Dominique Dambier

Molecular breeding in the genus Musa: a strong case for STMS marker technology

Musa species are among the tallest monocotyledons and include major food-producing species. The principal cultivars, derived from two major species Musa acuminata (‘A’ genome) and Musa balbisiana (‘B’ genome), are polyploid hybrids (mainly AAA, AAB and ABB triploids), medium to highly sterile, parthenocarpic and clonally propagated. Bananas and plantains are crops to which molecular breeding is expected to have a positive impact. In order to better understand banana genetics, more knowledge has to be accumulated about the complex genome structure of hybrids and cultivars. Therefore, the aim of our work is to develop molecular markers that are codominant, reliable, universal, highly polymorphic and that are applicable to collaborative Musa germplasm genotyping and mapping. Two size-selected genomic libraries have been screened for the presence of simple sequence repeats (SSR). Our data demonstrate that SSR are readily applicable to the study of Musa genetics. Our comprehensive analyses of a significant number of banana sequence tagged microsatellite sites (STMS) will add to our knowledge on the structure and phylogeny of genomes of the Musa species, and suggest that microsatellites be used as anchor markers for a banana genetic core map. Additional markers, such as e.g. CAPS have also been tested in order to increase the detection of polymorphisms exceeding that revealed by STMS technology. The utility of PCR-derived markers for collaborative genetic analyses of the banana genome, and the transferability of streamlined’ laboratory techniques and data analysis to Developing Countries are discussed.

Tetraploid citrus rootstocks are more tolerant to salt stress than diploid

Citrus trees are subject to several abiotic constraints such as salinity. Providing new rootstocks more tolerant is thus a requirement. In this article, we investigated salt stress tolerance of three tetraploid rootstock genotypes when compared to their respective diploid rootstocks (Poncirus trifoliata, Carrizo citrange, Cleopatra mandarin). Plant growth, leaf fall and ion contents were investigated. At the end of the experiment, leaf fall was observed only for diploid Poncirus trifoliata plants as well as chlorosis symptoms for Poncirus trifoliata and Carrizo citrange diploid plants. The diploid Cleopatra mandarin plants growth rate was not affected by salt stress and has even been increased for tetraploid Cleopatra mandarin. Ion contents investigation has shown lower accumulations of chloride ions in leaves of the tetraploid plants when compared to diploid plants. Our results suggest that citrus tetraploid rootstocks are more tolerant to salt stress than their corresponding diploid.

Characterization of microsatellite markers in mandarin orange (Citrus reticulata Blanco)

A dinucleotide‐enriched genomic library was obtained from mandarin orange (Citrus reticulata Blanco). A subset of 101 positive clones was sequenced and primers were designed. The loci were screened for levels of variation using 26–29 wild mandarin oranges collected in Vietnam. Forty‐three loci were polymorphic with the number of alleles ranging from two to 18. The observed heterozygosity (HO) and expected heterozygosity (HE) were from 0.03 to 0.96 and from 0.03 to 0.92, respectively.

Improvement of Citrus somatic embryo development by temporary immersion

Liquid medium improves and facilitates somatic embryo development from Citrus deliciosa Ten. suspension cultures. Three different culture conditions were compared to determine a means of overcoming poor somatic embryo development. Somatic embryos derived from suspension cultures were plated on solid medium, maintained in suspension culture or temporarily immersed. About 60% of somatic embryos plated on solid medium developed to the cotyledonary stage, but were hyperhydric. Continuous growth in suspension culture at 100 rpm hindered cotyledon and protoderm formation, and somatic embryos were unable to develop beyond the globular stage. Temporary immersion promoted somatic embryo development, i.e. 66% of the somatic embryos produced were cotyledonary, and were morphologically similar to nucellar embryos. This latter culture system also improved regeneration synchronization by hampering secondary embryogenesis at the onset of germination. Irrespective of the culture system used, most cotyledonary somatic embryos studied had no caulinary meristem or starch and protein reserves, thus explaining the low germination rates obtained.

Cytological and molecular characterization of three gametoclones of Citrus clementina

BackgroundThree gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (array-CGH).ResultsChromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control.ConclusionsThe presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes.

Inheritance of Characters Involved in Fruit Quality in a Citrus Interspecific Allotetraploid Somatic Hybrid

The main components of citrus fruit quality (organic acids, sugars, and aromatic compounds) were studied in fruits of a somatic hybrid allotetraploid between Willow leaf mandarin (Citrus deliciosa Ten.) + Eureka lemon [Citrus limon (L.) Burm.] and the two diploid parents. The somatic hybrid (WLM + EUR) combined both nuclear genomes of the parents, with chloroplasts and mitochondria of mandarin. Variations in sugar and acid content were studied in fruit pulp during the maturing period, and the chemical composition of peel oils was investigated by capillary gas chromatography (GC), GC/mass spectrometry (MS), and (13)C NMR. The somatic hybrid was close to the lemon parent in the synthesis of organic acids and close to the mandarin parent in fructose content, while sucrose and glucose contents were between the two parents. The aromatic compounds of WLM + EUR were close to mandarin with a non-negligible effect of lemon, which inhibits the methyl N-methylanthranilate, a mandarin-specific compound. Our results lead us to conclude that biosynthesis of compounds involved in citrus fruit quality is not inherited in an additive way in the allotetraploid hybrid. We observed mandarin dominance for fructose and most of the aromatic compounds, lemon dominance for organic acid and methyl N-methylanthranilate, and codominance for sucrose and glucose.

Evidence for spontaneous polyploidization in cassava Manihot esculenta Crantz.

Cassava, Manihot esculenta Crantz (Euphorbiaceae), is a major tropical crop. It is a diploid and sexually fertile, but confined to vegetative propagation in agriculture. While polyploidization is a major evolutionary process in higher plants and is of importance in the creation of improved crop varieties, spontaneous polyploidization is poorly documented for M. esculenta. Molecular and cytological evidence is provided here for two cases of intraspecific triploidization events that occurred in smallholders’ fields in Vanuatu, South Pacific. We then discuss the implications of triploidy for the crop and highlight the importance of this discovery for the evolution of this species in traditional cropping systems.

Somatic hybridization between diploid Poncirus and Citrus improves natural chilling and light stress tolerances compared with equivalent doubled-diploid genotypes

Key messageThe genome doubling of the allotetraploid somatic hybrid can confer greater tolerance to cold and light stress than the diploid parents and their respective tetraploid.AbstractAllopolyploids are generally known to display broader adaptation to abiotic stresses than their parental diploid species. In the Mediterranean area, Citrus species are subjected to abiotic constraints such as low temperature and high radiation. Tetraploids are known to resist these environmental constraints better, and so the use of new tetraploid rootstocks offers an alternative to overcome these threats to crop productivity. The objective of this study was to determine whether the use of an allotetraploid hybrid could provide greater tolerance to cold and light stresses than its diploid parents or respective doubled-diploid parents. We compared cold and light stress responses of the allotetraploid hybrid FlhorAG1 (FL-4x) with those of its diploid parents, the willow leaf mandarin (Citrus deliciosa Ten) (WLM-2x) and the Poncirus Pomeroy (Poncirus trifoliata (L.) Raf.) (POP-2x), and their respective doubled-diploids (WLM-4x and POP-4x, respectively) by measuring physiological and biochemical parameters. When subjected to cold and light stress, FL-4x showed lower photoinhibition (Fv/Fm) and less accumulation of oxidative markers (MDA and H2O2) than diploid and doubled-diploid WLM and POP genotypes. This was correlated with a greater increase for FL-4x in some antioxidant activities during cold stress (SOD, APX and GR) and light stress (SOD, APX and MDHAR mainly). Overall, our results suggest that greater antioxidant capability in FL-4x should make this allotetraploid hybrid more tolerant to low temperatures than the two WLM genotypes, and more tolerant to light stress than the two WLM and POP genotypes.