Molecular Biogeography of Prickly Lettuce (Lactuca serriola L.) Shows Traces of Recent Range Expansion.

Prickly lettuce (Lactuca serriola L., Asteraceae), a wild relative of cultivated lettuce, is an autogamous species which greatly expanded throughout Western and Northern Europe during the last 2 centuries. Here, we present a large-scale biogeographic genetic analysis performed on a dataset represented by 2622 individuals from 110 wild European populations. Thirty-two maternally inherited chloroplast RFLP-markers and 10 nuclear microsatellite loci were used. Microsatellites revealed low genetic variation and high inbreeding coefficients within populations, as well as strong genetic differentiation between populations, which was in accordance with the autogamous breeding system. Analysis of molecular variance based clustering indicated the presence of 3 population clusters, which showed strong geographical patterns. One cluster occupied United Kingdom and part of Northern Europe, and characterized populations with a single predominant genotype. The second mostly combined populations from Northern Europe, while the third cluster grouped populations particularly from Southern Europe. Kriging of gene diversity for L. serriola corroborated northwards and westwards spread from Central (Eastern) Europe. Significant lower genetic diversity characterized the newly colonized parts of the range compared to the historical ones, confirming the importance of founder effects. Stronger pattern of isolation by distance was assessed in the newly colonized areas than in the historical areas (Mantel's r = 0.20). In the newly colonized areas, populations at short geographic distances were genetically more similar than those in the historical areas. Our results corroborate the species' recent and rapid northward and westward colonization from Eastern Europe, as well as a decrease of genetic diversity in recently established populations.

Does insect netting affect the containment of airborne pollen from (GM-) plants in greenhouses?

Greenhouses are a well-accepted containment strategy to grow and study genetically modified plants (GM) before release into the environment. Various containment levels are requested by national regulations to minimize GM pollen escape. We tested the amount of pollen escaping from a standard greenhouse, which can be used for EU containment classes 1 and 2. More specifically, we investigated the hypothesis whether pollen escape could be minimized by insect-proof netting in front of the roof windows, since the turbulent airflow around the mesh wiring could avoid pollen from escaping. We studied the pollen flow out of greenhouses with and without insect netting of two non-transgenic crops, Ryegrass (Loliummultiflorum) and Corn (Zea Mays). Pollen flow was assessed with Rotorod(®) pollen samplers positioned inside and outside the greenhouse' roof windows. A significant proportion of airborne pollen inside the greenhouse leaves through roof windows. Moreover, the lighter pollen of Lolium escaped more readily than the heavier pollen of Maize. In contrast to our expectations, we did not identify any reduction in pollen flow with insect netting in front of open windows, even under induced airflow conditions. We conclude that insect netting, often present by default in greenhouses, is not effective in preventing pollen escape from greenhouses of wind-pollinated plants for containment classes 1 or 2. Further research would be needed to investigate whether other alternative strategies, including biotic ones, are more effective. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10453-011-9237-8) contains supplementary material, which is available to authorized users.

A Bayesian analysis of gene flow from crops to their wild relatives: cultivated (Lactuca sativa L.) and prickly lettuce (L. serriola L.) and the recent expansion of L. serriola in Europe.

Interspecific gene flow can lead to the formation of hybrid populations that have a competitive advantage over the parental populations, even for hybrids from a cross between crops and wild relatives. Wild prickly lettuce (Lactuca serriola) has recently expanded in Europe and hybridization with the related crop species (cultivated lettuce, L. sativa) has been hypothesized as one of the mechanisms behind this expansion. In a basically selfing species, such as lettuce, assessing hybridization in natural populations may not be straightforward. Therefore, we analysed a uniquely large data set of plants genotyped with SSR (simple sequence repeat) markers with two programs for Bayesian population genetic analysis, STRUCTURE and NewHybrids. The data set comprised 7738 plants, including a complete genebank collection, which provided a wide coverage of cultivated germplasm and a fair coverage of wild accessions, and a set of wild populations recently sampled across Europe. STRUCTURE analysis inferred the occurrence of hybrids at a level of 7% across Europe. NewHybrids indicated these hybrids to be advanced selfed generations of a hybridization event or of one backcross after such an event, which is according to expectations for a basically selfing species. These advanced selfed generations could not be detected effectively with crop-specific alleles. In the northern part of Europe, where the expansion of L. serriola took place, the fewest putative hybrids were found. Therefore, we conclude that other mechanisms than crop/wild gene flow, such as an increase in disturbed habitats and/or climate warming, are more likely explanations for this expansion.

Locus-dependent selection in crop-wild hybrids of lettuce under field conditions and its implication for GM crop development.

Gene escape from crops has gained much attention in the last two decades, as transgenes introgressing into wild populations could affect the latter's ecological characteristics. However, different genes have different likelihoods of introgression. The mixture of selective forces provided by natural conditions creates an adaptive mosaic of alleles from both parental species. We investigated segregation patterns after hybridization between lettuce (Lactuca sativa) and its wild relative, L. serriola. Three generations of hybrids (S1, BC1, and BC1S1) were grown in habitats mimicking the wild parent's habitat. As control, we harvested S1 seedlings grown under controlled conditions, providing very limited possibility for selection. We used 89 AFLP loci, as well as more recently developed dominant markers, 115 retrotransposon markers (SSAP), and 28 NBS loci linked to resistance genes. For many loci, allele frequencies were biased in plants exposed to natural field conditions, including over-representation of crop alleles for various loci. Furthermore, Linkage disequilibrium was locally changed, allegedly by selection caused by the natural field conditions, providing ample opportunity for genetic hitchhiking. Our study indicates that when developing genetically modified crops, a judicious selection of insertion sites, based on knowledge of selective (dis)advantages of the surrounding crop genome under field conditions, could diminish transgene persistence.

Existence of vigorous lineages of crop-wild hybrids in Lettuce under field conditions.

Plant to plant gene flow is a route of environmental exposure for GM plants specifically since crosses with wild relatives could lead to the formation of more vigorous hybrids, which could increase the rate of introgression and the environmental impact. Here, we test the first step in the process of potential transgene introgression: whether hybrid vigor can be inherited to the next generation, which could lead to fixation of altered, i.e., elevated, quantitative traits. The potential for a permanent elevated fitness was tested using individual autogamous progeny lineages of hybrids between the crop Lactuca sativa (Lettuce) and the wild species Lactuca serriola (Prickly Lettuce). We compared progeny from motherplants grown under either greenhouse or field conditions. The survival of young plants depended strongly on maternal environment. Furthermore, we observed that offspring reproductive fitness components were correlated with maternal fitness. Our study demonstrates that post-zygotic genotypic sorting at the young plants stage reduces the number of genotypes non-randomly, leading to inheritance of high levels of reproductive traits in the surviving hybrid lineages, compared to the pure wild relatives. Consequently, directional selection could lead to displacement of the pure wild relative and fixation of more vigorous genome segments originating from crops, stabilizing plant traits at elevated levels. Such information can be used to indentify segments which are less likely to introgress into wild relative populations as a target for transgene insertion.

A detailed linkage map of lettuce based on SSAP, AFLP and NBS markers.

Molecular markers based upon a novel lettuce LTR retrotransposon and the nucleotide binding site-leucine-rich repeat (NBS-LRR) family of disease resistance-associated genes have been combined with AFLP markers to generate a 458 locus genetic linkage map for lettuce. A total of 187 retrotransposon-specific SSAP markers, 29 NBS-LRR markers and 242 AFLP markers were mapped in an F(2) population, derived from an interspecific cross between a Lactuca sativa cultivar commonly used in Europe and a wild Lactuca serriola isolate from Northern Europe. The cross has been designed to aid efforts to assess gene flow from cultivated lettuce into the wild in the perspective of genetic modification biosafety. The markers were mapped in nine major and one minor linkage groups spanning 1,266.1 cM, with an average distance of 2.8 cM between adjacent mapped markers. The markers are well distributed throughout the lettuce genome, with limited clustering of different marker types. Seventy-seven of the AFLP markers have been mapped previously and cross-comparison shows that the map from this study corresponds well with the previous linkage map.

Population Size, Genetic Variation, and Reproductive Success in a Rapidly Declining, Self-Incompatible Perennial (Arnica montana) in The Netherlands.

Arnica montana is a rare and rapidly declining, self-incompatible plant species. In 26 populations in The Netherlands we investigated the relationship between population size and genetic variation using allozyme markers. Genetic variation was low in A. montana ( He = 0.088). There were positive correlations between population size and the proportion of polymorphic loci, the number of effective alleles, and expected heterozygosity, but not with observed heterozygosity. There was a significantly positive correlation between population size and the inbreeding coefficient. Generally, small populations showed heterozygote excess, which decreased with increasing population size. Possibly, the heterozygous individuals in small populations are survivors from the formerly larger populations with relatively high fitness. The F statistics showed a moderately high level of differentiation among populations ( FST = 0.140 ± 0.02), implying a low level of gene flow. For three out of four allozyme loci, we found significant inbreeding ( FIS = 0.104 ± 0.03). Only 14 of 26 populations were in Hardy-Weinberg equilibrium at all four polymorphic loci. In a subset of 14 populations of various size, we investigated natural seed production and offspring fitness. Population size was positively correlated with seed set, seedling size, number of flowering stems and flowerheads, adult survival, and total relative fitness, but not with the number of florets per flowerhead, germination rate, or the proportion of germination. Offspring performance in the greenhouse was not associated with genetic diversity measured on their mothers in the field. We conclude that the fitness of small populations is significantly reduced, but that there is as yet no evidence that this was caused by inbreeding. Possibly, the self-incompatibility system of A. montana has been effective in reducing selfing rates and inbreeding depression.

RESUMEN: Arnica montana es una especie de planta rara, en declinación rápida y autoincompatible. En 26 poblaciones de los Países Bajos investigamos la relación entre el tamaño poblacional y la variación genética mediante el uso de alozimas marcadoras. La variación genética fue baja en A. montana ( He = 0.088). Existió una correlación positiva entre el tamaño poblacional y la proporción de emplazamientos polimórficos, el número de alelos efectivos y la heterocigocidad esperada, pero no con la heterocigocidad observada. Existió una correlación positiva significativa entre el tamaño poblacional y el coeficiente de endogamia. Generalmente, las poblaciones pequeñas mostraron una heterocigocidad excesiva con disminuciones en el tamaño poblacional. Posiblemente, los individuos heterocigóticos de poblaciones pequeñas son sobrevivientes de poblaciones anteriormente grandes con una adaptabilidad relativamente alta. Las pruebas de F mostraron un nivel de diferenciación moderadamente alto entre poblaciones ( FST = 0.140 ± 0.02) lo que implica un nivel bajo de flujo de genes. Para tres de cuatro de los emplazamientos de alozimas encontramos una endogamia significativa ( FIS = 0.104 ± 0.03). Solamente 14 de las 26 poblaciones estuvieron en equilibrio Hardy-Weinberg para los cuatro emplazamientos polimórficos. En un subconjunto de 14 poblaciones de varios tamaños, investigamos la producción natural de semillas y la adaptabilidad de la descendencia. El tamaño poblacional estuvo positivamente correlacionado con el juego de semillas, el tamaño del almácigo, el número de tallos en flor y de inflorescencias, la supervivencia de adultos y la adaptabilidad total relativa, pero no con el número de flores por inflorescencia, la tasa de germinación ni la proporción de la germinación. El rendimiento de la descendencia en invernaderos no estuvo asociado con la diversidad genética medida en sus madres en el campo. Concluimos que la adaptabilidad de poblaciones pequeñas está significativamente reducida, pero no existe aún evidencia de que esto sea ocasionado por endogamia. Es posible que el sistema de autoincompatibilidad de A. montana haya sido efectivo en la reducción de tasas de autofecundación y depresión de la endogamia.