Inheritance pattern of tetraploids pummelo, mandarin, and their interspecific hybrid sour orange is highly influenced by their phylogenomic structure.

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.

Genotyping by Sequencing for SNP-Based Linkage Analysis and the Development of KASPar Markers for Male Sterility and Polyembryony in Citrus.

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.

Pollen Development and Viability in Diploid and Doubled Diploid Citrus Species.

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.

Strategies to Produce Grapefruit-Like Citrus Varieties With a Low Furanocoumarin Content and Distinctive Flavonoid Profiles.

Pummelos and hybrids, such as grapefruits, have high furanocoumarin and low flavonoid contents. Furanocoumarins interact negatively with certain drugs, while flavonoids are antioxidant compounds with health benefits. To obtain new grapefruit-like varieties with low furanocoumarin and high flavonoid contents, diploid and triploid hybrid populations from crosses between diploid and tetraploid "Clemenules" clementine and diploid "Pink" pummelo were recovered and analyzed. With regard to furanocoumarins, triploids produce less bergapten, bergamottin and 6,7-DHB than diploids. Regarding flavonoids, triploids yielded more eriocitrin, narirutin, hesperidin and neohesperidin than diploids, whereas no differences were observed in neoeriocitrin and naringin. These results indicate that, the strategy to recover triploid hybrids by 4x × 2x crosses is more appropriate than the recovery of diploid hybrids by 2x × 2x crosses for obtaining grapefruit-like varieties of citrus with lower furanocoumarin and higher flavonoid contents.

Analysis of Crossover Events and Allele Segregation Distortion in Interspecific Citrus Hybrids by Single Pollen Genotyping.

In citrus, a classical method of studying crossovers and segregation distortion (SD) is the genetic analysis of progenies. A new strategy combining fluorescence-activated cell sorting and whole genome amplification of haploid pollen nuclei with a large set of molecular markers, offers the opportunity to efficiently determine the frequency of crossovers and the identification of SD without the need to generate segregating populations. Here we have analyzed meiotic crossover events in a pollen nuclei population from "Eureka" lemon and the allelic SD was evaluated in a pollen nuclei population from a clementine × sweet orange hybrid ("CSO"). Data obtained from the "CSO" pollen nuclei population were compared to those obtained from genotyping of a segregating population ("RTSO") arising from a hand-made sexual hybridization between diploid non apomictic selected tangor (mandarin × sweet orange; "RTO" tangor) as female parent pollinated with "CSO" tangor as male parent. The analysis of crossovers rates on chromosome 1 revealed the presence of up to five crossovers events on one arm and four on the corresponding other arm, with an average of 1.97 crossovers per chromosome while no crossover events were observed in five "Eureka" lemon pollen nuclei. The rate of SD observed in "CSO" pollen nuclei (13.8%) was slightly lower than that recovered in the "RTSO" population (20.7%). In the pollen nuclei population, SD was found on linkage group (LG) 2, while the "RTSO" population showed SD on LGs 2 and 7. Potential male gametic selection mechanisms were distinguished in pollen grains, while in the population, mechanisms of gametophytic selection and/or zygotic selection were observed. This methodology is a very useful tool to facilitate research focused on the reproductive biology of citrus and study the mechanisms that affect crossovers and SD.

SILVOLIVE, a Germplasm Collection of Wild Subspecies With High Genetic Variability as a Source of Rootstocks and Resistance Genes for Olive Breeding.

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.

Consumer Description by Check-All-That-Apply Questions (CATA) of the Sensory Profiles of Commercial and New Mandarins. Identification of Preference Patterns and Drivers of Liking.

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.

Male and female inheritance patterns in tetraploid 'Moncada' mandarin.

KEY MESSAGE: Tetraploid `Moncada´ mandarin, used as male and female in interploidy hybridizations, displays mainly tetrasomic inheritance for most LGs, with slight variations according to the direction of the crossing. 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.

Assessing Ploidy Level Analysis and Single Pollen Genotyping of Diploid and Euploid Citrus Genotypes by Fluorescence-Activated Cell Sorting and Whole-Genome Amplification.

Flow cytometry is widely used to determine genome size and ploidy level in plants. This technique, when coupled with fluorescence-activated cell sorting (FACS), whole genome amplification and genotyping (WGA), opens up new opportunities for genetic studies of individualized nuclei. This strategy was used to analyze the genetic composition of single pollen nuclei of different citrus species. The flow cytometry and microscope observations allowed us to differentiate the populations of pollen nuclei present in the diploid and euploid genotypes analyzed, showing that citrus has binuclear pollen. We have identified in the "CSO" tangor an additional nuclei population composed by the vegetative plus generative nuclei. Genotyping of this nuclei population revealed that vegetative and generative nuclei show the same genetic configuration. In addition, we have demonstrated the presence of unreduced gametes in the diploid genotype "Mexican lime." Genomic amplification is a robust method for haploid nuclei genotyping with several molecular markers, whereas in diploid nuclei using heterozygous markers showed a bias towards one of the two alleles, limiting the use of this tool in this type of nuclei. We further discuss the importance and applications of single pollen genotyping in citrus genetic studies.

Molecular Characterization and Stress Tolerance Evaluation of New Allotetraploid Somatic Hybrids Between Carrizo Citrange and Citrus macrophylla W. rootstocks.

Polyploidy is one of the main forces that drives the evolution of plants and provides great advantages for breeding. Somatic hybridization by protoplast fusion is used in citrus breeding programs. This method allows combining the whole parental genomes in a single genotype, adding complementary dominant characters, regardless of parental heterozygosity. It also contributes to surpass limitations imposed by reproductive biology and quickly generates progenies that combine the required traits. Two allotetraploid somatic hybrids recovered from the citrus rootstocks-Citrus macrophylla (CM) and Carrizo citrange (CC)-were characterized for morphology, genome composition using molecular markers (SNP, SSR, and InDel), and their tolerance to iron chlorosis, salinity, and Citrus tristeza virus (CTV). Both hybrids combine the whole parental genomes even though the loss of parental alleles was detected in most linkage groups. Mitochondrial genome was inherited from CM in both the hybrids, whereas recombination was observed for chloroplastic genome. Thus, somatic hybrids differ from each other in their genome composition, indicating that losses and rearrangements occurred during the fusion process. Both inherited the tolerance to stem pitting caused by CTV from CC, are tolerant to iron chlorosis such as CM, and have a higher tolerance to salinity than the sensitive CC. These hybrids have potential as improved rootstocks to grow citrus in areas with calcareous and saline soils where CTV is present, such as the Mediterranean region. The provided knowledge on the effects of somatic hybridization on the genome composition, anatomy, and physiology of citrus rootstocks will be key for breeding programs that aim to address current and future needs of the citrus industry.

Unreduced Megagametophyte Production in Lemon Occurs via Three Meiotic Mechanisms, Predominantly Second-Division Restitution.

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 production was 4.9% for "Eureka Frost" and 8.3% for "Fino", with three meiotic mechanisms leading to 2n gamete formation. We performed the maximum-likelihood method at the individual level via centromeric marker analysis, finding that 88% of the hybrids arose from second-division restitution (SDR), 7% from first-division restitution (FDR) or pre-meiotic doubling (PRD), and 5% from post-meiotic genome doubling (PMD). The pattern of PHR along LG1 confirmed that SDR is the main mechanism for 2n gamete production. Recombination analysis between markers in this LG revealed partial chiasma interference on both arms. We discuss the implications of these restitution mechanisms for citrus breeding and lemon genetics.

Tetraploidy Enhances Boron-Excess Tolerance in Carrizo Citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.).

UNLABELLED: Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exodermis and earlier tissue maturation in root tips, where the Casparian strip was detected at a shorter distance from the root apex than in the 2x roots. The results presented herein suggest that the root anatomical characters of the 4x plants play a key role in their lower B uptake capacity and root-to-shoot transport. HIGHLIGHTS: Tetraploidy enhances B excess tolerance in citrange CarrizoExpression of NIP5 and BOR1 transporters and cell wall-bounded B are similar between ploidiesB tolerance is attributed to root anatomical modifications induced by genome duplicationThe rootstock 4x citrange carrizo may prevent citrus trees from B excess.

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding.

The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus-based vector (clbvINpr-AtFT and clbvINpr-CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4-6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr-AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.

Fine Mapping for Identification of Citrus Alternaria Brown Spot Candidate Resistance Genes and Development of New SNP Markers for Marker-Assisted Selection.

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 with a gene encoding a S-adenosyl-L-methionine-dependent-methyltransferase protein, should be considered as strong candidates for ABS-resistance. Moreover, the closest SNP was genotyped in 40 citrus varieties, revealing very high association with the resistant/susceptible phenotype. This new marker is currently used in our citrus breeding program for ABS-resistant parent and cultivar selection, at diploid, triploid and tetraploid level.

Maximum-likelihood method identifies meiotic restitution mechanism from heterozygosity transmission of centromeric loci: application in citrus.

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 conclusions at individual level. This new method was used to identify the meiotic restitution mechanism in nineteen mandarin genotypes used as female parents in triploid citrus breeding. SDR was identified for 85.3% of 543 triploid hybrids and FDR for 0.6%. No significant conclusions were obtained for 14.1% of the hybrids. At population level SDR was the predominant mechanisms for the 19 parental mandarins.

Genetic mapping of centromeres in the nine Citrus clementina chromosomes using half-tetrad analysis and recombination patterns in unreduced and haploid gametes.

BACKGROUND: Mapping centromere locations in plant species provides essential information for the analysis of genetic structures and population dynamics. The centromere's position affects the distribution of crossovers along a chromosome and the parental heterozygosity restitution by 2n gametes is a direct function of the genetic distance to the centromere. Sexual polyploidisation is relatively frequent in Citrus species and is widely used to develop new seedless triploid cultivars. The study's objectives were to (i) map the positions of the centromeres of the nine Citrus clementina chromosomes; (ii) analyse the crossover interference in unreduced gametes; and (iii) establish the pattern of genetic recombination in haploid clementine gametes along each chromosome and its relationship with the centromere location and distribution of genic sequences. RESULTS: Triploid progenies were derived from unreduced megagametophytes produced by second-division restitution. Centromere positions were mapped genetically for all linkage groups using half-tetrad analysis. Inference of the physical locations of centromeres revealed one acrocentric, four metacentric and four submetacentric chromosomes. Crossover interference was observed in unreduced gametes, with variation seen between chromosome arms. For haploid gametes, a strong decrease in the recombination rate occurred in centromeric and pericentromeric regions, which contained a low density of genic sequences. In chromosomes VIII and IX, these low recombination rates extended beyond the pericentromeric regions. The genomic region corresponding to a genetic distance < 5cM from a centromere represented 47% of the genome and 23% of the genic sequences. CONCLUSIONS: The centromere positions of the nine citrus chromosomes were genetically mapped. Their physical locations, inferred from the genetic ones, were consistent with the sequence constitution and recombination pattern along each chromosome. However, regions with low recombination rates extended beyond the pericentromeric regions of some chromosomes into areas richer in genic sequences. The persistence of strong linkage disequilibrium between large numbers of genes promotes the stability of epistatic interactions and multilocus-controlled traits over successive generations but also maintains multi-trait associations. Identification of the centromere positions will allow the development of simple methods to analyse unreduced gamete formation mechanisms in a large range of genotypes and further modelling of genetic inheritance in sexual polyploidisation breeding schemes.

Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication.

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes--a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes--and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement.

Genetically based location from triploid populations and gene ontology of a 3.3-mb genome region linked to Alternaria brown spot resistance in citrus reveal clusters of resistance genes.

Genetic analysis of phenotypical traits and marker-trait association in polyploid species is generally considered as a challenge. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from second division restitution (SDR) to map a genome region linked to Alternaria brown spot (ABS) resistance in triploid citrus progeny. ABS in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternata. This pathogen produces ACT-toxin, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a 3.3 Mb genomic region linked to ABS resistance near the centromere of chromosome III. Clusters of resistance genes were identified by gene ontology analysis of this genomic region. Some of these genes are good candidates to control the dominant susceptibility to the ACT-toxin. SSR and SNP markers were developed for efficient early marker-assisted selection of ABS resistant hybrids.

Cytological and molecular characterization of three gametoclones of Citrus clementina.

BACKGROUND: Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (array-CGH). RESULTS: Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control. CONCLUSIONS: The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes.

Assignment of SNP allelic configuration in polyploids using competitive allele-specific PCR: application to citrus triploid progeny.

BACKGROUND: Polyploidy is a major component of eukaryote evolution. Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole-genome coverage of single nucleotide polymorphism (SNP) markers, it is essential to implement a versatile SNP genotyping method to assign allelic configuration efficiently in polyploids. SCOPE: This work evaluates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration. Citrus was chosen as a model because of its economic importance, the ongoing worldwide polyploidy manipulation projects for cultivar and rootstock breeding, and the increasing availability of SNP markers. CONCLUSIONS: Fifteen SNP markers were successfully designed that produced clear allele signals that were in agreement with previous genotyping results at the diploid level. The analysis of DNA mixes between two haploid lines (Clementine and pummelo) at 13 different ratios revealed a very high correlation (average = 0·9796; s.d. = 0·0094) between the allele ratio and two parameters [θ angle = tan(-1) (y/x) and y' = y/(x + y)] derived from the two normalized allele signals (x and y) provided by KASPar. Separated cluster analysis and analysis of variance (ANOVA) from mixed DNA simulating triploid and tetraploid hybrids provided 99·71 % correct allelic configuration. Moreover, triploid populations arising from 2n gametes and interploid crosses were easily genotyped and provided useful genetic information. This work demonstrates that the KASPar SNP genotyping technique is an efficient way to assign heterozygous allelic configurations within polyploid populations. This method is accurate, simple and cost-effective. Moreover, it may be useful for quantitative studies, such as relative allele-specific expression analysis and bulk segregant analysis.