Gene flow from genetically modified rice to its wild relatives: Assessing potential ecological consequences

Pollen-mediated gene flow is the major pathway for transgene escape from GM rice to its wild relatives. Transgene escape to wild Oryza species having AA-genome will occur if GM rice is released to environments with these wild Oryza species. Transgenes may persist to and spread in wild populations after gene flow, resulting unwanted ecological consequences. For assessing the potential consequences caused by transgene escape, it is important to understand the actual gene flow frequencies from GM rice to wild relatives, transgene expression and inheritance in the wild relatives, as well as fitness changes that brought to wild relatives by the transgenes. This article reviews studies on transgene escape from rice to its wild relatives via gene flow and its ecological consequences. A framework for assessing potential ecological consequences caused by transgene escape from GM rice to its wild relatives is discussed based on studies of gene flow and fitness changes.     

An attempt to elucidate the origin of cultivated soybean via comparison of nucleotide sequences encoding glycinin B4 polypeptide of cultivated soybean,Glycine max,and its presumed wild progenitor,Glycine soja

Summary Nucleotide sequences of cDNAs encoding soybean glycinin B₄ polypeptide were compared for three soybean cultivars and two introductions of wild soybean, G. soja. For three G. max cultivars, only two nucleotide substitutions were found, while G. max and G. soja nucleotide sequences had four substitutions. These data give added proof that G. max originated from G. soja. On the other hand, the time required for the accumulation of four nucleotide substitutions (calculated from the parameters of 11S globulin molecular evolution) appeared to be longer than the duration of the soybean domestication period.     

QTL affecting fitness of hybrids between wild and cultivated soybeans in experimental fields

The objective of this study was to identify quantitative trait loci (QTL) affecting fitness of hybrids between wild soybean (Glycine soja) and cultivated soybean (Glycine max). Seed dormancy and seed number, both of which are important for fitness, were evaluated by testing artificial hybrids of G. soja × G. max in a multiple‐site field trial. Generally, the fitness of the F₁ hybrids and hybrid derivatives from self‐pollination was lower than that of G. soja due to loss of seed dormancy, whereas the fitness of hybrid derivatives with higher proportions of G. soja genetic background was comparable with that of G. soja. These differences were genetically dissected into QTL for each population. Three QTLs for seed dormancy and one QTL for total seed number were detected in the F₂ progenies of two diverse cross combinations. At those four QTLs, the G. max alleles reduced seed number and severely reduced seed survival during the winter, suggesting that major genes acquired during soybean adaptation to cultivation have a selective disadvantage in natural habitats. In progenies with a higher proportion of G. soja genetic background, the genetic effects of the G. max alleles were not expressed as phenotypes because the G. soja alleles were dominant over the G. max alleles. Considering the highly inbreeding nature of these species, most hybrid derivatives would disappear quickly in early self‐pollinating generations in natural habitats because of the low fitness of plants carrying G. max alleles.     

Normal expression of insect-resistant transgene in progeny of common wild rice crossed with genetically modified rice: its implication in ecological biosafety assessment

Transgene outflow from genetically modified (GM) rice to its wild relatives may cause undesirable ecological consequences. Understanding the level of transgene expression in wild rice following gene flow is important for assessing such consequences, providing that transgene escape from GM rice cannot be prevented. To determine the expression of a transgene in common wild rice (Oryza rufipogon), we analyzed the content of Cry1Ac protein in three GM rice lines containing a Bt transgene, their F₁ hybrids with common wild rice and F₂ progeny at different growth stages, using the sandwich enzyme-linked immunosorbent assay. The average content of Cry1Ac protein in leaf samples of the wild rice lines ranged between 0.016 and 0.069% during the entire growth period, whereas that in stems varied between 0.12 and 0.39%. A great variation in Cry1Ac protein content was detected among individuals of F₁ hybrids and F₂ progeny, with some wild individuals showing higher level of Bt toxin than the cultivated GM rice. The results suggest that the Bt transgene can express normally in the interspecific hybrids between insect-resistant GM rice and common wild rice, and may have similar effects on the target insects as in GM rice.     

Construction and comparison of three reference‐quality genome assemblies for soybean

We report reference‐quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single‐nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan‐gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40–42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.     

Loci underlying resistance to Race 3 of soybean cyst nematode in Glycine soja plant introduction 468916

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is an important soybean [Glycine max (L.) Merr.] pest in the U.S. and throughout the world. Genetic resistance is the primary method for controlling SCN and there is a need to identify new resistance genes. Glycine soja Sieb. and Zucc. is the wild ancestor of domesticated soybean and is a potential source of new SCN resistance genes. The goal of this research was to map quantitative trait loci (QTLs) that provide resistance to SCN Race 3 from the G. soja plant introduction (PI) 468916. Fifty seven F₂-derived lines from a cross between the G. soja PI 468916 and the G. max experimental line A81-356022 were tested for resistance to an SCN population with a Race-3 phenotype. These lines were also genotyped with 1,004 genetic markers and resistance genes were mapped by composite interval mapping with the computer program QTL-Cartographer. In the F₂ population, three significant (LOD > 3.0) QTLs were detected that explained from 5% to 27% of the variation for Race-3 resistance. The two most significant QTLs identified in the F₂ population were tested in a population of 100 BC1F₂ plants developed by crossing A81-356022 to a line from the F₂ population that carried the two resistance QTLs from G. soja. In the backcross population, both Race-3 resistance QTLs were significant, which confirms the existence of these QTLs. The QTLs identified in this experiment map to positions where SCN resistance genes have not been previously identified, suggesting that these are novel genes that could be useful for diversifying the resistance genes currently used in cultivar development. Received: 7 August 2000 / Accepted: 4 December 2000     

A genome-wide analysis of differentiation between wild and domesticated Phaseolus vulgaris from Mesoamerica

Lack of introgression or divergent selection may be responsible for the maintenance of phenotypic differences between sympatric populations of crops and their wild progenitors. To distinguish between these hypotheses, amplified fragment length polymorphism markers were located on a molecular linkage map of Phaseolus vulgaris relative to genes for the domestication syndrome and other traits. Diversity for these same markers was then analyzed in two samples of wild and domesticated populations from Mesoamerica. Differentiation between wild and domesticated populations was significantly higher in parapatric and allopatric populations compared to sympatric populations. It was also significantly higher near genes for domestication compared to those away from these genes. Concurrently, the differences in genetic diversity between wild and domesticated populations were strongest around such genes. These data suggest that selection in the presence of introgression appears to be a major evolutionary factor maintaining the identity of wild and domesticated populations in sympatric situations. Furthermore, alleles from domesticated populations appear to have displaced alleles in sympatric wild populations, thus leading to a reduction in genetic diversity in such populations. These results also provide a possible experimental framework for assessing the long-term risk of transgene escape and the targeting of transgenes inside the genome to minimize the survival of these transgenes into wild populations following introduction by gene flow.This article is dedicated to the memory of Epimaki M. K. Koinange.     

Uncovering signatures of selection in the soybean genome using SSR diversity near QTLs of agronomic importance

The cultivated soybean [Glycine max (L.) Merr.] is widely considered to descend from the wild soybean (G. soja Sieb. & Zucc.). This study was designed to evaluate the genetic variability and differentiation between G. soja and G. max, and to detect signatures of the selection that may have occurred during the domestication process from G. soja to G. max. A total of 192 G. soja accessions and 104 G. max accessions were genotyped using eight selected simple sequence repeat (SSR) markers assigned to three SSR groups. Four SSRs in group A were not located near any known QTL. Three SSRs in group B were associated with seed protein content, and an SSR in group C was associated with resistance to Sclerotinia stem rot. The number of alleles per locus and the level of genetic variability in G. soja were higher than those in G. max. A total of 122 out of 125 alleles were present in G. soja, but only 59 alleles were detected in G. max. The average gene diversity was 0.74 in G. soja and 0.64 in G. max. Four SSRs near QTLs of agronomic importance showed strong genetic differentiation and shift change in high frequency alleles in groups B and C between G. soja and G. max, revealing selection signatures that may reflect the domestication events and recent selective breeding. With reduced diversity in G. max, some undomesticated genes from G. soja should be prime candidates for introgression to increase the pool of diversity in G. max.     

Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T₁ progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.     

A Built-In Strategy to Mitigate Transgene Spreading from Genetically Modified Corn

Transgene spreading is a major concern in cultivating genetically modified (GM) corn. Cross-pollination may cause the spread of transgenes from GM cornfields to conventional fields. Occasionally, seed lot contamination, volunteers, mixing during sowing, harvest, and trade can also lead to transgene escape. Obviously, new biological confinement technologies are highly desired to mitigate transgene spreading in addition to physical separation and isolation methods. In this study, we report the development of a built-in containment method to mitigate transgene spreading in corn. In this method, an RNAi cassette for suppressing the expression of the nicosulfuron detoxifying enzyme CYP81A9 and an expression cassette for the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene G10 were constructed and transformed into corn via Agrobacterium-mediated transformation. The GM corn plants that were generated were found to be sensitive to nicosulfuron but resistant to glyphosate, which is exactly the opposite of conventional corn. Field tests demonstrated that GM corn plants with silenced CYP81A9 could be killed by applying nicosulfuron at 40 g/ha, which is the recommended dose for weed control in cornfields. This study suggests that this built-in containment method for controlling the spread of corn transgenes is effective and easy to implement.     

Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats

Concerns about genetically modified (GM) crops include transgene flow to compatible wild species and unintended ecological consequences of potential transgene introgression. However, there has been little empirical documentation of establishment and distribution of transgenic plants in wild populations. We present herein the first evidence for escape of transgenes into wild plant populations within the USA; glyphosate-resistant creeping bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS transgenes were found outside of cultivation area in central Oregon. Resident populations of three compatible Agrostis species were sampled in nonagronomic habitats outside the Oregon Department of Agriculture control area designated for test production of glyphosate-resistant creeping bentgrass. CP4 EPSPS protein and the corresponding transgene were found in nine A. stolonifera plants screened from 20,400 samples (0.04 +/- 0.01% SE). CP4 EPSPS-positive plants were located predominantly in mesic habitats downwind and up to 3.8 km beyond the control area perimeter; two plants were found within the USDA Crooked River National Grassland. Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggest that establishment resulted from both pollen-mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short-term production can result in establishment of transgenic plants at multi-kilometre distances from GM source fields or plants. Selective pressure from direct application or drift of glyphosate herbicide could enhance introgression of CP4 EPSPS transgenes and additional establishment. Obligatory outcrossing and vegetative spread could further contribute to persistence of CP4 EPSPS transgenes in wild Agrostis populations, both in the presence or absence of herbicide selection.     

Single nucleotide mutation leading to an amino acid substitution in the variant <Emphasis Type="Italic">Tik</Emphasis> soybean Kunitz trypsin inhibitor (SKTI) identified in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &; Zucc.)

The wild soybean (Glycine soja), which is the progenitor of cultivated soybean (Glycine max), is expected to offer more information about genetic variability and more useful mutants for evolutionary research and breeding applications. Here, a total of 1,600 wild soybean samples from China were investigated for genetic variation with regard to the soybean Kunitz trypsin inhibitor (SKTI). A new mutant SKTI, Tik, was identified. It was found to be a Tia-derived codominant allele caused by a transversion point mutation from C to G at nucleotide +171, leading to an alteration of one codon (AAC → AAG) and a corresponding amino acid substitution (Asn → Lys) at the ninth residue. Upon examination of this variant and others previously found in wild soybeans, it became clear that SKTI has undergone high-level evolutionary differentiation. There were more abundant polymorphisms in the wild than in the cultivated soybean.     

Genome-wide signatures of the geographic expansion and breeding of soybean

Soybean is a leguminous crop that provides oil and protein. Exploring the genomic signatures of soybean evolution is crucial for breeding varieties with improved adaptability to environmental extremes. We analyzed the genome sequences of 2,214 soybeans and proposed a soybean evolutionary route, i.e., the expansion of annual wild soybean (Glycine soja Sieb. & Zucc.) from southern China and its domestication in central China, followed by the expansion and local breeding selection of its landraces (G. max (L.) Merr.). We observed that the genetic introgression in soybean landraces was mostly derived from sympatric rather than allopatric wild populations during the geographic expansion. Soybean expansion and breeding were accompanied by the positive selection of flowering time genes, including GmSPA3c. Our study sheds light on the evolutionary history of soybean and provides valuable genetic resources for its future breeding.

     

Induction of cyclic electron flow around photosystem 1 and state transition are correlated with salt tolerance in soybean

We investigated the role of cyclic electron flow around photosystem 1 (CEF1) and state transition (ST) in two soybean cultivars that differed in salt tolerance. The CEF1 and maximum photochemical efficiency (F_v/F_m) were determined under control and NaCl (50 mM) stress and the NaCl-induced light-harvesting complex 2 (LHC2) phosphorylation in vitro was analysed in light and dark. NaCl induced the increase of CEF1 more greatly in wild soybean Glycine cyrtoloba (cv. ACC547) than in cultivated soybean Glycine max (cv. Melrose). The F_v/F_m was reduced less in G. cyrtoloba than in G. max after 10-d NaCl stress. In G. cyrtoloba, the increase of CEF1 was associated with enhancement of LHC2 phosphorylation in thylakoid membrane under both dark and light. However, in G. max the NaCl treatment decreased the LHC2 phosphorylation. Treatment with photosynthetic electron flow inhibitors (DCMU, DBMIB) inhibited LHC2 phosphorylation more in G. max than in G. cyrtoloba. Thus the NaCl-induced up-regulation in CEF1 and ST might contribute to salt resistance of G. cyrtoloba.     

Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F₂ segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.     

Molecular mapping of soybean aphid resistance genes in PI 567541B

The soybean aphid (Aphis glycines Matsumura) is an important pest of soybean [Glycine max (L.) Merr.] in North America since it was first reported in 2000. PI 567541B is a newly discovered aphid resistance germplasm with early maturity characteristics. The objectives of this study were to map and validate the aphid resistance genes in PI 567541B using molecular markers. A mapping population of 228 F₃ derived lines was investigated for the aphid resistance in both field and greenhouse trials. Two quantitative trait loci (QTLs) controlling the aphid resistance were found using the composite interval mapping method. These two QTLs were localized on linkage groups (LGs) F and M. PI 567541B conferred resistant alleles at both loci. An additive × additive interaction between these two QTLs was identified using the multiple interval mapping method. These two QTLs combined with their interaction explained most of the phenotypic variation in both field and greenhouse trials. In general, the QTL on LG F had less effect than the one on LG M, especially in the greenhouse trial. These two QTLs were further validated using an independent population. The effects of these two QTLs were also confirmed using 50 advanced breeding lines, which were all derived from PI 567541B and had various genetic backgrounds. Hence, these two QTLs identified and validated in this study could be useful in improving soybean aphid resistance by marker-assisted selection.     

A single origin and moderate bottleneck during domestication of soybean (Glycine max): implications from microsatellites and nucleotide sequences

Background and Aims

It is essential to illuminate the evolutionary history of crop domestication in order to understand further the origin and development of modern cultivation and agronomy; however, despite being one of the most important crops, the domestication origin and bottleneck of soybean (Glycine max) are poorly understood. In the present study, microsatellites and nucleotide sequences were employed to elucidate the domestication genetics of soybean.

Methods

The genomes of 79 landrace soybeans (endemic cultivated soybeans) and 231 wild soybeans (G. soja) that represented the species-wide distribution of wild soybean in East Asia were scanned with 56 microsatellites to identify the genetic structure and domestication origin of soybean. To understand better the domestication bottleneck, four nucleotide sequences were selected to simulate the domestication bottleneck.

Key Results

Model-based analysis revealed that most of the landrace genotypes were assigned to the inferred wild soybean cluster of south China, South Korea and Japan. Phylogeny for wild and landrace soybeans showed that all landrace soybeans formed a single cluster supporting a monophyletic origin of all the cultivars. The populations of the nearest branches which were basal to the cultivar lineage were wild soybeans from south China. The coalescent simulation detected a bottleneck severity of K′ = 2 during soybean domestication, which could be explained by a foundation population of 6000 individuals if domestication duration lasted 3000 years.

Conclusions

As a result of integrating geographic distribution with microsatellite genotype assignment and phylogeny between landrace and wild soybeans, a single origin of soybean in south China is proposed. The coalescent simulation revealed a moderate genetic bottleneck with an effective wild soybean population used for domestication estimated to be ≈2 % of the total number of ancestral wild soybeans. Wild soybeans in Asia, especially in south China contain tremendous genetic resources for cultivar improvement.     

Risk assessment of Bacillus thuringiensis in wild Brassica rapa: A field simulation of introgressed transgenes

Abstract

Brassica crops are able to hybridize with closely related wild and weedy species such as Brassica rapa and therefore these species could become recipients of transgenes from GM brassica crops, such as oilseed rape. Transgenes which protect against herbivory, such as a gene conferring the production of a Bt toxin, could increase the recipient's fitness and therefore enhance its competitiveness. We used microbial Bt and several other pesticides to exclude several guilds of herbivores and thus simulate the effect of transgene introgression on the performance of wild B. rapa. There were only minor negative effects of excluding insect herbivores on the performance of B. rapa and it appears that vertebrate herbivory has a more significant effect. The advantages and disadvantages of simulating the risks associated with GM plants are discussed. If we apply this simulation to GM risk assessment and make inferences from these findings, the implications of this for gene flow from insect-resistant transgenic plants are that it will be of little significance should it occur.