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PubMed2006

H22, a major resistance gene to the Hessian fly (Mayetiola destructor), is mapped to the distal region of wheat chromosome 1DS.

H22 is a major resistance gene conferring high-level of antibiosis to Hessian fly [Mayetiola destructor (Say)] larvae. It was previously assigned to wheat chromosome 1D through monosomic analysis (Raupp et al. in J Hered 84:142-145, 1993). The objective of this study was to identify molecular markers that can be used for marker-assisted selection for wheat breeding, and to further map this gene toward map-based cloning. Forty-five simple sequence repeat (SSR) and sequence-tagged site (STS) markers specific to chromosome 1D were evaluated for linkage to H22 using a segregating population consisting of 192 F(2:3) families, which were derived from the cross Tugela-Dn1 x KS85WGRC01(H22). The STS Xhor2kv and SSR Xgdm33 are two flanking markers that are tightly linked to H22 at genetic distances of 0.3 and 1.0 cM, respectively. Five other SSR markers including Xgpw7082, Xwmc147, Xcfd15, Xwmc432 and Xwmc336 were also linked to H22 at the distance from 0.8 to 20.8 cM. Analysis of Chinese Spring (CS) deletion lines revealed that all the H22-linked markers are located distal to the breakpoint of del 1DS-5, indicating that the H22 gene is located at the distal 30% region on the short arm of wheat chromosome 1D. Genomic comparison suggested that the H22 gene is located in the same or similar chromosomal region as the leaf rust resistance genes Lr21 and Lr40 on 1DS, and orthologous to the H9 gene cluster of 1AS.

Animals↗

PubMed2006

Genetic analysis of the cultivated potato Solanum tuberosum L. Phureja Group using RAPDs and nuclear SSRs.

The Solanum tuberosum L. Phureja Group consists of potato landraces widely grown in the Andes from western Venezuela to central Bolivia, and forms an important breeding stock due to their excellent culinary properties and other traits for developing modern varieties. They have been distinguished by short-day adaptation, diploid ploidy (2n = 2x = 24), and lack of tuber dormancy. This nuclear simple sequence repeat (nSSR or microsatellite) study complements a prior random amplified polymorphic DNA (RAPD) study to explore the use of these markers to form a core collection of cultivar groups of potatoes. Like this prior RAPD study, we analyzed 128 accessions of the Phureja Group using nuclear microsatellites (nSSR). Twenty-six of the 128 accessions were invariant for 22 nSSR markers assayed. The nSSR data uncovered 25 unexpected triploid and tetraploid accessions. Chromosome counts of the 102 accessions confirmed these nSSR results and highlighted seven more triploids or tetraploids. Thus, these nSSR markers (except 1) are good indicators of ploidy for diploid potatoes in 92% of the cases. The nSSR and RAPD results: (1) were highly discordant for the remaining 70 accessions that were diploid and variable in nSSR, (2) show the utility of nSSRs to effectively uncover many ploidy variants in cultivated potato, (3) support the use of a cultivar-group (rather than a species) classification of cultivated potato, (4) fail to support a relationship between genetic distance and geographic distance, (5) question the use of any single type of molecular marker to construct core collections.

Cell Nucleus↗

PubMed2006

Exploitation of pepper EST-SSRs and an SSR-based linkage map.

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR-ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum 'TF68' and C. chinense 'Habanero'. Dinucleotide SSRs and EST-SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST-SSRs were mapped onto the previously generated pepper linkage map, using 107 F(2) individuals from an interspecific cross of TF68 x Habanero. One-hundred and thirtynine EST-SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST-SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.

Capsicum↗

PubMed2006

Mapping of genes associated with leptine content of tetraploid potato.

High content of leptine glycoalkaloids present in Solanum chacoense has been associated with genetic resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]). From an unrecorded accession of S. chacoense, the North Dakota State University breeding program has developed a tetraploid genotype, ND4382-19, that contains foliar leptines. In this study, using a segregating population, ND5873 (ND4382-19 x Chipeta), and GC-MS to analyze foliar content of alkaloids, two loci, involved in the synthesis of leptines were identified. They segregated as two complementary epistatic genes that allowed the synthesis of leptinidine (Lep) and acetyl-leptinidine (AL), respectively. Partial AFLP maps for both parents were developed using 97 individuals from population ND5873. The total lengths mapped for ND4382-19 and Chipeta were 1,883 and 1,021 cM, respectively. The marker for Lep was located at the distal end of simplex-coupling linkage group R37. Expansion of the initial mapping population and analysis of Lep-containing individuals allowed us to identify the linkage group (R35) that enabled synthesis of AL. By the use of simple sequence repeat markers, linkage group R37 (Lep) and linkage group R35 (AL) have been identified as homologs of chromosomes II and VIII, respectively.

Animals↗

PubMed2006

The genetic map of finger millet, Eleusine coracana.

Restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), expressed-sequenced tag (EST), and simple sequence repeat (SSR) markers were used to generate a genetic map of the tetraploid finger millet (Eleusine coracana subsp. coracana) genome (2n = 4x = 36). Because levels of variation in finger millet are low, the map was generated in an inter-subspecific F(2) population from a cross between E. coracana subsp. coracana cv. Okhale-1 and its wild progenitor E. coracana subsp. africana acc. MD-20. Duplicated loci were used to identify homoeologous groups. Assignment of linkage groups to the A and B genome was done by comparing the hybridization patterns of probes in Okhale-1, MD-20, and Eleusine indica acc. MD-36. E. indica is the A genome donor to E. coracana. The maps span 721 cM on the A genome and 787 cM on the B genome and cover all 18 finger millet chromosomes, at least partially. To facilitate the use of marker-assisted selection in finger millet, a first set of 82 SSR markers was developed. The SSRs were identified in small-insert genomic libraries generated using methylation-sensitive restriction enzymes. Thirty-one of the SSRs were mapped. Application of the maps and markers in hybridization-based breeding programs will expedite the improvement of finger millet.

Chromosome Mapping↗

PubMed2006

Identification of QTL and association of a phytoene synthase gene with endosperm colour in durum wheat.

The yellow colour of durum wheat (Triticum turgidum L. var durum) semolina is due in part to the presence of carotenoid pigments found in the endosperm and is an important end-use quality trait. We hypothesized that variation in the genes coding for phytoene synthase (Psy), a critical enzyme in carotenoid biosynthesis, may partially explain the phenotypic variation in endosperm colour observed among durum cultivars. Using rice sequence information, primers were designed to PCR clone and sequence the Psy genes from Kofa (high colour) and W9262-260D3 (medium colour) durum cultivars. Sequencing confirmed the presence of four Psy genes in each parent, corresponding to a two member gene family designated as Psy1-1, Psy1-2 and Psy2-1 and Psy2-2. A genetic map was constructed using 155 F1-derived doubled haploid lines from the cross W9262-260D3/Kofa with 194 simple sequence repeat and DArT markers. Using Psy1-1 and Psy2-1 allele-specific markers and chromosome mapping, the Psy1 and Psy2 genes were located to the group 7 and 5 chromosomes, respectively. Four quantitative trait loci (QTL) underlying phenotypic variation in endosperm colour were identified on chromosomes 2A, 4B, 6B, and 7B. The Psy1-1 locus co-segregated with the 7B QTL, demonstrating an association of this gene with phenotypic variation for endosperm colour. This work is the first report of mapping Psy genes and supports the role of Psy1-1 in elevated levels of endosperm colour in durum wheat. This gene is a target for the further development of a molecular marker to enhance selection for endosperm colour in durum wheat breeding programs.

Alkyl and Aryl Transferases↗

PubMed2006

Genetic diversity analysis of traditional and improved Indonesian rice (Oryza sativa L.) germplasm using microsatellite markers.

The archipelago of Indonesia has a long history of rice production across a broad range of rice-growing environments resulting in a diverse array of local Indonesian rice varieties. Although some have been incorporated into modern breeding programs, the vast majority of these landraces remain untapped. To better understand this rich source of genetic diversity we have characterized 330 rice accessions, including 246 Indonesian landraces and 63 Indonesian improved cultivars, using 30 fluorescently-labeled microsatellite markers. The landraces were selected across 21 provinces and include representatives of the classical subpopulations of cere, bulu, and gundil rices. A total of 394 alleles were detected at the 30 simple sequence repeat loci, with an average number of 13 alleles per locus across all accessions, and an average polymorphism information content value of 0.66. Genetic diversity analysis characterized the Indonesian landraces as 68% indica and 32% tropical japonica, with an indica gene diversity of 0.53 and a tropical japonica gene diversity of 0.56, and a Fst of 0.38 between the two groups. All of the improved varieties sampled were indica, and had an average gene diversity of 0.46. A set of high quality Indonesian varieties, including Rojolele, formed a separate cluster within the tropical japonicas. This germplasm presents a valuable source of diversity for future breeding and association mapping efforts.

Chromosomes, Plant↗

PubMed2006

Mapping resistance to Southern rust in a tropical by temperate maize recombinant inbred topcross population.

Southern rust, caused by Puccinia polysora Underw, is a foliar disease that can severely reduce grain yield in maize (Zea mays L.). Major resistance genes exist, but their effectiveness can be limited in areas where P. polysora is multi-racial. General resistance could be achieved by combining quantitative and race-specific resistances. This would be desirable if the resistance alleles maintained resistance across environments while not increasing plant maturity. Recombinant inbred (RI) lines were derived from a cross between NC300, a temperate-adapted all-tropical line, and B104, an Iowa Stiff Stalk Synthetic line. The RI lines were topcrossed to the tester FR615 x FR697. The 143 topcrosses were scored for Southern rust in four environments. Time to flowering was measured in two environments. The RI lines were genotyped at 113 simple sequence repeat markers and quantitative trait loci (QTL) were mapped for both traits. The entry mean heritability estimate for Southern rust resistance was 0.93. A multiple interval mapping model, including four QTL, accounted for 88% of the variation among average disease ratings. A major QTL located on the short arm of chromosome 10, explained 83% of the phenotypic variation, with the NC300 allele carrying the resistance. Significant (P < 0.001), but relatively minor, topcross-by-environment interaction occurred for Southern rust, and resulted from the interaction of the major QTL with the environment. Maturity and Southern rust rating were slightly correlated, but QTL for the two traits did not co-localize. Resistance was simply inherited in this population and the major QTL is likely a dominant resistant gene that is independent of plant maturity.

Basidiomycota↗

PubMed2007

Quantitative trait loci and candidate gene mapping of aluminum tolerance in diploid alfalfa.

Aluminum (Al) toxicity in acid soils is a major limitation to the production of alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant alfalfa cultivars is one approach to overcome this constraint. Accessions of wild diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of useful genes for Al tolerance. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using restriction fragment length polymorphism (RFLP) markers and single marker analysis. This study was conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to identify simple sequence repeat (SSR) markers that flank the previously identified QTLs; to map candidate genes associated with Al tolerance from other plant species; and to test for co-localization with mapped QTLs. A genetic linkage map was constructed using EST-SSR markers in a population of 130 BC(1)F(1) plants derived from the cross between Al-sensitive and Al-tolerant genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III, explaining 38, 16 and 27% of the phenotypic variation, respectively, were identified. Six candidate gene markers designed from Medicago truncatula ESTs that showed homology to known Al-tolerance genes identified in other plant species were placed on the QTL map. A marker designed from a candidate gene involved in malic acid release mapped near a marginally significant QTL (LOD 2.83) on LG I. The SSR markers flanking these QTLs will be useful for transferring them to cultivated alfalfa via marker-assisted selection and for pyramiding Al tolerance QTLs.

Aluminum↗

PubMed2002

Microsatellite markers isolated in olive ( Olea europaea L.) are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars.

We have isolated and sequenced 52 microsatellites or simple sequence repeats (SSRs) from nearly 60 positive clones obtained from two 'Frantoio' olive genomic libraries enriched in (AC/GT) and (AG/CT) repeats, respectively. The repeat-containing fragments obtained from genomic DNA restricted with Tsp509I were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs which gave correct re-amplification in the source genome were used to assay the polymorphism of 12 olive cultivars, namely four well-known cultivars ('Coratina', 'Frantoio', 'Leccino', 'Pendolino') and eight ancient cultivars grown locally near Lake Garda ('Casaliva', 'Favarol', 'Fort', 'Grignan', 'Less', 'Raza', 'Rossanel', 'Trep'). The local cultivars were each re- presented by two to four long-lived individuals. The analysis was carried out using (33)P-labelled primers and 6% polyacrylamide sequencing gels. All except two microsatellites showed polymorphism, the number of alleles varying from 1 to 5. The average genetic diversity ( H) was 0.55. The power of discrimination ( PD) was 0.60. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individual plants of the same cultivar in four out of the eight local cultivars analysed. Several primer pairs (17%) amplified more than one locus.

Journal Article↗

PubMed2002

Chloroplast phylogeography and evolution of highly polymorphic microsatellites in lodgepole pine ( Pinus contorta).

We employed a novel set of six highly polymophic chloroplastic simple sequence repeat (cpSSR) loci to investigate the phylogeography of lodgepole pine ( Pinus contorta Dougl. Ex. Loud.), and to examine aspects of the evolutionary process operating on these repetitive DNA sequences. Chloroplast haplotypes of 500 trees, sampled throughout the range of lodgepole pine, were determined. We found a marked association of genetic distance with physical distance within the scale of 0 to 1,000 km, but no association beyond that range. Likewise, geographic clustering was observed only among recent clades in a dendrogram. These phylogeographic patterns are consistant with a rapid rangewide expansion ("big-bang") followed by recent, local population differentiation ("galaxy formation"). In support of this expansion, coalescent simulations of the genealogical process gave a long-term effective population size in the low thousands, and a time to common ancestry of about 1,500 generations (12,000 years), consistent with a post-Pleistocene population expansion as documented by previous pollen-sediment analyses. Two lines of evidence (mapping mutational events onto a phylogeny, and evaluation of observed versus expected gene diversity) suggest that five of the cpSSR loci evolve primarily by a stepwise model of evolution of single repeat changes (but with a small proportion of changes involving two or more repeats), and the coalescent simulations point to a mutation rate of about 10(-3).

Journal Article↗

PubMed2002

Microsatellites in Zea - variability, patterns of mutations, and use for evolutionary studies.

To evaluate the performance of microsatellites or simple sequence repeats (SSRs) for evolutionary studies in Zea, 46 microsatellite loci originally derived from maize were applied to diverse arrays of populations that represent all the diploid species of Zea and 101 maize inbreds. Although null phenotypes and amplification of more than two alleles per plant were observed at modest rates, no practical obstacle was encountered for applying maize microsatellites to other Zea species. Sequencing of microsatellite alleles revealed complex patterns of mutation including frequent indels in the regions flanking microsatellite repeats. In one case, all variation at a microsatellite locus came from indels in the flanking region rather than in the repeat motif. Maize microsatellites show great variability within populations and provide a reliable means to measure intraspecific variation. Phylogeographic relationships of Zea populations were successfully reconstructed with good resolution using a genetic distance based on the infinite allele model, indicating that microsatellite loci are useful in evolutionary studies in Zea. Microsatellite loci show a principal division between tropical and temperate inbred lines, and group inbreds within these two broad germplasm groups in a manner that is largely consistent with their known pedigrees.

Journal Article↗

PubMed2006

Molecular organization of 5S rDNAs in Rajidae (Chondrichthyes): Structural features and evolution of piscine 5S rRNA genes and nontranscribed intergenic spacers.

The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes.

Animals↗

PubMed2002

Molecular characterization of cytoplasmic and nuclear genomes in phenotypically abnormal Valencia orange (Citrus sinensis) + Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids.

Organelle DNA inheritance of four 10-year-old somatic hybrid trees between Valencia orange [Citrus sinensis (L.) Osbeck] and Meiwa kumquat (Fortunella crassifolia Swingle) was analyzed by cleaved amplified polymorphic sequence (CAPS) and restriction fragment length polymorphisms (RFLPs). Five chloroplast (cp) and three mitochondrial (mt) universal primer pairs were amplified, but no polymorphisms were detected. When the polymerase chain reaction products were digested by 15 restriction enzymes, four polymorphic cpDNA-CAPS and two mtDNA-CAPS markers were found. Both the cpDNA and mtDNA in the somatic hybrids were derived from Valencia orange (the embryogenic suspension parent). Genomic DNA of the somatic hybrids and corresponding parents was digested by five restriction endonucleases and hybridized with one chloroplast probe (RbcL- RbcL) and nine mitochondrial probes (coxI, coxII, c oxIII, c ob, atpA, tyr, proI, atp6 and atp9). The results indicated that three hybrid plants shared one strong cpDNA band with both parents and that the remaining one plant had two additional novel bands besides the shared band, while their mtDNA was identical to that of Valencia orange plus non-parental bands. When data on the mtDNA banding patterns were combined with observations on phenotypic performance in the field, it was found that the more complex mtDNA banding pattern coincided with increased vigor of the plant. The stability of the organelle genomes was studied by extracting the genomic DNA of one hybrid plant at monthly intervals for 1 year and then analyzing it using RFLPs. Before the dieback of the shoots, two fragments of the mtDNA were lost while the cpDNAs remained stable. Ploidy analysis by flow cytometry showed that all of the hybrids were stable tetraploids. Four simple sequence repeat primer pairs were applied to detect microsatellite alleles of the four hybrid plants, both parents and the 12 DNA samples from one plant. The results showed that all hybrids had biparental bands uniformly, which indicated that they had the same nuclear background. These results suggest that the mtDNA pattern is correlated with the phenotypic abnormality of Valencia and kumquat somatic hybrid plants and that nuclear-cytoplasm incompatibility may be the cause of dieback.

Cell Nucleus↗

PubMed2003

Genetic relatedness of Portuguese almond cultivars assessed by RAPD and ISSR markers.

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars. Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism. Out of 124 polymerase chain reaction fragments that were scored, 120 (96.8%) were polymorphic. All the plants could be discriminated and constitute a very heterogeneous group. Five unidentified almond plants found in the region of Foz Côa (north Portugal) and wild almond (P. webbii) from Italy and Spain were also included. Four main groups of plants could be distinguished: P. dulcis cultivars; one Foz Côa plant; P. webbii; and P. persica (outgroup). The segregating Foz Côa plant may represent a feral individual or a hybrid between P. dulcis and P. webbii.

Genetic Markers↗

PubMed2004

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye.

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments--cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.

Chimera↗

PubMed2004

Genetic stability of micropropagated almond plantlets, as assessed by RAPD and ISSR markers.

Almond shoots produced by axillary branching from clone VII derived from a seedling of cultivar Boa Casta were evaluated for somaclonal variation using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) analysis. To verify genetic stability we compared RAPD and ISSR patterns of plantlets obtained after 4 and 6 years of in vitro multiplication. A total of 64 RAPD and 10 ISSR primers gave 326 distinct and reproducible band classes, monomorphic across all 22 plantlets analysed. Thus, a total of 7,172 bands were generated, exhibiting homogeneous RAPD and ISSR patterns for the plantlets tested. These results suggest that the culture conditions used for axillary branching proliferation are appropriate for clonal propagation of almond clone VII, as they do not seem to interfere with the integrity of the regenerated plantlets. These results allowed us to establish the use of axillary branching plantlets (mother-plants) as internal controls for the analysis of somaclonal variation of shoots regenerated from other in vitro culture processes performed with clone VII (adventitious regeneration, regeneration from meristem culture, virus sanitation programs and genetic engineering).

Agriculture↗

PubMed2006

Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast-protoplast fusion.

Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4', 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata x C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast-protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast-protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast-protoplast fusion.

Cell Fusion↗