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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

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.

Self-incompatibility (SI) is present in around half of all species of flowering plants. SI limits endogamy and contributes to increased genetic diversity. SI is a very important trait in citrus because, when coupled with parthenocarpy, it allows seedless fruit production. Otherwise, SI is an impediment to genetic studies and breeding programs. Temperature stress, bud pollination and polyploidization can induce the breakdown of the SI mechanism in several species. In this work, we investigated how the SI mechanism can be broken down in two self-incompatible diploid citrus genotypes: 'Fortune' mandarin and 'Clemenules' clementine. The influence of temperature stress on the SI mechanism was assessed in self-pollinated flowers of 'Fortune' mandarins subjected to 2 temperature regimes (10 °C and 30 °C), whereas the bud pollination effect was investigated in the same genotype and in 'Clemenules' clementines cultivated under field conditions. The tetraploid 'Clemenules' clementine cultivated under field conditions was used to study if tetraploidization can bypass the SI reaction. Histological observations of pollen tube growth and seed production in self-pollinated flowers were used to evaluate the breakdown of SI, while the genetic analysis with SSR and SNP markers confirmed that all recovered plants were zygotic and had been originated by selfing. Our results confirm that the SI reaction can be surpassed by temperature stress, bud pollination and tetraploidy. To our knowledge, this is the first report in citrus in which the SI reaction breakdown by these three different strategies is demonstrated by molecular markers.

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.

Obtaining superior quality varieties are one of the main objectives of fruit breeding programs worldwide. In this study, we investigate employing check-all-that-apply (CATA) questions, which include the ideal product, as a method to select new varieties according to consumer organoleptic quality requirements. To this end, mandarin cultivars were used as a case study. Four new cultivars from the IVIA breeding program (‘Pri-88′, ‘Pri-89′, ‘Pri-90′ and ‘Tri-707′), and two commercial cultivars related to them (‘Clemenules’ and ‘Oronules’), were evaluated by a consumer panel using the CATA questions method, which include their ideal product. Our results reveal that this method is a useful tool for selecting varieties based on consumer descriptions of their sensory properties in relation to those of their ideal cultivar. This allows the different consumer preference patterns and differences in sample perceptions to be taken into account. A penalty analysis was performed, including the ideal product, to identify the “must-have” attributes; i.e., those that consumers included in their ideal mandarin description and, whenever present in samples, they significantly increased acceptance. For the mandarins herein evaluated, these attributes are “refreshing taste”, “very aromatic”, “sweet”, “juicy”, “very intense taste”, “sour” and “not very fibrous”. The fruit characteristics that contributed to improve the quality of the new varieties vs. the original varieties were also identified. ‘Pri-89′ and ‘Tri-707′, obtained from ‘Clemenules’, came closer to consumers’ ideal variety, because besides the aforementioned “must-have” attributes, these mandarins have small segments. ‘Pri-90′ implied improvement in relation to ‘Oronules’ and is an appropriate variety for those consumers sensitive to bitterness and who like mandarins with a certain level of sourness.

Wild subspecies of Olea europaea constitute a source of genetic variability with huge potential for olive breeding to face global changes in Mediterranean-climate regions. We intend to identify wild olive genotypes with optimal adaptability to different environmental conditions to serve as a source of rootstocks and resistance genes for olive breeding. The SILVOLIVE collection includes 146 wild genotypes representative of the six O. europaea subspecies and early-generations hybrids. These genotypes came either from olive germplasm collections or from direct prospection in Spain, continental Africa and the Macaronesian archipelago. The collection was genotyped with plastid and nuclear markers, confirming the origin of the genotypes and their high genetic variability. Morphological and architectural parameters were quantified in 103 genotypes allowing the identification of three major groups of correlative traits including vigor, branching habits and the belowground-to-aboveground ratio. The occurrence of strong phenotypic variability in these traits within the germplasm collection has been shown. Furthermore, wild olive relatives are of great significance to be used as rootstocks for olive cultivation. Thus, as a proof of concept, different wild genotypes used as rootstocks were shown to regulate vigor parameters of the grafted cultivar “Picual” scion, which could improve the productivity of high-density hedgerow orchards.

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 iden

Triploid-breeding programs in citrus are key tool to develop seedless cultivars. Obtaining triploid citrus hybrids may be achieved through different strategies, such as the exploitation of female unreduced gamete in crosses between diploid parents and diploid by tetraploid sexual hybridizations, in which tetraploid genotypes can be used as male or female parents. Genetic configuration of triploid populations from interploid crosses greatly depends on the chromosomic segregation mode of the tetraploid parent used. Here, we have analyzed the inheritance of the tetraploid 'Moncada' mandarin and compared the genetic structures of the resulting gametes when used as male and as female parent. The preferential chromosome pairing rate is calculated from the parental heterozygosity restitution (PHR) of codominant molecular markers, indicating the proportion between disomic and tetrasomic segregation. Tetraploid 'Moncada' both as female and male parent largely exhibited tetrasomic segregation. However, as female parent, one linkage group (LG8) showed intermediate segregation with tendency towards tetrasomic inheritance, while another linkage group (LG4) evidenced a clear intermediate segregation. On the other hand, when used as male parent two linkage groups (LG5 and LG6) showed values that fit an intermediate inheritance model with tetrasomic tendency. Significant doubled reduction (DR) rates were observed in five linkage groups as female parent, and in six linkage groups as male parent. The new knowledge generated here will serve to define crossing strategies in citrus improvement programs to efficiently obtain new varieties of interest in the global fresh consumption market.

Wild subspecies of Olea europaea constitute a source of genetic variability with huge potential for olive breeding to face global changes in Mediterranean-climate regions. We intend to identify wild olive genotypes with optimal adaptability to different environmental conditions to serve as a source of rootstocks and resistance genes for olive breeding. The SILVOLIVE collection includes 146 wild genotypes representative of the six O. europaea subspecies and early-generations hybrids. These genotypes came either from olive germplasm collections or from direct prospection in Spain, continental Africa and the Macaronesian archipelago. The collection was genotyped with plastid and nuclear markers, confirming the origin of the genotypes and their high genetic variability. Morphological and architectural parameters were quantified in 103 genotypes allowing the identification of three major groups of correlative traits including vigor, branching habits and the belowground-to-aboveground ratio. The occurrence of strong phenotypic variability in these traits within the germplasm collection has been shown. Furthermore, wild olive relatives are of great significance to be used as rootstocks for olive cultivation. Thus, as a proof of concept, different wild genotypes used as rootstocks were shown to regulate vigor parameters of the grafted cultivar “Picual” scion, which could improve the productivity of high-density hedgerow orchards.

In the last few years, the interest in developing new mandarin cultivars of superior quality has grown as a response to the increasing consumer demand of this appreciated fruit. This study evaluated the sensory profiles of five new late-season mandarin cultivars (‘Alborea‘, ’Coral‘, ’Omet‘, ’Matiz‘ and ’Tri-703‘) and six commercial cultivars found contemporarily in stores (‘Clemenules‘, ’Nova‘, ’Tango‘, ’Nadorcott‘, ‘Orri’ and ‘Ortanique’). The sensory profiles of the cultivars were described by consumers through CATA questions. Consumers’ acceptability and the main physico-chemical properties were also evaluated. Twenty-two out of 23 CATA terms differed significantly for the sensory profiles of the studied cultivars. The new cultivars shared a similar profile, described mainly as “very intense taste”, “refreshing taste”, “very aromatic” and “juicy”, and these characteristics were quite different from those of the commercial cultivars. By combining acceptability and CATA questions, drivers of liking for segments of consumers with different preference patterns were identified. This is the first time that the sensory profiles of mandarin cultivars have been described by consumers. A significant number of consumers preferred the new mandarin cultivars to the commercial ones, which allows them a promising introduction on the market