Origin of different ploidy levels of progeny from diploid x tetraploid somatic hybrid crosses in Citrus

World production of Citrus fruit is on a continuous growth, representing the first fruit crop in international trade. The main evolution during the last decades was the growth of request on small Citrus fruits (clementines and mandarins). Many breeding programs get started all over the world due to the evolution of consumer and market preferences. One of the consumer turn-off is the excessive seed number. One of the ways chosen, to resolve this problem is the creation of sterile triploids cultivars, which have a great commercial potential because of their seedlessness. A way for triploid creation is sexual cross between diploids and tetraploids. However the scarcity of natural tetraploid gene pool was a restriction for using this method. Citrus somatic hybridisation via protoplast fusion allowed the creation of tetraploid somatic hybrids that can be used as parents to generate triploids cultivars. Several crosses using diploids (female) and tetraploid somatic hybrids (male) were realised by CIRAD: · Fortune mandarin (C. reticulata Blanco) x (Willow leaf mandarin SRA 133 (C. deliciosa Ten.) (WLM) + Star ruby Pomelo (C. paradisi) Tetraploid somatic hybrid), · Eureka lemon SRA 4 (Citrus limon (L) Burm) x (Pumelo Star Ruby (C. paradisi Macfad) and Corsican citron (C. medica L.) tetraploid somatic hybrid) · Eureka lemon SRA 4 (Citrus limon (L) Burm) x (Mexican lime (C. aurantifolia) + Shamouti orange (C. sinensis L.) tetraploid somatic hybrid). After germination, 117 plantlets were analysed using flow cytometry for ploidy levels determination. Major part of progenies was triploid in the 3 crosses (61-76%). However diploid and tetraploid plantlets were also found for the 3 crosses. Molecular analysis with SSR markers revealed that: · Tetraploids were issued from a diploid male gamete and an unreduced female gamete. · Triploids were the result of a haploid ovule and diploid male gamete. · Diploids origin is a haploid ovule and a viable haploid male gamete in the cross with Fortune mandarin. · Diploids origin result of Eureka lemon apomixis in the crosses with lemon. This study reveals that these progeny ploidy variations were owed to meiotic dysfunction during meiosis of the female diploid parents leading to tetraploids. Diploids were issued from apomixes or from viable male haplogamete coming from the somatic hybrids. (Texte intégral

Preselection of promising triploid mandarin varieties in Corsica : S17P13

The selection of seedless mandarin varieties adapted to the European market is one of the main objectives of the CIRAD breeding program. Triploid hybrid creation is the method selected to develop seedless varieties. More than one thousand triploid progenies are currently evaluated at the San Giuliano Research Station in Corsica. There were created by 2x X 2x hybridization exploiting spontaneous 2n gametes. The objective of a first set of crosses with clementine as female parent was to select clementine-like varieties, in order to extend the production period of this crop in Corsica. Seven hybrids were preselected at the end of the first level of evaluation according to the visual and organoleptic fruit characteristics, the period of production and the yield. There are currently under the second level of evaluation to analyse their agronomic behaviour. One of this hybrids is under D.U.S. examination for the grant of Community plant variety rights. The objective of a more recent second set of crosses is to develop late mandarin varieties. Several progenies, with different mandarins as female parent, present interesting fruit characteristics. One of them, with short juvenile period and very high yield, is preselected for the second stage of evaluation. (Texte integral

Triploid seedless mandarin breeding in France : S02O02

Small citrus is an increasing component of the world citrus industry. New high quality, parthenocarpic, sterile mandarin varieties will play a pivotal role for its sustainable development. To produce seedless varieties, the CIRAD breeding program is focused on triploid hybrid selection. The first method to develop triploid progenies exploits 2n gametes that are naturally produced by diploid cultivars to obtain triploids in 2x X 2x crosses. The second method is based on interploid crossings (2x X 4x and 4x X 2x). For the last strategy the parental tetraploid gene pool has been diversified by selecting spontaneous tetraploids in apomictic cultivars, generating doubled diploids by colchicine treatments and production of allotetraploids by somatic hybridization. Several thousand triploid hybrids have been created by CIRAD using these strategies with the support of embryo rescue and ploidy evaluation by flow cytometry. This breeding program is supported by basic and methodological research performed in collaboration with IVIA (Spain), DAK (Morocco) and INRA (France) in the following topics: (1) citrus germplasm management and characterization, (2) studies of polyploidization mechanisms, tetraploid meiosis and its implications on the genetic and phenotypic structure of triploid progenies, and (3) studies of the implication of polyploidy on genomic and phenotypic expression. (Texte intégral

Microsatellite loci inheritance in a Citrus interspecific somatic hybrid

Polyploidy plays an important role in plant evolution. Many works were made to investigate the establishment, formation, genome organisation and evolution of polyploids. Allelic diversity and heterozygosity in polyploids may provide a genetic buffer against inbreeding depression. Many strategies have been developed for triploid citrus breeding. One of these strategies consists in sexual hybridization between diploids and allotetraploid somatic hybrids. Genetic structure of gametes of allotetraploids depends on the mode of chromosome association at meiosis. Microsatellite markers can provide many informations on the structure and genetic studies. In the present work, the allelic segregation is studied on a mandarin (Citrus reticulata Blanco) + lemon (Citrus limon L.) interspecific citrus somatic hybrid. A progeny derived from crossing pollen of allotetraploid hybrid (MC+EUR) with Citrus maxima (Burm.) Merr (CH) (2n=2x=18) was used in the present work for genetic analyses. 17 polymorphie markers have been selected among 200 microsatellites markers. Chi-square goodness-of-fit analysis test (X2) was carried to determine inheritance mode and which hypotheses described best the obtained frequencies. These results support a tetrasomic inheritance on a Citrus interspecific somatic hybrid. That means that chromosomes pairing on a Citrus interspecific somatic hybrid is the same as on autotetraploid. The ailelie inheritance mode of this somatic hybrid provided valuable information for their utilization in a citrus breeding program. (Texte intégral

Differences in ploidy levels of interploidal crosses progenies between diploids and tetraploid somatic hybrids in citrus : S02P19

One of the main objectives of citrus breeders is to obtain sterile mandarins that produce seedless fruits. Triploids are a response to these criteria. A way for triploid creation is sexual crosses between diploids and tetraploids. However, the scarcity of the natural tetraploid gene pool has been a restriction for using this method. Citrus somatic hybridisation via protoplast fusion allowed the creation of allotetraploid somatic hybrids that can be used as parents to generate triploid cultivars. Several crosses using diploids (female) and allotetraploid somatic hybrids (male) were conducted by CIRAD: a) 'Fortune' mandarin x ('Willow leaf' mandarin 'Star Ruby' grapefruit tetraploid somatic hybrid); b) 'Eureka' lemon x ('Star Ruby' grapefruit 'Corsican' citron tetraploid somatic hybrid); and c) 'Eureka' lemon x ('Mexican lime' 'Shamouti' orange tetraploid somatic hybrid). The majority of progenies were triploid from the 3 crosses (61-76%). However diploid and tetraploid progenies were also obtained in all of them. Molecular analysis of progenies with SSR markers revealed that tetraploids were issued from a diploid male gamete and an unreduced female gamete, while diploids could be issued from a haploid ovule and a haploid male gamete. This study reveals that ploidy variations were owed to meiotic dysfunction of either the tetraploid somatic hybrids or the diploid female parent. (Texte intégral

Efficient haploid production on 'Wilking' mandarin by induced gynogenesis

Seedlessness is a major criterion for the citrus fresh fruit market. Therefore, triploid breeding appears very promising for the selection of new citrus cultivars. CIRAD has developed a method to produce triploid hybrids by somatic hybridization between diploid and haploid lines, and demonstrates the potential for haploid citrus production by induced gynogenesis in clementine. In this work we analysed the efficiency of induced gynogenesis by irradiated pollen in 'Wilking' mandarin (Citrus reticulata). Five doses of gamma irradiation of 'Meyer' lemon pollen were tested (0, 150, 200, 250, 300 Grays). Embryos were extracted from small seeds, obtained in immature fruits, and cultivated in vitro. The ploidy level of plantlets was determined by flow cytometry analysis. Irradiated pollen appeared to be efficient for gynogenesis induction in 'Wilking'. A relatively high number of haploids (7 plantlets) were obtained, essentially after hybridisation with 'Meyer' lemon pollen irradiated at 250 Grays. The ploidy level of all haploid plantlets was confirmed by chromosome counts, and molecular markers demonstrated their gynogenetic origin. These haploid lines should be useful for triploid breeding by somatic hybridisation, and also constitute interesting resources for genomic studies. (Résumé d'auteur

New insights on limes and lemons origin from nuclear and cytoplasmic markers genenotyping and targeted nuclear gene sequencing

It is believed that Citrus medica, C. maxima, C. reticulate and C. micrantha have generated all cultivated citrus species. Depending on the classification, lemons and limes are classified either into two species, C. limon and C. aurantifolia (Swingle and Reece, 1967) or into more than 30 (Tanaka, 1977). In order to study the molecular phylogeny of this citrus group, we analyzed 23 targeted sequenced nuclear genes and used three mitochondrial and five chloroplastic markers for 15 lemons and limes compared with representatives of the four basic taxa. We observed three main groups, each one derived from direct interspecific hybridizations: (1) the Mexican lime group (C. aurantifolia), including C. macrophylla, arising from hybridizations between papeda (C. micrantha) and citron (C. medica); (2) the yellow lemon group (C. limon) that are hybrids between sour orange (C. aurantium, which is believed to be a hybrid between C. maxima and C. reticulata) and citron; and (3) a rootstock lemon/lime group (Rough lemon and Rangpur lime) that are hybrids between an acid mandarin and a citron. We also identified different probable backcrosses and genotypes with more complex origin. None of the analyzed limes and lemons shared the C. medica cytoplasm, while this taxon is the common nuclear contributor of all limes and lemons. Limes and lemons appear to be a very complex citrus varietal group with the contribution of the 4 basic taxa. Neither the Swingle and Reece classification nor the Tanaka fit with the genetic evidence. (Résumé d'auteur

Genetic strategy for identification or genes involved in citrus salt stress tolerance : Status of genome mapping program

Salt stress is one of the most obvious effects of high salinity on Citrus, which is classified among the most sensitive tree crops. Genetic and genomic analysis of tolerant and sensitive plants is a prerequisite for breeding programs and the selection of more adapted varieties to high salinity. The combined approach of genetic mapping and localization of candidate genes has been applied in plant genetics in the past decade with the objective of characterizing and cloning quantitative trait loci (QTLs). The segregation of the tolerancel sensitivity to salt character was initiated on the F2 population resulting from the crossing Cleopatra mandarin X Poncirus trifoliata. Citrus linkage map was derived from the segregation analysis of SSR markers from EST or BAC ends. From many polymorphic primers only few makers have Mendelian segregation. The skewed segregation can be due to abnormal meiosis at the intergeneric level. The excess of homozygous locus was observed only for the marker CiBE2626b. No difference was observed between frequency of alleles from Poncirus and alleles from mandarin in homozygous loci. Half ofF2 population (61 hybrids) has a percentage of homozygous loci lower than or equal to 20%. Other hybrids seem to have proportions of homozygous and heterozygous loci that follow a normal distribution although there is a slight shift of sorne homozygote individuals to represent the expected Gaussian curve (- 0.45 instead of 0.5). We have also studied the segregation and mapped several candidate genes putatively involved in salinity tolerance. We plan to confirm the role played by these genes by gene expression analysis from selected homozygous F2 genotypes under strong salt stress conditions. We will measure the effects of salt stress on physiological traits on the segregating population. (Texte intégral