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.

Pollen competition as a reproductive isolation barrier represses transgene flow between compatible and co-flowering citrus genotypes.

BACKGROUND/OBJECTIVE: Despite potential benefits granted by genetically modified (GM) fruit trees, their release and commercialization raises concerns about their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. Information compiled from field trials on GM trees is essential to propose measures to minimize the transgene dispersal. We have conducted a field trial of seven consecutive years to investigate the maximum frequency of pollen-mediated crop-to-crop transgene flow in a citrus orchard, and its relation to the genetic, phenological and environmental factors involved. METHODOLOGY/PRINCIPAL FINDINGS: Three different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM self-incompatible contiguous citrus genotype (recipient) were used in conditions allowing natural entomophilous pollination to occur. The examination of 603 to 2990 seeds per year showed unexpectedly low frequencies (0.17-2.86%) of transgene flow. Paternity analyses of the progeny of subsets of recipient plants using 10 microsatellite (SSR) loci demonstrated a higher mating competence of trees from another non-GM pollen source population that greatly limited the mating chance of the contiguous cross-compatible and flowering-synchronized transgenic pollen source. This mating superiority could be explained by a much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations. CONCLUSIONS/SIGNIFICANCE: Pollen competition strongly contributed to transgene confinement. Based on this finding, suitable isolation measures are proposed for the first time to prevent transgene outflow between contiguous plantings of citrus types that may be extendible to other entomophilous transgenic fruit tree species.

Recovery and characterization of a Citrus clementina Hort. ex Tan. 'Clemenules' haploid plant selected to establish the reference whole Citrus genome sequence.

BACKGROUND: In recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants. The use of homozygous lines presents a significant advantage for the accomplishment of sequencing projects. Commercial citrus species are characterized by high heterozygosity, making it difficult to assemble large genome sequences. Thus, the International Citrus Genomic Consortium (ICGC) decided to establish a reference whole citrus genome sequence from a homozygous plant. Due to the existence of important molecular resources and previous success in obtaining haploid clementine plants, haploid clementine was selected as the target for the implementation of the reference whole genome citrus sequence. RESULTS: To obtain haploid clementine lines we used the technique of in situ gynogenesis induced by irradiated pollen. Flow cytometry, chromosome counts and SSR marker (Simple Sequence Repeats) analysis facilitated the identification of six different haploid lines (2n = x = 9), one aneuploid line (2n = 2x+4 = 22) and one doubled haploid plant (2n = 2x = 18) of 'Clemenules' clementine. One of the haploids, obtained directly from an original haploid embryo, grew vigorously and produced flowers after four years. This is the first haploid plant of clementine that has bloomed and we have, for the first time, characterized the histology of haploid and diploid flowers of clementine. Additionally a double haploid plant was obtained spontaneously from this haploid line. CONCLUSION: The first haploid plant of 'Clemenules' clementine produced directly by germination of a haploid embryo, which grew vigorously and produced flowers, has been obtained in this work. This haploid line has been selected and it is being used by the ICGC to establish the reference sequence of the nuclear genome of citrus.

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets.

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of 'Meyer' lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of 'Fortune' (Citrus clementina Hort ex Tan. x Citrus tangerina Hort ex Tan.) and 'Ellendale' (Citrus reticulata Blanco x Citrus sinensis L. Osb) is reported here for the first time.

Carotenoid diversity in cultivated citrus is highly influenced by genetic factors.

Citrus fruits are complex sources of carotenoids with more than 100 kinds of pigments reported in this genus. To understand the origin of the diversity of carotenoid compositions of citrus fruit, 25 genotypes that belong to the 8 cultivated Citrus species were analyzed. Juice extracts of mature fruit were analyzed by high-performance liquid chromatography using a C30 column. The 25 citrus genotypes presented different carotenoid profiles with 25 distinct compounds isolated. Statistical analyses revealed a strong impact of genotype on carotenoid compositions. Two kinds of classifications of genotypes were performed: on qualitative data and on quantitative data, respectively. The results showed that variability in carotenoid compositions was more interspecific than intraspecific. Two carotenoids, cis-violaxanthin and the beta-cryptoxanthin, strongly determined the classification on qualitative data, which was also in agreement with previous citrus variety classifications. These findings provide evidence that, as for other phenotypical traits, the general evolution of cultivated Citrus is the main factor of the organization of carotenoid diversity among citrus varieties. To the authors' knowledge this is the first study that links the diversity of carotenoid composition to the citrus genetic diversity. These results lead to the proposed major biosynthetic steps involved in the differential carotenoid accumulation. Possible regulation mechanisms are also discussed.

Leaf volatile compounds of seven citrus somatic tetraploid hybrids sharing willow leaf mandarin (Citrus deliciosa Ten.) as their common parent.

Volatile compounds were extracted by a pentane/ether (1:1) mixture from the leaves of seven citrus somatic tetraploid hybrids sharing mandarin as their common parent and having lime, Eurêka lemon, lac lemon, sweet orange, grapefruit, kumquat, or poncirus as the other parent. Extracts were examined by GC-MS and compared with those of their respective parents. All hybrids were like their mandarin parent, and unlike their nonmandarin parents, in being unable to synthesize monoterpene aldehydes and alcohols. The hybrids did retain the ability, although strongly reduced, of their nonmandarin parents to synthesize sesquiterpene hydrocarbons, alcohols, and aldehydes. These results suggest that complex forms of dominance in the mandarin genome determine the biosynthesis pathways of volatile compounds in tetraploid hybrids. A down-regulation of the biosynthesis of methyl N-methylanthranilate, a mandarin-specific compound, originates from the genomes of the nonmandarin parents. Statistical analyses showed that all of the hybrids were similar to their common mandarin parent in the relative composition of their volatile compounds.