Citrus breeding for biotic and abiotic stress tolerances; new tools and strategies for biodiversity Mobilization : S04T01

Citrus industry is affected worldwide by several fungal, bacterial and viral diseases. Some production areas also suffer of strong abiotic constraints such as water deficit, salt stress and iron chlorosis. Resistance or tolerance traits for most of these constraints are present in the citrus germplam but their mobilization by conventional breeding is hampered by the complex interspecific genome structures of the modern citrus, the partial apomixis of most genotypes and the long juvenile phase. Beside these constrainst the vegetative propagation of grafted plants is an important advantage. Indeed it allow dividing breeding objective between scions and rootstocks and to select any complex elite genotype that will be then asexually propagated. A fundamental progress of the last years, to improve citrus breeding efficiency, was the elaboration and public release, by the International Citrus Genome Consortium, of the first citrus reference genome anchored with a medium density genetic map and the increasing availability of resequencing data. A first application was to study the interspecific genomic structures of the main cultivated species. The origins of sweet oranges (C. sinensis), sour oranges (C. aurantium), grapefruits (C. paradisi), limes (C. aurantifolia) and lemons (C. limon) and their interspecific genome structure along the genome are already deciphered. A huge number of DNA polymorphism have been identified from resequencing data and genome wide association studies are going on using GBS approaches. When useful SNP polymorphisms are encountered they are easely converted in genetic markers with tools adapted to (i) highthroughputs studies by using arrays systems or (ii) more targeted studies using competitive allele PCR methods such Kaspar methodology allowing for allele dosis evaluation in polyploid genotypes. Molecular markers studies have also provided useful information on segregation and recombination at diploid and polyploid levels. It will allow improving the progenitor choice and the inheritance of the parental value to their progenies particularly for project aiming to create seedless triploid varieties or tetraploid rootstock cumulating tolerances to abiotic and biotic constraints. Joining genetic and genomic approaches, a cluster of resistance genes, genetically associated with resistance to Alternaria alternata, was located in chromosome 3. Alternaria resistance is actually one of the rare traits for witch SAM is routinely applied in citrus. New breeding approaches are also developed or possible, including (i) tetraploid rootstock breeding to improve abiotic stress adaptation, (ii) the reconstruction of modern species ideotypes from germplasm or (iii) the introgression of useful traits from citrus relatives combining MAS and GMO with early flowering genes. By the whole, the new genetic and genomic insights and tools will change greatly the potential for biodiversity mobilization in future breeding projects for biotic and abiotic stress tolerances. Most of the presented data results from CIRAD/INRA/IVIA collaboration. (Texte intégral

Origin of 2N gametes in C. reticulata cv fortune mandarin

Citrus are most important fruit crop worldwide. Seed1essness is a key characteristic for the fresh fruit market and the development of triploid hybrids is one strategy developed by several groups over the world. Indeed, triploid hybrids are generally sterile and produce seedless fruits and do not pollinate other varieties. Triploid citlllS hybrids can be obtained by several strategies, including hybridization betwe6n diploid parents. Mechanism of 2n gamete formation and its implication on parental heterozygosity restitution is a main parameter determining the genetic and phenotypic structure of the triploid population. In the case of Citrus it has been shown that the 2n gametes are of maternaI origin. It has been proposed that the origin of 2n gametes is from the second division restitution (SDR) in Clementines and from the first division restitution (FDR) in sweet oranges. No data is available for other genotypes and particularly 'Fortune', a mandarin hybrid producing very high rate of triploids in 2x x 2x crosses and massively used to create triploid progenies. The aim of this work was to analyse the mechanism of 2n gamete formation in 'Fortune' mandarin genotype. One hundred and five triploid hybrids from the crosses between 'Fortune' as female diploid parent and 'Murcott' or 'Mandarino ComÛIl' as male diploid parents were genotyped for twenty- four codominant molecular SSRs (Simple Sequence Repeat) markers using a capillary genetic fragment analyzer. Estimation of ailelic doses from relative peaks area ailowed inferring the female and male gamete structures and thus the heterozygosity restitution in the 2n gametes.; this demonstrated that ail triploid arise from 2n megaspores. The unimodal distribution of heterozygosity restitution in the 2n megaspores among the analyzed genotypes suggests that an these 2n gametes arise from a same mechanism. Restitution of maternaI heterozygosity for the used rnarkers makes suppose that underlying mechanism in the 2n gamete formation is SDR. Indeed there are six markers with less than 50% of maternaI heterozygosity restitution, which is incompatible with FDR hypothesis. SDR hypothesis is coherent with the results published in case of the clementine, which is one of the parents of the 'Fortune' variety. Under this hypothesis, the relatively high global heterozygosity restitution level (60,95%) should indicate that a majority of the analyzed markers are far frorn the centromeres. This genetic struc turation will soon be confronted with phenotypic variability and compared with structuration obtained with other triploid creation strategies such as 2x x 4x hybridization. (Texte intégral

Parental heterozygosity and origin of 2n gametes in mandarin: Two key parameters for Citrus triploid breeding projects

Citrus are the most important fruit crop in Spain and worldwide. Spain is the fifth producing country and the first in fresh fruit exportation. Seedlesness is a key characteristic for the fresh fruit market. Mandarin varietal structure in Spain causes important commercial problems because of cross-pollination between Clementines and late maturing mandarin hybrids, which produces seeds in both groups of varieties, in spite of their self-incompatibility. Development of triploid hybrids could be a solution for this problem, since they are sterile and they produce seedless fruits, and do not pollinate other varieties (Navarro et al., 2002). Triploid citrus hybrids can be obtained by several strategies (Ollitrault et al., 2008), including hybridization between diploid parents. In the case of Citrus it has been shown that the 2n gametes producing the triploid are of maternaI origin. It has been proposed that the origin of 2n gametes is from the second division restitution (SDR) in Clementines and from the first division restitution (FDR) in sweet oranges. No data is available for other genotypes and particularly Fortune, a mandarin hybrid producing very high rate of triploids in 2x x 2x crosses and massively used to create triploid progenies. Mechanism of 2n gametes formation and its implication on parental heterozygosity restitution, as weIl as the male parent heterozygosity and parental differentiations, are the main parameters determining the genetic and phenotypic structure of the triploid population. Thus, the aim of this work was to evaluate heterozygosity level and differentiation between several male parents and Fortune mandarin and to analyse the mechanism of 2n gamete formation in Fortune. Fifteen parental genotypes used in the IVIA triploid breeding project have been analyzed with 35 codominant molecular markers SSRs (Simple Sequence Repeat), 25 of them being polymorphic for the analyzed genotypes. Heterozygosity levels vary between 37% for Fremont and Ellendale and 54% for Pineapple 4n. Likewise, the same SSR markers were used for analyzing 67 triploid hybrids from the cross between Fortune as female parent and Murcott as male diploid parent. This later was the more genetically distant to Fortune according to information of the previous study (D=0.49). This analysis allows us to study the mechanism of formation of the Fortune 2n gametes. The obtained results are in favour ofthe SDR hypothesis, rather than FDR. The SDR hypothesis is coherent with the results published in case of the clementine (Luro et al., 2004), which is one of the parents of the Fortune variety. (Texte intégral

Selection of spontaneous autotetraploid plants from Citrus polyembryonic cultivars

Seedlessness is one of the most important characteristics for mandarin fresh fruit market. Mandarin triploid hybrids allow implementing this trait in commercial varieties. Indeed triploids plants are generally sterile and they do not pollinate other varieties. Triploid plants can he recovered by crossing diploid and tetraploid parents. Most citrus genotypes are apomictic. The origin of apomixis is determined by adventitious embryony in polyembryonic seeds (Koltunow, 1996). The adventitious embryos originate from nucellar cells (Kobayashi et. al., 1981). Tetraploid plants are found with variable frequency in seedling populations of polyembryonic citrus genotypes. This allows the recovery of citrus tetraploid genotypes that can he used for triploid breeding. In the framework of the IVIA triploid breeding program (Navarro et. al., 2(02), we have searched for tetraploids plants in seedling populations of 'Anana', 'Fairchild', 'Kara', 'Page', 'Saltemta', 'Simeto', 'Sunburst' and 'Tardivo di Ciaculli' mandarins, 'Afourer', 'Murcott' and 'Ortanique' tangors, 'Mapo'and 'Minneola' tangelos, 'Duncan' and 'Star Ruby' grapefruits and 'Sanguinelli' orange. Determination of ploidy level was made by flow cytometry and genetic analysis to confirm their genetic origin with 31 SSRs markers. Tetraploid plants were found in ail genotypes analyzed, except 'Salteiiita' and 'Simeto' mandarins, but the frequency of tetraploids varied with the genotype. 'Kinnow' mandarin produced the larger number of tetraploids plants (9,7%), whereas 'Page' mandarin produced the smaller percentage (0,5%). Ali the tetraploid plants showed the same ploidy level in ail parts of the plant, indicating that they were not chimeras. They presented the sarne molecular profile as their maternai parents for alllaci analyzed, thus indicating that these plants originated as a result of the duplication of the chromosome number in nucellar cells. The new tetraploid plants have been included in the collection of tetraploid genotypes of the IVIA Germplasm Bank for their use as male parents in the triploid breeding program. (Texte intégral

Development of SSR markers from Citrus clementina (Rutaceae) BAC end sequences and interspecific transferability In Citrus (P148)

Microsatellite primers were developed from bacterial artificial chromosome (BAC) end sequences (BES) of Citrus clementina and their transferability and polymorphism tested in the genus Citrus for future anchorage of physical and genetic maps and comparative interspecific genetic mapping. Using PAGE and DNA silver staining, 79 primer pairs were selected for their transferability and polymorphism among 526 microsatellites mined in BES. A preliminary diversity study in Citrus was conducted with 18 of them, in C. reticulata , C. maxima , C. medica , C. sinensis , C. aurantium , C. paradisi , C. lemon , C. aurantifolia, and some papedas (wild citrus), using a capillary electrophoresis fragment analyzer. Intra and interspecific polymorphism was observed, and heterozygous markers were identified for the different genotypes to be used for genetic mapping. These results indicate the utility of the developed primers for comparative mapping studies and the integration of physical and genetic maps. (Résumé d'auteur

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