Citrus reproductive biology is complex. One of its characteristic features is parthenocarpy that enables seedless fruit production. Citrus parthenocarpy and self-incompatibility knowledge is only partial and sometimes discrepant. Increasing such knowledge is relevant for better managing cultivated varieties and improving the selection of parents in breeding strategies to recover seedless varieties such as mandarins. This work develops an efficient protocol to characterize self-incompatibility and different parthenocarpy types based on emasculation, hand self-pollination, and hand cross-pollination. It analyzes fruit setting and seed production coupled with histological pollen performance observations. We analyzed the reproductive behavior of nine mandarin varieties with relevant characteristics as parents for seedless mandarin breeding. ‘Clemenules’ clementine and ‘Moncada’ mandarins were strictly self-incompatible with facultative and vegetative parthenocarpy; ‘Imperial’ mandarin and ‘Ellendale’ tangor displayed no strict self-incompatibility associated with facultative and vegetative parthenocarpy; ‘Fortune’ mandarin was self-incompatible with facultative and stimulative parthenocarpy; ‘Campeona’ and ‘Salteñita’ mandarins were self-compatible with vegetative parthenocarpy; ‘Serafines’ satsuma was associated with male sterility together with facultative and vegetative parthenocarpy; and ‘Monreal’ clementine was self-compatible and nonparthenocarpic. Our protocol can be applied for screening of mandarin germplasm and to characterize new parents. Reproductive behavior knowledge is important for optimizing seedless mandarin breeding programs based on diploidy, triploidy, or induced mutagenesis.

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 as male parent showed distorted segregation toward the susceptible hybrids. The second method is based on a cleaved amplified polymorphic sequences (CAPS) marker that was developed using the same primers as the nested PCR at the SNP08 locus, coupled with BccI restriction enzyme digestion. To verify the reliability of the two genotyping methods, in vitro leaf phenotyping was carried out by inoculating A. alternata spores onto young leaves of 101 hybrids, randomly chosen among the susceptible and resistant progenies. The phenotyping confirmed the SNP08 genotyping results, so the proposed method of selection based on HRM or CAPS genotyping could be routinely used as an alternative to KBioscience competitive allele specific polymerase chain reaction (KASPar) single nucleotide polymorphism (SNP) genotyping system to improve citrus breeding programs. While the study confirmed that the SNP08 marker is a reliable tool for MAS of new citrus hybrids with different genetic backgrounds, it also identified a small group of genotypes where the resistance mechanism requires further investigation.

Seedlessness is one of the most important agronomic traits in mandarins on the fresh fruit market. Creation of triploid plants is an important breeding strategy for development of new commercial varieties of seedless citrus. To this end, one strategy is to perform sexual hybridizations, with tetraploid genotypes as male parents. However, while seed development has been widely studied in citrus, knowledge of key steps such as microsporogenesis and microgametogenesis, is scarce, especially in polyploids. Therefore, we performed a study on the effect of ploidy level on pollen development by including diploid and tetraploid (double diploid) genotypes with different degrees of pollen performance. A comprehensive study on the pollen ontogeny of diploid and doubled diploid “Sanguinelli” blood orange and “Clemenules” clementine was performed, with focus on pollen grain germination in vitro and in planta, morphology of mature pollen grains by scanning electron microscopy (SEM), cytochemical characterization of carbohydrates by periodic acid–Shiff staining, and specific cell wall components revealed by immunolocalization. During microsporogenesis, the main difference between diploid and doubled diploid genotypes was cell area, which was larger in doubled diploid genotypes. However, after increase in size and vacuolization of microspores, but before mitosis I, doubled diploid “Clemenules” clementine showed drastic differences in shape, cell area, and starch hydrolysis, which resulted in shrinkage of pollen grains. The loss of fertility in doubled diploid “Clemenules” clementine is mainly due to lack of carbohydrate accumulation in pollen during microgametogenesis, especially starch content, which led to pollen grain abortion. All these changes make the pollen of this genotype unviable and very difficult to use as a male parent in sexual hybridization with the objective of recovering large progenies of triploid hybrids.

Polyembryony and male sterility (MS) are essential characters for citrus breeding. MS, coupled with parthenocarpy, allows for addressing the diversification of diploid seedless mandarin varieties, and nucleocytoplasmic MS is the most prevalent system. Polyembryony limits the use of seed parents in scion breeding programs, and the recovery of monoembryonic hybrids to be used as female parents is a crucial pre-breeding component. The objectives of this work were the identification of SNPs closely linked with the genes implied in these traits for marker-assisted selection. Genotyping by sequencing was used to genotype 61 diploid hybrids from an F1 progeny recovered from crossing ‘Kiyomi’ and ‘Murcott’ tangors. A total of 6444 segregating markers were identified and used to establish the two parental genetic maps. They consisted of 1374 and 697 markers encompassing 1416.287 and 1339.735 cM for ‘Kiyomi’ and ‘Murcott’, respectively. Phenotyping for MS and polyembryony was performed. The genotype–trait association study identified a genomic region on LG8 which was significantly associated with MS, and a genomic region on LG1 which was significantly associated with polyembryony. Annotation of the identified region for MS revealed 19 candidate genes. One SNP KASPar marker was developed and fully validated for each trait.

Citrus polyploidy is associated with a wide range of morphological, genetic, and physiological changes that are often advantageous for breeding. Citrus triploid hybrids are very interesting as new seedless varieties. However, tetraploid rootstocks promote adaptation to different abiotic stresses and promote resilience. Triploid and tetraploid hybrids can be obtained through sexual hybridizations using tetraploid parents (2x × 4x, 4x × 2x, or 4x × 4x), but more knowledge is needed about the inheritance pattern of tetraploid parents to optimize the efficiency of triploid varieties and tetraploid rootstock breeding strategies. In this work, we have analyzed the inheritance pattern of three tetraploid genotypes: ‘Chandler’ pummelo (Citrus maxima) and ‘Cleopatra’ mandarin (Citrus reticulata), which represent two clear examples of autotetraploid plants constituted by the genome of a single species, and the ‘Sevillano’ sour orange, which is an allotetraploid interspecific hybrid between C. maxima and C. reticulata. Polymorphic simple sequence repeat (SSR) and single-nucleotide polymorphism (SNP) markers were used to estimate parental heterozygosity restitution, and allele frequencies for centromeric loci were used to calculate the preferential pairing rate related to the proportion of disomic and tetrasomic segregation. The tetraploid pummelo and mandarin displayed tetrasomic segregation. Sour orange evidenced a clear intermediate inheritance for five of the nine chromosomes (1, 2, 5, 7, and 8), a slight tendency toward tetrasomic inheritance on chromosome 3, and intermediate inheritance with a tendency toward disomy for chromosomes 4, 6, and 9. These results indicate that the interspecific versus intraspecific phylogenomic origin affects preferential pairing and, therefore, the inheritance patterns. Despite its high level of heterozygosity, the important preferential chromosome pairing observed in sour orange results in a limited diversity of the genotypic variability of its diploid gametes, and consequently, a large part of the genetic value of the original diploid sour orange is transferred to the tetraploid progenies.

Tetraploid non-apomictic citrus genotypes are key female parents for 4x × 2x hybridizations aimed at producing seedless triploid hybrids. However, the extent to which different tetraploidization methods affect genome integrity remains insufficiently characterized at a genome-wide scale. In this study, genotyping-by-sequencing (GBS) was used to evaluate marker-based genomic stability in ten tetraploid plants of ‘Clemenules’, ‘Fina’, and ‘Marisol’ clementines obtained via colchicine treatment, in vitro adventitious organogenesis, or somatic cybridization. Diploid parental plants, two haploid plants of ‘Clemenules’ and ‘Fina’ clementines, and one doubled haploid plant of ‘Clemenules’ clementine were included, being the haploid and double haploid essential to resolve allelic phases. After quality filtering, 3333 SNP (Single Nucleotide Polymorphism) markers distributed across the nine citrus chromosomes were identified and used to compare allele dosage patterns along the genome. Across all GBS-covered regions, no major marker-based genomic gains or losses were detected in any tetraploid plant. These results indicate that, at the resolution provided by GBS, all three tetraploidization methods largely preserve chromosome structure, supporting their suitability for citrus triploid breeding programs based on 4x × 2x sexual hybridizations.