Jose Cuenca

Unreduced (2n) gametes have played a pivotal role in polyploid plant evolution and are useful for sexual polyploid breeding in various species, particularly for developing new seedless citrus varieties. The underlying mechanisms of 2n gamete formation were recently revealed for Citrus reticulata but remain poorly understood for other citrus species, including lemon (C. limon [L.] Burm. f.). Here, we investigated the frequency and causal meiotic mechanisms of 2n megagametophyte production in lemon. We genotyped 48progeny plants of two lemon genotypes, "Eureka Frost" and "Fino", using 16 Simple Sequence Repeat (SSR) and 18 Single Nucleotide Polymorphism (SNP) markers to determine the genetic origin of the progenies and the underlying mechanisms for 2n gamete formation. We utilized a maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric markers and analysis of PHR patterns along the chromosome. The frequency of 2n gamete product...

Polyploidy appears to have played a limited role in citrus germplasm evolution. However, today, ploidy manipulation is an important component of citrus breeding strategies. For varieties, the main objective is to develop triploid seedless cultivars. For rootstock, the aim is to cumulate interesting traits in tetraploid hybrids and to improve adaptation to biotic and abiotic stresses. This chapter starts with a review of the recent knowledge acquired on the natural mechanisms of citrus polyploidization and tetraploid meiosis. Chromosome doubling of nucellar cells is frequent in apomictic citrus and results in tetraploid seedling production. Unreduced gametes are also frequently produced, mainly by second division restitution for ovules. First division restitution was described for pollen as well as alternative mechanisms for both ovules and pollen. Tetraploid plants display tetrasomic to disomic segregations in relation to their genome structure (autotetraploid versus allotetraploid) and the divergence of the parental species. The implications of the origin of diploid gametes, on the genetic diversity of polyploid progenies, are discussed. The biotechnological tools (haplomethods, chromosome doubling by chemical treatments, somatic hybridization, and cytogenetic/molecular tools for polyploid genome studies) used to optimize ploidy manipulation are presented. The interest of haploid and polyploid genotypes for basic genetic and genomic studies is discussed. The research areas reviewed are as follows: haploids and doubled haploids for genome sequencing and haplotyping, centromere mapping from unreduced gametes, marker–trait association study in polyploids, and phenome and gene expression in polyploids with a special focus on polyploidy and adaptation. Finally, we give an overview of the recent advances of concrete polyploid citrus breeding programs in China, Florida, and the Mediterranean Basin.

Alternaria alternata is a fungus that causes a serious disease in susceptible genotypes of citrus, particularly in mandarins. The Alternaria citri toxin (ACT) produced by the pathogen induces necrotic lesions on young leaves and fruits, defoliation and fruit drop. Here, we describe two methods of marker-assisted selection (MAS) that could be used for the early identification of Alternaria brown spot (ABS)-resistant mandarin hybrids. The first method is based on a nested PCR coupled to high resolution melting (HRM) analysis at the SNP08 locus, which is located at 0.4 cM from the ABS resistance locus, and was previously indicated as the most suitable for the selection of ABS-resistant hybrids. The method was validated on 41 mandarin hybrids of the CREA germplasm collection, and on 862 progenies generated from five crosses involving different susceptible parents. Four out of five populations showed Mendelian segregation at the analyzed locus, while a population involving Murcott tangor...

Alternaria brown spot (ABS) is a serious disease affecting susceptible citrus genotypes, which is a strong concern regarding citrus breeding programs. Resistance is conferred by a recessive locus (ABSr) previously located by our group within a 3.3 Mb genome region near the centromere in chromosome III. This work addresses fine-linkage mapping of this region for identifying candidate resistance genes and develops new molecular markers for ABS-resistance effective marker-assisted selection (MAS). Markers closely linked to ABSr locus were used for fine mapping using a 268-segregating diploid progeny derived from a heterozygous susceptible × resistant cross. Fine mapping limited the genomic region containing the ABSr resistance gene to 366 kb, flanked by markers at 0.4 and 0.7 cM. This region contains nine genes related to pathogen resistance. Among them, eight are resistance (R) gene homologs, with two of them harboring a serine/threonine protein kinase domain. These two genes along wi...

Polyploidisation is a key source of diversification and speciation in plants. Most researchers consider sexual polyploidisation leading to unreduced gamete as its main origin. Unreduced gametes are useful in several crop breeding schemes. Their formation mechanism, i.e., First-Division Restitution (FDR) or Second-Division Restitution (SDR), greatly impacts the gametic and population structures and, therefore, the breeding efficiency. Previous methods to identify the underlying mechanism required the analysis of a large set of markers over large progeny. This work develops a new maximum-likelihood method to identify the unreduced gamete formation mechanism both at the population and individual levels using independent centromeric markers. Knowledge of marker-centromere distances greatly improves the statistical power of the comparison between the SDR and FDR hypotheses. Simulating data demonstrated the importance of selecting markers very close to the centromere to obtain significant...