Genetic analysis of gliadin components in winter wheat using two-dimensional polyacrylamide gel electrophoresis

Summary Blocks of gliadin components found both in a number of varieties and in single F₂ grains of winter wheat intervarietal hybrids have been studied by two-dimensional electrophoresis combining electrophoresis in acidic aluminium-lactate buffer (pH3.1) and SDS-electrophoresis. Gliadin components (spots) have been shown to be inherited as linked groups (blocks), codominantly and in accordance with a gene dosage in triploid endosperm. Blocks include components differing in their electrophoretic mobility and molecular weight. Some allelic variants of blocks differ only in presence of few additional components or in the electrophoretic mobility of components with similar molecular weights; other variants may contain no similar components. Apparently, in the course of evolution, mutations in individual genes of gliadin-coding loci and processes changing the number of expressing genes and the sizes of their structural part occurred.     

Blocks of gliadin components in winter wheat detected by one-dimensional polyacrylamide gel electrophoresis

Summary Inheritance of gliadin components in winter wheat has been studied by one-dimensional polyacrylamide gel electrophoresis. Single F₂ grains from 36 intervarietal hybrid combinations have been analysed. The genetic analysis has revealed blocks, including 1–6 gliadin components, which are inherited as individual mendelian traits. About 80 variants of blocks have been detected. On the basis of the allelism test they are grouped into 6 series in accordance with the number of known gliadin-coding loci located on chromosomes of the homoeologous groups 1 and 6. Each series includes 8–18 blocks controlled by different alleles of one gliadin-coding locus. Blocks of components have been confirmed to be inherited codominantly in accordance to the gene dose in the triploid endosperm. The highest similarity between members of one series is observed in groups of blocks controlled by chromosomes ID and 6D. On the contrary, many blocks controlled by chromosomes 1A and 1B have no bands in common. The presented catalogue of blocks of components may be used to make up gliadin genetic formulae and to compare electrophoregrams obtained by different authors. Blocks of gliadin components are suitable genetic markers for use in revealing and studying heterogeneity of wheat varieties, in tracing their origin, in identifying recombinations, translocations and substitutions of the genetic material and in solving many other problems of the origin, evolution and selection of hexaploid wheat.     

Revision of SAT chromosome number in Triticum monococcum with respect to nucleolar organizers activity

Summary Six varieties of Triticum monococcum were analysed by means of the nucleolar test; i.e., estimation of the maximum number of primary nucleoli per nucleus. All of the varieties exhibited 4 primary nucleoli in telophase and early interphase. Following detailed karyological analysis four SAT chromosomes in all six karyotypes were found in accordance with the maximum nucleolar number. Secondary constrictions and microsatellites were localised on the short arms of chromosome pairs 3 and 5. A new order of the chromosomes in the idiogram of Tr. monococcum is proposed.     

Genetic aspects of wheat gliadin proteins

Inheritance of gliadin components unique to three different varieties of common wheat (Triticum aestivum L.) was studied in F₁ and F₂ seeds of intervarietal crosses using protein patterns obtained by polyacrylamide gel electrophoresis in aluminum lactate buffer (pH 3.2). The patterns of F₁ seeds of the crosses Cheyenne × Justin and INIA 66R × Justin evidenced all the bands present in the patterns of the parents; band intensities reflected gene dosage levels dependent on whether the contributing parent was maternal or paternal in accordance with the triploid nature of endosperm tissue. Most of the gliadin components examined segregated in accordance with control by a single dominant gene, but in two instances single bands in the one-dimensional electrophoretic patterns segregated in the F₂ as expected if controlled by two genes. A method of two-dimensional electrophoresis was developed that resolved these apparently single bands into two components each, which could segregate independently. Linkage analysis provided evidence of codominant alleles and closely linked genes coding for gliadin protein components in both coupling and repulsion situations. The gliadin protein components seem to be coded for by clusters of genes located on chromosomes of homoeologous groups 1 and 6 in hexaploid wheats.Reference to a company or product name does not imply approval by the U.S. Department of Agriculture to the exclusion of others which may also be suitable.     

A simple method for estimating recombination percentages and linkage intensities from F2 data: examples from Triticum monococcum and other self-fertilizing diploid plant species

Summary We offer an alternative approach to the extensively used maximum likelihood and product methods for calculating recombination values and linkage intensities from F₂ data. This new method which we designate as the square root approach is simpler than the ones in current use in that it obviates the need for formulae and tables. It can be applied to autosomal F₂ data from F₁'s heterozygous in both the coupling and repulsion phases. It has greater applicability than the product method in that it can be used in all cases involving 2-, 3-, 4-, 6- and 9-class segregations regardless whether gene interaction occurs or not, provided the double recessive and other specific phenotypes are each determined by one particular genotype. The proposed method is based on the same well established genetic facts as the other two approaches. Percent recombinant gametes and therefore percent recombination are calculated by deriving the square root of the proportion of the F₂ population that expresses the double recessive or equivalent phenotype. The recombination values obtained by our method are compared with those derived by product method for 17 crosses in 7 different species and were found to be insignificantly different from the latter. The advantages and disadvantages of the square root method compared with the two most used ones are discussed.     

Cytological analysis on the distribution and origin of the alien chromosome pair conferring blue aleurone color in several European common wheat (Triticum aestivum L.) strains

Summary Meiotic chromosome pairing and Giemsa C-banding analyses in crosses of several European blue-grained wheat strains with Chinese Spring double ditelosomic and other aneuploid lines showed that Triticum aestivum Blaukorn strains Berlin, Probstdorf, Tschermak, and Weihenstephan are chromosome substitutions, in which the complete wheat chromosome 4A pair is replaced, whereas the strains Brünn and Moskau are 4B substitutions. The alien chromosome pair in all of these strains is an A genome chromosome (4A) from diploid Triticum monococcum or T. boeoticum not present in common tetraploid and hexaploid cultivated wheats. The Blaukorn strain Weihenstephan W 70a86 possesses, in addition to a rye chromosome pair 5R compensating for the loss of part of chromosome 5D, a 4A/5DL translocation replacing chromosome pair 4B of wheat.     

Quantitative inheritance and barriers to crossability in Cicer arietinum L.

Summary Three different diallel crosses were studied in Cicer arietinum; two of size 6×6, one within each of the two botanical groups macrosperma and microsperma of the cultivated subspecies, and one of 9×9 involving lines covering most of the morphological variation of chickpea. Barriers to crossability present neither a botanical nor a geographical pattern, being probably a direct consequence of interactions between genotypes. The genetic systems of twelve quantitative characters were analysed. Full dominance in a negative sense (small values dominant) is shown by leaflet length, width and shape index, rachis length, leaflet density on the rachis and pod length. Full dominance in a positive sense is shown by seeds per pod. Overdominance (in a positive sense) is evident for pods, seeds and yield per plant. Weak reciprocal differences were manifested by pod length, and pods, seeds and yield per plant. The system controlling number of leaflets per leaf is not clear. Dominance of primitive over selected characters seems to be the rule. As far as the environmental effects have permitted the analysis, no differences in genetic systems were observed between botanical groups.     

Fractionation of wheat gliadin and glutenin subunits by two-dimensional electrophoresis and the role of group 6 and group 2 chromosomes in gliadin synthesis

Summary Subunits of wheat endosperm proteins have been fractionated by two-dimensional electrophoresis. To determine which subunits in the two-dimensional electrophoretic pattern belong to gliadin or glutenin the endosperm proteins have also been fractionated by a modified Osborne procedure and by gel filtration on Sephadex G-100 and Sepharose CL-4B prior to separation by two-dimensional electrophoresis.The control of production of five major grain protein subunits is shown to be determined by chromosomes 6A, 6B and 6D by comparing two-dimensional electrophoretic protein subunit patterns of aneuploid lines of the variety Chinese Spring. From these and previous studies it is concluded that some , and gliadins (molecular weights by SDS-PAGE 30,000 to 40,000) are specified by genes on the short arms of homoeologous Group 6 chromosomes, the gliadins (molecular weights by SDS-PAGE 50,000 to 70,000) are specified by genes on the short arms of homoeologous Group 1 chromosomes and the glutenin subunits (molecular weights by SDS-PAGE > 85,000) are specified by genes on the long arms of homoeologous Group 1 chromosomes.No major gliadins or glutenin subunits were absent when any of the chromosomes in homoeologous Groups 2, 3, 4, 5 or 7 were deleted. However two gliadins whose presumed structural genes are on chromosome 6D were absent in aneuploid stocks of Chinese Spring carrying two additional doses of chromosome 2A. Two out of thirty-three intervarietal or interspecific chromosome substitution lines examined, involving homoeologous Group 2 chromosomes, lacked the same two gliadins. All the subunits in the other thirty-one chromosome substitution lines were indistinguishable from those in Chinese Spring. It is therefore concluded that the major variation affecting gliadin and glutenins in wheat is concentrated on the chromosomes of homoeologous Groups 1 and 6 but Group 2 chromosomes are candidates for further study.An endosperm protein controlled by chromosome 4D in Chinese Spring is shown to be a high molecular weight globulin.     

Variation and inheritance of the arachin polypeptides of groundnut (Arachis hypogaea L.)

Summary Variation in the arachin polypeptides of groundnut genotypes was observed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Three regions could be observed on the electropherogram. Region 1, corresponding to conarachin, did not show any variation; region 2, consisting of arachin acidic subunits, showed variation; region 3, containing the arachin basic subunits, did not show any variation. There are four varietal classes of arachin polypeptide patterns: class A comprised three acidic subunits of arachin of molecular weights 47.5, 45.1 and 42.6 kd and a basic subunit of 21.4 kd; class B, with three acidic subunits of molecular weights 47.5, 45.1 and 41.2 kd and a basic subunit of 21.4 kd; class C of an additive pattern of class A and class B; class D, of two acidic polypeptides of 47.5, 45.1 kd and the basic 21.4 kd subunit. Of the 90 genotypes studied, 73% belong to class A, 15% to class B and 6% each to class C and D. Analysis of F₂ seeds from a cross between class A and class B genotypes showed that the two polypeptides (42.6 kd and 41.2 kd) are coded by nonallelic genes and also revealed that class C and class D patterns arose as a result of hybridisation between class A and class B. A. monticola, the progenitor of A. hypogaea, showed a pattern similar to the additive pattern of class A and class B while some diploid Arachis species had the 41.2 kd polypeptide. Based on arachin polypeptide patterns the probable origin of A. hypogaea has been suggested.     

Chromosomal control of wheat gliadin protein epitopes: analysis with specific monoclonal antibodies

Summary The genetic relationships between small clusters of monomeric alcohol-soluble wheat (Triticum aestivum L.) grain storage proteins (gliadins) were studied using a panel of monoclonal antibodies and immunoblotting, ELISA, and RIA methods. Use of Chinese Spring nullisomic-tetrasomic lines showed that several narrow-specificity antibodies bound specifically to gliadins encoded by genes located on a single chromosome. In at least one case, antibodies bound to genetic blocks of gliadins, indicating that these block members have structural homology. However, often not all gliadins of a block were recognized by an antibody. For broad-specificity antibodies and some narrow-specificity antibodies, structural genes on several chromosomes were important. Studies with several primitive wheat species indicated that, while antibodies usually bound gliadins from the same genome in bread and primitive wheats, antibodies sometimes bound proteins of quite differing mobilities in the two wheat types. Use of antibodies to identify gliadin blocks is simpler than block analysis based on performing crosses, and should be of value in monitoring genotype/end-use quality relationships.     

Use of RAPD markers to determine the genetic diversity of diploid,wheat genotypes

Summary The genetic diversity of two diploid wheat species, Triticum monococcum and Triticum urartu (2n=2x=14), was assessed using random primers and the polymerase chain reaction (PCR). Electrophoretic analysis of the amplification products revealed a higher incidence of polymorphism in T. urartu than T. monococcum. Pair-wise comparisons of unique and shared polymorphic amplification products, were used to generate Jaccard's similarity coefficients. These were employed to construct phenograms using an unweighted pair-group method with arithmetical averages (UPGMA). The UPGMA analysis indicated a higher similarity among T. monococcum than T. urartu. Analysis of RAPD data appears to be helpful in determining the genetic relationships among genotypes.Contribution of the College of Agricultural Sciences, Texas Tech University Journal No. T-4-334. This work was supported by a grant from the National Science Foundation (BSR-8552915)     

Leaf development and phenology of Triticum aestivum and T. durum under different moisture regimes

Wheat grain yield production in the rain-fed areas is limited by water deficits during crop growth. A greenhouse experiment was conducted during spring 1992 at ICARDA, Tel Hadya, Syria, with eight genotypes representing two Triticum species (Triticum turgidum var. durum and Triticum aestivum L.) under four soil-moisture regimes (95%, 75%, 55%, and 35% field capacity) to study the effect of water deficit on leaf development. The phyllochron was similar in the two species across the watering regimes. The range in variation in phyllochron among the genotypes was similar in the two species. Phyllochron response to water stress among genotypes was distinct in the driest regime in both species. Cham 6 (T. aestivum) and Gallareta (T. turgidum var. durum) had similar phyllochron across all moisture regimes whereas in other genotypes phyllochron was higher in the dries regime. Leaf area decreased with increasing moisture stress. Triticum turgidum var. durum genotypes were later in flowering as they had, on average, one leaf more than Triticum aestivum genotypes with similar leaf appearance rates.     

Immuno-localization of ferritin polypeptides in oocytes and somatic tissue of the freshwater snails Lymnaea stagnalis L. and Planorbarius corneus L.

Summary Antibodies were raised in rabbits against the 19000 Mr and 24000 Mr polypeptides of snail ferritin from Lymnaea stagnalis L. Anti-24000 Mr polypeptide antibodies were purified by an affinity-purification step and were made monospecific for their antigen by preabsorption with the 19000 Mr antigen. These purified antibodies were then used for in situ detection of their respective antigens by the indirect immunofluorescence method. The 19000 Mr polypeptide was found widely distributed in tissues of both pulmonate snails investigated (Lymnaea stagnalis L. and Planorbarius corneus L.) with the most intense antigen-directed fluorescence in certain connective tissue cells, secretory cells of the midgut gland and Sertoli cells and epithelia of the gonadal acini. In contrast, the 24000 Mr polypeptide could be detected only in yolk platelets of vitellogenic oocytes. The results indicate that yolk and somatic cell ferritins differ in immunoreactivity and structure and, accordingly may differ in function.This investigation was supported by the Deutsche Forschungsgemeinschaft. I greatly appreciate the advice given to me by Drs. U. Mays and V. Riedel, Münster.     

RFLP patterns of gliadin alleles in Triticum aestivum L.: implications for analysis of the organization and evolution of complex loci

A correspondence between RFLP patterns and gliadin alleles at the Gli-1 and Gli-2 loci was established in a set of 70 common wheat (T.aestivum L.) cultivars using -gliadin (K32) and -gliadin (pTU1) specific probes. All Gli-B1 and Gli-D1 alleles which differed in encoded -gliadins showed definite RFLP patterns after hybridization with the K32 probe. Two groups of Gli-B1 alleles, Gli-B1b-like and Gli-B1e-like, were identified, and these could originate from distinct genotypes of the presumptive donor of the B-genome. Intralocus recombination and/or gene conversion as well as small deletions, gene silencing and gene amplification were assumed to be responsible for the origin of new gliadin alleles. Silent -gliadin sequences were shown to exist in all of the genotypes studied. K32 also differentiated Gli-A1a from all other Gli-A1 alleles as well as the Gli-B11 allele in cultivars carrying the 1B/1R (wheat/rye) translocation. PTU1 was shown to recognize several Gli-A2 alleles, but not the Gli-B2 or Gli-D2 alleles. Moreover, this probe hybridized to chromosome 1R sequences suggesting the existence of rye gene(s), probably silent, for -gliadin-like proteins on chromosome 1R.     

The inheritance of cyanoglucoside content in Trifolium repens L.

A four generation backcross breeding program was undertaken. Analysis of the levels of cyanoglucoside in Acac progeny shows that the level of cyanoglucoside (linamarin and lotaustralin) is inherited and that part of the inherited variation in cyanoglucoside levels is attributable to the existence of different Ac alleles in the parent plant. In vitro microsomal cyanoglucoside biosynthetic activity was measured in a high-level and a low-level parent plant. There was no evidence for the presence of microsomes with different qualitative properties in the two plants. The Ac locus was shown to segregate independently of the S incompatibility locus.This research was supported in part by SERC Grant GRA 95550.     

Genetic diversity in wild diploid wheats Triticum monococcum var. boeoticum and T. urartu (Poaceae)

Summary The genetic diversity of two wild diploid wheat species, Triticum monococcum var. boeoticum and T. urartu, was assessed using starch gel electrophoresis. Genetic diversity is uniformly low in both species. Number of alleles per locus was very low with a mean of 1.22 for T. monococcum var. boeoticum and 1.19 in T. urartu. Percentage of polymorphic loci was also low, with a mean of 19.71 for T. monococcum var. boeoticum and a mean of 18.35 for T. urartu. Mean gene diversity was low with a mean of 0.052 in populations of T. monococcum var. boeoticum and a mean of 0.040 in populations of T. urartu. Genetic affinities of the species and of populations were computed using Nei's identity index (NI). Overall genetic affinities of the two species are NI=0.697. The genetic affinities of different populations of a species are uniformly high with NIs ranging from 0.894 to 1.000 in T. monococcum var. boeoticum and from 0.898 to 1.000 in T. urartu.Research supported by the California Agricultural Experiment Station and the International Board of Plant Genetic Resources     

Electrophoretic analysis of the high-molecular-weight glutenin subunits of Triticum monococcum,T. urartu,and the A genome of bread wheat (T. aestivum)

Summary The high molecular weight (HMW) subunit composition of glutenin was analysed by sodium dodecyl sulphate, polyacrylamide gel electrophoresis (SDS-PAGE) in the A genome of 497 diploid wheats and in 851 landraces of bread wheat. The material comprised 209 accessions of wild Triticum monococcum ssp. boeoticum from Greece, Turkey, Lebanon, Armenia, Iraq, and Iran; 132 accessions of the primitive domesticate T. monococcum ssp. monococcum from many different germplasm collections; one accession of free-threshing T. monococcum ssp. sinskajae; 155 accessions of wild T. urartu from Lebanon, Turkey, Armenia, Iraq, and Iran; and landraces of T. aestivum, mainly from the Mediterranean area and countries bordering on the Himalayan Mountains. Four novel HMW glutenin sub-units were discovered in the landraces of bread wheat, and the alleles that control them were designated Glu-Ald through Glu-Alg, respectively. The HMW subunits of T. monococcum ssp. boeoticum have a major, x subunit of slow mobility and several, less prominent, y subunits of greater mobility, all of which fall within the mobility range of HMW subunits reported for bread wheat. In T. monococcum ssp. monococcum the range of the banding patterns for HMW subunits was similar to that of ssp. boeoticum. However, two accessions, while containing y subunits were null for x subunits. The single accession of Triticum monococcum ssp. sinskajae had a banding pattern similar to that of most ssp. boeoticum and ssp. monococcum accessions. The HMW subunit banding patterns of T. urartu accessions were distinct from those of T. monococcum. All of them contained one major x and most contained one major y subunit. In the other accessions a y subunit was not expressed. The active genes for y subunits, if transferred to bread wheat, may be useful in improving bread-making quality.     

Polymorphism and Mendelian inheritance of photosystem II 23-kilodalton polypeptide

Soluble proteins from leaves of Nicotiana glauca Grah., N. langsdorffii Weinm., their reciprocal hybrids and amphiploid hybrid (N. glaucaxN. langsdorffii) were resolved by two-dimensional gel electrophoresis. Among a group of well-resolved polypeptides, in the isoelectric-point range of 5–5.5 and relative-molecular-mass (M_r) range of 18–23 kilodaltons (kDa), species-specific variation was observed. Polypeptides designated L and l are specific to N. langsdorffii, and G and g to N. glauca, while C is common to both species. Polypeptides L, G and C are localized in the chloroplasts and associated with thylakoid membranes. Polypeptide L is more acidic than polypeptide G, and both polypeptides have an M_r of 23 kDa. They were isolated from two-dimensional gels and their first 13 N-terminal amino-acid sequences were determined. These were found to be identical to the 13N-terminal amino acids of the photosystem II (PSII) 23-kDa polypeptide from spinach (T. Jansen et al. (1987) FEBS Lett. 216, 234–240) and, except for one change, to those from pea (R. Wales et al. (1989) Plant Molec. Biol., in press). Polypeptides G and L cross-react with antiserum against the PSII 23-kDa polypeptide from pea. Therefore, polypeptides G and L are extrinsic PSII 23-kDa polypeptides. They appear jointly and in equal amounts in the reciprocal hybrids. Since chloroplasts in Nicotiana are maternally inherited, these results demonstrate that polypeptides G and L are encoded by nuclear genes, are polymorphic variants of the PSII 23-kDa polypeptide, and are inherited in a Mendelian manner.Abbreviations kDa kilodalton - LS large subunit of Rubisco - M_r relative molecular mass - NEPHGE non-equilibrium pH gradient gel electrophoresis - PSII photosystem II - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SS small subunit of Rubisco