Fluorescence in situ hybridization mapping of Avena sativa L. cv. SunII and its monosomic lines using cloned repetitive DNA sequences.

Fluorescent in situ hybridization (FISH) employing multiple probes was used with mitotic or meiotic chromosome spreads of Avena sativa L. cv. SunII and its monosomic lines to produce physical chromosome maps. The probes used were Avena strigosa pAs120a (which hybridizes exclusively to A-genome chromosomes), Avena murphyi pAm1 (which hybridizes exclusively to C-genome chromosomes), A. strigosa pAs121 (which hybridizes exclusively to A- and D-genome chromosomes), and the wheat rDNA probes pTa71 and pTa794. Simultaneous and sequential FISH employing two-by-two combinations of these probes allowed the unequivocal identification and genome assignation of all chromosomes. Ten pairs were found carrying intergenomic translocations: (i) between the A and C genomes (chromosome pair 5A); (ii) between the C and D genomes (pairs 1C, 2C, 4C, 10C, and 16C); and (iii) between the D and C genomes (pairs 9D, 11D, 13D, and 14D). The existence of a reciprocal intergenomic translocation (10C-14D) is also proposed. Comparing these results with those of other hexaploids, three intergenomic translocations (10C, 9D, and 14D) were found to be unique to A. sativa cv. SunII, supporting the view that 'SunII' is genetically distinct from other hexaploid Avena species and from cultivars of the A. sativa species. FISH mapping using meiotic and mitotic metaphases facilitated the genomic and chromosomal identification of the aneuploid chromosome in each monosomic line. Of the 18 analyzed, only 11 distinct monosomic lines were actually found, corresponding to 5 lines of the A genome, 2 lines of the C genome, and 4 lines of the D genome. The presence or absence of the 10C-14D interchange was also monitored in these lines.     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Common wheat is one of the most important cereal crops in the world. The improvement of its yield and quality by the introduction of heterologous gene(s) is very significant. Avena sativa L. (2n = 42), belonging to the Avena tribe, possesses resistance to drought, coldness and many dis-eases. Its contents of proteins and fat in seed, especially lysine and unsaturated fatty acid are highest in crops, therefore it is regarded as healthy food. Sexual hybridization between wheat and Avena sativa…     

Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids

Genomic in situ hybridization (GISH) methods were used to detect different genome components within Brassica amphidiploid species and to identify donor chromatin in hybrids between Brassica napus and Raphanus sativus. In Brassica juncea and Brassica carinata the respective diploid donor genomes could be reliably distinguished by GISH, as could all R-genome chromosomes in the intergeneric hybrids. The A- and C-genome components in B. napus could not be clearly distinguished from one another using GISH, confirming the considerable homoeology between these genomes. GISH methods will be extremely beneficial for monitoring chromatin transfer and introgression in interspecific Brassica hybrids. Received: 20 May 1997 / Accepted: 28 July 1997     

C-Glycosylflavones from Avena sativa

Avena sativa leaves, stems and inflorescences contain a range of new C-glycosylflavone 2″-O-glycosides, including vitexin and isoswertisin 2″-rhamnosides, isovitexin and isoorientin 2″-arabinosides. The structure of ‘vitexin 4′-rhamnoside’ from Crataegus oxyacantha is revised in vitexin 2″-rhamnoside.     

Spectrophotometric phytochrome measurements in light-grown Avena sativa L.

Phytochrome was studied spectrophotometrically in Avena sativa L. seedlings that had been grown for 6 d in continous white fluorescent light from lamps. Greening was prevented through the use of the herbicide San 9789. When placed in the light, phytochrome (P_tot) decreased with first order kinetics (_1/2 2 h) but reached a stable low level (2.5% of the dark level) after 36 h. This concentration of phytochrome remained constant in the light and during the initial hours of a subsequent dark period, but increased significantly after a prolonged dark period. Evidence suggests that the constant pool of phytochrome in the light is achieved through an equilibrium between synthesis of the red absorbing (P_r) and destruction of the far-red absorbing form (P_fr) of phytochrome. It is concluded that the phytochrome system in light-grown oat seedlings is qualitatively the same as that known from etiolated monocotyledonous seedlings, but different than that described for cauliflower florets.Abbreviations P_fr the far-red light absorbing form of phytochroma - P_r the red light absorbing form of phytochrome - P_tot P_r+P_fr - k_s rate constant of P_r synthesis - k_d rate constant of P_fr destruction - MOPS N-morpholino-3-propane-sulfonic acid - IRIS Tris (hydroxymethyl) amino methane - San 9789 4-chloro-5-(methyl amino)-2-(,,-trifluoro-m-tolyl)-3(2H)pyridazinone     

Genomic in situ hybridization differentiates between A/D- and C-genome chromatin and detects intergenomic translocations in polyploid oat species (genus Avena).

The genomic in situ hybridization (GISH) technique was used to discriminate between chromosomes of the C genome and those of the A and A/D genomes in allopolyploid oat species (genus Avena). Total biotinylated DNA from A. strigosa (2n = 2x = 14, AsAs genome) was mixed with sheared, unlabelled total DNA from A. eriantha (2n = 2x = 14, CpCp) at a ratio of 1:200 (labelled to unlabelled). The resulting hybridization pattern consisted of 28 mostly labelled and 14 mostly unlabelled chromosomes in the hexaploids. Attempts to discriminate between chromosomes of the A and D genomes in A. sativa (2n = 6x = 42, AACCDD) were unsuccessful using GISH. At least eight intergenomic translocation segments were detected in A. sativa 'Ogle', several of which were not observed in A. byzantina 'Kanota' (2n = 6x = 42, AACCDD) or in A. sterilis CW 439-2 (2n = 6x = 42, AACCDD). At least five intergenomic translocation segments were observed in A. maroccana CI 8330 'Magna' (2n = 4x = 28, AACC). In both 'Ogle' and 'Magna', positions of most of these translocations matched with C-banding patterns.     

Genomic in situ hybridization analysis of a trigenomic hybrid involving Solanum and Lycopersicon species.

A 4x potato (+) tomato fusion hybrid (2n = 4x = 48) was successfully backcrossed with a diploid Lycopersicon pennellii (2n = 2x = 24). Genomic in situ hybridization (GISH) on somatic and meiotic chromosomes confirmed that the progenies were triploids (2n = 3x = 36) and possessed three different genomes: potato, tomato, and L. pennellii. Therefore, they have been called trigenomic hybrids. Total genomic probes of both Lycopersicon species were found to hybridize mutually, whereas the potato genome was clearly differentiated. During metaphase I, bivalents were formed predominantly between tomato and L. pennellii chromosomes and the univalents of potato chromosomes were most common. Trivalents in all cases included homoeologous chromosomes of potato, tomato, and L. pennellii. However, the triploids were totally sterile as determined from extensive crossing. On chromosome doubling of triploids by shoot regeneration from callus, hexaploids (2n = 6x = 72) were obtained. Despite exhibiting clear allohexaploid behaviour by forming 36 bivalents at meiosis, these were also completely sterile like their triploid counterparts. In spite of this drawback, the prospects of chromosome pairing between potato L. pennellii and Solanum genomes does open the possibilities for bringing the two genera close.     

Intergenomic translocations of polyploid oats (genus Avena) revealed by genomic in situ hybridization

Wild and cultivated hexaploid oats share the same genomes (AACCDD) and display a considerable level of interspecific variation in both plant and chromosome morphology. The GISH was utilized to detect the interspecific genomic compositions in four hexaploid and two tetraploid oats using total genomic DNA of Avena eriantha (a C-genome diploid) as probe. Intergenomic translocations between A/D and C-genome chromosomes were frequently observed in hexaploid and tetraploid species. In the hexaploid, two pairs of A/D genome segments on C-genome chromosome (A/D-C) translocation and four to six pairs of C-genome segments on A/D genome chromosome (C-A/D) translocation were clearly identified whilst the number of A/D-C translocations was constant among species. In the tetraploid A. maroccana (AACC), a pair of A-C and four pairs of C-A translocations were observed. Moreover, the A/D translocation segments on chromosome 5C was detected only in A. byzantina and A. maroccana, whilst A/D-C translocations were observed on the 1C and 7C of A. sativa, A. fatua and A. sterilis. A. byzantina did however also carry the 1C rearrangement. This result shows that A. byzantina has retained a similar genomic constitution to the tetraploid ancestor of hexaploid oats, A. maroccana. Three pairs of A-C translocations were detected only in A. murphyi (AACC), and two pairs of those were the 1C and 7C as well as the three hexaploid species except A. byzantina.     

Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

Summary Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe, together with excess amounts of unlabelled blocking DNA from wheat, for DNA:DNA in-situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root-tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in-situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.     

Phytochrome-controlled extension growth of Avena sativa L. seedlings

The effects of continuous red and far-red light and of brief light pulses on the growth kinetics of the mesocotyl, coleoptile, and primary leaf of intact oat (Avena sativa L.) seedlings were investigated. Mesocotyl lengthening is strongly inhibited, even by very small amounts of P_fr, the far-red light absorbing form of phytochrome (e.g., by [P_fr]0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by P_fr, but apparently inhibited later. This inhibition is correlated in time with the rupturing of the coleoptile tip by the primary leaf, the growth of which is also promoted by phytochrome. The growth responses of all three seedling organs are fully reversible by far-red light. The apparent lack of photoreversibility observed by some previous investigators of the mesocotyl inhibition can be explained by an extremely high sensitivity to P_fr. Experiments with different seedling parts failed to demonstrate any further obvious interorgan relationship in the light-mediated growth responses of the mesocotyl and coleoptile. The organspecific growth kinetics, don't appear to be influenced by P_fr destruction. Following an irradiation, the growth responses are quantitatively determined by the level of P_fr established at the onset of darkness rather than by the actual P_fr level present during the growth period.Abbreviation P_fr far-red light absorbing form of phytochrome     

Cyclic GMP-Binding Activity in Avena sativa Seedlings

Binding of ³H-labeled cyclic GMP (³H-cGMP) to the structural components of subcellular fractions was studied in oat seedlings. The binding was found to depend on the conditions of incubation and illumination of growing plants. In the green seedlings, the binding activity was lower than in the etiolated seedlings. The highest binding was observed in soluble cytosolic fraction where two types of specific sites for cGMP binding (with high and low affinity to the cyclic mononucleotide) were detected. The binding activity was found to increase as a result of red light influence via phytochrome, as well as in the presence of calcium ions and calcium–calmodulin complex.     

Metabolism of diclofop-methyl in cell cultures of Avena sativa and Avena fatua

The metabolism of the herbicide, diclofop-methyl (methyl-2-[4-(2', 4'-dichlorophenoxy) phenoxy]propanoate), in cell suspension cultures of Avena sativa L. (cv. Garry) and in callus of Avena fatua L. (transferred to liquid) was determined as a function of time (8 h to about 3 weeks) and was compared to previous metabolism data from intact plants. A. fatua metabolized ¹⁴C-labeled diclofop-methyl more rapidly than A. sativa, but the metabolites formed were similar if not identical. Within 2 days, approximately 50% of the total ¹⁴C recovered was in A. fatua cells whereas less than 15% was in A. sativa cells. In older cultures of A. fatua, the amounts of ¹⁴C in the cells and in the medium were about 45% each; 10 to 12% was in the non-extractable cell residue. The ¹⁴C recovered from A. sativa cells increased to a maximum of about 35% at 7 days and then slowly decreased to about 18% by 21 days, whereas the ¹⁴C in the medium of A. sativa decreased to about 60% at 7 days and then increased to over 75% by 21 days. The nonextractable ¹⁴C residue was 5% or less even after 21 days. Major metabolites in methanolic extracts of cells of both A. sativa and A. fatua were diclofop (2-[4-(2', 4'-dichlorophenoxy)phenoxy] propanoate), diclofop hydroxylated at an undetermined position on the 2,4-dichlorophenyl ring (ring OH-diclofop), and conjugates of diclofop and ring-OH diclofop.     

Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed.     

Genomic in situ hybridization reveals the allopolyploid nature ofMilium montianum (Gramineae)

Molecular techniques that paint chromosomes offer exciting new opportunities for testing genome relationships.Milium montianum (2n=22) is a grass whose distinctive bimodal karyotype comprises 8 large (L-) and 14 smaller (S-) chromosomes. The proposal thatM. montianum is an allotetraploid, with diploidMilium vernale (2n=8) as the L-chromosome genome donor, has been impossible to confirm by classical means. To test this hypothesis, biotinylated total genomic DNA of diploidM. vernale (2n=8) was hybridized in situ to root tip chromosomes ofM. montianum. TheM. vernale probe hybridized preferentially to all L-chromosomes, but not to the S-chromosomes. These results (i) confirm the allopolyploid nature ofM. montianum, (ii) strongly support the theory that the L-chromosomes ofM. montianum were donated byM. vernale, or a closely related genotype and (iii) show that subsequently the L-chromosomes have largely retained their genomic integrity in the new allopolyploid backgroud. Clearly, genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics. It will often be useful for investigating the origins of wild and cultivated polyploid plant species, especially where conventional methods have failed, for studying introgression, and for understanding the mechanism(s) of origin of bimodal karyotypes.by D. Schweizer     

Rapid Phytochrome-Controlled Protein Phosphorylation and Dephosphorylation in Avena sativa L.

The time course for in vivo changes in the protein phosphorylationpattern was measured after red and red/far-red light. Avenacoleoptile tips were incubated in ³²P-labeled phosphate andirradiated. The supernatant fractions of homogenates were subjectedto SDS-poly-acrylamide gel electrophoresis and then autoradiographed.Within seconds, the radioactive label of two proteins decreasedand the radioactive label of one protein increased. These datasuggest that the phosphorylation states for these proteins maybe under phytochrome control. (Received July 20, 1987; Accepted July 20, 1988)     

Analysis of phytochrome kinetics in light-grown Avena sativa L. seedlings

The phytochrome content, the rate of phytochrome accumulation after a light/dark transition and the rate of phytochrome destruction after a 1.5 d reaccumulation period in darkness were measured in light grown Avena sativa L. seedlings. The results using spectrophotometrical methods (Norflurazon treated seedlings) and the radio-immunoassay (RIA) (green seedlings) were almost identical. The rate of phytochrome synthesis was analysed by measuring the activity of poly(A⁺)-RNA coding for the phytochrome apoprotein. It was demonstrated that the rate of phytochrome synthesis is different in light and in dark. These results were confirmed by measuring the incorporation of radioactive label in vivo. Five minutes red (and 5 min far-red) light strongly reduces the rate of phytochrome synthesis. Even after prolonged dark periods only 50% of the initial rate of phytochrome synthesis is recovered for light and dark grown seedlings which received one red light pulse.     

Genomic relationships between Medicago murex Willd. and Medicago lesinsii E. Small. investigated by in situ hybridization

Medicago murex Willd. is an annual species (2n = 14) widespread in the wild and of remarkable interest for pastures in regions with a mediterranean climate. It is considered closely related to Medicago lesinsii E. Small (2n = 16) but, up to now, there is no evidence demonstrating their genetic affinity. This research was undertaken to investigate the genomic relationships between M. murex and M. lesinsii by using genomic in situ hybridization (GISH). In this study GISH experiments were performed using both species as sources of chromosomes and genomic probes. To better evaluate the results of the hybridization, the labelled DNA of each species was hybridized to chromosomes of the same species and to chromosomes of the diploid Medicago littoralis (2n = 16). Strong hybridization signals were found on chromosomes of M. murex and M. lesinsii after GISH. Differences in the hybridization strength were not observed when slides from interspecific hybridization were compared with the control preparations. These results suggest that consistent divergences of the DNA sequences did not occur after the separation of the two species. Instead very reduced cross hybridization was found on chromosome spreads of M. littoralis hybridized with the DNA of M. lesinsii or M. murex. The distribution of the ribosomal genes (rDNA) investigated by fluorescent in situ hybridization (FISH) appeared similar in both M. murex and M. lesinsii. The GISH technique may be a valuable approach to obtain information on evolution of the 2n = 14 species and on the origin of the polyploids Medicago rugosa (2n = 30) and Medicago scutellata (2n = 30). The first attempt to investigate the genomic composition of M. scutellata using a genomic probe is reported in this paper.