Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation. Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat SSR markers to analyse their genetic origin. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes.
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Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation.
Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat SSR markers to analyse their genetic origin. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric.
Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates.
Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.
Polyploidy is a major component of eukaryote evolution and particularly in angiosperms Grant, ; Soltis and Soltis, ; Wendel and Doyle, Many plant species result from autopolyploidization or allopolyploidization events and polyploidization should be considered as the most common sympatric speciation mechanism Otto and Whitton, According to Thompson and Lumaret , the dynamics of polyploid plants is based on three processes: the origin polyploidization events , the establishment and the persistence of new polyploids.
The mechanisms leading to polyploidy were variously discussed during the s, and for a long time chromosome doubling was considered the major cause. Most authors e. Stebbins, considered that meiotic restitution played only a minor role in the evolution of polyploid complexes. However, Harlan and De Wet argued that spontaneous chromosome doubling must be relatively rare in nature while polyploidization arising from 2 n gametes seems quite common.
Bretagnolle and Thompson, ; Ramsey and Schemske, , However, some higher euploid genotypes have been found in the citrus germplasm. The most common euploid variations are triploid and tetraploids Lee, Despite the scarcity of polyploid genotypes in citrus germplasm banks, it appears that polyploidization events are relatively frequent when seedling populations are analysed.
It appears that most of the spontaneous triploids arising from diploid parents are found in small and abnormal seeds Esen and Soost, , ; Geraci et al.
Therefore, 2 n gametes cannot have played an important role in citrus evolution. In citrus germplasm, apomictic nucellar polyembryony and non-apomictic genotypes are found Frost and Soost, In non-apomictic citrus genotypes, cases of sexual polyembryony have been reported, probably originating by cleavage of the zygotic embryo or from two or more functional embryo sacs in a single ovule Frost, ; Bacchi, ; Aleza et al.
The majority of citrus genotypes are apomictic, with the exception of all citron C. Tetraploidization seems to occur frequently in apomictic citrus genotypes. In these pioneering studies, ploidy variation was estimated by observation of morphological traits and some tetraploids were confirmed by chromosome counts.
The reliability of the estimated rates of tetraploidy is thus questionable. Chromosome doubling in somatic tissues was observed by Raghuvanshi ; however, very few tetraploid budsports have been identified as a consequence of unfavourable competition between diploid and tetraploid cells in the meristem Iwamasa et al. Esen and Soost suggested that they originated from unreduced gametes fertilized by diploid pollen. However, it appears that most of the natural tetraploid lines of citrus arise from apomictic genotypes.
Frost and Soost and Kobayashi et al. However, this hypothesis was not formally demonstrated for a large range of genetic diversity. Today, there is renewed interest in citrus tetraploid lines as parents for seedless triploid breeding programmes for a bibliographic review see Ollitrault et al. Several research groups are working to expand the tetraploid gene pool by somatic hybridization Grosser et al. Within this context, the origin and genetic structure of tetraploid seedlings must be clearly identified for their rational use in citrus breeding programmes.
The aim of the present work was to answer three basic questions. The taxonomic classification of Swingle and Reece is used in this paper. Seeds of seven apomictic genotypes including one sour orange C. Seedlings were grown as described above. For this experiment, tetraploid rates were evaluated in three samples of 80 seeds from each geographical origin, except for the South African origin, for which three samples of seeds were used.
Meteorological data were collected from the different sites during the period covering floral induction and the flowering period August—November for the Southern Hemisphere and February—May for the Northern Hemisphere.
Origin of the seeds used for the analysis of environmental influence and mean temperatures during the blooming period on tetraploidization. Ploidy level was determined by flow cytometry according to the methodology described by Aleza et al.
Each sample comprised a small piece of leaf of the analysed plant approx. For each genotype, one of the seedlings identified as tetraploid was subjected to detailed analysis. Ten samples of four mixed leaves harvested from the entire canopy of the tetraploid plants were studied without a diploid control to check their possible cytochimeric nature.
The preparations were then treated for 20 min in 5 m HCl and washed with distilled water. Finally, the tissue was deposited on microscope slides, stained with a drop of DAPI 4—6-diaminephenylindol and squashed. Observations were carried out with UV light using an E eclipse Nikon microscope. These markers display a broad distribution in the clementine genetic map Ollitrault et al. Silver staining was performed according to Benbouzas et al.
The number of heterozygous loci analysed n was recorded for each genotype. The recombination rate r for each pair of adjacent SSR markers in a single linkage group was evaluated from their genetic distance Kosambi map function in the clementine genetic map Ollitrault et al. The global probability was calculated by multiplying the probability per each linkage group.
As four of the markers used are not mapped and not taken into account, this probability is an overestimation of the risk to consider a zygotic plant as apomictic. To synthesize the genotype data, a cluster analysis was done with the Darwin v. Chi square tests of homogeneity were used to evaluate the significance of annual and genotypic factors in the genotypic and inter-annual variation of the frequency of tetraploid seedlings.
The Duncan test was used for comparisons between means. Seedlings of 30 apomictic diploid genotypes of the Citrus genus were analysed to investigate the genetic origin of tetraploid seedlings. Tetraploid seedlings were obtained from fruits producing a majority of diploid seedlings. For 15 genotypes, a more detailed analysis of the ploidy level of plantlets arising from seeds producing at least one tetraploid seedling was performed.
Twenty-eight seeds produced only one tetraploid plant. Number of seeds that germinated producing different combinations of tetraploid and diploid plantlets for 15 accessions of Citrus. One tetraploid plant of each cultivar was thoroughly analysed using ten samples of four mixed leaves coming from different branches covering the entire canopy.
All the samples analysed displayed a single peak corresponding to the tetraploid level. This result demonstrates that these plants were not chimeric for ploidy level Fig. All the tetraploid plants and their parental diploid lines were characteriZed by using 24 SSR markers.
These SSR markers showed considerable polymorphism among the parental lines and the observed segregation confirmed the monolocus status of the markers and thus the heterozygosity of the cultivars displaying two allelic bands. For example, all the cultivars analysed in Fig. According to positions of the heterozygous markers for each parental genotype, we have estimated the probability of a diploid zygotic seedling from self-fertilization being identical to the mother plant.
This is also the rate for a potential doubled diploid zygotic plant. Regardless, the probability of a zygotic plant originating from outcrossing being identical to the mother plant would be lower than it would from selfing. All the tetraploid plants were found to be identical to their diploid parent for all the markers analysed, as shown in the neighbour-joining tree Fig.
Cluster analysis of tetraploid plants and their diploid parental lines based on 24 SSR markers: neighbour-joining analysis using simple-matching dissimilarity index. The numbers of diploid and tetraploid plants analysed are indicated, and the last number corresponds to the number of heterozygotic SSR markers used for genetic analysis. Under greenhouse conditions the rate of germination of apomictic embryos is low because many embryos are very small Fig.
These data indicate that the germination rate of both diploid and tetraploid embryos is much higher in vitro than in vivo , as expected. However, in vitro we found that 40 of 96 seeds contained at least one tetraploid embryo while only six of 73 seeds produced a tetraploid plant in the greenhouse. The number of successful tetraploidization events producing tetraploid embryos per seed is therefore at least more than four times higher than that estimated by analysing seedlings produced under greenhouse conditions.
All seed containing tetraploid embryos also contained diploid embryos. Among the seeds containing tetraploid embryos, most contained a single tetraploid 25 , while ten, four and one seed contained two, three and four tetraploid embryos, respectively Fig. All the plants were identical to the parental diploid line, demonstrating they were all of nucellar origin. It is also clear that tetraploid plants were obtained from nucelli that predominantly comprised diploid cells.
Systematic analysis of all tetraploid seedlings with five SSR markers selected to be heterozygotic for each diploid parental genotype confirmed their identity with their diploid parents data not shown. Variability between genotypes for tetraploid production during the same year was highly significant. Significant variations were also observed between years for a single genotype, and consistent results were observed for all genotypes.
Tetraploid rates were highest in and were lowest for all genotypes in Given the inter-annual variation of tetraploid rates and thus the impact of environmental conditions on tetraploidization events, we compared tetraploid rates between seed samples of a single genotype, harvested in different countries.
Rates for California were also intermediate but did not differ significantly from regions producing the highest rates Valencia, Corsica and Uruguay or from South Africa. Overall, it appears that tropical regions produced fewer tetraploid seedlings than the Mediterranean and sub-tropical regions.
Moreover, tropical samples produced no more than one tetraploid plant per seed. Tetraploid seedlings may result from chromosome doubling of somatic or zygotic tissue or from the fertilization of non-reduced ovules by diploid pollen.
Non-reduced pollen seems to be extremely rare in citrus Esen and Soost, ; Luro et al. Therefore, the potential origin of the tetraploids obtained could be: 1 nucellar seedlings from fruits of tetraploid branches of the diploid mother tree produced by chromosome doubling in somatic tissues of the diploid parental tree, 2 nucellar seedlings from doubled primordium cells of nucellar tissue, 3 chromosome doubling during the nucellar embryo development, and 4 chromosome doubling of the zygotic embryo at early or later stages of development.
Flow cytometry analysis of numerous leaves and roots of tetraploid seedlings clearly demonstrated that these plants were not chimeric for ploidy. Thus the tetraploid plants did not originate by chromosome doubling at a later stage of embryo development.
Citrus taxonomy refers to the botanical classification of the species , varieties , cultivars , and graft hybrids within the genus Citrus and related genera, found in cultivation and in the wild. Citrus taxonomy is complex. Some are only selections of the original wild types, while others are hybrids between two or more ancestors. Citrus plants hybridize easily between species with completely different morphologies, and similar-looking citrus fruits may have quite different ancestries.
Obtencion de hidricos triploides de citricos
Citrus is a genus of flowering trees and shrubs in the rue family, Rutaceae. Plants in the genus produce citrus fruits , including important crops such as oranges , lemons , grapefruits , pomelos , and limes. Various citrus species have been utilized and domesticated by indigenous cultures in these areas since ancient times. From there its cultivation spread into Micronesia and Polynesia by the Austronesian expansion c. Citrus plants are native to subtropical and tropical regions of Asia, Island Southeast Asia , Near Oceania , and northeastern Australia. Domestication of citrus species involved much hybridization and introgression , leaving much uncertainty about when and where domestication first happened. It diverged from a common ancestor with Poncirus trifoliata.