Genetic Structure of Diploid Gametes for the Production of Triploid Citrus Hybrids

Abstract

The citrus industry is an important source of incomes for both individual growers and producing countries. Therefore, breeding for quality especially seedlessness has a pivotal role for the market, since consumers demand seedless fruits. Recovery of triploid hybrids through ploidy manipulation is a very valuable methodology to recover seedless citrus varieties. Citrus triploid hybrids can be recovered through 2x x 2x taking advantage of the unreduced (2n) gametes formation. 2x x 4x and 4x x 2x sexual hybridizations are also widely exploited. Underlying the production mechanisms and genetic structures of diploid gametes is a key for optimizing polyploid breeding strategies. Two main mechanisms have been found in angiosperm for production of unreduced gametes: First Division Restitution (FDR) and Second Division Restitution (SDR). On the other hand, although tetraploid rootstocks display promising agronomic traits, their meiotic behavior and their segregation analysis is still mostly unknown in citrus. Disomic and tetrasomic models were defined as extreme models for tetraploid segregation, however, an intermediate inheritance model has been described for several crops. In this framework, this thesis aimed to study three main aspects: (i) the mechanisms underlying unreduced pollen gamete formation in the diploid 'CSO' tangor hybrid used as male parent in 4x x 2x triploid breeding programs, (ii) the frequencies and mechanism s involved in the unreduced 2n female gametes production for 'Eureka Frost' and 'Fino' lemon genotypes, and (iii) the interspecific recombination and the resulting diploid gamete structures of doubled-diploid 'Mexican' lime to evaluate the possibility that natural interploid hybridization maybe the origin of C. latifolia ('Tahiti' lime type) and C. aurantifolia ('Tanepao' lime type) triploid varieties. The production of 54 tetraploid hybrids from 4x x 2x sexual hybridizations allowed the analysis of the mechanisms underlying unreduced pollen gamete formation. SSR and SNP molecular markers revealed that the majority of these plants were obtained from unreduced 2n pollen of the diploid tangor parent. Then, the maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric loci revealed that both FDR with predominant occurrence and to a lower extend SDR were the mechanisms leading to unreduced male gamete formation in the tangor studied. These observations were confirmed by the analysis of PHR pattern along the linkage group (LG) 2. To our knowledge, this is the first report of tetraploid citrus progenies arising from unreduced pollen and the first description of the coexistence of two meiotic restitution mechanisms (SDR and FDR) producing unreduced pollen in citrus. In order to study the frequencies and the mechanisms involved in the unreduced 2n female gametes production in two different genotypes of lemon, we produced 43 triploid and tetraploid hybrids from 2x x 2x and 2x x 4x sexual hybridizations using 'Eureka Frost' and 'Fino' as female parents. The frequencies of 2n gamete production were respectively 4.9% and 8.3%. The maximum-likelihood analysis and pattern of PHR along LG1 revealed that SDR is the main mechanism of unreduced female lemon gametes (88%), followed by FDR or pre-meiotic doubling (PRD) (7%) and post-meiotic genome doubling (PMD) mechanisms (5%). This is the first report of the production of a large number of lemon progenies from 2n gametes and the first identification of a new mechanism, PMD that has never been observed in citrus and has rarely been described in other herbaceous or woody species. Across both studies, we demonstrated at the methodological level the effectiveness of using two complementary approaches, the analysis of the PHR pattern in one LG and the maximum-likelihood method based on centromeric loci for distinguishing between the different mechanisms of unreduced gamete production. We analyzed the meiotic mechanisms of a doubled diploid 'Mexican' lime, the interspecific recombination and the resulting diploid gamete structures combining a segregation analysis of SSR and SNPs markers, a cytogenetic study and pollen viability evaluation. We concluded that the doubled-diploid 'Mexican' lime had a predominantly disomic segregation for three LGs, intermediate inheritance with disomic tendency was found for five LGs and intermediate models for one LG. The resulting interspecific diploid gamete structures displayed high C. medica / C. micrantha heterozygosity. The revealed genetic structures of the diploid gametes produced by the doubled–diploid 'Mexican' lime are compatible with the hypothesis that 'Tahiti' and 'Tanepao' triploid varieties results from interploid hybridization involving a doubled-diploid 'Mexican' like lime. This disomic tendency limits the recombination and the diversity of the diploid gamete population; however the observed pollen viability restoration at tetraploid level could be advantageous for intensive breeding projects. The implications for triploid breeding projects of the meiotic behavior leading to unreduced pollen in 'CSO' tangor, unreduced ovules in lemons and diploid gametes of the DD 'Mexican' lime are discussed. (Resume d'auteur)