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.

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.

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.

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.