The Application of Fluorescence In Situ Hybridization in Different Ploidy Levels Cross-Breeding of Lily

21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH). FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids) hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid ‘Freya’ had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true.     

In Situ Localization of Parental Genomes in a Wide Hybrid

In situ hybridization enabled DNA originating from the two parentalgenomes to be distinguished in plant hybrids. A probe of biotinylatedtotal genomic DNA from Secale africanum labelled the chromosomesof S. africanum origin but not those from Hordeum chilense inroot-tip chromosome spreads of the sexual hybrid between thetwo species. Hybridization of total genomic DNA from S. africanumto DNA on filters (dot blots) confirmed the distinction betweenDNA from Hordeum and Secale. The total genomic probe hybridizedto the whole length of the chromosomes from S. africanum remarkablyuniformly, labelling both euchromatin and heterochromatin, exceptat the centromeric region. The probe binding was visualizedas a yellow colour by the fluorescein-coupled detection systemwhich contrasted with the red fluorescing counterstain of theunlabelled chromatin. The chromosomes originating from bothparents could be seen and distinguished as red and yellow fluorescenceat all stages of the cell cycle. At interphase and prophase,the chromatin originating from the two parental genomes didnot mix. Chromosomes or groups of chromosomes occupied distinctdomains and also tended to be arranged in a Rabl configurationwith the centromeres clustered at one end of the nucleus. Wepropose calling the technique using total genomic DNA as a probe‘genomic in situ hybridization.’ Hordeum chilense, Secale africanum, hybrids, genomic in situ hybridization, DNA, repetitive sequences, chromosomes, chromosome disposition, nuclear order     

LOCALIZATION OF RIBOSOMAL DNA WITHIN OOCYTES OF THE HOUSE CRICKET, ACHETA DOMESTICUS (ORTHOPTERA: GRYLLIDAE)

A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass.     

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH,PCR-RFLP and RFLP

BackgroundThe intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang''s Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.Methods/ResultsGISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.ConclusionsBoth GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.     

Genomic Characterization of Interspecific Hybrids between the Scallops Argopecten purpuratus and A. irradians irradians

The Peruvian scallop (Argopecten purpuratus) has been introduced to China and has successfully been hybridized with the bay scallop (A. irradians irradians). The F1 hybrids of these two scallops exhibited a large increase in production traits and some other interesting new characteristics. To understand the genetic basis of this heterosis, nuclear gene and partial mtDNA sequences, and genomic in situ hybridization (GISH) were employed to analyze the genomic organization of the hybrids. Amplification of the ribosomal DNA internal transcribed spacer (ITS) showed that the parental ITS sequences were present in all the hybrid individuals, illustrating that the hybrid offspring inherited nuclear DNA from both parents. Sequence analyses of the ITS region further confirmed that the hybrids harbored alleles from their parents; some recombinant variants were also detected, which revealed some alterations in the nuclear genetic material of the hybrids. The analysis of mitochondrial 16S rDNA showed that the hybrids possessed sequences that were identical to the 16S rDNA of the female parents, proving a matrilineal inheritance of mitochondrial genes in scallops. In addition, GISH clearly discriminated between the parental chromosomes and indicated a combination of haploid genomes of duplex parents in the hybrids. The genetic analyses in our study illustrated that the F1 hybrids inherited nuclear material from both parents and cytoplasmic genetic material maternally, and some variations occurred in the genome, which might contribute to a further understanding of crossbreeding and heterosis in scallop species.     

Identification of the lampbrush chromosome loops which transcribe 5S ribosomal RNA in Notophthalmus (Triturus) viridescens

The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. — The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.     

CHROMOSOMAL LOCALIZATION OF REPETITIVE DNA IN THE NEWT, TRITURUS

The repetitive DNA sequences of the newt, Triturus viridescens, have been studied by nucleic acid hybridization procedures. Complementary RNA was synthesized enzymatically from unfractionated newt DNA. This RNA hybridized strongly to the centromeric regions of both somatic and lampbrush chromosomes It also bound to other loci scattered along the lengths of the chromosomes The amplified ribosomal DNA in the multiple oocyte nucleoli was demonstrated by in situ hybridization     

Identification of the Genomic Constitution ofMusaL. Lines (Bananas, Plantains and Hybrids) Using Molecular Cytogenetics

The genomic constitutions of someMusaL. lines (bananas, plantainsand artificial hybrids) were identified using molecular cytogenetictechniques. Double targetin situDNA:DNA hybridization to chromosomespreads using as probes, total genomic DNA isolated from diploidMusalinesof known AA (labelled with biotin-11-dUTP) and BB (labelledwith digoxigenin-11-dUTP) genome constitution was carried out.The use of 60% acetic acid combined with heating over a flamegave high quality chromosome spreads free of cytoplasm forinsituhybridization. Total genomic A DNA labelled broad centromericregions of all 22 chromosomes of the diploid line, Calcutta4 (M. acuminataColla. ssp.burmanniccoides; A genome) with somechromosomes showing stronger hybridization. Labelled DNA fromthe B genome hybridized strongly to the centromeric regionsof all 22 chromosomes of Butohan 2 (M. balbisianaColla; B genome).The two satellited chromosomes of genome B labelled stronglywith genomic A DNA.In situhybridization of labelled A and Bgenomic DNA to metaphase chromosomes of triploid AAB and ABBcultivars discriminated between A and B genome chromosomes.The plantains Agbagba, Obino l'Ewai and Mbi Egome showed 22genome A and 11 genome B chromosomes while the cooking bananasBluggoe and Fougamou showed 11 genome A and 22 genome B chromosomes.Hybridization of labelled A and B genomic DNA to chromosomesof the hybrids showed that TMP2x 2829-62 has all 22 genome Achromosomes while TMPx 4698-1 has 33 genome A and 11 genomeB chromosomes.In situhybridization of labelled total genomicDNA to chromosomes has immense potential for identificationof chromosome origin and can be used to characterize cultivarsand hybrids produced inMusabreeding.Copyright 1997 Annals ofBotany Company Genomicin situhybridization; banana; plantain; hybrids; plant breeding; genome organization; biodiversity     

Distribution of 18+28S ribosomal genes in mammalian genomes

In situ hybridization with ³H 18S and 28S ribosomal RNA from Xenopus laevis has been used to study the distribution of DNA sequences coding for these RNAs (the nucleolus organizing regions) in the genomes of six mammals. Several patterns of distribution have been found: 1) A single major site (rat kangaroo, Seba's fruit bat), 2) Two major sites (Indian muntjac), 3) Multiple sites in centromeric heterochromatin (field vole), 4) Multiple sites in heterochromatic short arms (Peromyscus eremicus), 5) Multiple sites in telomeric regions (Chinese hamster). — The chromosomal sites which bind ³H 18S and 28S ribosomal RNA correspond closely to the sites of secondary constrictions where these are known. However, the correlation is not absolute. Some secondary constrictions do not appear to bind ³H ribosomal RNA. Some regions which bind ribosomal RNA do not appear as secondary constrictions in metaphase chromosomes. — Although the nucleolus organizing regions of most mammalian karyotypes are found on the autosomes, the X chromosomes in Carollia perspicillata and C. castanea carry large clusters of sequences complementary to ribosomal RNA. In situ hybridization shows that the Y chromosome in C. castanea also has a large nucleolus organizing region.     

Chromosome location of the genes for 28S, 18S and 5S ribosomal RNA in Triturus marmoratus (Amphibia Urodela)

The localization of the 28S, 18S and 5S rRNA genes in the mitotic chromosomes, and of the 5S rRNA genes in the lampbrush chromosomes of Triturus marmoratus has been studied by RNA/DNA in situ hybridization. The 28S and 18S genes are located in a subterminal position, and the 5S genes in an intermediate position, on the long arm of mitotic chromosome X. In situ hybridization on lampbrush chromosomes has shown that the 5S genes are located at or near a dense matrix loop landmark. The cytogenetic implications of these findings are briefly discussed.     

A comparative study of in situ hybridization procedures using cRNA applied to Drosophila hydei salivary gland chromosomes

The effect of different denaturation and hybridization procedures on the efficiency of in situ ³H-cRNA hybridization with DNA in the polytene chromosomes of Drosophila hydei was investigated.Denaturation of the DNA in the squash preparations with 90% formamide in 0.1 × SSC at 65 °C for 2.5 h gave a significantly higher retention of radioactivity following in situ hybridization than did denaturation by 30 sec incubation in boiling 0.1 × SSC.A comparison of the effect of various SSC concentrations in the hybridization mixture revealed that among the SSC concentrations tested, 3 × SSC or 4 × SSC gave the highest efficiency of hybrid formation.Hybridization in 50% formamide at 20 °C resulted in continuing hybrid formation over a period of 3.5 h, the majority of the cRNA/DNA hybrids being formed within the first 10 min of the incubation period. The thermal dissociation profile of in situ cRNA/DNA hybrids formed in 50% formamide, 4 × SSC at 20 °C, as determined in 0.1 × SSC indicated a T_m of 66 °C. The shape of the profile and the results of competition experiments suggested a high fidelity of base-matching in the in situ ³H-cRNA/DNA hybrids.Non-chromosomal background labeling in autoradiographs of polytene chromosomes hybridized with ³H-cRNA was effectively reduced by adding a 200–1000 fold excess of cold 28S + 18S RNA.     

Localization of repetitive DNA in the chromosomes of <Emphasis Type="Italic">Microtus agrestis</Emphasis> by means of <Emphasis Type="Italic">in situ</Emphasis> hybridization

Heterochromatin in the European field vole, Microtus agrestis, was studied using a special staining technique and DNA/RNA in situ hybridization. The heterochromatin composed the proximal 1/4 of the short arm and the entire long arm of the X chromosome, practically the entire Y chromosome and the centromeric areas of the autosomes. By using the DNA/RNA in situ hybridization technique, repeated nucleotide sequences are shown to be in the heterochromatin of the sex chromosomes.     

Development of a genomic in situ hybridization method using Technovit 7100 sections of early wheat embryo

It is difficult to observe the behavior of chromosomes in early wheat embryos because they are wrapped in several cell layers of the ovary. Here we conducted genomic in situ hybridization on sections of ovary embedded in Technovit 7100, a resinous compound suitable for in situ hybridization of mRNA in sectioned tissues. With this resin it is possible to make thin sections with high resolution, no autofluorescence, and good water permeability. These features enable histochemical study using fluorescence microscopy. We established the most suitable conditions for the denaturation of target DNA embedded in Technovit resin, and performed GISH on them. Using this method, we identified Leymus mollis chromosomes in the young ovary of F₁ hybrids between wheat and L. mollis. Furthermore, we observed the behavior of maize chromosomes in early wheat × maize hybrid embryos.     

Karyotype Analysis of Gossypium arboreum × G. bickii by Genome in situ Hybridization

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum × G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid GarboreumxG. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreumxG. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

Balbiani ring nucleotide sequences in cytoplasmic 75 S RNA of Chironomus tentans salivary gland cells

The giant puffs, the Balbiani rings (BR) 1 and 2 of Chironomus tentans polytene chromosomes synthesize large RNA molecules sedimenting at about 75S. An RNA fraction of approximately the same size is present in nuclear sap and cytoplasm. In situ hybridization of cytoplasmic 75S RNA and other electrophoretically defined cytoplasmic RNA fractions showed BR RNA to be confined to the 75S RNA, and absent in other high molecular-weight cytoplasmic RNA fractions, which indiates that BR RNA is transferred from the nucleus into the cytoplasm without an appreciable size reduction.     

Somatic hybrid plants of Nicotiana x sanderae (+) N. debneyi with fungal resistance to Peronospora tabacina

Background and Aims

The genus Nicotiana includes diploid and tetraploid species, with complementary ecological, agronomic and commercial characteristics. The species are of economic value for tobacco, as ornamentals, and for secondary plant-product biosynthesis. They show substantial differences in disease resistance because of their range of secondary products. In the last decade, sexual hybridization and transgenic technologies have tended to eclipse protoplast fusion for gene transfer. Somatic hybridization was exploited in the present investigation to generate a new hybrid combination involving two sexually incompatible tetraploid species. The somatic hybrid plants were characterized using molecular, molecular cytogenetic and phenotypic approaches.

Methods

Mesophyll protoplasts of the wild fungus-resistant species N. debneyi (2n = 4x = 48) were electrofused with those of the ornamental interspecific sexual hybrid N. × sanderae (2n = 2x = 18). From 1570 protoplast-derived cell colonies selected manually in five experiments, 580 tissues were sub-cultured to shoot regeneration medium. Regenerated plants were transferred to the glasshouse and screened for their morphology, chromosomal composition and disease resistance.

Key Results

Eighty-nine regenerated plants flowered; five were confirmed as somatic hybrids by their intermediate morphology compared with parental plants, cytological constitution and DNA-marker analysis. Somatic hybrid plants had chromosome complements of 60 or 62. Chromosomes were identified to parental genomes by genomic in situ hybridization and included all 18 chromosomes from N. × sanderae, and 42 or 44 chromosomes from N. debneyi. Four or six chromosomes of one ancestral genome of N. debneyi were eliminated during culture of electrofusion-treated protoplasts and plant regeneration. Both chloroplasts and mitochondria of the somatic hybrid plants were probably derived from N. debneyi. All somatic hybrid plants were fertile. In contrast to parental plants of N. × sanderae, the seed progeny of somatic hybrid plants were resistant to infection by Peronospora tabacina, a trait introgressed from the wild parent, N. debneyi.

Conclusions

Sexual incompatibility between N. × sanderae and N. debneyi was circumvented by somatic hybridization involving protoplast fusion. Asymmetrical nuclear hybridity was seen in the hybrids with loss of chromosomes, although importantly, somatic hybrids were fertile and stable. Expression of fungal resistance makes these somatic hybrids extremely valuable germplasm in future breeding programmes in ornamental tobacco.     

The Hybrid Origin of Two Cultivars of Crocus (Iridaceae) Analysed by Molecular Cytogenetics including Genomic Southern and in situ Hybridization

The origin of the two common cultivars of Crocus, C. 'Stellaris'(2n = 2x = 10) and C. 'Golden Yellow' (2n = 3x = 14) was investigatedby fluorescent in situ hybridization using both total genomicDNA and cloned DNA sequences as probes. The clear differentiationbetween the chromosomes after genomic in situ hybridizationsupports the proposals of a hybrid origin of the cultivars andshows that they have the same parental genomes originating fromC. flavus (2n = 8) and C. angustifolius (2n = 12). C. 'Stellaris'has four chromosomes of C. flavus origin and six chromosomesof C. angustifolius origin. C. 'Golden Yellow' has eight chromosomesof C. flavus origin and six chromosomes of C. angustifoliusorigin. The number and location of 18S-5·8S-26S rRNAgenes on the chromosomes of the hybrids and of the parentalspecies agree with the results from the genomic probings. Hybridizationto Southern membranes also supports the hybrid origin of C.'Golden Yellow'.Copyright 1995, 1999 Academic Press Taxonomy, cytology, rDNA sites, in situ hybridization, Southern hybridization, Crocus     

Molecular cytogenetic evidence of hybridization in the “purple corolla clade of the genus Capsicum” (C. eximium × C. cardenasii)

Abstract

A hybrid individual between two taxa from “the Purple Corolla clade of the genus Capsicum”, C. eximium × C. cardenasii (2n = 24) was found during a cytogenetic study of a population belonging to C. cardenasii cytotype 1, from Bolivia. 5S and 45S rDNA probes were located on mitotic chromosomes by fluorescent in situ hybridization. The hybrid haploid karyotype length was an average value of the two taxa; the hybrid presented 24 45S loci in the diploid complement, 18 45S sites belonged to C. cardenasii cytotype 2, and six came from C. eximium cytotype 2, although this cytotype of C. eximium has been mentioned only for Argentina. Whereas hybrids were previously reported in the purple flower group, their existence has not been cytologically corroborated. This finding constitutes the first molecular cytogenetic evidence of hybridization between two taxa from this group.     

Chimeric Genomes of Natural Hybrids of Saccharomyces cerevisiae and Saccharomyces kudriavzevii

Recently, a new type of hybrid resulting from the hybridization between Saccharomyces cerevisiae and Saccharomyces kudriavzevii was described. These strains exhibit physiological properties of potential biotechnological interest. A preliminary characterization of these hybrids showed a trend to reduce the S. kudriavzevii fraction of the hybrid genome. We characterized the genomic constitution of several wine S. cerevisiae × S. kudriavzevii strains by using a combined approach based on the restriction fragment length polymorphism analysis of gene regions, comparative genome hybridizations with S. cerevisiae DNA arrays, ploidy analysis, and gene dose determination by quantitative real-time PCR. The high similarity in the genome structures of the S. cerevisiae × S. kudriavzevii hybrids under study indicates that they originated from a single hybridization event. After hybridization, the hybrid genome underwent extensive chromosomal rearrangements, including chromosome losses and the generation of chimeric chromosomes by the nonreciprocal recombination between homeologous chromosomes. These nonreciprocal recombinations between homeologous chromosomes occurred in highly conserved regions, such as Ty long terminal repeats (LTRs), rRNA regions, and conserved protein-coding genes. This study supports the hypothesis that chimeric chromosomes may have been generated by a mechanism similar to the recombination-mediated chromosome loss acting during meiosis in Saccharomyces hybrids. As a result of the selective processes acting during fermentation, hybrid genomes maintained the S. cerevisiae genome but reduced the S. kudriavzevii fraction.The genus Saccharomyces consists of seven biological species: S. arboricolus, S. bayanus, S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus (29, 59) and the partially allotetraploid species S. pastorianus (46, 58).The hybrid species S. pastorianus, restricted to lager brewing environments, arose from two or more natural hybridization events between S. cerevisiae and a S. bayanus-like yeast (7, 16, 28, 46). Recent studies of S. bayanus have also revealed the hybrid nature of certain strains of this species, which has subsequently been subdivided into two groups, S. bayanus var. bayanus, containing a variety of hybrid strains, and S. bayanus var. uvarum, also referred to as S. uvarum, that contains nonhybrid strains (45, 46).New hybrids of other species from the genus Saccharomyces have recently been described. Hybrid yeasts of S. cerevisiae and S. kudriavzevii have been characterized among wine (6, 20, 33) and brewing yeasts (21); even triple hybrids of S. cerevisiae, S. bayanus, and S. kudriavzevii have been identified (20, 41).The first natural Saccharomyces interspecific hybrid identified, the lager brewing yeast S. pastorianus (S. carlsbergensis) (42, 57), has become one of the most investigated types of yeast hybrids. The genome structure of these hybrids has been examined by competitive array comparative genome hybridization (aCGH) (5, 16, 28), complete genome sequencing (28), and PCR-restriction fragment length polymorphism (RFLP) analysis of 48 genes and partial sequences of 16 genes (46). The aCGH analyses of several S. pastorianus strains with S. cerevisiae-only DNA arrays (5, 28) revealed the presence of aneuploidies due to deletions of entire regions of the S. cerevisiae fraction of the hybrid genomes. A recent aCGH analysis of S. pastorianus strains with S. cerevisiae and S. bayanus DNA arrays (16) showed two groups of strains according to their genome structure and composition. These groups arose from two independent hybridization events, and each one is characterized by a reduction and an amplification of the S. cerevisiae genome fraction, respectively.The genetic characterization of the wine S. cerevisiae and S. kudriavzevii hybrids by restriction analysis of five nuclear genes located in different chromosomes, 5.8S-ITS rDNA region and the mitochondrial COX2 gene, revealed the presence of three types of hybrids in Swiss wines, thus indicating the presence of different hybrid genomes (20). In a recent study (21), we identified six new types of S. cerevisiae and S. kudriavzevii hybrids among brewing strains, which were compared to wine hybrids by a genetic characterization based on RFLP analysis of 35 protein-encoding genes. This analysis confirmed the presence of three different genome types among wine hybrids that contain putative chimeric chromosomes, probably generated by a recombination between homeologous chromosomes of different parental origins.The aim of the present study is to investigate the genome composition and structure of wine hybrids of S. cerevisiae and S. kudriavzevii. This has been achieved by a combined approach based on the RFLP analysis of 35 gene regions from our previous study, comparative genome hybridizations using S. cerevisiae DNA macroarrays, a ploidy analysis by flow cytometry, and gene dose determinations by quantitative real-time PCR. This multiple approach allowed us to confirm the presence of chimeric chromosomes and define the mechanisms involved in their origins.