Open Access Open Badges Research article

Cytological and molecular characterization of three gametoclones of Citrus clementina

Maria Antonietta Germana1, Pablo Aleza9, Esther Carrera2, Chunxian Chen103, Benedetta Chiancone1, Gilles Costantino7, Dominique Dambier4, Xiuxin Deng5, Claire T Federici6, Yann Froelicher4, Wenwu Guo5, Victoria Ibáñez2, José Juárez9, Kevin Kwok6, François Luro7, Marcos A Machado8, Miguel Angel Naranjo2, Luis Navarro9, Patrick Ollitrault4, Gabino Ríos2, Mikeal L Roose6, Manuel Talon2, Qiang Xu5 and Fred G Gmitter3*

Author Affiliations

1 Università degli Studi di Palermo, Dipartimento di Scienze Agrarie e Forestali, Viale delle Scienze, 11, Palermo 90128, Italy

2 IVIA, Centro de Genómica, Moncada, Valencia, Spain

3 University of Florida, Citrus Research and Education Center, Lake Alfred, FL, USA

4 CIRAD, Département “Systèmes Biologiques” Unité de Recherche ‘Multiplication Végétative’ Montpellier, Paris, France

5 Huazhong Agricultural University, Wuhan, Hubei, China

6 University of California, Department of Botany and Plant Sciences, Riverside, CA, USA

7 INRA, UR GEQA, San Giuliano, France

8 Instituto Agronômico de Campinas, Centro APTA Citros Sylvio Moreira, Cordeirópolis, SP, Brazil

9 IVIA, Centro de Proteccion Vegetal y Biotecnologia, Moncada, Valencia, Spain

10 USDA-ARS, Southeastern Fruit and Tree Nut Research Laboratory, Byron, GA, USA

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BMC Plant Biology 2013, 13:129  doi:10.1186/1471-2229-13-129

Published: 10 September 2013



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).


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.


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.

Anther culture; Gynogenesis; Gametoclonal variation; Genome sequencing