Roberto Guadagnuolo

Gene flow from wheat (Triticum aestivum L.) to jointed goatgrass (Aegilops cylindrica Host.), as revealed by RAPD and microsatellite markers

Abstract  In order to estimate the potential of gene flow between wheat (Triticum æstivum L.) and jointed goatgrass (Aegilops cylindrica Host.), we carried out mixed pollinations in experimental and natural conditions. A set of species-specific RAPD (random amplified polymorphic DNA) and microsatellite markers were used to detect the presence of parental markers in the progeny of the plants used in these experiments. No hybrids were found within the offsprings of the plants used for the greenhouse experiments, while 85 Ae. cylindrica×T. æstivum hybrids were found within 2400 analyzed F1 plants resulting from the field pollinations. The hybridization rates for individuals of different populations of the wild species differed considerably: 1% for two populations known for more than 90 years versus 7% for a newly discovered population. Most of the hybrids were completely sterile, but five of them produced 13 seeds (BC1) by backcross with Ae. cylindrica. Twelve seeds germinated and generated viable and partly fertile plants. About 25% of the wheat specific RAPD markers were found in the BC1 plants, indicating that introgression of wheat DNA into Ae. cylindrica is possible. In addition, one microsatellite marker, known to be situated on the D genome (a genome shared by both species), was also found in the BC1 plants.

Origin and expansion of the allotetraploid Aegilops geniculata, a wild relative of wheat

*This study reconstructs the phylogeography of Aegilops geniculata, an allotetraploid relative of wheat, to discuss the impact of past climate changes and recent human activities (e.g. the early expansion of agriculture) on the genetic diversity of ruderal plant species. *We combined chloroplast DNA (cpDNA) sequencing, analysed using statistical parsimony network, with nonhierarchical K-means clustering of amplified fragment length polymorphism (AFLP) genotyping, to unravel patterns of genetic structure across the native range of Ae. geniculata. The AFLP dataset was further explored by measurement of the regional genetic diversity and the detection of isolation by distance patterns. *Both cpDNA and AFLP suggest an eastern Mediterranean origin of Ae. geniculata. Two lineages have spread independently over northern and southern Mediterranean areas. Northern populations show low genetic diversity but strong phylogeographical structure among the main peninsulas, indicating a major influence of glacial cycles. By contrast, low genetic structuring and a high genetic diversity are detected in southern Mediterranean populations. Finally, we highlight human-mediated dispersal resulting in substantial introgression between resident and migrant populations. *We have shown that the evolutionary trajectories of ruderal plants can be similar to those of wild species, but are interfered by human activities, promoting range expansions through increased long-distance dispersal and the creation of suitable habitats.

Gene flow between wheat and wild relatives: empirical evidence from Aegilops geniculata, Ae. neglecta and Ae. triuncialis.

Gene flow between domesticated species and their wild relatives is receiving growing attention. This study addressed introgression between wheat and natural populations of its wild relatives (Aegilops species). The sampling included 472 individuals, collected from 32 Mediterranean populations of three widespread Aegilops species (Aegilops geniculata, Ae. neglecta and Ae. triuncialis) and compared wheat field borders to areas isolated from agriculture. Individuals were characterized with amplified fragment length polymorphism fingerprinting, analysed through two computational approaches (i.e. Bayesian estimations of admixture and fuzzy clustering), and sequences marking wheat‐specific insertions of transposable elements. With this combined approach, we detected substantial gene flow between wheat and Aegilops species. Specifically, Ae. neglecta and Ae. triuncialis showed significantly more admixed individuals close to wheat fields than in locations isolated from agriculture. In contrast, little evidence of gene flow was found in Ae. geniculata. Our results indicated that reproductive barriers have been regularly bypassed during the long history of sympatry between wheat and Aegilops.

Hybridization rates between lettuce (Lactuca sativa) and its wild relative (L. serriola) under field conditions.

Hybridization and introgression between crops and wild relatives may have important evolutionary and ecological consequences such as gene swamping or increased invasiveness. In the present study, we investigated hybridization under field conditions between crop lettuce (Lactuca sativa) and its wild relative prickly lettuce (L. serriola), two cross-compatible, predominantly autogamous and insect pollinated species. In 2003 and 2004, we estimated the rates of hybridization between L. sativa and L. serriola in close-to-reality field experiments carried out in two locations of Northern Switzerland. Seeds set by the experimental wild plants were collected and sown (44 352 in 2003 and 252 345 in 2004). Progeny was screened morphologically for detecting natural hybrids. Prior to the experiment, specific RAPD markers were used to confirm that morphological characters were reliable for hybrid identification. Hybridization occurred up to the maximal distance tested (40 m), and hybridization rates varied between 0 to 26%, decreasing with distance. More than 80% of the wild plants produced at least one hybrid (incidence of hybridization, IH) at 0 m and 1 m. It equaled 4 to 5% at 40 m. In sympatric crop-wild populations, cross-pollination between cultivated lettuce and its wild relative has to be seen as the rule rather than the exception for short distances.

Molecular Analysis, Cytogenetics and Fertility of Introgression Lines From Transgenic Wheat to Aegilops cylindrica Host

Natural hybridization and backcrossing between Aegilops cylindrica and Triticum aestivum can lead to introgression of wheat DNA into the wild species. Hybrids between Ae. cylindrica and wheat lines bearing herbicide resistance (bar), reporter (gus), fungal disease resistance (kp4), and increased insect tolerance (gna) transgenes were produced by pollination of emasculated Ae. cylindrica plants. F1 hybrids were backcrossed to Ae. cylindrica under open-pollination conditions, and first backcrosses were selfed using pollen bags. Female fertility of F1 ranged from 0.03 to 0.6%. Eighteen percent of the sown BC1s germinated and flowered. Chromosome numbers ranged from 30 to 84 and several of the plants bore wheat-specific sequence-characterized amplified regions (SCARs) and the bar gene. Self fertility in two BC1 plants was 0.16 and 5.21%, and the others were completely self-sterile. Among 19 BC1S1 individuals one plant was transgenic, had 43 chromosomes, contained the bar gene, and survived glufosinate treatments. The other BC1S1 plants had between 28 and 31 chromosomes, and several of them carried SCARs specific to wheat A and D genomes. Fertility of these plants was higher under open-pollination conditions than by selfing and did not necessarily correlate with even or euploid chromosome number. Some individuals having supernumerary wheat chromosomes recovered full fertility.

Introgression of wheat DNA markers from A, B and D genomes in early generation progeny of Aegilops cylindrica Host × Triticum aestivum L. hybrids

Introgression from allohexaploid wheat (Triticum aestivum L., AABBDD) to allotetraploid jointed goatgrass (Aegilops cylindrica Host, CCDD) can take place in areas where the two species grow in sympatry and hybridize. Wheat and Ae. cylindrica share the D genome, issued from the common diploid ancestor Aegilops tauschii Coss. It has been proposed that the A and B genome of bread wheat are secure places to insert transgenes to avoid their introgression into Ae. cylindrica because during meiosis in pentaploid hybrids, A and B genome chromosomes form univalents and tend to be eliminated whereas recombination takes place only in D genome chromosomes. Wheat random amplified polymorphic DNA (RAPD) fragments, detected in intergeneric hybrids and introgressed to the first backcross generation with Ae. cylindrica as the recurrent parent and having a euploid Ae. cylindrica chromosome number or one supernumerary chromosome, were assigned to wheat chromosomes using Chinese Spring nulli-tetrasomic wheat lines. Introgressed fragments were not limited to the D genome of wheat, but specific fragments of A and B genomes were also present in the BC1. Their presence indicates that DNA from any of the wheat genomes can introgress into Ae. cylindrica. Successfully located RAPD fragments were then converted into highly specific and easy-to-use sequence characterised amplified regions (SCARs) through sequencing and primer design. Subsequently these markers were used to characterise introgression of wheat DNA into a BC1S1 family. Implications for risk assessment of genetically modified wheat are discussed.

Search for evidence of introgression of wheat (Triticum aestivum L.) traits into sea barley (Hordeum marinum s.str. Huds.) and bearded wheatgrass (Elymus caninus L.) in central and northern Europe, using isozymes, RAPD and microsatellite markers

Abstract Seeds of English and Austrian populations of bearded wheatgrass (Elymus caninus L.) and sea barley (Hordeum marinum Huds.) growing in the vicinity of wheat (Triticum aestivum L.) fields were collected in order to search for evidence of the introgression of wheat traits into these wild relatives. Seeds were sown and plants grown for subsequent analyses using morphological and genetic (isozymes, RAPD and wheat microsatellites) markers. No F1 hybrids were found within the individuals of the two species grown, neither with morphological nor with genetic markers. Also, no evidence of introgression of wheat traits into E. caninus was observed. However, in one individual of H. marinum which had the typical morphology of this species, numerous species-specific DNA markers of wheat were amplified, thereby demonstrating previous hybridization. Consequently, the hybridization between wheat and H. marinum under natural conditions and the introgression of wheat traits into this wild relative seems to be possible. Our results contribute to the risk assessment of transgenic wheat cultivation.

Genetic and Ecological Consequences of Transgene Flow to the Wild Flora

Gene flow from crops to wild relatives by sexual reproduction is one of the major issues in risk assessment for the cultivation of genetically engineered (GE) plants. The main factors which influence hybridization and introgression, the two processes of gene flow, as well as the accompanying containment measures of the transgene, are reviewed. The comparison of risks between Switzerland and Europe highlights the importance of regional studies. Differences were assessed for barley, beet and wheat. Moreover, transgene flow through several wild species acting as bridge (bridge species) has been up to now poorly investigated. Indeed, transgene flow may go beyond the closest wild relative, as in nature several wild species complexes hybridize. Its importance is assessed by several examples in Poaceae. Finally, the transgene itself has genetic and ecological consequences that are reviewed. Transgenic hybrids between crops and wild relatives may have lower fitness than the wild relatives, but in several cases, no cost was detected. On the other hand, the transgene provides advantages to the hybrids, in the case of selective value as a Bt transgene in the presence of herbivores. Genetic and ecological consequences of a transgene in a wild species are complex and depend on the type of transgene, its insertion site, the density of plants and ecological factors. More studies are needed for understanding the short and long term consequences of escape of a transgene in the wild.

Phylogenetics and phylogeography of the monocot genus Baldellia (Alismataceae): Mediterranean refugia, suture zones and implications for conservation

Aquatic plants, and especially the emblematic genus Baldellia (Alismataceae), are among the most threatened organisms, due to unprecedented human-driven habitat destructions. Therefore protection plans are crucially needed and call for thoroughly documenting the genetic diversity and clarifying the taxonomy of this endangered genus. Our sampling included 282 individuals from 42 natural populations and covered the whole geographical range of the genus, across Europe and the Mediterranean. We combined sequencing of nuclear internal transcribed spacer (ITS) and chloroplastic trnL-ndhF regions with amplified fragment length polymorphism (AFLP) genotyping to investigate the Alismataceae phylogeny, and produce a phylogeography of Baldellia. Our phylogeny strongly supported the monophyly of Baldellia and placed it as the sister clade to Luronium and Alisma, therefore excluding, as previously supposed, a close genetic relatedness to the predominantly neotropical genus Echinodorus. The phylogeography of Baldellia outlined patterns consistent with a hypothesis considering glacial refugia located in the Iberian Peninsula and the Italy/Balkan region from which two distinct genetic lineages re-colonized Europe. These two lineages corresponded respectively to Baldellia ranunculoides (Italy/Balkan derived populations) and Baldellia repens (populations recovered from the Iberian Peninsula refuge), therefore supporting differences outlined between the two taxa in previous ecological and morphological studies. These results allowed clarifying taxonomic uncertainties by confirming the genetic distinctness of B. repens according to B. ranunculoides. A third lineage, Baldellia alpestris, originated and remained endemic to the mountainous regions of the Iberian Peninsula. Unexpectedly, B. repens populations collected in northern Africa, appeared to be genetically distinct from their European counterparts, this calls for further investigation to fully address their genetic and conservation status. Finally, we detected a large hybridization zone in northwestern Europe between B. repens and B. ranunculoides. These results were discussed in light of conservation approaches for Baldellia populations.

Spontaneous gene flow and population structure in wild and cultivated chicory, Cichorium intybus L.

Spontaneous gene flow between wild and cultivated chicory, Cichorium intybus L., may have implications for the genetic structure and evolution of populations and varieties. One aspect of this crop-wild gene flow is the dispersal of transgenes from genetically modified varieties, e.g. gene flow from GM chicory to natural chicory could have unwanted consequences. With the purpose to identify and quantify crop-wild gene flow in chicory, we analysed introgression in 19 wild chicory populations and 16 accessions of chicory varieties and landraces distributed across Northern, Central and Mediterranean Europe. The analysis used 281 AFLP markers and 75 SSAP markers giving a total of 356 polymorphic markers. Results from model based assignments with the program STRUCTURE indicated many incidents of recent gene flow. Gene flow was observed both between cultivars and wild populations, between landraces and wild populations, between different wild populations as well as between cultivars. Population structure visualized by distance-based clustering showed a North–South geographical structuring of the wild populations, and a general grouping of the cultivars corresponding to known origin. The results indicated, however, that the structuring between the two groups of wild and cultivated types was weak. As crop and wild recipients are genetically close and genes are transferred between the two types rather frequently, focus on mitigating crop-wild gene flow should be increased, before transgenic varieties are cultivated openly.