Characterization of spelt (Triticum spelta L.) forms by gel-electrophoretic analyses of seed storage proteins. II. The glutenins

 Seed proteins of 28 spelt cultivars (Triticum spelta L.), 16 cross combinations between spelt forms or between spelt and English winter wheat cultivars, and ten winter wheat varieties (Triticum aestivum L.) were analysed by SDS (sodium dodecyl sulphate)-PAGE (polyacrylamide-gel electrophoresis). Different wheat types were chosen for distinct purposes: five popular German wheat cultivars were used for a comparison of wheat and spelt protein band patterns, two of them are old varieties (‘Kanzler’ and ‘Jubilar’) and one is a modern wheat standard (‘Orestis’); five English winter wheat varieties with short straw were used for the crosses with spelt cultivars to improve seed yield and especially the lodging resistance of spelt. The objectives of these studies were the adaptation of existing SDS-PAGE methods, which have been successfully applied in other crops, for the analysis of seed proteins in spelt, and the characterization and differentiation of spelt varieties from corresponding cross combinations with other spelt forms or with winter wheat cultivars using gel-electrophoretic methods (SDS-PAGE). Considerable differences in protein band patterns were found between spelt and winter wheat varieties, especially in three distinct lanes of the electropherogrammes where the molecular weights range from 40 to 49 , 53 to 62 and 74 to 115 kDa. Spelt cross combinations, and especially crosses between spelt and winter wheat cultivars, were easily distinguishable particularly after a preceding extraction in chlorethanol. Received: 4 December 1996 1 / Accepted: 6 December 1996     

Characterization of spelt (Triticum spelta L.) forms by gel-electrophoretic analyses of seed storage proteins. III. Comparative analyses of spelt and Central European winter wheat (Triticum aestivum L.) cultivars by SDS-PAGE and acid-PAGE

 Seed storage proteins of a few selected spelt forms and crosses have already been electrophoretically analysed by SDS-PAGE and acid-PAGE and compared with a few winter wheat cultivars. In the analyses presented here further important Central European spelt varieties were included, as well as modern winter wheat cultivars which were chosen as standards. In this study gliadin and glutenin band patterns of modern Central European winter wheat cultivars were analysed, in particular for a comparison with spelt varieties. An improved differentiation within and between the two species was obtained. Received: 27 April 1998 / Accepted: 26 May 1998     

Gel electrophoretic investigations of prolamins in eu- and alloplasmatic octoploid primary triticale forms

 Unreduced prolamin fractions of eu- and alloplasmatic octoploid triticale forms were investigated by means of gel electrophoresis. The electrophoretic separation of the prolamin fractions was carried out by using one-dimensional, horizontal, native acidic polyacrylamide gel electrophoresis (APAGE) and gradient gels. A comparison of the electropherogram patterns of triticale forms with those of the genome donors showed that most of the parent prolamins can be found again in the corresponding triticale. However, the bands of the triticale forms exhibited lower intensities than those of the genome donors. On average the gliadin bands of the triticale forms showed equal reductions in intensities, thereby preserving the relationships of the band intensities in most cases. On the other hand, secalin bands which came from the rye parent showed varying reductions in intensities. A comparative analysis of the band patterns revealed that differences could often be found between triticale forms with the same genomic constitution. Often it concerned differences in intensity. In some cases the bands showed changes in mobility or a splitting up into two sub-bands. Starting with the plant material presented here we developed a new nomenclature of the prolamin band patterns of the triticale forms. Received: 15 July 1997 / Accepted: 23 July 1997     

Gliadin polymorphism in wild and cultivated einkorn wheats

To study the relationships between different species of the Einkorn group, 408 accessions of Triticum monococcum, T. boeoticum, T. boeoticum ssp. thauodar and T. urartu were analyzed electrophoretically for their protein composition at the Gli-1 and Gli-2 loci. In all the species the range of allelic variation at the loci examined is remarkable. The gliadin patterns of T. monococcum and T. boeoticum were very similar to one another but differed substantially from those of T. urartu. Several accessions of T. boeoticum and T. monococcum were shown to share the same alleles at the Gli-1 and Gli-2 loci, confirming the recent nomenclature that considers these wheats as different subspecies of the same species, T. monococcum. The gliadin composition of T. urartu resembled that of the A genome of polyploid wheats more than did T. boeoticum or T. monococcum, supporting the hypothesis that T. urartu, rather than T. boeoticum, is the donor of the A genome in cultivated wheats. Because of their high degree of polymorphism the gliadin markers may help in selecting breeding parents from diploid wheat germ plasm collections and can be used both to search for valuable genes linked to the gliadin-coding loci and to monitor the transfer of alien genes into cultivated polyploid wheats. Received: 8 July 1996 / Accepted: 12 July 1996     

Variation and classification of B low-molecular-weight glutenin subunit alleles in durum wheat

 The B low-molecular-weight (LMW) glutenin subunit composition of a collection of 88 durum wheat cultivars was analyzed. Extensive variation has been found and 18 different patterns were detected. Each cultivar exhibited 4–8 subunits, and altogether 20 subunits of different mobility were identified. The genetic control of all these subunits was determined through the analysis of nine F₂ populations and one backcross. Five subunits were controlled at the Glu-A3 locus, 14 at Glu-B3 and 1 at Glu-B2. At the Glu-A3 locus each cultivar possessed from zero to three bands and eight alleles were identified. At the Glu-B3 locus each cultivar showed four or five bands and nine alleles were detected. Only one band was encoded by the Glu-B2 locus. A nomenclature for these alleles is proposed and the relationship between them and the commonly used LMW-model nomenclature is discussed. Received: 10 February 1997 / Accepted: 25 April 1997     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

Genetic diversity among European cultivated spelt revealed by microsatellites

Spelt and common wheat constitute two of the six groups of the hexaploid wheats with an AABBDD genome. Spelt culture has been progressively replaced by that of common wheat which out-yields spelt under high-input conditions. In the last decades, spelt breeders intended to introduce the yield-potential and bread-making qualities of common wheat into spelt, by frequent crossings between accessions of these two different groups. The present study aims at determining the genetic basis of modern spelt cultivars in terms of intra-group variability and inter-group (spelt vs common wheat) distances, by using microsatellite markers developed for common wheat. The allelic composition of 30 spelt and nine common wheat accessions was determined at 17 microsatellite loci. The coefficient of co-ancestry (ƒ) and the genetic distances (1 - proportion of shared alleles) based upon allelic composition were calculated for all pairs of accessions. Two dendrograms were constructed using the UPGMA method. Amplification products were found for all loci on most accessions. A total of 113 alleles was identified, of which 60.2% were specific to spelt or common wheat. The correlation between (1 –ƒ ) and the genetic distance was high (0.701***). The mean pairwise genetic distance was 0.656 ± 0.181 over the 39 accessions, 0.706 ± 0.14 among common wheat and 0.573 ± 0.172 among spelt. The mean genetic distance between spelt and wheat was 0.782 ± 0.113. The two dendrograms were in accordance with each other and clearly separated the spelt from the common wheat accessions. It is concluded that microsatellites developed for common wheat and distances based on the proportion of shared alleles are powerful tools for reconstructing phylogenies in spelt, and that the genetic basis of modern spelt cultivars is narrow despite frequent crosses made with bread wheat. Received: 26 January 2000 / Accepted: 26 May 2000     

Reliability of an electrophoretic and image processing analysis of human skeletal muscle taken from m. vastus lateralis

The relative content of myosin heavy chain (MHC) isoforms IIb, IIa and I in human skeletal muscle taken from the m. vastus lateralis of 30 healthy male subjects was analysed using mini-gel electrophoresis. Repeated electrophoretic gels utilizing the same methods were produced for all subjects and the determination of MHC protein bands was performed using a digital scanner and National Institutes of Health (NIH) Image software and laser densitometry. A comparison between the NIH Image processing technique and laser densitometry revealed differences of 6.47%, 6.35% and 6.84% between these measurement techniques for MHC-IIb, -IIa and -I isoforms, respectively. The percentage technical error of measurement (TEM%) between electrophoretic gels was shown to be 19.1%, 17.8% and 14.2%, with regard to percentage of occurrence of MHC-IIb, -IIa and -I isoforms respectively. The variation in electrophoretic gel analyses was shown to be 5.7%, 7.3% and 5.5%, with regard to the percentage of MHC-IIb, -IIa and -I isoforms respectively. Intra-class correlations comparing NIH Image and laser densitometry produced r values in the range 0.38–0.63. Comparisons between and within gel analyses produced r values in the range 0.59–0.94 and 0.93–0.98, respectively. Analyses of variance revealed no significant differences (P < 0.05) between analysis techniques, between␣gels or within gels for the measurement of MHC-IIb, -IIa and -I isoforms. The inter-gel error between fibre subgroups was moderate for the two type-II MHC populations and less for type-I MHC; the intra-individual error in the measuring technique used for classifying the MHC-IIb, -IIa and -I protein bands was small. The results obtained in this investigation showed consistent trends which may reflect a false classification of the type-II MHC populations for the inter-gel and intra-individual analyses. The NIH Image software and digitizing process was shown to be a valid and reliable method for distinguishing between MHC protein bands of human skeletal tissue as separated by mini-gel electrophoretic techniques. Accepted: 6 January 1997     

Polymorphism, distribution, and segregation of AFLP markers in a doubled haploid rice population

We exploited the newly developed amplified fragment length polymorphism (AFLP) technique to study the polymorphism, distribution and inheritance of AFLP markers with a doubled haploid rice population derived from ‘IR64’/‘Azucena’. Using only 20 pairs of primer combinations, we detected 945 AFLP bands of which 208 were polymorphic. All 208 AFLP markers were mapped and distributed over all 12 chromosomes. When these were compared with RFLP markers already mapped in the population, we found the AFLP markers to be highly polymorphic in rice and to follow Mendelian segregation. As linkage map of rice can be generated rapidly with AFLP markers they will be very useful for marker-assisted backcrossing. Received: 11 April 1996 / Accepted: 14 June 1996     

Chromosome mapping of low-temperature induced Wcs120 family genes and regulation of cold-tolerance expression in wheat

 Low-temperature (LT) induced genes of the Wcs120 family in wheat (Triticum aestivum) were mapped to specific chromosome arms using Western and Southern blot analysis on the ditelocentric series in the cultivar Chinese Spring (CS). Identified genes were located on the long arms of the homoeologous group 6 chromosomes of all 3 genomes (A, B, and D) of hexaploid wheat. Related species carrying either the A, D, or AB genomes were also examined using Southern and Western analysis with the Wcs120 probe and the WCS120 antibody. All closely related species carrying one or more of the genomes of hexaploid wheat produced a 50 kDa protein that was identified by the antibody, and a Wcs120 homoeologue was detected by Southern analysis in all species. In the absence of chromosome arm 6DL in hexaploid CS wheat no 50 kDa protein was produced and the high-intensity Wcs120 band was missing, indicating 6DL as the location of Wcs120 but suggesting silencing of the Wcs120 homoeologue in the A genome. Levels of proteins that cross-reacted with the Wcs120 antibody and degrees of cold tolerance were also investigated in the Chinese Spring/Cheyenne (CS/CNN) chromosome substitution series. CNN chromosome 5A increased the cold tolerance of CS wheat. Densitometry scanning of Western blots to determine protein levels showed that the group 5 chromosome 5A had a regulatory effect on the expression of the Wcs120 gene family located on the group 6 chromosomes of all three hexaploid wheat genomes. Received: 10 July 1996 / Accepted: 30 September 1996     

A gene-for-gene model to explain interactions between cultivars of strawberry and races of Phytophthora fragariae var. fragariae

 A gene-for-gene model is postulated to explain the observed interactions between cultivars of strawberry and races of Phytophthora fragariae. Five interacting resistance (R1–R5) and avirulence (Avr1–Avr5) factors explain all the available data involving 15 host genotypes, including the USA and Canadian differential series, and 12 pathogen isolates from North America. Interactions between pathogen isolates and UK and German differentials are also explained by the proposed model. The model makes it possible to develop a universally applicable differential series, to present a systematic, unequivocal nomenclature of races, and to increase the efficiency of breeding programs. Received: 26 April 1996 / Accepted: 19 July 1996     

The pattern of sensory discharge can determine the motor response in young Xenopus tadpoles

Young Xenopus tadpoles were used to test whether the pattern of discharge in specific sensory neurons can determine the motor response of a whole animal. Young Xenopus tadpoles show two main rhythmic behaviours: swimming and struggling. Touch-sensitive skin sensory neurons in the spinal cord of immobilised tadpoles were penetrated singly or in pairs using microelectrodes to allow precise control of their firing patterns. A single impulse in one Rohon-Beard neuron (= light touch) could sometimes trigger “fictive” swimming. Two to six impulses at 30–50 Hz (= a light stroke) reliably triggered fictive swimming. Neither stimulus evoked fictive struggling. Twenty-five or more impulses at 30–50 Hz (= pressure) could evoke a pattern of rhythmic bursts, distinct from swimming and suitable to drive slower, stronger movements. This pattern showed some or all the characteristics of “fictive” struggling. These results demonstrate clearly that sensory neurons can determine the pattern of motor output simply by their pattern of discharge. This provides a simple form of behavioural selection according to stimulus. Accepted: 28 November 1996     

The α-gliadin gene family. II. DNA and protein sequence variation, subfamily structure, and origins of pseudogenes

 The derived amino-acid sequences of all reported α-gliadin clones are compared and analyzed, and the patterns of sequence change within the α-gliadin family are examined. The most variable sequences are two polyglutamine domains. These two domains are characteristic features of the α-gliadin storage proteins and account for most of the variation in protein size of this otherwise highly conserved protein family. In addition, their encoding DNA sequences form microsatellites. Single-base substitutions in the α-gliadin genes show a preponderance of transitions, including the C to T substitution which contributes to the generation of stop codons, and consequently to the observation that approximately 50% of the α-gliadin genes are pseudogenes. In one unusual gene, a microsatellite has expanded to 321 bp as compared to the normal 36–72 bp, and may result from similar mechanisms that produce polyglutamine-associated genetic diseases in humans. A comparison of the 27 reported sequences show several α-gliadin gene subfamilies, at least some of which are genome specific. Received: 1 October 1996/Accepted: 20 December 1996     

RFLP mapping of resistance to chlorosis induction by Pyrenophora tritici-repentis in wheat

Tan spot, caused by Pyrenophora tritici-repentis, is an economically important disease in major wheat production areas. The fungus can produce two genetically distinct symptoms on leaves of susceptible wheat genotypes: tan necrosis (nec) and extensive chlorosis (chl). Our objectives were to determine the number of genes conditioning resistance to tan spot in a population of wheat recombinant inbred lines, and map the chromosomal location of the resistance genes using RFLPs. Conidia produced by the P. tritici-repentis isolate Pti2 (nec+chl+) were used to inoculate seedlings of 135 recombinant inbred lines derived from the cross of the synthetic hexaploid wheat W-7984 with Opata 85. A subset of the population was inoculated with conidia produced by the isolates D308 (nec−chl+) and 86-124 (nec+chl−). Inoculated seedlings were rated on a scale of 1 to 5 based on lesion type. Necrosis-inducing culture filtrate produced by the isolate 86-124 was also used to screen the entire population. A map consisting of 532 markers was employed to identify significant associations between marker loci and tan spot resistance. The entire population was insensitive to culture filtrate produced by the isolate 86-124, and the entire subset was resistant to conidial inoculation of the same isolate. The population segregated for reaction to isolates D308 and Pti2, indicating that this population segregates for resistance to extensive chlorosis only, and not to tan necrosis. RFLP analysis indicated the presence of a gene with a major effect in 1AS, a gene with a minor effect in 4AL, and an interaction between the 1AS gene and a gene in 2DL. Together, these loci explained 49.0% of the variation in this population for resistance to tan spot produced by the isolate Pti2. Two regions one in 1BL and one in 3BL, were significantly associated with resistance to extensive chlorosis, but were not significant in the multiple regression model. It should be feasible to introgress these resistance loci into adapted genetic backgrounds by using a marker-assisted selection scheme. Received: 30 March 1996 / Accepted: 31 May 1996     

Genetic diversity of French common wheat germplasm based on gliadin alleles

 Analysis of gliadin electrophoretic (APAGE) patterns made it possible to identify 79 alleles at six Gli-1 and Gli-2 loci (from 9 to 18 per locus) and 173 gliadin genotypes in the 187 French common wheat cultivars considered. Six new alleles were registered in the catalogue of gliadin alleles. The genetic diversity of French common wheats was found to be high (H=0.714) and had not changed much during the last 25–50 years. Analysis of genetic distances showed some gradual changes in French wheat germplasm over the course of time. Genetic distances between French and several European wheat germplasm were analysed; genotypes of European wheats were found to relate very distantly to Canadian genotypes. The considerable differentiation of wheat genotypes from different countries and cereal companies might be caused by breeders’ personal preferences and by hidden natural selection specific to each local environment. In French cultivars, genetic variation in earliness, and in the North/South habit of the cultivars studied, correlated significantly with allelic variation at Gli-B1, Gli-A2 and Gli-D2 for earliness, and at Gli-D2 for the North/ South habit. Early and late cultivars are grown mainly in Southern and Northern France, respectively (r ²=0.30). Cultivars having either the 1B/1R translocation or allele Gli-D2g are, on average, later and more resistant to cold; they hence are grown in the North of France. Alternatively, cultivars with the allele Gli-D2m are earlier and cold-sensitive, and are grown in the South of France. Received: 5 February 1997 / Accepted: 19 September 1997     

The α-gliadin gene family. I. Characterization of ten new wheat α-gliadin genomic clones, evidence for limited sequence conservation of flanking DNA, and Southern analysis of the gene family

 A detailed study of the organization of the wheat α-gliadin gene family is described. In the first stage of this study recombinant libraries of sufficient size and quality were constructed to allow examination of large, closely-related, gene families. A large number of α-gliadin clones were then isolated in a partial screen of these libraries. A set of these clones were sequenced and ten new gene sequences are now reported. Including these new sequences, 20 of the 27 known α-gliadin genomic and cDNA sequences are from cv Cheyenne, making this cultivar the best studied for gliadin family organization. An analysis of the DNA flanking the coding regions shows divergences which may indicate the functional ends of the α-gliadin genes. High-resolution Southern analyses on DNA from aneuploid and chromosome-substitution lines allow most of the 20 distinct α-gliadin HindIII restriction fragments in cvs Cheyenne and Chinese Spring to be assigned to specific genomes. It is estimated that as many as 150 α-gliadin genes occur in the cultivar Cheyenne, and the limitations of such estimates are discussed. Received: 1 October 1996 / Accepted: 20 December 1996     

Effects of plant hybridization on herbivore-parasitoid interactions

We studied the effects of host plant hybridization on the survival and mortality of the leaf-mining moth Phyllonorycter salicifoliella on hybrid and parental willow plants in the field and in a common garden experiment. P. salicifoliella survival differed significantly among three willow taxa in the field in 1994 but not in the field in 1995 or in the common garden. Parasitism by eulophid wasps differed significantly among taxa in 1994 and appeared to account for the variation in their survival. In the field in 1995, host feeding predation varied significant among taxa. The theory of tritrophic interactions predicts that plant genotype can affect natural enemy impact, and this study supports this prediction. Significant variation in survival and eulophid parasitism was also found among genotypes within taxa in the field in both years and in the common garden experiment. The common garden results show that genetic differences in plants affect the herbivore-parasitoid interaction. Variation among years in the patterns of survival and causes of mortality among field plants suggest that genotype by environment interactions may be important. Received: 1 March 1996 / Accepted: 4 November 1996     

Genetic diversity evaluation of some elite cotton varieties by RAPD analysis

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity of elite commercial cotton varieties. Twenty two varieties belonging to Gossypium hirsutum L. and one to G. arboreum L. were analyzed with 50 random decamer primers using the polymerase chain reaction (PCR). Forty nine primers detected polymorphism in all 23 cotton varieties, while one produced monomorphic amplification profiles. A total of 349 bands were amplified, 89.1% of which were polymorphic. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that 17 varieties can be placed in two groups with a similarity ranging from 81.51% to 93.41%. G. hirsutum L. varieties S-12, V3 and MNH-93 showed a similarity of 78.12, 74.46 and 69.56% respectively with rest of the varieties. One variety, CIM-1100, showed 57.02% similarity and was quite distinct. The diploid cotton G. arboreum L. var. Ravi was also very distinct from rest of its tetraploid counterparts and showed only 55.7% similarity. The analysis revealed that the intervarietal genetic relationships of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The results obtained can be used for the selection of possible parents to generate a mapping population. The results also reveal the genetic relationship of elite commercial cotton varieties with some standard “Coker” varieties and the diploid G. arboreum L. var. Ravi (old world cotton). Received: 12 July 1996 / Accepted: 26 July 1996     

PCR primed with minisatellite core sequences yields DNA fingerprinting probes in wheat

 Four minisatellite core sequences were used as primers in a polymerase chain reaction (PCR) technique, known as the directed amplification of minisatellite-region DNA (DAMD), to detect polymorphisms in three pairs of hexaploid/tetraploid wheat cultivars. In each pair, the tetraploid cultivar (genomic formula AABB) was extracted from its corresponding hexaploid (genomic formula AABBDD) parent. Reproducible profiles of the amplified products revealed characteristic bands that were present only in the hexaploid wheats but not in their extracted tetraploids. Some polymorphisms were observed among the hexaploid cultivars. Twenty-three DAMD-PCR amplified fragments were isolated and screened as molecular probes on the genomic DNA of wild wheat species, hexaploid wheat and triticale cultivars. Subsequently, 8 of the fragments were cloned and sequenced. The DAMD-PCR clones revealed various degrees of polymorphism among different wild and cultivated wheats. Two clones yielded individual-specific DNA fingerprinting patterns which could be used for species differentiation and cultivar identification. The results demonstrated the use of DAMD-PCR as a tool for the isolation of informative molecular probes for DNA fingerprinting in wheat cultivars and species. Received: 13 May 1996/Accepted: 11 October 1996