Identification of parental species of the Alstroemeria cv. ‘Jubilee’ using AFLP marker technique

Twelve Alstroemeria species, two hybrids, one cv. ‘Jubilee’, an anther-cultured plant from cultivar ‘Jubilee,’ and Bomarea salsilla and Leontochir ovallei (the latter two were chosen as outgroup) were evaluated using the AFLP marker technique in order to identify putative parental genotypes of the Alstroemeria cv. ‘Jubilee’ and of known interspecific hybrids, and to estimate their genetic relationships within the genus Alstroemeria. A total of 297 AFLP markers were scored by using the primer combination (E + ACCA/M + CTAG). In order to discriminate all Alstroemeria genotypes, cluster analysis (UPGMA) and principal coordinates analysis were performed. The Alstroemeria cv. ‘Jubilee’, of which the parents are unknown, had genetic distance (GD) 0.54 from Alstroemeria exserens, GD 0.57 from Alstroemeria garaventae, GD 0.62 from Alstroemeria gayana, and GD 0.66 from Alstroemeria hookeri cumminghiana. Thus, these four species are considered as putative parental genotypes. An interspecific hybrid (Alstroemeria aurea × Alstroemeria inodora), showed the smallest genetic distance from A. aurea (GD 0.56) and A. inodora (GD 0.45). The Alstroemeria ligtu group was distantly allocated from other Chilean species. We conclude that the AFLP marker technique appears to be a satisfactory tool for identifying the parental genotypes of interspecific hybrids in Alstroemeria.     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

Genetic characterization of asparagus doubled haploids collection and wild relatives

The genus Asparagus is very large consisting of around 150 species found as herbaceous perennials, tender woody shrubs and vines. The cultivated species (Asparagus officinalis L., diploid) is a highly prized vegetable, grown in different environments ranging from cool temperate zones to deserts, Mediterranean climates and tropical areas. As a consequence, Asparagus breeders have developed different cultivars that differ for their morpho-agronomic traits, habit and ploidic status (few triploid and tetraploid cultivars are used). Several breeding methods are used for developing cultivars, among which a well developed in vitro anther culture technique produces homozygous clones useful for F₁ hybrids constitution. A fluorescent based AFLP (amplified fragment length polymorphism) technique were applied with the aim to assess genetic diversity among a collection of 173 doubled haploid (DH) androgenetic clones, five Asparagus wild species and interspecific hybrids obtained among the cultivated species and two wild relatives. The average number of AFLP fragments generated per primer set was 105, varying in size from 50 to 550 bp. A total of 1054 AFLP fragments were detected, 20% of which were polymorphic. Genetic similarity based on DNA polymorphisms, showed that a few number of AFLP primer combinations are able to distinguish the cultivated DH clones from the wild species. Indeed, from one DH clone for each anther donors and the wild species were used to construct a dendrogram using Dice's coefficient and the unweighted pair group method with the arithmetic mean (UPGMA). Genetic distances among all DH clones were calculated using the C.S. Chord distance; and a neighbour-joining (NJ) consensus tree was constructed in order to support the breeder for parental genotype choice for asparagus hybrid constitution.     

Genetic characterization of different demes in Prunus persica revealed by RAPD markers

DNAs of 180 accessions in 10 demes in Prunus persica were amplified with twenty-two, 10-base primers selected from 200 arbitrary primers using Randomly Amplified Polymorphic DNA (RAPD) technology. One hundred and eighty loci were observed and recorded. With statistical analyses of the data from the study, genetic diversity of the demes was expressed as follow: yellow peach group > honey peach group > flat peach group > red leaf peach group > crisp peach group > bitao group and juicy peach group > nectarine group > shouxingtao group > weeping peach group. Genetic variations among and within groups by AMOVA analyses were 11.9, 88.1%, respectively. Demes clustered by UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5, the edible peaches of which were combined as a section, while the ornamental species were classified into separate sections. Through analyses of genetic diversity and genetic structure, the results could provide molecular biological evidence for conservation and utilization of P. persica germplasm.     

Genetic diversity and population structure of an endangered Orchid (Dendrobium loddigesii Rolfe) from China revealed by SRAP markers

Dendrobium loddigesii Rolfe is an endangered perennial herb with ornamental and medicinal value. Due to habitat deterioration and human over-exploitation, it has suffered a significant decline in abundance. Determining the level of genetic diversity and pattern of population genetic structure of this species would be helpful for its conservation and management. In this paper, sequence-related amplified polymorphism (SRAP) markers were applied to assess the level and pattern of genetic diversity in seven populations of D. loddigesii. Seventeen SRAP primer combinations generated a total of 231 clear amplification bands encompassing 187 (80.95%) polymorphic bands. A high level of genetic diversity was detected (PPB = 80.52%, H = 0.2743, I = 0.4113) at the species level. There was a moderate genetic differentiation (G_st = 0.304) among populations. Two main clusters were detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Mantel test revealed that no significant positive correlation was found between genetic distances and geographic distances (r = 0.2302; P > 0.05). Recommendations for conservation of the endangered species resources are proposed.     

Using bulked AFLP analysis to assess genetic diversity in Echinacea species

Echinacea is an allogamous genus, thus its cultivars or populations are genetically heterogeneous. Using amplified fragment length polymorphism (AFLP) to estimate the genetic diversity of Echinacea is generally limited by the large number of individual plants and the higher cost that need to be processed. In the present study, effectiveness of several sizes of DNA bulking (10, 15, 20, 25 and 30 individuals) with 20, 36 and 55 primer pairs was compared using AFLP in determining the genetic diversity of Echinacea species. The results indicated that the use of bulked DNA-based AFLP analysis by using the selected eight primer pairs was capable of detecting genetic diversity between the tested Echinacea species, provided that the potential presence of low frequency variants was ignored and a possible bias in the estimates of genetic similarity was accepted. The assessments showed that a bulk of 15 individuals could detect sufficient AFLP variations at most genomic sites. Additionally, 20 primer pairs could generate sufficient polymorphic fragments to achieve high resolving power of AFLP for the tested Echinacea species.     

Molecular polymorphism of cytoplasmic DNA in Ficus carica L.: Insights from non-coding regions of chloroplast DNA

The trnL (UAA) intron and the intergenic spacer between the 3′ exon of trnL (UAA) and trnF (GAA) sequences were used as genetic markers for differentiating Ficus carica cultivars and establishing refined genetic relationships. The study was based on 20 fig cultivars, collected from south and centre of Tunisia. Since, the intron was thought to be more variable among close relatives than is the chloroplast spacer. The size of these non-coding regions varied from 554 to 589 and from 989 to 1022 bases pairs for the intron and the combined sequences correspondingly. The average of GC content was 33.9% and 34.6% in the intron and the combined intron and spacer respectively. High values of A + T contents were detected in both data sets and may explain the high proportions of transversions founded. The observed variation pattern of plastid DNA provides evidence of an important genetic diversity. The overall transition/transversion bias (R) was 0.202 in the intron and 0.27 in the combined regions. The RI index of 0.592 indicates that these combined sequences have clearly more homoplasy then the intron (RI = 0.705) and spacer (RI = 0.777) sequences separately. Phylogenetic trees were generated based on maximum parsimony (MP) and neighbor-joining (NJ) analysis of the chloroplast sequences data. Results proved that a typically continuous genetic diversity characterizes the local fig germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical or tree sex correspondence. Although the level of apparent diversity is considerable, we may conclude that non-coding regions of chloroplast genome provide a new and practical opportunity to evaluate genetic diversity and to discriminate fig cultivars. Revealed cytoplasmic DNA markers are reliable to elaborate a molecular data base to conduct management and breeding programs on local fig germplasm.     

Phylogenetic analysis in some Diospyros spp. (Ebenaceae) and Japanese persimmon using chloroplast DNA PCR-RFLP markers

Ten universal primer pairs of chloroplast genome were used to amplify non-coding regions of chloroplast DNA (cpDNA) in 7 Diospyros L. species including 29 genotypes and approximately 20.4 kb, 12.6% of the chloroplast genome were analyzed. The amplified products were digested by seven restriction enzymes. The results showed that there were abundant polymorphisms in inter-specific cpDNA within the genus Diospyros. However, it was not observed intra-species variations in 22 tested genotypes of Diospyros kaki (Japanese persimmon), except for ‘Male strain No. 9’. Persimmon had close relationship with Diospyros lotus and Diospyros glaucifolia, but distantly with Diospyros virginiana and Diospyros rhombifolia in diagram based on principal coordinates analysis and Wagner parsimony method. The discrepancy of digesting pattern in cpDNA suggested that the genotype Jinzaoshi was distinct with the remaining Diospyros spp., which revealed that Jinzaoshi may be a new species of the genus Diospyros.     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

Aneuploid strawberry (2n = 8x + 2 = 58) was developed from homozygous unreduced gamete (8x) produced by second division restitution in pollen

Unreduced gamete formation is significant in the evolutionary development of complex polyploidy series found in wild strawberry, genus Fragaria (Rosaceae). Also, it is important for genetics and breeding in strawberry plants to elucidate the mechanism of unreduced gamete formation. The objective of this study was to search for ploidy anomalies resulting from artificial diploid × octoploid crosses, and examine the mechanism through which these unreduced gametes were produced. Five everbearing cultivars of Fragaria vesca L. diploid (2n = 2x = 14) were crossed with pollen from six June-bearing cultivars of Fragaria × ananassa Duch., octoploid (2n = 8x = 56). A total of 3000 mature seeds, 100 from each of the 30 parental combinations were sown at 23 °C/20 °C (day/night) under artificial lighting with a 16 h day. The seedlings were transplanted to pots and grown in a greenhouse. Reproductive and morphological observations, flow cytometry analyses, chromosome counts and DNA analyses using CAPS markers were performed to identify the genetic background of the offspring. Most of the seed (79%) did not germinate or died soon after germination. Of the seedlings produced, 7% seemed to be pure F. vesca based on morphological characteristics, flow cytometry analyses and chromosome counts; 14% were pentaploids (2n = 5x = 35), 0.1% were hexaploids (2n = 6x = 42), and 0.03% (one individual) was aneuploid (2n = 8x + 2 = 58). Electrophoresis banding patterns obtained by CAPS marker analysis were heterozygotic in the 8x pollen parent but homozygotic in the aneuploid progeny. Judging from the chromosome counts and the CAPS marker analysis, the aneuploid was the result of a homozygous unreduced pollen grain (8x) crossed with an incomplete chromosome compliment from the egg. Because of the homozygosity, the unreduced male gamete must have been derived from second division restitution (SDR) in the octoploid pollen parent.     

DNA content,morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus

This work investigated the fingerprinting and phenotyping of Citrus germplasm; species selected were of historical importance belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus. An integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs, was employed. We studied a core collection of 54 distinct accessions, including 43 genotypes of the Citrus species (18 species or supposed species) and 11 genotypes of the Poncirus genus, which was used as the reference outgroup. Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny. In particular, our experiments aimed at estimating the genetic variation within and the genetic relatedness among C limon (L.) Burm., C. limonimedica and C. medica L. to shed light on the hybrid origin hypothesis of C. limonimedica. The results of the multidisciplinary analyses allowed us to confirm a remarkable differentiation between Poncirus and Citrus genera and to highlight a close relationship among the three investigated Citrus species but a distinct difference between these three species and other species in the Citrus genus. RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species. Some accessions of C. medica and C. limonimedica that deviate from such a trend suggest recurrent introgression and/or hybridisation with other species of Citrus.     

Genetic relationships among nine cultivated taxa of Calliandra Benth. (Leguminosae: Mimosoideae) using random amplified polymorphic DNA (RAPD) markers

Random amplified polymorphic DNA (RAPD) marker was used to assess genetic diversity and inter-specific relationships among nine taxa of Calliandra (Leguminosae: Mimosoideae) grown in Indian gardens. DNA from leaf sample was isolated and RAPD analysis was performed using 22 primers. The genetic similarities were analyzed from the dendrogram constructed by the RAPD data using a similarity index which supported the segregation of the nine taxa of the genus into two groups; the sect. Androcallis with seven taxa, viz. C. haematocephala, C. haematocephala var. alba, C. surinamensis, C. tweedii, C. tergemina var. emarginata and C. selloi and sect. Calliandra having two species namely, C. inermis and C. calothyrsus. The intra-generic classification and phylogeny inferred from molecular markers supported the traditional classification of the genus based on morphological characters at the level of sections and series except in case of C. selloi (C. brevipes) which did not show much genetic similarity with C. tweedii and C. surinamensis; all the three species being members of the sect. Androcallis series Androcallis.     

Stoloniferous shoot protoplast,an efficient cell system in potato for somatic cell genetic manipulations

The present study reports that protoplasts isolated from stoloniferous shoots (SS) of potato represent an efficient system for somatic cell genetic manipulations. SS were established from single-node cuttings on MS medium supplemented with either 0.1 or 0.2 M sucrose (Suc), and protoplasts were isolated and cultured within the alginate strip, following an improved method. SS induced by 0.1 M Suc yielded 8–22 × 10⁵ protoplasts g⁻¹ fresh mass, with a high morphogenic competence. However, 0.2 M Suc-induced SS yielded protoplasts that contained large amounts of starch grains, resulting in their high degree of fragility, delayed cell division and poor morphogenic competence. For symmetric somatic hybridization (electrofusion) between Solanum tuberosum Gp. Tuberosum androgenic (di)haploid (2n = 2x = 24) ‘C-13’ and diploid (2n = 2x = 24) wild species S. pinnatisectum, protoplasts isolated from 0.1 M Suc-induced SS were also found to be most responsive. Out of several putative somatic hybrids, there were two tetraploids and five diploids, with 48 and 24 chromosomes, respectively at all the three shoot layers (L₁–L₃). This precluded the occurrence of mixoploidy vis-à-vis chimaerism in regenerants, as common in somatic fusion involving mesophyll protoplasts of S. pinnatisectum. Nuclear microsatellite analyses based on the two single-locus nSSR loci (STM0037 and STM2030) confirmed that one of the tetraploids was a true nuclear hybrid (heterokaryon), while the other a homokaryon of the Tuberosum parent ‘C-13’. The use of 0.2 M Suc-induced SS protoplasts for fundamental studies on tissue- and/or cell type-specific transient gene expression underlying tuberization has been discussed.     

Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It is native to Iran and spread from Iran to other areas. In this study amplified fragment length polymorphism (AFLP) was used to detect intra- and inter-population genetic diversity of pomegranate. A group of 67 accessions belonged to 4 populations from Iran was studied using eight primer combinations. A total of 221 scorable bands were amplified, of which, 118 (54.13%) were polymorphic. Resolving power (Rp) ranged from 5.70 to 9.21, and the average of polymorphism information content (PIC) per primer pair was 0.40. According to Nei's gene diversity and allelic statistics, Isfahan population had a highest genetic diversity (H = 0.3646, I = 0.5327, N_e = 1.6467). Coefficient of gene differentiation between populations (GST) was 0.124, indicated that mainly proportion of genetic variation (87.6%), was within populations and the remaining (12.4%) of the variation was among populations that, also supported by analysis of molecular variance (AMOVA). The gene flow (Nm) varied from 0.969 to 10.404 between pair-wise populations and was 3.504 among all of the populations. The Jaccard similarity coefficient between individuals ranged from 0.26 to 0.88. The UPGMA dendrogram clustered all 67 accessions into 6 groups. In some cases accessions from same region were grouped together but in most cases, there was gene exchange. To study the genetic relationships among populations, a principal coordinate analysis (PCoA) based on Nei's genetic distances was performed. Results of this study showed that AFLP marker can be a useful tool for investigating the genetic diversity of pomegranate genotypes.     

AFLP-based genetic relationships in the Mediterranean myrtle (Myrtus communis L.)

Myrtle is an important plant species of the Mediterranean maquis, and is widely exploited for its aromatic properties. It is used in Italy for the production of a typical liqueur, for cut foliage and as an ornamental pot plant. We report the use of amplified fragment length polymorphism (AFLP) profiling to estimate genetic similarities within myrtle germplasm collected from six Italian regions, and from the Botanical Gardens of six other Mediterranean countries (including the outgroup Myrtus communis subsp. tarentina). Five AFLP primer combinations identified 122 polymorphic fragments analysing 92 individual samples, most of them (56%) were informative in discriminating among the populations. The AFLP patterns indicated that the majority of the variation occurs among rather than within populations (G_ST = 0.61). A neighbour-joining (NJ) tree separated the populations into two main branches: the first one grouped some of the Italian populations with those from Spain and Portugal; the second one included a Southern Italian subcluster together with samples from Greece, Israel, France and Croatia. A principal coordinate analysis supported the two major branches identified in the NJ analysis and showed the separation of Western and Eastern Mediterranean populations along the first axis. The Italian populations did not cluster in a single clade, but rather form distinct regional groups. The present analysis suggests that Italy represents a botanical transition zone between the Western and Eastern Mediterranean region in Myrtus communis.     

Fingerprinting of three typical macrosperma Italian lentil (Lens culinaris Medik.) landraces using fluorescence-based AFLP markers

Italian lentil landraces are principally cultivated for self or local consumption. Most of them are disappearing, particularly macrosperma types by being less required by the market. A pre-requisite for the conservation and the efficient use of genetic resources is the better understanding of the extent and the distribution of the existing genetic variation, useful for future breeding programmes. Our study was undertaken to analyse and quantify the genetic diversity within and among three macrosperma Italian lentil landraces (Onano, Altamura and Villalba), using fluorescent AFLP markers. AFLP markers generated information to differentiate among closely related genotypes and group within the same cluster individuals belonging to the same landrace. The total genetic diversity (H_T), the genetic diversity within population (H_S) and the extent of differentiation between populations (D_ST) were 0.198, 0.155 and 0.043, respectively. The fixation index (G_ST = 0.219) showed that about 78% of the observed total genetic variation can be attributed to within population differences and around 22% is due to differences among populations. The gene flow estimate (N_m = 1.774) and the mean genetic distance value (0.077) suggested narrow genetic base among the analysed populations, confirming the tendency of Italian lentil landraces to group together. The present study showed that fluorescence-based AFLP technique is a biotechnological tool that can provide significant insights for research in genetic diversity of lentil landraces and their subsequent conservation and utilization in breeding programs.     

Genetic diversity of Dimocarpus longan in China revealed by AFLP markers and partial rbcL gene sequences

Amplified fragment length polymorphism (AFLP) and partial rbcL gene sequencing were used to investigate genetic diversity among various longan (Dimocarpus longan Lour) accessions as well as a presumed closely related species Dimocarpus confinis How et Ho and litchi (Litchi chinensis Sonn). No significantly shared AFLP fragment was found between the three species, indicating that D. confinis and litchi are very far in genetic distance from any longan accession studied. Partial rbcL sequences of 501 bp from the first coding site in these species were obtained, which revealed several substitutes. One such DNA base pair substitute resulted in an amino acid difference between longan and litchi. Furthermore, another 4 bp resulted in a two amino acid difference between longan and D. confinis, which was consistent with AFLP results and indicated that D. confinis should be excluded from the longan genus, Dimocarpus. Within the longan species, no DNA substitute was found. Using nine primer combinations, a total of 66 AFLP markers were obtained from 41 longan accessions. One non-Chinese longan accession ‘Miaoqiao’ was distinctly different from all other longan cultivars collected in China, indicating that more genetic resources of longan might be collected also from longan production regions outside of China. AFLP markers might be developed to identify longan cultivars as well as expedite progeny screening in breeding programs of this perennial fruit tree.     

Genetic diversity in mainland and island populations of the endangered Pancratium maritimum L. (Amaryllidaceae) in Tunisia

Tunisian Pancratium maritimum L. populations are at present endangered and represented by scattered individuals as a result of coastal habitat destruction caused by urbanization and overharvesting for its significant ornamental interest. Nineteen populations growing in mainland and island habitats were sampled for allozyme diversity to assess their genetic diversity and structuration using seven isozymes revealed by starch gel electrophoresis. The species exhibited relatively high levels of genetic diversity (the mean A_p = 1.37, P = 37.4%, and H_e = 0.100), indicates a preferentially outcrossing mating system.     

Can a post-harvest ripening treatment extend the longevity of Rhododendron L. seeds?

Pre-dehiscent capsules were collected from two Rhododendron griersonianum (Balf.f. & Forrest) trees and either immediately dried in a dry-room (15% relative humidity, 15 °C) or placed in a high humidity room (80% relative humidity, 15 °C) for 30, 60, or 90 d. Further capsules were also collected from the trees at 30 and 60 d, but seeds had been dispersed by 90 d. Seed ageing experiments (60% relative humidity, 45 °C) carried out on these seed-lots and on seeds from a further 10 Rhododendron (L.) species confirmed that short seed lifespans is a trait of the genus, with a mean P₅₀ value of ca. 20 d for this storage environment.     

Cybrid/hybrid plants regenerated from somatic fusions between male sterile Satsuma mandarin and seedy tangelos

Somatic hybridization provides an alternative for transferring mitochondria-encoded cytoplasmic male sterility (CMS). Herein, symmetric protoplast electrofusion was conducted between embryogenic callus protoplasts of Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a CMS cultivar, and mesophyll-derived protoplasts of seedy ‘Page’ tangelo [C. reticulata Blanco × (C. reticulata Blanco × C. paradisi Macf.)] or ‘Nova’ tangelo [C. reticulata Blanco × (C. reticulata Blanco × C. paradisi Macf.)], to transfer CMS trait. Flow cytometry analysis showed that 14 plants recovered from G1 + ‘Page’ tangelo that displayed typical morphological character of ‘Page’ were diploid, and 6 plants regenerated from G1 + ‘Nova’ tangelo were tetraploid. Genetic compositions of regenerated plants from the two fusions were determined by SSR, CAPS and chloroplast-SSR analysis. Cybrid nature of diploids from G1 + ‘Page’ tangelo with nuclear DNA from ‘Page’, mitochondrial DNA (mtDNA) from the G1 and chloroplast DNA (cpDNA) derived from either parent was confirmed. Tetraploid plants from G1 + ‘Nova’ tangelo were identified as somatic hybrids with random cpDNA inheritance. The regenerated cybrid and hybrid plants hold great potential for Citrus seedless breeding at diploid or triploid levels.