Chromosome identification and nuclear architecture in triticale tritordeum F1 hybrids

In situ hybridization with cloned, repetitive DNA probes andtotal genomic DNA enables the parental origin of all chromosomesto be established in metaphases of triticale tritordeum F¹hybrids (2n=6x=42). Nuclei contain seven chromosomes of Hordeumchilense origin, seven from Secale cereale and 28 of wheat origin.When used as a probe, total genomic rye DNA labelled the ryechromosomes strongly and uniformly along their lengths, withbrighter regions coincident with the terminal heterochromatin.The probe labelled the wheat-origin chromosomes weakly and wasalmost undetectable on the H. chilense-origin chromosomes. Incontrast, under the same conditions, H. chilense DNA hybridizedstrongly to the H. chilense- and, with intermediate strength,to the S. cereale-origin chromosomes, excluding the subtelomericheterochromatin: it hybridized only weakly to the wheat chromosomes,in some experiments revealing characteristic bands on wheatchromosomes. Cloned repetitive DNA probes from rye and H. chilensewere used as probes to identify the linkage groups of all oftheir own-species chromosomes. Analysis of hybridization patternsof various probes to prophase and interphase nuclei indicatedthat there are many non-random features in the localizationof both repetitive DNA and whole chromosomes, although generalpatterns of nuclear organization have yet to emerge. Both theparticular lines used and the techniques developed here arelikely to be valuable for production and characterization ofplant breeding material. Key words: In situ hybridization, triticale, cytogenetics, plant breeding, Hordeum chilense     

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     

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Mapping and introgression of a tomato yellow leaf curl virus tolerance gene,TY-1

The whitefly-transmitted tomato yellow-leaf curl gemini-virus (TYLCV) is a major pathogen of tomatoes. The wild tomato species Lycopersicon chilense, which is resistant to the virus, was crossed to the cultivated tomato, L. esculentum. The backcross-1 selfed (BC1S1) generation was inoculated and a symptomless plant was selected. This plant was analyzed using 61 molecular markers, which span the tomato genome, to determine which L. chilense chromosome segments were introgressed. A BC2S1 population was cage-inoculated with viroliferous whiteflies (Bemisia tabaci), the natural insect vector of the virus, and subjected to RFLP analysis. Markers on chromosomes 3 and 6 were significantly associated with the level of tolerance; the association of chromosome-6 markers was further substantiated in two additional BC2S1 populations. A tolerant BC2S1 plant which was homozygous for L. chilense introgressions in chromosomes 3, 6 and 7 was crossed to generate a BC3S1 population which was planted in an infested field. A TYLCV-tolerance gene with partial dominance, TY-1, was mapped to chromosome 6; two modifier genes were mapped to chromosomes 3 and 7. Field and whitefly-mediated cage inoculations of nearly-isogenic lines in BC3S3 supported our conclusion that TY-1 is the major TYLCV-tolerance locus.     

Cloning and Characterization of the Ribosomal Protein Genes in the spc Operon of a Prokaryotic Endosymbiont of the Pea Aphid, Acyrthosiphon kondoi

To correlate a prokaryotic endosymbiont in the pea aphid, Acyrthosiphonkondoi, with the endosymbionts in related aphid species as wellas with free-living bacteria and subcellular organelles, andto study the mode of its gene expression within aphid cells,we have cloned and characterized the genes encoding ribosomalproteins S3, L16, L29, S17, L14, L24, L5, S14, S8, L6, L18,S5, L30, L15 and secretion protein Y (Sec Y) from the S10 andspc ribosomal protein gene operons of this endosymbiont. Theorganization of these genes is identical to that in Escherichiacoli, and their nucleotide sequences are highly similar (87%identity) to the corresponding E. coli genes. They are muchless similar to the corresponding chloroplast and mitochondrialgenes. The guanine plus cytosine G+C content of the genes ofthe A. kondoi endosymbiont is much higher than those of theendosymbionts in related aphid species reported so far. It appearseither that the A. kondoi endosymbiont is derived from an ancestralbacterium different from those in other aphids or that its G+Ccontent increased in a relatively short time after the evolutionarydivergence of its host.     

Molecular and cytological characterization of an extra acrocentric chromosome that restores male fertility of wheat in the msH1 CMS system

A new CMS system designated as ‘msH1’ has been reported in bread wheat using the cytoplasm of H. chilense. While testing this system in different wheat backgrounds, a highly fertile line with chromosome number 42 plus an extra acrocentric chromosome was obtained. The extra chromosome did not pair with any wheat chromosome at meiosis, and progeny from this line which lack the acrocentric chromosome showed pollen abortion and male sterility. In order to establish the origin of this chromosome, FISH using H. chilense genomic DNA as probe was used and showed that it had originated from H. chilense chromosome(s). The novel chromosome did not possess sequences similar to wheat rDNA; however, the probe pSc119.2 from S. cereale containing the 120 bp family was found to occur at the end of its long arm. Data obtained from FISH and EST molecular markers confirm that the long arm of the acrocentric chromosome is indeed, the short arm of chromosome 1H^ch from H. chilense. We suggest that the novel chromosome originated from a deletion of the distal part of the long arm of chromosome 1H^ch. Neither the 1H^chS short arm, nor the whole chromosome 1H^ch restores pollen fertility of the alloplasmic wheat. Therefore, the restorer gene on the acrocentric chromosome must be located on the retained segment from the hypothetical 1H^chL, while some pollen fertility inhibitor could be present on the deleted 1H^chL distal segment. Disomic addition of the acrocentric chromosome was obtained and this line resulted fully stable and fertile.     

Study of the three-genome hybridLycopersicon esculentum Mill. —L. chilense Dun. —L. peruvianum var ‘humifusum’ Mill. and its use as a source for resistance

L. peruvianum var humifusum is reproductively the most isolated of the species of the genusLycopersicon. It can be crossed with the cultivated tomato usingL. chilense as an intermediary. After a series of backcrosses of the three-genome hybrid F₁ (L. esculentum ×L. chilense) ×L. peruvianum var humifusum withL. esculentum, accompanied by selection for resistance to some economically important diseases, several lines were established. One of these lines, Cm 180, which showed resistance toClavibacter michiganensis subsp.michiganensis, was subjected to genetic analysis. This resistance was found to be controlled by a single dominant gene (Cm) that was not allelic to the gene originating fromL. hirsutum f.glabratum. ThisCm gene was genetically mapped on chromosome 4. The germ plasm ofL. peruvianum var humifusum in combination withL. chilense was transferred intoL. esculentum. Different breeding lines possessing resistance to various diseases and pests could be developed from this material.     

Inheritance and genetic mapping of cucumber mosaic virus resistance introgressed from Lycopersicon chilense into tomato

Cucumber mosaic virus (CMV) infects a wide variety of crop plants and in tomato (Lycopersicon esculentum Mill.) causes significant economic losses in many growing regions, particularly the Mediterranean. The objective of the present study was to identify the number and map locations of genes controlling resistance to CMV in breeding lines (BC₁–inbreds) derived from the related wild species L. chilense. These lines also carried the gene Tm-2 ^a for resistance to ToMV, which facilitated the interpretation of disease symptoms. The segregation for CMV resistance in the BC₂F₁ and BC₂F₂ generations, following mechanical inoculation with subgroup-I isolates, was consistent with expectations for a single dominant gene, for which the symbol Cmr (cucumber mosaic resistance) was given. Resistant and susceptible BC₁-inbreds were analyzed with RFLP and isozyme markers to identify genomic regions introgressed from L. chilense. The only L. chilense-specific markers found were on chromosome 12; some resistant lines contained a single introgression comprising the entire short arm and part of the long arm of this chromosome, while others contained a recombinant derivative of this introgression. The chromosome 12 markers were significantly associated with CMV resistance in both qualitative and quantitative models of inheritance. The qualitative analysis, however, demonstrated that CMV resistance was not expressed as a reliable monogenic character, suggesting a lack of penetrance, significant environmental effects, or the existence of additional (undetected) resistance factors. In the quantitative analysis, the marker interval TG68 – CT79 showed the most significant association with CMV resistance. No association between CMV resistance and the Tm-2 ^a gene was observed. These breeding lines are potentially useful sources of CMV resistance for tomato improvement, in which context knowledge of the map location of Cmr should accelerate introgression by marker-assisted selection. Received: 9 August 1999 / Accepted: 22 December 1999     

Phylogenetic Analysis of Molecular Lineages in a Species-Rich Subgenus of Sea Stars (Leptasterias Subgenus Hexasterias)

Among diverse marine invertebrate taxa, the evolution of a non-planktotrophiclarva is often associated with increased rates of cladogenesis,compared to related taxa that retain the ancestral planktotrophiclarval form. Molecular phylogenetic analyses of non-planktotrophic(and, presumably species-rich) clades of marine invertebratesare rare. Here we analyze 1542 base pairs of mitochondrial DNAsequence comprising two gene regions, the cytochrome oxidaseI gene and the putative control region and flanking sequences,for 23 molecular lineages in the obligately brood-protectingasteroid genus Leptasterias. Using maximum likelihood, minimumevolution, and maximum parsimony methods, five major cladeswere identified that corresponded to five taxa (species or speciescomplexes) in the subgenus Hexasterias, section camtschatica(following the taxonomy of Walter K. Fisher). Two clades (L.aequalis> complex and L. aleutica/L. camtschatica complex) werecomposed of numerous molecular lineages (7–8 lineages/clade),and several clades had multiple shallow nodes, suggestive ofrecent radiations. Two of the clades (L. aleutica/L. camtschaticacomplex and L. hexactis complex), with geographic ranges restrictedto latitudes higher than 48°N, were lacking deep phylogeneticnodes. This pattern is consistent with the hypothesis that high-latitudetaxa have high rates of extinction due to repeated climaticcrises. A log-likelihood ratio test performed on the camtschaticasection, including a member (Leptasterias polaris) of the polarissection and using a representative (L. mülleri) of Leptasteriassubgenus Leptasterias as an outgroup, demonstrated that thecamtschatica section is monophyletic.     

Expression of Vernalization Genes in Near-lsogenic Wheat Lines: Duration of Vernalization Period

Vernalization periods ranging from 0 to 11 weeks at 4 °Cwere used to study the reproductive development of four near-isogeniclines of wheat (Triticum aestivum L. em. Thell). From the resultsfor days to anthesis two types of gene action were identified,a threshold (all-or-nothing) response (vrn3 and/or vrn4) anda cumulative (graded) response (vrn1). The action of anothergene (vrn2) intensified these two responses. Based on the actionof genes, a model relating days to anthesis to genotype wasderived. Final leaf number and days to anthesis were shown tobe closely related after adjusting for differences due to theduration of vernalization treatment. No relationship betweendays to anthesis and spikelet number was observed. This studyemphasises the need to understand vernalization at the levelof the gene in terms of responses and interactions. Such knowledgeshould enable the plant breeder to predict and more preciselycontrol reproductive development. Triticum aestivum L., wheat, vernalization, gene action, isogenic lines     

Cell Cycle Timing of Replication of the Nuclear Gene Encoding Chloroplastic NADP-Specific Glutamate Dehydrogenase Isoenzymes in Chlorella sorokiniana

In synchronized Chlorella sorokiniana cells, the NH₄⁺ inducibleNADP-specific glutamate dehydrogenase enzyme (NADP-GDH) accumulatedin a linear manner throughout the first cell cycle. Early inthe following second cell cycle, an increase in its rate ofaccumulation occurred that was proportional to the increasein total cellular DNA in the previous cell cycle. In synchronizedbacterial cells, increases in rate of linear accumulation ofinducible enzymes coincide with the time of replication of theirstructural genes. To determine whether the rate change in NADPGDHaccumulation resulted from a delay in replication of its nuclearstructural gene (gdhN) in fully induced C. sorokiniana cells,the cell cycle timing of replication of this gene was comparedto that of another nuclear gene, nitrate reductase (nia), andof a chloroplast gene, ribulose bisphosphate carboxylase large-subunit(rbcL), in synchronized cells cultured in NH₄⁺ or NO₃^–(uninduced) medium. The gdhN and nia genes replicated withinthe period of nDNA synthesis and rbcL within the period of ctDNAsynthesis in cells growing in either nitrogen source. Therefore,the delayed rate change in enzyme accumulation results froma process that regulates expression of the gdhN gene after itsreplication. (Received July 16, 1994; Accepted November 28, 1994)     

In situPCR on Plant Material with Sub-cellular Resolution

In situPCR and reverse transcribedin situPCR have been testedon leaves of sugar cane fixed in FAA, 4% PFA or 2% PFA+2.5%GA.In situPCR amplification of the gene coding for ribulose-1,5-bisphosphatecarboxylase/oxygenase (rbcL) was successfully performed followingall three fixation protocols. As expected the PCR product wasrestricted to the plastids of all cells. Reverse transcribedinsituPCR was performed onrbcL mRNA and in this case the PCR productwas restricted to the plastids of the bundle sheath cells. Thisis the first report ofin situPCR on plant material and onlythe second report ofin situPCR with sub-cellular resolution.InsituPCR onrbcL may prove to be a valuable positive control forfuturein situPCR studies on plant material.Copyright 1997 Annalsof Botany Company In situPCR; reversed transcribedin situPCR; rbcL; C₄-plants     

Tissue-Dependent Expression of the Rice wx+ Gene Promoter in Transgenic Rice and Petunia

The waxy (wx) locus, which controls the amylose synthesis, isknown to be expressed specifically in the endosperm and pollen.To study the tissue-specific regulation of the wx⁺ gene, weintroduced a fusion gene that consisted of the upstream sequenceof the wx⁺ gene and the gene for rß-glucuronidase(GUS) into cells of rice (Oryza sativa L.) and petunia (Petuniahybrida L.). GUS activity was examined in the regenerated transgenicrice and petunia plants. In transgenic rice, the upstream sequenceof the wx⁺ gene was sufficient to direct the tissue-specificexpression of GUS in the endosperm and pollen, and the controlof expression was quantitative. By contrast, in transgenic petunia,the same fusion gene was expressed in pollen but not in theendosperm. These results suggest that the putative cis-actingelements that direct pollen-specific expression are common toor similar in both monocotyledonous and dicotyledonous plants,whereas ciy-elements responsible for the endosperm-specificexpression of the rice wx⁺ gene do not function in petunia,in which development of the endosperm differs from that in rice. ⁴Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan     

Differences in Esterase Distribution Detected by Electrophoresis as Evidence of Continuing Interspecific Hybridization in the Genus Senecio

The distribution of esterases of different mobility, locatedby acrylamide gel electrophoresis, was compared for differentpopulations of plants of the genus Senecio in Central Scotland.Evidence was obtained of continued interspecific hybridization,with gene flow from Senecio squalidus L. to Senecio vulgarisL.     

Morphological Description of Transgenic Soybean Chimeras Created by the Delivery, Integration and Expression of Foreign DNA Using Electric Discharge Particle Acceleration

Transgenic soybean (Glycine max L.) plants derived from electricdischarge particle acceleration experiments exhibited varyingdegrees of chimerism which was followed by the expression ofthe introduced ß-glucuronidase (gus) gene. Degreesof chimerism in transgenic plants were established by determiningexpression of the gus gene observed as blue spots, streaks orsectors in stem and leaf tissues in in vitro grown plantletsand greenhouse plants. Clonal plants were also obtained. Presenceof the gene was confirmed by Southern blot analysis. These studiespermitted the reconstruction of a partial picture for the developmentof the soybean plant. Glycine max L. cv. Williams 82, soybean, transformation, ß-glucuronidase, chimeric plant phenotypes, development     

Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense

We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24 943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.     

Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense

We have identified one osmotic stress- and abscisic acid-responsive member of the endochitinase (EC 3.2.1.14) gene family from leaves of drought-stressed Lycopersicon chilense plants, a natural inhabitant of extremely arid regions in South America. The 966-bp full-length cDNA (designated pcht28) encodes an acidic chitinase precursor with an amino-terminal signal peptide. The mature protein is predicted to have 229 amino acid residues with a relative molecular mass of 24 943 and pI value of 6.2. Sequence analysis revealed that pcht28 has a high degree of homology with class II chitinases (EC 3.2.1.14) from tomato and tobacco. Expression of the pcht28 protein in Escherichia coli verified that it is indeed a chitinase. Northern blot analysis indicated that this gene has evolved a different pattern of expression from that of other family members reported thus far. It is highly induced by both osmotic stress and the plant hormone abscisic acid. Southern blot analysis of genomic DNA suggested that the pcht28-related genes may form a small multigene family in this species. The efficiency of induction of the gene by drought stress, in leaves and stems, is significantly higher in L. chilense than in the cultivated tomato. It is speculated that, besides its general defensive function, the pcht28-encoded chitinase may play a particular role in plant development or in protecting plants from pathogen attack during water stress.     

Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines. Received: 20 November 2000 / Accepted: 12 April 2001