Inheritance of grain esterase genes in rye populations (Secale cereale L.)

Genetic analysis of four tow straw rye populations has been carried out basing on the trait electrophoretic spectrum of grain esterase isozymes. Expression of seven independent Est genes has been shown. Three of them are clustered and their intrapopulation polymorphism exceeds two alleles. Comparative analysis of the frequences of different genes in parental populations and in F2 with literature data about localization of Est genes makes it possible to carry out chromosomal localization of the genes identified according to the fact of presence or absence of linking with the genes of rye self-incompatibility.     

Embryogenesis and germination in rye (Secale cereale L.)

Summary Fine structural investigations of non-viable rye grains indicate recognisible abnormalities in the plasmalemma and mitochondrial membranes of the unimbibed embryo. Once such grains are wetted there is rapid and progressive disorganisation of the tissue. Biochemical studies show a reduced uptake of water, lack of respiratory activity and a failure in nucleic acid and protein synthesis. Whereas total DNA, RNA and protein levels are unchanged on loss of viability, the integrity of DNA and RNA is impaired and ribosomal RNA and soluble protein levels are reduced.     

Molecular analysis of mitochondrial DNA from rye (Secale cereale L.)

Summary Molecular characterization of mitochondrial (mt) DNA of rye (Secale cereale L.), free of significant amounts of contaminating chloroplast (cp) DNA, was initiated using the open-pollinated cultivar Halo as a source of mtDNA. Based on the compilation of data from restriction patterns, the molecular size of the mtDNA was estimated to be 410 Kb and its buoyant density was determined as 1.705 g/ml. Southern hybridization, using labelled cp genes (P700 and ribulosebiphosphate-carboxylase large subunit), indicated the presence of cpDNA-homologous regions on putative mtDNA fragments. Mt DNAs of inbred lines with fertile and cytoplasmic male sterile (CMS) Pampa cytoplasm were also analysed. Whereas the restriction patterns of mtDNAs of Halors and the fertile line turned out to be identical, Pampa mtDNA showed a unique restriction pattern, indicating (as in most other CMS systems) the involvement of mtDNA rearrangements in the expression of male sterility in rye. All 3 mtDNAs investigated contain regions homologous to the plasmid S1 of the CMS-S cytoplasm of Maize (Zea mays), as indicated by hybridization experiments. In Pampa cytoplasm the S-homologous sequence is located within a rearranged region of mtDNA.     

Embryogenesis and germination in rye (Secale cereale L.)

Summary When rye embryos imbibe water they rapidly return to a condition of biochemical and structural complexity. Three stages of imbibition can be recognised: Phase I a short period (10 min) of physical wetting; Phase II a longer period (1 h) when little further imbibition occurs, followed by Phase III a continuous phase of active water uptake. The latter coincides with an increase in respiration rate and an increase both in the number of mitochondria and of cristae within them. Changes in fine structure become evident in all organelles in Phase III, after 2 h of imbibition. In the unimbibed embryo endoplasmic reticulum is present only as short crescents associated with electron lucent bodies, but in Phase III the endoplasmic reticulum proliferates to form many surrounding cirlets. After 6 h these circlets become fewer and instead the endoplasmic reticulum is seen in close association with the nuclear membrane. Concurrently incorporation of radioactive uridine and thymidine is first detectible. This suggests that the large increase in protein synthesis occurs on new ribosomes present on the reticulum associated with the nuclear membrane. For the first 6 h protein synthesis must occur either on polysomes within the dense packing of ribosomes or on these circlets of endoplasmic reticulum associated with electron lucent bodies.     

Embryogenesis and germination in rye (Secale cereale L.)

Summary Cells of the young embryo contain highly differentiated organelles. During maturation and dehydration, complexity is reduced, the many layers of endoplasmic reticulum associated with electron lucent bodies become reduced to a few residual crescents, lipid droplets distributed in the cytoplasm migrate to and become closely appressed to the plasmalemma, mitochondrial cristae are reduced in number and dictyosomes are compacted.     

Molecular linkage mapping in rye (Secale cereale L.)

A rye linkage map containing clones from rye, wheat, barley, oat and rice genomic and cDNA libraries, known-function genes and microsatellite markers, was created using an F₂ population consisting of 110 F₂-derived F₃ families. Both co-dominant and dominant markers were added to the map. Of all probes screened, 30.8% were polymorphic, and of those polymorphic 79.3% were mapped. The current map contains 184 markers present in all seven linkage groups covering only 727.3 cM. This places a marker about every 3.96 cM on average throughout the map; however, large gaps are still present. The map contains 60 markers that have been integrated from previous rye maps. Surprisingly, no markers were placed between the centromere and C1–1RS in the short arm of 1R. The short arm of chromosome 4 also lacked an adequate number of polymorphic markers. The population showed a remarkable degree of segregation distortion (72.8%). In addition, the genetic distance observed in rye was found to be very different among the maps created by different mapping populations. Received: 10 January 2000 / Accepted: 26 May 2000     

Genetic linkage map of rye (Secale cereale L.)

Communicated by G. Wenzel     

B-chromosomes in inbred lines of rye (Secale cereale L.)

B chromosomes from an experimental population of the Japanese JNK strain of rye, isogenic for its Bs, have been backcrossed into twelve different inbred lines. The experiment is a way of studying the effects of the Bs against a range of different homozygous A chromosome backgrounds. This publication deals with pairing effects of both the As and the Bs, and their interactions, and with pollen mitosis. At meiosis there is a genotypic component to B effects, and they do not appear to act solely through a physical disturbance within the nucleus. In pollen the Bs are always present in more than 50% of the grains regardless of their pairing behaviour during meiosis; this result fits with a parasitic model of the activity of rye Bs.     

B-chromosomes in inbred lines of rye (Secale cereale L.)

B-chromosomes from an experimental population of the Japanese JNK strain of rye, isogenic for its Bs, have been backcrossed into twelve different inbred lines. The experiment provides a way to study the effects of the Bs against a range of homozygous A-chromosome backgrounds. This publication deals with vigour and fertility: it shows that the rye Bs fit a parasitic model, and that they interact in their effects with the A-chromosome background genotype.     

A genetic linkage map of rye (Secale cereale L.)

 A genetic linkage map of rye composed of 91 loci (88 RFLP, two morphological and one isozyme markers) has been developed using two reciprocal crosses. The RFLP loci covering all seven chromosomes were detected by a selection of rye, wheat, barley and oat cDNA and genomic DNA probes. The level of polymorphism was dependent on the source of the clones, with a ranking of rye>wheat>barley>oat. Distorted segregations were detected in linkage groups of chromosomes 1R, 4R, 5R and 7R. When the recombination of the two reciprocal crosses was compared, no systematic increase or decrease in one or the other direction was observed suggesting that a combination of populations of reciprocal crosses is possible. Received: 5 August 1997/Accepted: 2 September 1997     

Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.)

A system for the genetic transformation of rye by co-cultivation with Agrobacterium tumefaciens is described. A total of 45 independent transgenic plants were regenerated with a transformation efficiency of 1 to % of the inoculated explants. The co-cultivation of Agrobacterium-strain AGL0, harboring plasmid pJFnptII and rye im-mature embryos in liquid medium allowed a high throughput and facilitated washing of the cultures to avoid Agrobacterium overgrowth. Transgenic plants were phenotypically normal and fully fertile, which might be aconsequence of the short time in tissue culture. The selection with paromomycin exclusively during the regen-eration allowed the efficient recovery of transgenic events without interfering with somatic embryogenesis. Southern blot analysis confirmed the independent nature of the analyzed plants and indicated single copy inserts in more than 50% of them. Segregation analysis confirmed single locus integration and stable transgene expression in most of the lines, while one line with multiple locus integration was also observed. The analysis of T-DNA:: plant DNA boundary sequences revealed examples of exclusion of vector sequences, deletion of a few bases of the T-DNA or insertion of up to 29 bases of the vector backbone. This stresses the importance of detailed analysis of the inserted transgenes in order to identify events with the desired integration profile.     

Linkage relationships of esterase loci in rye (Secale cereale L.)

Summary Genetic analysis of esterase polymorphism in rye inbred lines with isoelectric focusing in polyacrylamide flat gels yielded evidence for the existence of at least ten esterase loci, Est 1–Est 10. The loci can be attributed to four different linkage groups (Est 1/ Est 2/Est 3/Est 5/Est 6/Est 7), (Est 4), (Est 8/Est 9), and (Est 10). Loci Est 5/Est 6/Est 7 and Est 8/Est 9, respectively, are tightly linked with a maximum recombination frequency of 0.2% and can therefore be regarded as compound loci which possibly originated in tandem duplications.     

Genetic control of aluminium tolerance in rye (Secale cereale L.)

 Aluminium (Al) tolerance in roots of two cultivars (“Ailés” and “JNK”) and two inbred lines (“Riodeva” and “Pool”) of rye was studied using intact roots immersed in a nutrient solution at a controlled pH and temperature. Both the cultivars and the inbred lines analysed showed high Al tolerance, this character being under multigenic control. The inbred line “Riodeva” was sensitive (non-telerant) at a concentration of 150 μM, whereas the “Ailes” cultivar showed the highest level of Al tolerance at this concentration. The segregation of aluminium-tolerance genes and several isozyme loci in different F₁s, F₂s and backcrosses between plants of “Ailés” and “Riodeva” were also studied. The segregation ratios obtained for aluminium tolerance in the F₂s analysed were 3 : 1 and 15 : 1 (tolerant : non-tolerant) while in backcrosses they were 1 : 1 and 3 : 1. These results indicated that Al tolerance is controlled by, at least, two major dominant and independent loci in rye (Alt1 and Alt3). Linkage analyses carried out between Al-tolerance genes and several isozyme loci revealed that the Alt1 locus was linked to the aconitase-1 (Aco1), nicotinamide adenine dinucleotide dehydrogenase-2 (Ndh2), esterase-6 (Est6) and esterase-8 (Est8) loci, located on chromosome arm 6RL. The order obtained was Alt1-Aco1-Ndh2-Est6-Est8. The Alt3 locus was not linked to the Lap1, Aco1 and Ndh2 loci, located on chromosome arms, 6RS, 6RL and 6RL respectively. Therefore, the Alt3 locus is probably on a different chromosome. Received: 18 March 1997 / Accepted: 21 March 1997     

Linkage mapping of mutant loci in rye (Secale cereale L.)

Eight mutant loci determining the traits waxy plant (w and wa1), brown culm (cb), multiple pistils (mp), weak plant with reduced plant height (np), monoculm growth habit (mc), compactum growth habit (ct3) and anthocyaninless (an) were mapped on rye chromosomes 4R (w, np), 6R (cb, mc) and 7R (mp, wa1, ct3, an). For five mutants (w, wa1, cb, mp, np) molecular and biochemical markers were applied, whereas for mc, ct3 and an a classical linkage analysis was performed. Furthermore, it could be demonstrated that homoeologous relationships exist between most of the mapped rye loci and comparable mutants in wheat and barley. It was confirmed not only that genes controlling fundamental aspects of plant biology are highly conserved across the Triticeae species but so also were many mutant loci. Received: 19 June 2000 / Accepted: 18 October 2000     

Studies on enzymatic polymorphism in rye (Secale cereale L.) cultivars

An electrophoretic study of seed peroxidases of eight rye (Secale cereale L.) cultivars was carried out in order to estimate the polymorphism and the variability degree of this highly variable enzymatic system. Analysis of the Cathodal Peroxidase System (CPX) were performed using polyacrylamide gel electrophoresis. Thirteen loci were recorded in embryo plus scutellum and endosperm. The percentage of polymorphic loci ranged from 53.84% to 92.30% when the 99% polymorphism criterion was used; these high values were possibly due to the studied system. The average heterozygosity values ranged from 0.179 to 0.378; these values fell into the expected for open-pollinated species. Genetic distance values fitted well with those expected for conspocific plants with a little of diversification.     

Chromosome condensation in mitosis and meiosis of rye (Secale cereale L.)

Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids.     

Isolated microspore culture and plant regeneration in rye (Secale cereale L.)

 An isolated microspore culture and green plant regeneration method for rye (Secale cereale L.) was established. Rye isolated microspore androgenesis was genotype-dependent. PG-96M medium supplemented with 6% maltose gave the highest microspore survival rate after 48 h of culture and the highest embryo/callus yield (930 embryos/calli per 100 anthers from cv. Florida 401). Osmotic pressure in the induction medium played an important role. Pretreatment of the anthers with mannitol was beneficial for the microspore culture. Embryos/calli of a relatively younger age and smaller size had a higher regeneration ability, with the best green plant regeneration rate being 6%. Over 150 microspore-derived green plants have been obtained so far. About 90% of the regenerated plants were spontaneous doubled haploids. This is the first report of isolated microspore culture in true rye resulting in androgenic embryogenesis and plant regeneration. Received: 26 April 1999 / Accepted: 23 November 1999     

Microdissection and microcloning of rye (Secale cereale L.) chromosome 1R

Chromosome 1R was microdissected and collected from mitotic metaphase spreads of rye (Secale cereale L.) by using glass needles. The isolated chromosomes were amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (PCR). After amplification, the presence of rye-specific DNA was verified by Southern hybridization. The second-round PCR products from five 1R chromosomes were cloned into a plasmid vector to create a chromosome-specific library, which produced approximately 220,000 recombinant clones. Characterization of the microclone library showed that the 172 clones evaluated ranged in size from 300–1800 bp with an average size of 950 bp, of which approximately 42% were medium/high copy and 58% were low/unique copy clones. Chromosome in situ hybridization confirmed that the PCR products from microdissected chromosomes originated from chromosome 1R, indicating that many chromosome 1R-specific sequences were present in the library. Received: 5 December 1998; in revised form: 15 April 1999 / Accepted: 29 April 1999