Y. Froelicher

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900xa0Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan.xa0×xa0Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blancoxa0×xa0Citrus sinensis L. Osb) is reported here for the first time.

Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment

BACKGROUND AND AIMSnPolyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation.nnnMETHODSnTetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of Tardivo di Ciaculli mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype (Carrizo citrange; Citrus sinensis × Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries.nnnKEY RESULTSnTetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area.nnnCONCLUSIONSnTetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.

Sensitivity to high salinity in tetraploid citrus seedlings increases with water availability and correlates with expression of candidate genes

We investigated tolerance to high salinity in well-irrigated diploid and tetraploid citrus. Comparisons were made between two diploids (2×) of trifoliate orange (Poncirus trifoliata (L.) Raf.) and willow leaf mandarin (Citrus deliciosa Ten), their respective doubled diploids (4×) and the allotetraploid (FLHORAG1) obtained from the protoplast fusion of trifoliate orange and Willow leaf mandarin. Salinity stress was applied by progressively increasing the concentration of NaCl from 50u2009mM to 400u2009mM for 8 weeks. Two-year-old plants were watered daily. Maximum quantum yield of PSII, and leaf and root chloride and sodium content were monitored. We previously reported that under moderate saline stress, citrus 4× genotypes were more tolerant that the 2×, but under these experimental conditions, 4× seedlings were certainly more sensitive to salt stress than 2×, as they accumulated more toxic ions and were more affected than 2×. Chloride accumulation in 4× leaves was greater and the maximum quantum yield of PSII was more reduced in 4× than in 2×. The expression of several candidate genes involved in signal transduction, sodium and chloride transport, osmotic adjustment, regulation of the stomata opening and detoxification processes were also investigated by quantitative real-time reverse transcription-PCR. A high correlation was observed between phenotype of sensitivity to stress and gene expression changes.

Non-additive phenotypic and transcriptomic inheritance in a citrus allotetraploid somatic hybrid between C. reticulata and C. limon: the case of pulp carotenoid biosynthesis pathway

Allopolyploidy is known to induce novel patterns of gene expression and often gives rise to new phenotypes. Here we report on the first attempt to relate phenotypic inheritance in an allotetraploid somatic hybrid with gene expression. Carotenoid compounds in the fruit pulp of the two parental species and the hybrid were evaluated quantitatively by HPLC. Only very low levels of β-carotene and β-cryptoxanthin were observed in Citrus limon, while β-cryptoxanthin was a major component of C. reticulata, which also displayed high levels of phytoene, phytofluene, β-carotene, lutein, zeaxantin and violaxanthin. Total carotenoid content in mandarin juice sacs was 60 times greater than that in lemon. The allotetraploid hybrid produced all the same compounds as mandarin but at very low levels. Transgressive concentration of abscisic acid (ABA) was observed in the somatic hybrid. Real-time RT-PCR of total RNA from juice sacs was used to study expression of seven genes (CitDxs, CitPsy, CitPds, CitZds, CitLcy-b, CitChx-b, and CitZep) of the carotenoid biosynthetic pathway and two genes (CitNced1 and CitNced2) involved in abscisic acid synthesis from carotenoid. Gene expression was significantly higher for mandarin than lemon for seven of the nine genes analyzed. Lemon under expression was partially dominant in the somatic hybrid for three upstream steps of the biosynthetic pathway, particularly for CitDxs. Transgressive over expression was observed for the two CitNced genes. A limitation of the upstream steps of the pathway and a downstream higher consumption of carotenoids may explain the phenotype of the somatic hybrid.

Influence of mitochondria on gene expression in a citrus cybrid

The production of cybrids, combining nucleus of a species with alien cytoplasmic organelles, is a valuable method used for improvement of various crops. Several citrus cybrids have been created by somatic hybridization. These genotypes are interesting models to analyze the impact of cytoplasmic genome change on nuclear genome expression. Herein, we report genome-wide gene expression analysis in leaves of a citrus cybrid between C. reticulata cv ‘Willowleaf mandarin’ and C. limon cv ‘Eureka lemon’ compared with its lemon parent, using a Citrus 20K cDNA microarray. Molecular analysis showed that this cybrid possesses nuclear and chloroplast genomes of Eureka lemon plus mitochondria from Willowleaf mandarin and, therefore, can be considered as a lemon bearing foreign mitochondria. Mandarin mitochondria influenced the expression of a large set of lemon nuclear genes causing an over-expression of 480 of them and repression of 39 genes. Quantitative real-time RT–PCR further confirmed the credibility of microarray data. Genes over-expressed in cybrid leaves are predominantly attributed to the functional category “cellular protein metabolism” whereas in the down-regulated none functional category was enriched. Overall, mitochondria replacement affected different nuclear genes including particularly genes predicted to be involved in mitochondrial retrograde signaling. Mitochondria regulate all cell structures even chloroplast status. These results suggest that nuclear gene expression is modulated with respect to new information received from the foreign organelle, with the final objective to suit specific needs to ensure better cell physiological balance.

Origin of C. latifolia and C. aurantiifolia triploid limes: the preferential disomic inheritance of doubled-diploid ‘Mexican’ lime is consistent with an interploid hybridization hypothesis

Background and AimsnTwo main types of triploid limes are produced worldwide. The Tahiti lime type (Citrus latifolia) is predominant, while the Tanepao type (C. aurantiifolia) is produced to a lesser extent. Both types result from natural interspecific hybridization involving a diploid gamete of C. aurantiifolia Mexican lime type (itself a direct interspecific C. micrantha × C. medica hybrid). The meiotic behaviour of a doubled-diploid Mexican lime, the interspecific micrantha/medica recombination and the resulting diploid gamete structures were analysed to investigate the possibility that Tahiti and Tanepao varieties are derived from natural interploid hybridization.nnnMethodsnA population of 85 tetraploid hybrids was established between a doubled-diploid clementine and a doubled-diploid Mexican lime and used to infer the genotypes of Mexican lime diploid gametes. Meiotic behaviour was studied through combined segregation analysis of 35 simple sequenbce repeat (SSR) and single nucleotide polymorphismn (SNP) markers covering the nine citrus chromosomes and cytogenetic studies. It was supplemented by pollen viability assessment.nnnKey ResultsnPollen viability of the doubled-diploid Mexican lime (64 %) was much higher than that of the diploid. On average, 65 % of the chromosomes paired as bivalents and 31.4 % as tetravalents. Parental heterozygosity restitution ranged from 83 to 99 %. Disomic inheritance with high preferential pairing values was deduced for three chromosomes. Intermediate inheritances, with disomic trend, were found for five chromosomes, and an intermediate inheritance was observed for one chromosome. The average effective interspecific recombination rate was low (1.2 cM Mb-1).nnnConclusionnThe doubled-diploid Mexican lime had predominantly disomic segregation, producing interspecific diploid gamete structures with high C. medica/C. micrantha heterozygosity, compatible with the phylogenomic structures of triploid C. latifolia and C. aurantiifolia varieties. This disomic trend limits effective interspecific recombination and diversity of the diploid gamete population. Interploid reconstruction breeding using doubled-diploid lime as one parent is a promising approach for triploid lime diversification.