Mechanisms of sexual polyploidization and inheritance in triploid citrus populations

Abstract

Citrus is the main fruit crop in the world and Spain is the 6th producer and the major exporter for the fresh fruit market. Seedlessness is one of the most important fruit quality traits for this market since consumers do not accept seedy fruits. Recovery of triploid hybrids has become an important breeding strategy to develop new seedless citrus varieties and several of them have been already released from citrus breeding programs worldwide. Despite the undisputable importance of polyploidy in plant species, their genetics are much less well known than those of their diploid counterparts. Citrus triploid hybrids can be routinely recovered from sexual polyploidization (2x × 2x) or interploid crosses (2x × 4x and 4x × 2x). In 2x × 2x sexual crosses, spontaneous triploid hybrids arise from the union of an unreduced (2n) megagametophyte with haploid pollen. In the case of interploid sexual crosses (2x × 4x and 4x × 2x), triploid hybrids result from the fecundation of a diploid gamete arising from the tetraploid parent and a haploid gamete arising from the diploid parent. The genetic and phenotypic structures of triploid populations greatly depend on the parental heterozygosity restitution (HR) in the diploid gamete at each locus, which is mainly affected by the triploid recovery strategy. In 2x × 2x crosses, HR depends on the underlying mechanism leading to the unreduced gamete formation, which are genetically equivalent to First Division Restitution (FDR) or Second Division Restitution (SDR) mechanisms. Moreover, under each restitution mechanism, HR also depends on the locus-centromere genetic distance. In the case of interploid crosses, parental heterozygosity restitution from tetraploid parents depends on the double reduction frequency. In citrus, the unreduced gamete formation mechanism is still controversial; FDR has been the mechanism proposed for sweet orange, whereas SDR has been proposed for clementine. On the other hand, inferring the allelic configurations of genetic markers is a main challenge in polyploidy crops to infer genotypic and gametic structures with the objective to analyze meiosis and inheritance mechanisms. According to this scientific context, the objectives of the thesis where: (i) to develop a new approach for allele dosis assignation when using co-dominant markers, (ii) to implement and apply methods for the analysis of 2n gametes origin and locate centromeres, and (iii) to take advantage of this knowledge to locate a major gene of resistance to Alternaria Brown Spot (ABS) which is a major constraint for triploid mandarin breeding. For microsatellite (SSR) markers, we have demonstrated that triploid progeny genotyping can be successfully performed using the microsatellite allele-counting peak ratio (MAC-PR) method. However, SSR analysis remains relatively costly and time consuming compared with actual SNP genotyping methods. Moreover, with the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers that can be selected along the entire genome. In this thesis, a new method based on competitive allele-specific PCR has been developed to assign SNP allele dosage in an accurate, simple, and cost effective way. Combining the MAC-PR and the new developed SNP genotyping methods offers the possibility to utilize a broad range of molecular markers in genotyping triploid genotypes. Both methods have been used in further works included in this thesis. SDR has been demonstrated as the mechanism underlying unreduced gamete production in 'Fortune' mandarin by genotyping triploid progenies with SSR markers. In addition, a new method to locate the centromere, based on the best fit between observed heterozygosity restitution within a linkage group and theoretical functions under either partial or no chiasma interference hypotheses has been developed and successfully applied. To expand the knowledge of the mechanism underlying unreduced game