Cytological and molecular characterization of three gametoclones of Citrus clementina
- Maria Antonietta Germana1,
- Pablo Aleza9,
- Esther Carrera2,
- Chunxian Chen3, 10,
- 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 GmitterJr3Email author
© Germana et al.; licensee BioMed Central Ltd. 2013
Received: 17 April 2013
Accepted: 24 August 2013
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.
KeywordsAnther culture Gynogenesis Gametoclonal variation Genome sequencing
Haploid plants or their derivatives, e.g. doubled haploid (DH) or tri-haploid (TH), are valuable in conventional breeding and genetic studies. However, most Citrus genomes are highly heterozygous, and it is practically impossible to develop homozygous lines through conventional hybridization, due to sexual incompatibility, nucellar embryony, severe inbreeding depression, and long juvenility. Gametic embryogenesis is a single-step approach to produce homozygous clones from heterozygous parents [1–7], from which most Citrus haploids were generated.
In situ parthenogenesis induced by irradiated pollen, followed by in vitro embryo culture has been reported in Citrus[8–11]. Haploid plantlets have been recovered by anther culture from Poncirus trifoliata L. Raf.  and C. madurensis Lour. . A doubled haploid plantlet has been obtained from the hybrid No. 14 of C. ichangensis × C. reticulata. Haploid plantlets and highly embryogenic haploid calli were recovered from C. clementina Hort. ex Tan. [15–17]. Haploid, but albino embryos, arose from cultures of ‘Mapo’ tangelo (C. deliciosa × C. paradisi) . In other reports, haploid and diploid calli, embryos and leafy structures, but no green plants, were produced from culture of C. limon L. Burm. f. anthers . Haploid embryos of Clausena excavata and homozygous short-lived plantlets of Rhode Red Valencia sweet orange  have also been reported.
The objective of this work was to elucidate the effect of haploidization in the genome structure of three different gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules. To compare their genomes, the three gametoclones obtained by gynogenesis or by pollen embryogenesis, were freely provided by research groups in Spain (Navarro), France (Ollitrault), and Italy (Germanà). The tissues and DNA samples from the three candidate plants have been analyzed and characterized using various technologies and methods by laboratories in several institutions worldwide to assure that they are free of large deletions or other defects, as well as to confirm their homozygosity (mono-allelic at any locus analyzed). Specifically, candidate tree chromosome numbers were verified for their ploidy levels. The candidate genomes were evaluated using genomic or EST-derived SSR markers and microarray technology. The collaborative results on the three materials are reported here in detail.
Three gametoclonal plants, designated ESP, FRA, and ITA respectively acquired in the lab of Navarro (Spain), Ollitrault (France), and Germanà (Italy), were all derived from Citrus clementina Hort. ex Tan., cv. Nules and preliminarily shown to be homozygous based on some selected loci. They were obtained by in situ parthenogenesis induced by irradiated pollen followed by in vitro embryo culture, or by pollen embryogenesis. Specifically, ESP was through in vivo-induced gynogenesis by pollination of Nules Clementine with irradiated pollen of Fortune mandarin followed by in vitro embryo rescue , FRA also through gynogenesis by pollination in the field with irradiated Meyer lemon (Citrus meyeri Y. Tan.) pollen , and ITA was obtained through anther culture of C. clementina cv. Nules [15, 17]. ESP was previously characterized as a haploid . All three plants were much less vigorous than the heterozygous mother plant, as revealed by leaf size and growth habit (Additional file 1: Figure S1). Samples from all three plants were sent to the respective laboratories of the collaborators for the specific analyses to which each group had committed.
Chromosome number determination
Root- and shoot-tip chromosome counting was conducted using DAPI (4,6-diamidino-2-phenylindole) and hematoxylin staining techniques , respectively.
SSR genotyping and analysis
Summary of SSR marker analysis of haploids from Clementine
No. of PCR products in diploid clementine
No. of PCR products in ITA, ESP, and FRA
0 or 1
1 or 2
Array-CGH was performed as described in Rios et al. . Genomic DNA was isolated from leaves . Four Cy3- or Cy5-labelled samples from each gametoclonal plant were co-profiled on four 20 K Citrus cDNA microarrays containing 21240 EST, using Cy5- or Cy3-labelled control genomic DNA, respectively . To prepare labelled probes, Cy3- or Cy5-dCTP fluorescent nucleotides (Amersham Biosciences) were incorporated directly in control and gametoclonal genomic DNA (2 μg) using BioPrime Array CGH Genomic Labelling System (Invitrogen). Each pair of purified Cy5 and Cy3 probes (about 50 μl each) was combined and mixed with 30 μg Cot-1 DNA (Invitrogen), 100 μg yeast tRNA (Invitrogen), and 346 μl TE buffer pH 7.4. Samples were concentrated with a microcon YM-30 filter (Millipore), and SSC buffer and SDS were added to reach a final volume of 60 μl containing 3.4× SSC and 0.3% SDS. For microarray hybridization, the probe mixture was denatured by heating at 97°C for 5 minutes, and immediately incubated at 37°C during 30 minutes to block repetitive DNA sequences. Hybridization mixture was applied to a 37°C pre-warmed hybrid-slip (Sigma), and a pre-warmed array slide was lowered onto the mix. Microarrays were hybridized in darkness at 65°C overnight (16–20 hours) using a glass array cassette following manufacturer’s instructions (Ambion). To prevent evaporation of hybridization solution during incubation, 5 μl of 3× SCC were poured into the reservoir inside the cassette chamber. Following hybridization, microarray slides were placed in a rack and the cover slip removed by 10 minutes immersion in a washing chamber containing 2× SSC and 0.03% SDS at room temperature (RT). Microarray slides were passed through a series of washes on a shaking platform. Wash series were as follow: 2× SSC, 0.03% SDS for 5 min at 65°C, followed by 1× SSC for 5 min at RT, and 3× 15 min washes in 0.2× SSC at RT. Microarray slides were dried by centrifugation for 5 min at 300 rpm. Arrays were immediately scanned at 5 μm. Cy3 and Cy5 fluorescence intensity was collected by using a ScanArray Gx (Perkin Elmer). The resulting images were overlaid and spots identified by the ScanArray Express program (Perkin Elmer). Spot quality was first measured by the signal-to-background method with parameters lower limit (200) and multiplier (2), and subsequently confirmed by visual test. The results were normalized for labeling and detection efficiencies of the two fluorescence dyes, prior to determining differential gene expression between haploid and diploid citrus samples. Intensities of selected spots were transformed into log2 (Cy3/Cy5) and data were normalized by the locally weighted linear regression (LOWESS) method. Genespring vs 7.3 software (Agilent, http.//http://www.agilent.com) was used to normalize values for each gene and for data analysis. Differentially regulated genes were ranked on the basis of signal intensity, normalized ratio, flag value and variance across 4 replicate experiments. Filtered genes identified to be differentially expressed by haploid/diploid signals lower than 0.3 with a P-value not higher than 0.05 were considered for subsequent gene dosage measurements. One-way ANOVA, parametric test without the assumption of equal variances was used to define differentially expressed genes.
Gene dosage measurement
Primer sequences for each gene
Results and discussion
SSR marker analysis
A total of 237 SSR markers were selected, in many cases from previous mapping exercises, to represent as broad and unbiased coverage of the citrus genome as possible, and plant materials were genotyped (Table 1). No SSR alleles were detected in the gametoclones that were not present in diploid Clementine. At 232 loci the three gametoclones had one SSR allele also found in diploid Clementine. The gametoclones had the same allele as diploid Clementine at 45 of the 47 loci tested at which Clementine appeared homozygous. At two “anomalous” loci, the Clementine allele was observed in one or more of the gametoclones, and no allele was observed in the others. These two loci segregated for a null (no amplification) allele in a Clementine hybrid population, so these markers are also consistent with all gametoclones having complete, homozygous genomes. The gametoclones contained one of the two Clementine alleles at 183 of the 187 loci that were heterozygous in diploid Clementine. For three loci, the same two PCR products amplified from diploid Clementine were also observed in one or more of the gametoclones. Segregation of one of these loci was studied in a Clementine hybrid population and it was shown that these two PCR products segregated as a single Mendelian unit. This pattern could be caused by a tandem duplication of the amplified region, or by annealing of one PCR primer to nearly adjacent sites in the template DNA. Segregation of the other two loci has not been examined, but they could be explained possibly by a similar mechanism. Only one SSR locus revealed anomalous results in FRA, while the remainder revealed only a single allele product at all other loci surveyed.
Underrepresented ESTs after array-CGH of haploid genomic DNA
Normalized ratio (*)
Normalized ratio (*)
Normalized ratio (*)
No annotation available
No annotation available
Cu/Zn-superoxide dismutase copper chaperone
Cu/Zn-superoxide dismutase copper chaperone
Thaumatin-like protein isoform 3
Nematode resistance-like protein
No annotation available
No annotation available
Copine I-like protein
No annotation available
No annotation available
No annotation available
Gene dosage experiment
In this study, chromosome counting confirmed that ESP and FRA were haploid and ITA tri-haploid. Among a total of 237 SSR markers, most were selected from previous mapping exercises and represented broad and unbiased coverage of the citrus genome. 231 markers detected a single allele in ITA, ESP and FRA; each allele also existed in the diploid Clementine genome. Of the six SSR loci with anomalous results, segregation in Clementine was studied for three loci and in these cases the anomalous results in the haploids were shown to be caused by similar anomalies in Clementine. The array-CGH experiment revealed that only 13 cDNAs had anomalous results among more than 20,000 cDNAs on the array. After real-time PCR of 7 of these genes, only four showed a gene dosage close to zero in one or two candidates, so no relevant gene loss was detected in any of the three genomes. Consequently array-CGH, in addition to all other characterization methods employed, provided compelling evidence that haploidization of citrus through in situ parthenogenesis induced by irradiated pollen followed by in vitro embryo culture, or by pollen embryogenesis, does not generate substantial genome rearrangements. Therefore, these three gametoclones can be used, with no concerns regarding their genomic integrity, for genetic studies as well as for citrus improvement, for example, through di-haploidization. In addition, it is noteworthy that the conclusions reached in this study, that haploidization does not disrupt the natural citrus genome structure, provided the major basis for the selection of the target citrus genome for producing the reference sequence for the international citrus research community .
Work at the Centro de Genómica (IVIA) was supported by the Spanish Ministerio de Ciencia e Innovación-FEDER grants PSE-060000-2009-008 and IPT-010000-2010-43, and grants [AGL2011-26490] from the Ministry of ‘Economía y Competivida-Fondo Europeo de Desarrollo Regional (FEDER)’ and [PrometeoII/2013/008] from the Generalitat Valenciana, Spain. Work performed by Elena Blázquez, Ángel Boix, Isabel Sanchis is gratefully acknowledged. Work at the UF-Citrus Research and Education Center was supported by grants from the Florida Citrus Production Research Advisory Council, and the Citrus Research and Development Foundation, on behalf of the Florida citrus growers. Work at the CIRAD was supported by the French Genomic ANR 2008 CITRUSSEQ project. Work at IAC was supported by INCT Citrus by FAPESP and CNPq.
Requesting deposition of data
The array data have been submitted to ArrayExpress. Experiment name: Molecular Characterization of Three Gametoclones of Citrus clementine. ArrayExpress accession: E-MEXP-3926. Specified release date: 2104-01-10.
- Germanà MA: Anther culture for haploid and doubled haploid production. Special issue: “In vitro Ploidy Manipulation in the Genomics Era”. Plant Cell Tiss Organ Cult. 2011, 104: 283-300. 10.1007/s11240-010-9852-z. doi:10.1007/s11240-010-9852-zView ArticleGoogle Scholar
- Germanà MA: Gametic embryogenesis and haploid technology as valuable support to plant breeding. Special issue “Plant Biotechnology in support of the Millenium Development Goals”. Plant Cell Rep. 2011, 30: 839-857. 10.1007/s00299-011-1061-7.PubMedView ArticleGoogle Scholar
- Germanà MA: Haploidy. Citrus Genetics, Breeding and Biotechnology. Edited by: Khan I. Oxfordshire, UK: CABI: 2007, 167-196.View ArticleGoogle Scholar
- Germanà MA: Doubled haploid production in fruit crops. Plant Cell Tiss Org Cult. 2006, 86: 131-146. 10.1007/s11240-006-9088-0.View ArticleGoogle Scholar
- Germanà MA: Protocol of somatic embryogenesis from Citrus spp. anther culture. Protocol of Somatic Embryogenesis-Woody Plants. Edited by: Jain SM, Gupta PK. Netherlands: Dordrecht Springer: 2005, 191-207. 1-4020-2984-5View ArticleGoogle Scholar
- Germanà MA: Haploids and doubled haploids in Citrus spp. Doubled Haploid Production in Crop Plants. A Manual. Edited by: Maluszynsky M, Kasha KJ, Forster BP, Szaejko I. Netherlands: FAO-IAEA, Dordrecht Kluwer Academic Publisher: 2003, 303-308.View ArticleGoogle Scholar
- Germanà MA: Haploidy in Citrus. In vitro Haploid Production in Higher Plants volume 5th edition. Edited by: Jain SM, Sopory SK, Veilleux RE. Dordrecht, The Netherlands: Kluwer Academic Publishers: 1997, 195-217.View ArticleGoogle Scholar
- Chiancone B, Tassoni A, Bagni N, Germanà MA: Effect of polyamines on in vitro anther culture of Citrus clementina Hort ex Tan. Plant Cell Tissue and Organ Culture. ISSN: 0167–6857. 2006, 87: 145-153.View ArticleGoogle Scholar
- De Lange JH, Vincent AP: Studies on Citrus pollination using gamma irradiated pollen. South African Journal of Botany. 1988, 54: 257-264.Google Scholar
- Froelicher Y, Bassene JB, Jedidi-Neji E, Dambier D, Morillon R, Bernardini GG, et al: Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets. Plant Cell Rep. 2007, 23: 2007-Google Scholar
- Ollitrault P, Allent V, Luro F: Production of haploid plants and embryogenic calli of clementine (Citrus reticulata Blanco) after in situ parthenogenesis induced by irradiated pollen. Proceeding International Society Citriculture. South Africa. 1996, 2: 913-917.Google Scholar
- Hidaka T, Yamada Y, Shichijo T: In vitro differentiation of haploid plants by anther culture in Poncirus trifoliata (L.) Raf. Japanese Journal Breeding. 1979, 29: 248-254. 10.1270/jsbbs1951.29.248.View ArticleGoogle Scholar
- Chen Z, Wang H, Liao H: The induction of Citrus pollen plants in artificial media. Acta Genetica Sinica. 1980, 7: 189-192.Google Scholar
- Deng XX, Deng ZA, Xiao SY, Zhang WC: Pollen derived plantlets from anther culture of Ichang papeda hybrids No.14 and Trifoliate orange. Proceedings of the International Society of Citriculture. Acireale, Italy: 1992, 190-192.Google Scholar
- Germanà MA, Chiancone B, Lain O, Testolin R: Anther culture in Citrus clementina: a way to regenerate tri-haploids. Aust J Agr Res. 2005, 56: 839-845. 10.1071/AR05025. ISSN: 0004–9409View ArticleGoogle Scholar
- Germanà MA, Chiancone B: Improvement of the anther culture protocol in Citrus clementina Hort. ex Tan. Plant Cell Rep. 2003, 22 (3): 181-187. 10.1007/s00299-003-0669-7. ISSN: 0721–7714PubMedView ArticleGoogle Scholar
- Germanà MA, Wang YY, Barbagallo MG, Iannolino G, Crescimanno FG: Recovery of haploid and diploid plantlets from anther culture of Citrus clementina Hort. ex Tan. and Citrus reticulata Blanco. J Hort Science. 1994, 69: 473-480.Google Scholar
- Germanà MA, Reforgiato G: Haploid embryos regeneration from anther culture of ‘Mapo’ tangelo (Citrus deliciosa x C. paradisi). Advances in Horticultural Science. 1997, 11: 147-152.Google Scholar
- Germanà MA, Crescimanno FG, De Pasquale F, Wang YY: Androgenesis in 5 cultivars of Citrus limonL. Burm. f. Acta Horticulturae. 1991, 300: 315-324.Google Scholar
- Froelicher Y, Ollitrault P: Effects of the hormonal balance on Clausena excavata androgenesis. Proc. of the First Inter. Symp. on Citrus Biotechnology 535th edition. Edited by: Goren R, Goldschmidt EE. Eilat, Israel: Acta Horticulturae: 2000, 139-146.Google Scholar
- Cao H, Kumar Biswas M, Lu Y, Hamdy Amar M, Tong Z, Xu Q, Xu J, Guo W, Deng X: Doubled haploid callus lines of Valencia sweet orange recovered from anther culture. Plant Cell Tiss Organ Cult. 2011, 104: 415-423. 10.1007/s11240-010-9860-z.View ArticleGoogle Scholar
- Aleza P, Juarez J, Hernandez M, Pina JA, Ollitrault P, Navarro L: Recovery and characterization of a Citrus clementina Hort ex Tan Clemenules haploid plant selected to establish the reference whole Citrus genome sequence. BMC Plant Biol. 2009, 9: 110-10.1186/1471-2229-9-110. doi:10.1186/1471-2229-9-110PubMedPubMed CentralView ArticleGoogle Scholar
- Guo WW, Deng XX, Shi YZ: Optimization of electrofusion parameters and interspecific somatic hybrids regeneration in Citrus (in Chinese with English summary). Acta Bot Sin. 1998, 40: 417-424.Google Scholar
- Froelicher Y, Dambier D, Costantino G, Lotfy S, Didout C, Beaumont V, Brottier P, Risterucci AM, Luro F, Ollitrault P: Characterization of microsatellite markers in Citrus reticulata Blanco. Molecular Ecology Notes. 2008, 8 (1): 119-122.View ArticleGoogle Scholar
- Luro F, Costantino G, Billot C, Froelicher Y, Morillon R, Ollitrault P, Terol J, Talon M, Gmitter FG Jr, Chen C: Genetic maps of Clementine mandarin and intergeneric hybrid Clementine X Poncirus using genomic and EST microsatellite markers. Plant & Animal Genome XV, San Diego, CA. 2007, 13–17: 2007-Google Scholar
- Luro F, Costantino G, Argout X, Froelicher Y, Terol J, Talon M, Wincker P, Ollitrault P, Morillon R: Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other Citrus species and their effectiveness for genetic mapping. BMC Genomics. 2008, 9: 287-10.1186/1471-2164-9-287.PubMedPubMed CentralView ArticleGoogle Scholar
- Chen C, Zhou P, Choi YA, Huang S, Gmitter FG: Mining and characterizing microsatellites from citrus ESTs. Theor Appl Genet. 2006, 112: 1248-1257. 10.1007/s00122-006-0226-1.PubMedView ArticleGoogle Scholar
- Chen C, Bowman KD, Choi YA, Dang PM, Rao MN, Huang S, Soneji JR, McCollum TG, Gmitter FG: EST-SSR genetic maps for Citrus sinensis and Poncirus trifoliata. Tree Genetics and Genomes. 2008, 4: 1-10.View ArticleGoogle Scholar
- Chalhoub BA, Thibault S, Laucoou V, Rameau C, Höfte H, Cousin R: Silver staining and recovery of AFLP amplification products on large denaturing polyacrylamide gels. BioTech. 1997, 22: 216-220.Google Scholar
- Barkley NA, Roose ML, Krueger RR, Federici CT: Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). Theor Appl Genet. 2006, 112: 1519-1531. 10.1007/s00122-006-0255-9.PubMedView ArticleGoogle Scholar
- Oetting WS, Lee HK, Flanders DJ, Wiesner GL, Sellers TA, King RA: Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers. Genomics. 1995, 30: 450-458. 10.1006/geno.1995.1264.PubMedView ArticleGoogle Scholar
- Rios G, Naranjo MA, Iglesias DJ, Ruiz-Rivero O, Geraud M, Usach A, Talon M: Characterization of hemizygous deletions in Citrus using array-comparative genomic hybridization and microsynteny comparisons with the poplar genome. BMC Genomics. 2008, 9: 381-10.1186/1471-2164-9-381.PubMedPubMed CentralView ArticleGoogle Scholar
- Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: version II. Plant Molecular Biology Reports. 1983, 1: 19-21. 10.1007/BF02712670.View ArticleGoogle Scholar
- Martinez-Godoy MA, Mauri N, Juarez J, Marques MC, Santiago J, Forment J, Gadea J: A genome-wide 20 K citrus microarray for gene expression analysis. BMC Genomics. 2008, 9: 318-10.1186/1471-2164-9-318.PubMedPubMed CentralView ArticleGoogle Scholar