Pollen Competition as a Reproductive Isolation Barrier Represses Transgene Flow between Compatible and Co-Flowering Citrus Genotypes

Background/Objective: Despite potential benefits granted by genetically modified (GM) fruit trees, their release and commercialization raises concerns about their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. Information compiled from field trials on GM trees is essential to propose measures to minimize the transgene dispersal. We have conducted a field trial of seven consecutive years to investigate the maximum frequency of pollen-mediated crop-to-crop transgene flow in a citrus orchard, and its relation to the genetic, phenological and environmental factors involved. Methodology/Principal Findings: Three different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM self-incompatible contiguous citrus genotype (recipient) were used in conditions allowing natural entomophilous pollination to occur. The examination of 603 to 2990 seeds per year showed unexpectedly low frequencies (0.17-2.86%) of transgene flow. Paternity analyses of the progeny of subsets of recipient plants using 10 microsatellite (SSR) loci demonstrated a higher mating competence of trees from another non-GM pollen source population that greatly limited the mating chance of the contiguous cross-compatible and flowering-synchronized transgenic pollen source. This mating superiority could be explained by a much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations. Conclusions/Significance: Pollen competition strongly contributed to transgene confinement. Based on this finding, suitable isolation measures are proposed for the first time to prevent transgene outflow between contiguous plantings of citrus types that may be extendible to other entomophilous transgenic fruit tree species. (Résumé d'auteur

Recovery of citrus triploid hybrids by embryo rescue and flow cytometry from 2x x 2x sexual hybridisation and its application to extensive breeding programs

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market and mandarin triploid hybrids have this trait. Citrus triploid plants can be recovered by 2x x 2x sexual hybridisations as a consequence of the formation of unreduced gametes at low frequency. Triploid embryos are found in small seeds that do not germinate under greenhouse conditions. Extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation. In this work, we describe an effective methodology to recover triploid hybrids from 2x x 2x hybridisations based on in vitro embryo rescue and ploidy level determination by means of flow cytometry. The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed. The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. The methodology described here has enabled us to obtain over 4,000 triploid hybrids so far, of which 13 have been protected in the European Union and two are being extensively planted by citrus growers to establish new commercial plots. These triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents, and thus molecular identification will help defend plant breeders' rights

AFLP markers closely linked to a major gene essential for nucellar embryony (apomixis) in Citrus maxima × Poncirus trifoliata

Some citrus varieties express a form of apomixis termed nucellar embryony in which the adventive embryos develop from nucellus tissue surrounding the embryo sac. This trait results in many seeds containing multiple embryos (polyembryony). Inheritance of the frequency of polyembryony was studied in 88 progeny from a cross of Citrus maxima (monoembryonic) × Poncirus trifoliata (polyembryonic). The frequency of polyembryonic seed produced by each progeny was determined by scoring 100–500 seeds for the number of seedlings to emerge from each seed. Two groups of eight individuals from each extreme of the population were chosen for bulked segregant analysis with amplified fragment length polymorphism markers amplified with 256 primer combinations. Candidate markers identified in the bulks as linked to the trait were tested on the 32 individuals used to create the bulks and then on the remaining plants in the population. Five candidate markers tightly linked to polyembryony in P. trifoliata were identified. Specific marker alleles were present in nearly all progeny that produced polyembryonic seed, and alternate alleles were present in nearly all progeny that produced only monoembryonic seed. The region defined by these markers very likely contains a gene that is essential for the production of polyembryonic seeds by apomixis, but also shows segregation distortion. The proportion of polyembryonic seeds varied widely among the hybrid progeny, probably due to other genes. Scoring 119 progeny of a P. trifoliata selfed population for the closely linked markers and the proportion of polyembryonic seeds confirmed close linkage between these markers and polyembryony