fitness-related traits of plants. We produced segregating T2 and T3Arabidopsis thaliana lineages with or without a transgene overexpressing EPSPS isolated from rice or Agrobacterium (CP4). For each of the three transgenes, we compared glyphosate tolerance, some fitness-related traits, and auxin (indole-3-acetic acid) content in transgene-present, transgene-absent, empty vector (EV), and parental lineages in a common-garden experiment. We detected substantially increased glyphosate tolerance in T2 plants of transgene-present lineages that overproduced EPSPS. We also documented significant increases in fecundity, which was associated with increased auxin content in T3 transgene-present lineages containing rice EPSPS genes, compared with their segregating transgene-absent lineages, EV, and parental controls. Our results from Arabidopsis with nine transgenic events provide a strong support to the hypothesis that transgenic plants overproducing EPSPS can benefit from a fecundity advantage in glyphosate-free environments. Stimulated biosynthesis of auxin, an important plant growth hormone, by overproducing EPSPS may play a role in enhanced fecundity of the transgenic Arabidopsis plants. The obtained knowledge is useful for assessing environmental impact caused by introgression of transgenes overproducing EPSPS from any GE crop into populations of its wild relatives.

Introgression from cultivated rice alters genetic structures of wild relative populations: implications for in situ conservation.

Maintaining genetic integrity is essential for in situ and ex situ conservation of crop wild relative (CWR) species. However, introgression of crop alleles into CWR species/populations may change their genetic structure and diversity, resulting in more invasive weeds or, in contrast, the extinction of endangered populations. To determine crop-wild introgression and its consequences, we examined the genetic structure and diversity of six wild rice (Oryza rufipogon) populations under in situ conservation in China. Thirty-four simple sequence repeat (SSR) and 34 insertion/deletion markers were used to genotype the wild rice populations and two sets of rice cultivars (O. sativa), corresponding to the two types of molecular markers. Shared alleles and STRUCTURE analyses suggested a variable level of crop-wild introgression and admixture. Principal coordinates and cluster analyses indicated differentiation of wild rice populations, which was associated with the spatial distances to cultivated rice fields. The level of overall genetic diversity was comparable between wild rice populations and rice cultivars, but a great number of wild-specific alleles was detected in the wild populations. We conclude based on the results that crop-wild introgression can considerably alter the pattern of genetic structure and relationships of CWR populations. Appropriate measures should be taken for effective in situ conservation of CWR species under the scenario of crop-wild introgression.

Non-random transmission of parental alleles into crop-wild and crop-weed hybrid lineages separated by a transgene and neutral identifiers in rice.

It is essential to assess environmental impact of transgene flow from genetically engineered crops to their wild or weedy relatives before commercialization. Measuring comparative trials of fitness in the transgene-flow-resulted hybrids plays the key role in the assessment, where the segregated isogenic hybrid lineages/subpopulations with or without a transgene of the same genomic background are involved. Here, we report substantial genomic differentiation between transgene-present and -absent lineages (F2-F3) divided by a glyphosate-resistance transgene from a crop-wild/weed hybrid population in rice. We further confirmed that such differentiation is attributed to increased frequencies of crop-parent alleles in transgenic hybrid lineages at multiple loci across the genome, as estimated by SSR (simple sequence repeat) markers. Such preferential transmission of parental alleles was also found in equally divided crop-wild/weed hybrid lineages with or without a particular neutral SSR identifier. We conclude that selecting either a transgene or neutral marker as an identifier to create hybrid lineages will result in different genomic background of the lineages due to non-random transmission of parental alleles. Non-random allele transmission may misrepresent the outcomes of fitness effects. We therefore propose seeking other means to evaluate fitness effects of transgenes for assessing environmental impact caused by crop-to-wild/weed gene flow.

Genetically engineered rice endogenous 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) transgene alters phenology and fitness of crop-wild hybrid offspring.

Genetically engineered (GE) rice endogenous epsps (5-enolpyruvoylshikimate-3-phosphate synthase) gene overexpressing EPSPS can increase glyphosate herbicide-resistance of cultivated rice. This type of epsps transgene can enhance the fecundity of rice crop-weed hybrid offspring in the absence of glyphosate, stimulating great concerns over undesired environmental impacts of transgene flow to populations of wild relatives. Here, we report the substantial alteration of phenology and fitness traits in F1-F3 crop-wild hybrid descendants derived from crosses between an epsps GE rice line and two endangered wild rice (Oryza rufipogon) populations, based on the common-garden field experiments. Under the glyphosate-free condition, transgenic hybrid lineages showed significantly earlier tillering and flowering, as well as increased fecundity and overwintering survival/regeneration abilities. In addition, a negative correlation was observed between the contents of endogenous EPSPS of wild, weedy, and cultivated rice parents and fitness differences caused by the incorporation of the epsps transgene. Namely, a lower level of endogenous EPSPS in the transgene-recipient populations displayed a more pronounced enhancement in fitness. The altered phenology and enhanced fitness of crop-wild hybrid offspring by the epsps transgene may cause unwanted environmental consequences when this type of glyphosate-resistance transgene introgressed into wild rice populations through gene flow.

Reduced weed seed shattering by silencing a cultivated rice gene: strategic mitigation for escaped transgenes.

Transgene flow form a genetically engineered (GE) crop to its wild relatives may result in unwanted environmental consequences. Mitigating transgenes via introducing a gene that is disadvantageous to wild relatives but beneficial to crops, and is tightly-linked with the target transgenes, may provide a promising solution to limit the spread of transgenes in wild/weedy populations. Here we demonstrate a novel system with significantly reduced seed shattering in crop-weed hybrid descendants by partially silenced expression of the seed-shattering gene SH4 in cultivated rice, using artificial microRNA and antisense RNA techniques. Accordingly, fewer seeds were found in the soil of the field plots where transgenic hybrid lineages were grown. However, no differences in productivity-related traits were detected between GE and non-GE cultivated rice. To silence seed-shattering genes provides a useful strategy to reduce the potential environmental impacts caused by transgene flow from commercial GE rice to weedy rice, in addition to the control of weedy rice.

Ambient insect pressure and recipient genotypes determine fecundity of transgenic crop-weed rice hybrid progeny: Implications for environmental biosafety assessment.

Transgene introgression into crop weedy/wild relatives can provide natural selective advantages, probably causing undesirable environmental impact. The advantages are likely associated with factors such as transgenes, selective pressure, and genetic background of transgene recipients. To explore the role of the environment and background of transgene recipients in affecting the advantages, we estimated the fitness of crop-weed hybrid lineages derived from crosses between marker-free insect-resistant transgenic (Bt/CpTI) rice with five weedy rice populations under varied insect pressure. Multiway anova indicated the significant effect of both transgenes and weedy rice genotypes on the performance of crop-weed hybrid lineages in the high-insect environment. Increased fecundity was detected in most transgene-present F1 and F2 hybrid lineages under high-insect pressure, but varied among crop-weed hybrid lineages with different weedy rice parents. Increased fecundity of transgenic crop-weed hybrid lineages was associated with the environmental insect pressure and genotypes of their weedy rice parents. The findings suggest that the fitness effects of an insect-resistant transgene introgressed into weedy populations are not uniform across different environments and genotypes of the recipient plants that have acquired the transgene. Therefore, these factors should be considered when assessing the environmental impact of transgene flow to weedy or wild rice relatives.

Fitness correlates of crop transgene flow into weedy populations: a case study of weedy rice in China and other examples.

Whether transgene flow from crops to cross-compatible weedy relatives will result in negative environmental consequences has been the topic of discussion for decades. An important component of environmental risk assessment depends on whether an introgressed transgene is associated with a fitness change in weedy populations. Several crop-weed pairs have received experimental attention. Perhaps, the most worrisome example is transgene flow from genetically engineered cultivated rice, a staple for billions globally, to its conspecific weed, weedy rice. China's cultivated/weedy rice system is one of the best experimentally studied systems under field conditions for assessing how the presence of transgenes alters the weed's fitness and the likely impacts of that fitness change. Here, we present the cultivated/weedy rice system as a case study on the consequences of introgressed transgenes in unmanaged populations. The experimental work on this system reveals considerable variation in fitness outcomes - increased, decreased, and none - based on the transgenic trait, its introgressed genomic background, and the environment. A review of similar research from a sample of other crop-wild pairs suggests such variation is the rule. We conclude such variation in fitness correlates supports the case-by-case method of biosafety regulation is sound.

High-Resolution Gene Flow Model for Assessing Environmental Impacts of Transgene Escape Based on Biological Parameters and Wind Speed.

Environmental impacts caused by transgene flow from genetically engineered (GE) crops to their wild relatives mediated by pollination are longstanding biosafety concerns worldwide. Mathematical modeling provides a useful tool for estimating frequencies of pollen-mediated gene flow (PMGF) that are critical for assessing such environmental impacts. However, most PMGF models are impractical for this purpose because their parameterization requires actual data from field experiments. In addition, most of these models are usually too general and ignored the important biological characteristics of concerned plant species; and therefore cannot provide accurate prediction for PMGF frequencies. It is necessary to develop more accurate PMGF models based on biological and climatic parameters that can be easily measured in situ. Here, we present a quasi-mechanistic PMGF model that only requires the input of biological and wind speed parameters without actual data from field experiments. Validation of the quasi-mechanistic model based on five sets of published data from field experiments showed significant correlations between the model-simulated and field experimental-generated PMGF frequencies. These results suggest accurate prediction for PMGF frequencies using this model, provided that the necessary biological parameters and wind speed data are available. This model can largely facilitate the assessment and management of environmental impacts caused by transgene flow, such as determining transgene flow frequencies at a particular spatial distance, and establishing spatial isolation between a GE crop and its coexisting non-GE counterparts and wild relatives.

Multiple tissue-specific expression of rice seed-shattering gene SH4 regulated by its promoter pSH4.

BACKGROUND: Rice seed shattering is an important domestication syndrome encoded by a gene named as SH4. The coding region of SH4 has been well studied regarding its function and roles in evolution. However, its promoter has not been identified, which limited our understanding of the detailed regulatory mechanisms of this gene. It is therefore critical to characterize the promoter and study its expression pattern. RESULTS: We analyzed the 5' upstream sequences of this gene and identified a ~2.6 kb fragment with typical promoter features, which was designated as pSH4. The promoter contained a number of cis-acting elements related to abscisic acid (ABA) and a CpG island that were characteristics of multiple tissue-specific expression. We isolated and ligated pSH4 to the ß-glucuronidase (GUS) reporter gene, and transformed it into a japonica rice cultivar to determine the multiple expression pattern of SH4. Histochemical location and fluorescence analyses of GUS activity of transgenic plants indicated multiple tissue-specific expression of pSH4 in the seed-pedicel junction region of mature panicles (with highest level), stems, coleoptiles of germinated seeds, and scutella of mature seeds. CONCLUSIONS: The multiple tissue-specific expression pSH4 is categorized as a spatiotemporal promoter that drives the expression of the SH4 gene in different rice tissues, in addition to the seed-pedicel junction region. Our findings suggest that SH4 may have additional functions in the growth and development of rice, apart from its major role in seed shattering.

Seed-mediated gene flow promotes genetic diversity of weedy rice within populations: implications for weed management.

Increased infestation of weedy rice-a noxious agricultural pest has caused significant reduction of grain yield of cultivated rice (Oryza sativa) worldwide. Knowledge on genetic diversity and structure of weedy rice populations will facilitate the design of effective methods to control this weed by tracing its origins and dispersal patterns in a given region. To generate such knowledge, we studied genetic diversity and structure of 21 weedy rice populations from Sri Lanka based on 23 selected microsatellite (SSR) loci. Results indicated an exceptionally high level of within-population genetic diversity (He = 0.62) and limited among-population differentiation (Fst = 0.17) for this predominantly self-pollinating weed. UPGMA analysis showed a loose genetic affinity of the weedy rice populations in relation to their geographical locations, and no obvious genetic structure among populations across the country. This phenomenon was associated with the considerable amount of gene flow between populations. Limited admixture from STRUCTURE analyses suggested a very low level of hybridization (pollen-mediated gene flow) between populations. The abundant within-population genetic diversity coupled with limited population genetic structure and differentiation is likely caused by the considerable seed-mediated gene flow of weedy rice along with the long-distance exchange of farmer-saved rice seeds between weedy-rice contaminated regions in Sri Lanka. In addition to other effective weed management strategies, promoting the application of certified rice seeds with no weedy rice contamination should be the immediate action to significantly reduce the proliferation and infestation of this weed in rice ecosystems in countries with similar rice farming styles as in Sri Lanka.