Cytological evidence for preservation of mitochondrial and plastid DNA in the mature generative cells of Chlorophytum spp. (Liliaceae)

Summary.  Following 4′,6-diamidino-2-phenylindole staining of mature pollen grains of Chlorophytum comosum, fluorescence microscopy confirmed that cytoplasmic nucleoids (DNA aggregates) were present in the generative cells, which indicated the possibility of biparental cytoplasmic inheritance. Electron and immuno-electron microscopy showed that both plastids and mitochondria were present in the generative cells, and both organelles contained DNA. These results indicate that mitochondria and plastids of C. comosum have the potential for biparental inheritance. Similar results were obtained with mature pollen grains of C. chinense. Therefore, we conclude the coincident biparental inheritance for mitochondria and plastids in the members of the genus Chlorophytum. Received June 28, 2002; accepted September 26, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: College of Life Science, Peking University, Bejing 100871, People's Republic of China.     

Paternal inheritance of mitochondria in Chlamydomonas

To analyze mitochondrial DNA (mtDNA) inheritance, differences in mtDNA between Chlamydomonas reinhardtii and Chlamydomonas smithii, respiration deficiency and antibiotic resistance were used to distinguish mtDNA origins. The analyses indicated paternal inheritance. However, these experiments raised questions regarding whether paternal inheritance occurred normally. Mitochondrial nucleoids were observed in living zygotes from mating until 3 days after mating and then until progeny formation. However, selective disappearance of nucleoids was not observed. Subsequently, experimental serial backcrosses between the two strains demonstrated strict paternal inheritance. The fate of mt+ and mt− mtDNA was followed using the differences in mtDNA between the two strains. The slow elimination of mt+ mtDNA through zygote maturation in darkness was observed, and later the disappearance of mt+ mtDNA was observed at the beginning of meiosis. To explain the different fates of mtDNA, methylation status was investigated; however, no methylation was detected. Variously constructed diploid cells showed biparental inheritance. Thus, when the mating process occurs normally, paternal inheritance occurs. Mutations disrupting mtDNA inheritance have not yet been isolated. Mutations that disrupt maternal inheritance of chloroplast DNA (cpDNA) do not disrupt inheritance of mtDNA. The genes responsible for mtDNA inheritance are different from those of chloroplasts.     

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle. Received: 7 April 1998 / Accepted: 10 June 1998     

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F₁ hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F₁ hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.     

The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance

Organellar DNA in mature pollen grains of eight angiosperm species (Actinidia deliciosa Lindl., Antirrhinum majus L., Arabidopsis thaliana (L.) Heynh., Medicago sativa L., Musa acuminata Colla, Pelargonium zonale (L.) L'Hér, Petunia hybrida Vilm. and Rhododendron mucronatum (Blume) G. Don, in which the modes of organellar inheritance have been determined genetically, was observed by fluorescence microscopy using Technovit 7100 resin sections double-stained with 4′,6-diamidino-2-phenylindole (DAPI) and 3,3′-dihexyloxacarbocyanine iodide (DiOC₆). The eight species were classified into four types, based on the presence or absence of organellar DNA in mature generative cells: namely (1) type “m+p+”, which has both mitochondrial and plastid DNA (P. zonale), (2) type “m+p–”, which only has mitochondrial DNA (M. acuminata), (3) type “m−p+”, which only has plastid DNA (A. deliciosa, M. sativa, R. mucronatum), and (4) type “m−p−”, which has neither mitochondrial nor plastid DNA (A. majus, A. thaliana, P. hybrida). This classification corresponded to the mode of organellar inheritance determined by genetic analysis. The presence or absence of mitochondrial and plastid DNA corresponded to paternal/biparental inheritance or maternal inheritance of the respective organelle, respectively. When organellar DNA was present in mature generative cells (m+ or p+), the DNA content of the organelles in the generative cells started to increase immediately after pollen mitosis one (PMI). In contrast, the DNA content of organelles in generative cells decreased rapidly after PMI when organellar DNA was absent from mature generative cells (m− or p−). These results indicate that the modes of inheritance (paternal/biparental inheritance or maternal inheritance) of mitochondria and plastids are determined independently of each other in young generative cells just after PMI. Received: 22 December 1998 / Accepted: 8 February 1999     

The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining

Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination.The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates (‘nucleoids’) which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote.Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 × as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8–16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.     

Nucleotide diversity in the mitochondrial and nuclear compartments of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: investigating the origins of genome architecture

Background  

The magnitude of intronic and intergenic DNA can vary substantially both within and among evolutionary lineages; however, the forces responsible for this disparity in genome compactness are conjectural. One explanation, termed the mutational-burden hypothesis, posits that genome compactness is primarily driven by two nonadaptive processes: mutation and random genetic drift – the effects of which can be discerned by measuring the nucleotide diversity at silent sites (π_silent), defined as noncoding sites and the synonymous sites of protein-coding regions. The mutational-burden hypothesis holds that π_silent is negatively correlated to genome compactness. We used the model organism Chlamydomonas reinhardtii, which has a streamlined, coding-dense mitochondrial genome and an noncompact, intron-rich nuclear genome, to investigate the mutational-burden hypothesis. For measuring π_silent we sequenced the complete mitochondrial genome and portions of 7 nuclear genes from 7 geographical isolates of C. reinhardtii.     

Existence of traveling wave solutions in a diffusive predator-prey model

 We establish the existence of traveling front solutions and small amplitude traveling wave train solutions for a reaction-diffusion system based on a predator-prey model with Holling type-II functional response. The traveling front solutions are equivalent to heteroclinic orbits in R ⁴ and the small amplitude traveling wave train solutions are equivalent to small amplitude periodic orbits in R ⁴ . The methods used to prove the results are the shooting argument and the Hopf bifurcation theorem. Received: 25 May 2001 / Revised version: 5 August 2002 / Published online: 19 November 2002 RID="*" ID="*" Research was supported by the National Natural Science Foundations (NNSF) of China. RID="*" ID="*" Research was partially supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada. On leave from the Department of Mathematics and Statistics, Dalhousie University, Halifax, Nova Scotia B3H 3J5, Canada. Mathematics Subject Classification (2000): 34C35, 35K57 Key words or phrases: Traveling wave solution – Wazewski set – Shooting argument – Hopf bifurcation Acknowledgements. We would like to thank the two referees for their careful reading and helpful comments.     

Mitochondrial fusion in Chlamydomonas reinhardtii zygotes

Mitochondria are dynamic organelles that were found to fuse and divide in many different cell types. Mitochondrial fusion plays important roles in maintenance of respiratory capacity, dissipation of metabolic energy, and inheritance of mitochondrial DNA. While the molecular machinery of mitochondrial fusion has been characterized in great detail in yeast and mammals, only little is known about mitochondrial fusion in higher plants and algae. We asked whether mitochondrial fusion can be observed in the unicellular green alga Chlamydomonas reinhardtii. Mitochondria were stained with fluorescent dyes in gametes, and mixing of fluorescent markers was detected by fluorescence microscopy in zygotes indicating fusion. Mitochondrial fusion was observed in wild type zygotes, and also in respiratory mutants, albeit with less efficiency. We conclude that mitochondria readily fuse in green algae.     

Complete elimination of maternal mitochondrial DNA during meiosis resulting in the paternal inheritance of the mitochondrial genome in Chlamydomonas species

Summary. The non-Mendelian inheritance of organellar DNA is common in most plants and animals. In the isogamous green alga Chlamydomonas species, progeny inherit chloroplast genes from the maternal parent, as paternal chloroplast genes are selectively eliminated in young zygotes. Mitochondrial genes are inherited from the paternal parent. Analogically, maternal mitochondrial DNA (mtDNA) is thought to be selectively eliminated. Nevertheless, it is unclear when this selective elimination occurs. Here, we examined the behaviors of maternal and paternal mtDNAs by various methods during the period between the beginning of zygote formation and zoospore formation. First, we observed the behavior of the organelle nucleoids of living cells by specifically staining DNA with the fluorochrome SYBR Green I and staining mitochondria with 3,3′-dihexyloxacarbocyanine iodide. We also examined the fate of mtDNA of male and female parental origin by real-time PCR, nested PCR with single zygotes, and fluorescence in situ hybridization analysis. The mtDNA of maternal origin was completely eliminated before the first cell nuclear division, probably just before mtDNA synthesis, during meiosis. Therefore, the progeny inherit the remaining paternal mtDNA. We suggest that the complete elimination of maternal mtDNA during meiosis is the primary cause of paternal mitochondrial inheritance. Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 901-0213, Japan.     

Linkage of a RAPD marker with powdery mildew resistance <Emphasis Type="Italic">er-1</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

The aim of this study was to investigate the inheritance of powdery mildew disease and to tag it with a DNA marker to utilize for the marker-assisted selection (MAS) breeding program. The powdery mildew resistant genotype Fallon ^er and susceptible genotype 11760-3 ^ER were selected from 177 genotypes by heavy infestation of germplasm with Erysiphe pisi through artificial inoculation The F₁ plants of the cross Fallon/11760-3 indicated the dominance of the susceptible allele, while F₂ plants segregated in 3: 1 ratio (susceptible: resistant) that fit for goodness of fitness by χ² (P > 0.07), indicating monogenic recessive inheritance for powdery mildew resistance in Pisum sativum. A novel RAPD marker OPB18 (5′-CCACAGCAGT-3′) was linked to the er-1 gene with 83% probability with a LOD score of 4.13, and was located at a distance of 11.2 cM from the er-1 gene.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.     

Alpha and beta subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ⁻) and can survive complete loss of the organellar genome (ρ^o), the genetic factor(s) that permit(s) survival of ρ⁻ and ρ^o mutants remain(s) unknown. In this report we show that a function associated with the F₁-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ɛ subunit of F₁, renders cells petite-negative. The F₁ complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρ^o cells and those impaired in electron transport. The activity of F₁ that suppresses ρ^o lethality is independent of the membrane F_o complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F₁ subunits to suppress ρ^o lethality has been achieved by simultaneous expression of S. cerevisiae F₁α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ⁻ or ρ^o mutations in S. cerevisiae. Received: 20 March 1999 / Accepted: 18 July 1999     

RFLP marker analysis supports tetrasonic inheritance in Lotus corniculatus L.

Lotus corniculatus is a tetraploid (2n=4x=24) perennial forage legume and has been reported to have tetrasomic inheritance for several traits, although it has also been reported to show disomic inheritance. Molecular markers were used to clarify whether tetrasomic inheritance, disomic inheritance, or a combination of both, was found within an F₂ population arising from a cross between two diverse L. corniculatus accessions. The inheritance of ”tetra-allelic” RFLP markers (markers with four segregating bands) indicated that disomic inheritance could not account for the phenotypic F₂ classes observed, and that only tetrasomic inheritance would explain the observed results. Goodness of fit tests for ”tetra-allelic” and ”tri-allelic” (three segregating bands) RFLP marker data suggested support for chromosomal-type tetrasomic inheritance. RFLP genotypes interpreted from autoradiographic signal intensity provided additional support for tetrasomic inheritance and the occurrence of preferential pairing between parental chromosomes. Bivalent pairing was predominant in the two parental lines and their F₁ hybrid in cytological analyses. L. corniculatus has been classified as both an autotetraploid and an allotetraploid species. RFLP evidence of tetrasomic inheritance gives support for L. corniculatus being classified as an autotetraploid species. Even though bivalent pairing occurs, as seen in other autotetraploid species, pairing between any of the four homologous chromosomes is possible. Preferential pairing in the F₁ hybrid suggests that genome differentiation appears to be minimal between homologs within an accession, while genome differentiation is greater between homologs from different accessions of this genetically diverse species. Received: 16 November 1999 / Accepted: 14 July 2000     

Inheritance of high palmitic acid content in the seed oil of sunflower mutant CAS-5

 Sunflower genotypes with increased levels of palmitic acid (C16 : 0) in the seed oil could be useful for food and industrial applications. The objective of the present study was to determine the inheritance of the high C16 : 0 content in the sunflower mutant line CAS-5 (>25% of the total oil fatty acids). This mutant was reciprocally crossed with the lines HA-89 (5.7% C16 : 0) and BSD-2-691 (5.4% C16 : 0), the latter being the parental line from which CAS-5 was isolated. No maternal effect for the C16 : 0 content was observed from the analysis of F₁ seeds in any of the crosses. The inheritance study of the C16 : 0 content in F₁, F₂ and BC₁F₁ seeds from the crosses of CAS-5 with its parental line BSD-2-691 indicated that the segregation fitted a model of two alleles at one locus with partial dominance for the low content. The analysis of the fatty acid composition in the F₂ populations from the crosses with HA-89 revealed a segregation fitting a ratio 19 : 38 : 7 for low (<7.5%), middle (7.5–15%), and high (>25%) C16 : 0 content, respectively. This segregation was explained on the basis of three loci (P1, P2, P3) each having two alleles showing partial dominance for low content. The genotypes with a high C16 : 0 content were homozygous for the recessive allele p1 and for at least one of the other two recessive alleles, p2 or p3. This model was further confirmed with the analysis of the F₃ and the BC₁F₁ generations. It was concluded that both the recessive alleles p2 and p3 were already present in the BSD-2-691 line, the allele p1 being the result of a mutation from P1. This genetic study will facilitate breeding strategies associated with the incorporation of the high C16 : 0 trait into agronomically acceptable sunflower hybrids. Received: 30 March 1998 / Accepted: 13 August 1998     

Localization of p210-related proteins in green flagellates and analysis of flagellar assembly in the green alga Dunaliella bioculata with monoclonal anti-p210

Summary.  Recently, p210 was identified as a component of the flagellar basal apparatus in the green flagellate Spermatozopsis similis. In a search for potential homologues to p210, isolated cytoskeletons of several green flagellates were probed with a monoclonal antibody, BAS4.13, against p210. In Western blots, cross-reacting bands in the molecular-mass range of 210 kDa were detected only in the quadriflagellate Spermatozopsis exsultans. As described earlier for S. similis, the flagellar transition region was decorated in Chlamydomonas reinhardtii and several other green flagellates, whereas in the marine alga Dunaliella bioculata the antigen was present in the proximal part of the axoneme. Double immunofluorescence of D. bioculata with an antitubulin antibody further revealed dotlike signals at sites where the probasal bodies are located. Since most of the antigen in D. bioculata was located in the axoneme, deflagellation offered a possibility to study the kinetics of its incorporation during flagellar regeneration. The antigen was only detected after a flagellum reached a length of 3–4 μm and its integration into the growing flagellar proceeded from proximal to distal. A similar delay in the incorporation of the antigen was also observed during flagellar assembly on new basal bodies during cell division. Thus, the antigen of BAS4.13 was incorporated late and from proximal to distal into the growing flagellum. We conclude that the pace and site by which individual proteins are integrated into the flagellum differ greatly. Received February 18, 2002; accepted May 17, 2002; published on line October 31, 2002 RID="*" ID="*" Correspondence and reprints: Botanisches Institut, Universit?t zu K?ln, Gyrhofstrasse 15, 50931 K?ln, Federal Republic of Germany     

Mitochondrial fusion and inheritance of the mitochondrial genome

Although maternal or uniparental inheritance of mitochondrial genomes is a general rule, biparental inheritance is sometimes observed in protists and fungi, including yeasts. In yeast, recombination occurs between the mitochondrial genomes inherited from both parents. Mitochondrial fusion observed in yeast zygotes is thought to set up a space for DNA recombination. In the last decade, a universal mitochondrial fusion mechanism has been uncovered, using yeast as a model. On the other hand, an alternative mitochondrial fusion mechanism has been identified in the true slime mold Physarum polycephalum. A specific mitochondrial plasmid, mF, has been detected as the genetic material that causes mitochondrial fusion in P. polycephalum. Without mF, fusion of the mitochondria is not observed throughout the life cycle, suggesting that Physarum has no constitutive mitochondrial fusion mechanism. Conversely, mitochondria fuse in zygotes and during sporulation with mF. The complete mF sequence suggests that one gene, ORF640, encodes a fusogen for Physarum mitochondria. Although in general, mitochondria are inherited uniparentally, biparental inheritance occurs with specific sexual crossing in P. polycephalum. An analysis of the transmission of mitochondrial genomes has shown that recombinations between two parental mitochondrial genomes require mitochondrial fusion, mediated by mF. Physarum is a unique organism for studying mitochondrial fusion.     

Inheritance of spontaneous male sterility in pigeonpea

 A male-sterile plant was observed in the UPAS-120 cultivar of pigeonpea (Cajanus cajan). The plant was about 5–7 days late-flowering and had white translucent anthers with complete pollen sterility. The inheritance of this spontaneous male sterility was studied in a cross involving the mutant and fertile UPAS-120, including their F₁, F₂, BC₁F₁ and BC₂F₁ generations. The results suggested that the male sterility was genetic and due to a recessive gene. Received: 12 November 1996/Accepted: 17 January 1997     

Inheritance of chloroplast and mitochondrial DNA in alloplasmic forms of the genus Daucus

The inheritance of mitochondrial (mt) and chloroplast (ct) DNA in the progeny from interspecific crosses between the cultivated carrot (Daucus carota sativus) and wild forms of the genus Daucus was investigated by analysis of mt and ct RFLPs in single plants of the parental and filial generations. We observed a strict maternal inheritance of the organellar DNAs in all interspecific crosses examined. Previous studies on putative F₂ plants from a cross between Daucus muricatus x D. carota sativus suggested paternal inheritance of ctDNA. Our reinvestigation of this material revealed that the mtDNA of the putative F₂ plants differed from the mtDNA of both putative parents. Therefore, our data suggest that the investigated material originated from other, not yet identified, parents. Consequently, the analysis of this material cannot provide evidence for a paternal inheritance of ctDNA.     

Analysis of hybrids of durum wheat with Thinopyrum junceiforme using RAPD markers

 The objective of this study was to detect the presence of alien chromatin in intergeneric hybrids of durum wheat (Triticum turgidum, 2n=4x=28; AABB genomes) with the perennial grass Thinopyrum junceiforme (2n=4x=28; J₁J₁J₂J₂) using RAPD markers. The first step was to identify amplification of species-specific DNA markers in the parental grass species and durum wheat cultivars. Initially, the genomic DNA of five grass species (Thinopyrum junceiforme, Th. bessarabicum, Lophopyrum elongatum, Leymus karataviensis and Elytrigia pycnantha) and selected durum cultivars (‘Langdon’, ‘Durox’, ‘Lloyd’, ‘Monroe’, and ‘Medora’) was screened with 40 oligonucleotide primers (nano-mers). Three oligonucleotides that amplified DNA fragments specific to a grass species or to a durum cultivar were identified. Primer PR21 amplified DNA fragments specific to each of the five durum cultivars, and primers PR22 and PR23 amplified fragments specific to each of the grass species. Intergeneric hybrids between the durum cultivars ‘Langdon’, ‘Lloyd’ and ‘Durox’ and Th. junceiforme, and their backcross (BC) progeny were screened with all 40 primers. Six primers amplified parent-specific DNA fragments in the F₁ hybrids and their BC₁ progeny. Three primers, PR22, PR23 and PR41, that amplified Th. junceiforme DNA fragments in both F₁ and BC₁ were further analyzed. The presence of an amplified 1.7-kb Th. junceiforme DNA fragment in the F₁ hybrids and BC₁ progeny was confirmed using Southern analysis by hybridization with both Th. junceiforme genomic DNA and Th. junceiforme DNA amplified with primer PR41. With the exception of line BC₁F₂ no. 5, five selfed progeny of BC₁ and a BC₂ of line 3 (BC₁F₂ no. 3בLloyd’) from a cross of ‘Lloyd’×Th. junceiforme showed the presence of the 1.7-kb DNA fragment. All selfed BC₁ and BC₂ lines retained the 600-bp fragment that was confirmed after hybridization with Th. junceiforme DNA amplified with primer PR22. Other experiments using RFLP markers also showed the presence of up to seven Th. junceiforme DNA fragments in the F₁ hybrids and their BC progeny after hybridization with Th. junceiforme DNA amplified with primer PR41. These studies show the usefulness of molecular markers in detecting alien chromatin/DNA fragments in intergeneric hybrids with durum wheat. Received: 21 November 1996 / Accepted: 21 March 1997