Allelic variation of the D-prolamin subunits encoded at the Hch genome in a collection of primary hexaploid tritordeums

 Hexaploid tritordeum is the amphiploid derived from the cross between Hordeum chilense and durum wheat. The storage proteins synthesised by the H^ch genome have an influence on the gluten strength of this amphiploid. The D-prolamins of H. chilense are glutenin-like proteins. The variability has been analysed electrophoretically and up to 20 different patterns have been detected in a world collection of this species. This genetic variability of H. chilense could be a source of additional variation in tritordeum and in breeding wheat for quality. Received: 29 November 1998 / Accepted: 19 December 1998     

Variability in monomeric prolamins in Hordeum chilense

Eighty-eight lines of the species Hordeum chilense Roem. et Schult., representative of the variability found in the latter’s distribution zone, were analysed. Monomeric prolamins (protein fraction corresponding to wheat gliadins) were studied using the SDS-PAGE technique. The analysis of the different bands was performed using the programme BioImage ^™ Whole Band Analyzer. Jaccard’s coefficient of similarity was calculated, and the lines were grouped by cluster analysis using UPGMA. A great variability was found between the different lines studied. Forty-two different bands were identified, all of which were polymorphic. Sixty-eight different patterns of monomeric prolamins were identified within the 82 lines studied. A dendrogram was obtained from the analysis of the groups. No relationship between the distribution of the variability in the dendrogram and the geographical origin or the ecological characteristics of the species could be detected. It is concluded that H. chilense is an important pool of variability for storage proteins that could be used in cereal breeding. Received: 10 January 2000 / Accepted: 21 March 2000     

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     

Development and characterization of Hordeum chilense chromosome-specific STS markers suitable for wheat introgression and marker-assisted selection

 RAPD markers were developed for octoploid×Tritordeum (amphiploid Hordeum chilense×Triticum aestivum) and its parents. Addition lines were used to identify specific RAPD markers for the Hordeum chilense chromosomes detectable in a wheat background. Twelve RAPD fragments have been cloned, sequenced and converted into STS markers. Eleven of these STSs have maintained both the chromosome specificity and the possibility of detection in a wheat background. The use of these markers in multiplexed PCRs facilitates both the efficient and reliable screening of new addition lines as well as the monitoring of introgression of H. chilense in bread and durum wheat. Received: 5 June 1998 / Accepted: 17 September 1998     

Variation in the HMW and LMW glutenin subunits from Spanish accessions of emmer wheat (Triticum turgidum ssp. dicoccum Schrank)

Emmer wheat (Triticum turgidum ssp. dicoccum Schrank) is hulled wheat that survives in marginal areas of the Mediterranean Region. The HMW and LMW glutenin subunit composition of 97 accessions of emmer wheat from Spain have been analysed by SDS-PAGE. For the HMW glutenin subunits, four allelic variants were detected for the Glu-A1 locus; one of them has not been previously described. For the Glu-B1 locus, three of the nine alleles detected have not been found before. A high degree of variation was evident for the LMW glutenin subunits, and up to 23 different patterns were detected for the B-LMW glutenin subunits. Considering both types of proteins (HMW and LMW), 30 combinations were found between all the evaluated lines. This wide polymorphism can be used to transfer new quality genes to wheat, and to widen its genetic basis. Received: 13 June 2000 / Accepted: 3 July 2000     

Conservation in wheat high-molecular-weight glutenin gene promoter sequences: comparisons among loci and among alleles of the GLU-B1-1 locus

 The high-molecular-weight glutenin (HMW) genes and encoded subunits are known to be critical for wheat quality characteristics and are among the best-studied cereal research subjects. Two lines of experiments were undertaken to further understand the structure and high expression levels of the HMW-glutenin gene promoters. Cross hybridizations of clones of the paralogous x-type and y-type HMW-glutenin genes to a complete set of six genes from a single cultivar showed that each type hybridizes best within that type. The extent of hybridization was relatively restricted to the coding and immediate flanking DNA sequences. Additional DNA sequences were determined for four published members of the HMW-glutenin gene family (encoding subunits Ax2^*, Bx7, Dx5, and Dy10) and showed that the flanking DNA of the examined genes diverge at approximately −1200 bp 5′ to the start codon and 200–400 bp 3′ to the stop codon. These divergence sites may indicate the boundaries of sequences important in gene expression. In addition, promoter sequences were determined for alleles of the Bx gene (Glu-B1-1), a gene reported to show higher levels of expression than other HMW-glutenin genes and with variation among cultivars. The sequences of Bx promoters from three cultivars and one wild tetraploid wheat indicated that all Bx alleles had few differences and contained a duplicated portion of the promoter sequence “cereal-box” previously suspected as a factor in higher levels of expression. Thus, the “cereal-box” duplication preceeded the origin of hexaploid wheat, and provides no evidence to explain the variations in Bx subunit synthesis levels. One active Bx allele contained a 185-bp insertion that evidently resulted from a transposition event. Received: 5 August 1997 / Accepted: 6 November 1997     

Chromosomal location of structural genes controlling isozymes in Hordeum chilense

Summary Polyacrylamide and starch gel electrophoresis of esterase (EST), glutamate oxaloacetate transaminase (GOT) and phosphoglucomutase (PGM) isozymes in Hordeum chilense, Triticum turgidum conv. durum, the amphiploid H. chilense X T. turgidum (Tritordeum), and the durum wheat/H. chilense monosomic addition lines revealed the chromosomal location of one EST locus, two GOT loci and one PGM locus. Loci Est-H ^ch1 and Got-H ^ch2 were found on chromosome 6H^ch,Got-H ^ch3 on chromosome 3H^ch, and Pgm-H ^ch1 on chromosome 4H^ch. These results lend evidence for the assumed homoeology relationships between chromosomes of Triticeae species.     

Chromosomal location of structural genes controlling isozymes in Hordeum chilense

Summary A study of 6-phosphogluconate dehydrogenase and malate dehydrogenase isozyme expression in Triticum turgidum conv. durum /Hordeum chilense monosomic addition lines has revealed the location of two structural genes, 6-pgd-H ^ch 2 and Mdh-H ^ch 1, on chromosome 1H^ch of H. chilense. The homoeology between 1H^ch and other chromosome of Triticeae related species is discussed on the basis of isozyme gene analysis.     

PCR analysis of genes encoding allelic variants of high-molecular-weight glutenin subunits at the Glu-D1 locus

Genes encoding high-molecular-weight (HMW) glutenin subunits, present in bread-wheat lines and cultivars, were studied by RFLP (restriction fragment length polymorphism) and PCR (polymerase chain reaction) analyses. In particular, allelic subunits of the x-or y-type, encoded at the Glu-D1 locus present on the long arm of chromosome 1D, were investigated. The variation in size, observed in different allelic subunits, is mainly due to variation in the length of the central repetitive domain, typical of these proteins. Deletions or duplications, probably caused by unequal crossingover, have given rise to the size heterogeneity currently observed. The possibility of using the PCR technique for a detailed analysis of HMW glutenin genes in order to obtain a more accurate estimation of the molecular weight of their encoded subunits, and the detection of unexpressed genes, is also described.     

Allelic variation of the HMW glutenin subunits in Spanish accessions of spelt wheat (Triticum aestivum ssp. spelta L. em. Thell.)

Spelt wheat (Triticum aestivum ssp. spelta L. em. Thell.) is a hulled wheat of Germanic origin that survives at marginal areas in Asturias (Spain). The HMW glutenin subunit composition of 403 accessions of spelt wheat from Spain has been analysed by SDS-PAGE. Three allelic variants were detected for Glu-A1. For the Glu-B1 locus, two of seven alleles detected have not been found before; while four of nine alleles detected for the Glu-D1 are not previously described. Considering the three loci, twenty five combinations were found among all the evaluated lines. This wide polymorphism could be used to transfer new quality genes to wheat, and widen the genetic basis of them. Received: 19 September 2000 / Accepted: 20 October 2000     

An RFLP map of diploid Hordeum bulbosum L. and comparison with maps of barley (H. vulgare L.) and wheat (Triticum aestivum L.)

This paper describes the first extensive genetic map of Hordeum bulbosum, the closest wild relative of cultivated barley. H. bulbosum is valuable for haploid production in barley breeding, and because of desirable agronomic characteristics, it also has potential for trait introgression into barley. A H. bulbosum map will assist introgression and provide a basis for the identification of QTLs for crossability with barley and other potentially useful genes. The present study used a population of 111 individuals from a PB1×PB11 cross to develop a genetic linkage map of diploid H. bulbosum (2n=2x=14) based on barley, wheat and other ”anchor” cereal RFLP markers previously mapped in other species. Because of the cross-pollinating and highly polymorphic nature of H. bulbosum, up to four alleles showed segregation at any one locus, and five different segregation types were found. This enabled maps to be developed for the PB1 and PB11 parents, as well as a combined map. In total, 136 RFLP loci were mapped with a marker coverage of 621 cM. The markers were generally colinear with barley but H. bulbosum had less recombination in the centromeric regions and similar or more in the distal regions. Cytological studies on pollen mother cells at metaphase-I showed marked distal localization of chiasmata and a frequency consistent with the genetic map length. This study showed that H. bulbosum was highly polymorphic, making it suitable for trait analysis and supplementing maps of barley. Received: 20 November 2000 / Accepted: 5 January 2001     

Transgenic tomato plants expressing a Lycopersicon chilense chitinase gene demonstrate improved resistance to Verticillium dahliae race 2

 An acidic endochitinase gene (pcht28) isolated from Lycopersicon chilense was introduced into tomato (L. esculentum) through Agrobacterium-mediated transformation, using the CAMV 35S promoter. Transgenic plants demonstrated a high level of constitutive expression of pcht28 and chitinase enzyme activity. Kanamycin-resistant R1 plants (resulting from self-pollination of transgenic plants) as well as R2 plants were evaluated for their tolerance to Verticillium dahliae (race 1 and 2 for R1 plants and race 2 for R2 plants) in the greenhouse. They demonstrated a significantly (P<0.05) higher level of tolerance to the fungi compared to the nontransgenic plants, as measured by foliar disease symptoms, vascular discoloration, and vascular discoloration index. The transgenic plants produced in this study represent a source of genetic resistance to Verticillium dahliae. Received: 18 August 1998 / Revision received: 22 March 1999 / Accepted: 14 April 1999     

Recombination mapping of some chromosome 1A-, 1B-, 1D- and 6B-controlled gliadins and low-molecular-weight glutenin subunits in common wheat

 Inheritance of low-molecular-weight glutenin subunits (LMW GS) and gliadins was studied in the segregating progeny from several crosses between common wheat genotypes. The occurrence of a few recombinants in the F₂ grains of the cross Skorospelka Uluchshennaya×Kharkovskaya 6 could be accounted for by assuming that the short arm of chromosome 1D contains two tightly linked loci each coding for at least one gliadin plus one C-type LMW GS. These loci were found to recombine at a frequency of about 2%, and to be linked to the Glu-D3 locus coding for B-type LMW GS. Some proteins showing biochemical characteristics of D-type or C-type LMW GS were found to be encoded by the Gli-B1 and Gli-B2 loci, respectively. Strongly stained B-type LMW GS in cvs Skorospelka Uluchshennaya and Richelle were assigned to the Glu-B3 locus, but recombination between this locus and Gli-B1 was not found. Analogously, in the cross Bezostaya 1×Anda, no recombination was found between Gli-A1 and Glu-A3, suggesting the maximum genetic distance between these loci to be 0.97% (P=0.05). A B-type LMW GS in cv Kharkovskaya 6 was assigned to the Glu-B2 locus, with about 25% recombination from the Gli-B1 locus. The present results suggested that alleles at Gli loci may relate to dough quality and serve as genetic markers of certain LMW GS affecting breadmaking quality. Received: 9 July 1996/Accepted: 15 November 1996     

PCR detection of Hordeum bulbosum introgressions in an H. vulgare background using a retrotransposon-like sequence

Retrotransposon-like sequences are ideal tools for initial screening assays to distinguish between closely related species because of their ubiquitous presence, high copy number, chromosome coverage and rapid sequence evolution. A retrotransposon-like sequence, pSc119.1, cloned from Secale cereale (rye) has been used to obtain PCR primers that are capable of detecting small introgressions of Hordeum bulbosum (bulbous barley grass) chromatin in a Hordeum vulgare (cultivated barley) background. Combining this PCR-based assay with a crude but effective high-throughput DNA extraction has enabled the rapid identification of plants possessing H. bulbosum introgressions from large numbers of progeny from H. vulgare×H. bulbosum crosses. These plants are then further characterized by more-refined cytological, molecular and pathological techniques to locate and map the introgressed chromatin and to evaluate their disease resistance. Received: 18 April 2001 / Accepted: 23 August 2001     

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     

Interaction between genes controlling a new group of glutenin subunits in bread wheat

Summary One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced total protein extracts from the endosperm of hexaploid wheat revealed a new set of faintly-stained bands, having slower electrophoretic mobility than the high-molecular-weight (HMW) glutenin subunits. These new bands have been termed the E group of glutenin subunits. Analysis of aneuploid stocks of Chinese Spring wheat has shown that three of the E bands, in order of increasing electrophoretic mobility, are controlled by genes on the short arms of chromosomes 1B, 1A and 1D, respectively. The E bands are expressed only in the presence of the long arm of chromosome 1B indicating an interaction between two or more genes involved in their production in wheat endosperm. The gene on the short arm of chromosome 1D controlling an E subunit recombined freely with Tri-D1 and the centromere but not at all with Gli-D1, indicating additional complexity at the Gli-DI locus in wheat.     

Sequence variations at a complex microsatellite locus in rice and its conservation in cereals

In an attempt to study changes associated with microsatellites in rice, the DNAs of cultivated rice, including indica and japonica varieties, and wild rice genotypes were amplified by the polymerase chain reaction with primers flanking the (GATA) _n and (AC) _n repeats at a microsatellite-containing locus OS1E6 (Genebank accession number AFO16647) previously reported from a PstI rice (var. Malkolam) genomic library in pUC18. Eight alleles of varying sizes were obtained which were cloned and sequenced. Sequencing data indicated that the size variations of the different alleles were due to differences in the repeat number as well as to sequence variations in the region flanking the microsatellite motifs. In order to study the presence of this complex microsatellite-containing locus of rice in different cereals, their DNAs were amplified using primers flanking the OS1E6 locus. It was found that this locus was present in the various cereal genotypes analyzed, indicating its conservation across different cereal members. Received: 10 March 2000 / Accepted: 14 April 2000     

Localising QTLs for leaf rust resistance and agronomic traits in barley (Hordeum vulgare L.)

The Hordeum vulgare accession ’HOR 1063’ was crossed with the barley cultivar Krona, and 220 doubled haploid lines were produced based on this cross. A molecular map was constructed based on RFLP markers. Field trials were performed over 2 years and at two locations. In field trials, resistance to leaf rust by means of artificial infection, heading date, plant height and Kernel weight were assessed. For leaf rust resistance, 4 QTLs were localised, that explained 96.1% of the genetic variation. One QTL on chromosome 4H confirmed a position found in another genetic background and one mapped to the same position as Rph16 on chromosome 2H. All digenic effects decreased the effects of the respective QTLs. In addition to the denso-locus and the hex-v locus, other QTLs influencing heading date, plant length and kernel weight were found in this cross. Received: 16 July 1999 / Accepted: 7 September 1999     

High-molecular-weight glutenin subunits in the Cylindropyrum and Vertebrata section of the Aegilops genus and identification of subunits related to those encoded by the Dx alleles of common wheat

The high-molecular-weight (HMW) glute-nin subunit composition of seven species from the Cylindropyrum and Vertebrata sections of the Aegilops genus was studied using SDS-PAGE and Western blot analysis. Two subunits were detected in Ae. caudata and three in Ae. cylindrica. In both species, subunits showing electrophoretic mobility similar to that of 1Dx2 were present. Western blot analysis using a monoclonal antibody (IFRN 1602) specific for the 1Ax and 1Dx subunits of bread wheat showed that the 1Dx-like subunit of Ae. caudata gave only a weak reaction. This indicates that Ae. caudata expresses subunits which are more distantly related to the 1Dx subunits. Two subunits were detected in each of the 60 accessions of Ae. tauschii, including several 1D^tx subunits showing different electrophoretic mobilities from those of the 1Dx subunits commonly found in bread wheat. All of the 1D^tx subunits reacted strongly with IFRN 1602, confirming their close relationship to the 1Dx subunits of bread wheat. Three subunits were found in Ae. crassa (6 x), four in Ae. ventricosa and Ae. juvenalis and five in Ae. vavilovii. In these four species, the subunits that showed electrophoretic mobility similar, or close, to that of 1Dx2 all reacted with IFRN 1602. In addition, Ae. ventricosa contained a subunit showing electrophoretic mobility slower than that of 1Dx2.2, which also reacted with IFRN 1602. These results suggest that the D-genome component in the multiploid Aegilops species express at least one HMW glutenin subunit that is structurally related to the 1Dx subunits of bread wheat. Received: 5 November 1999 / Accepted: 12 February 2000     

Identification of random amplified polymorphic DNA (RAPD) markers linked to the v locus in barley, Hordeum vulgare L.

 Recombinant backcross lines of barley were produced from a cross between Kanto Nakate Gold (KNG; two-rowed) and Azumamugi (AZ; six-rowed) after backcrosses of F₁ plants with AZ as the recurrent parent. Each of these lines had an introgressed segment from chromosome 2 of KNG. Two recombinant backcross lines, L1 and M3-13, were used for an initial screening of polymorphism. After screening a total of 888 oligonucleotides as arbitrary primers, we identified eight random amplified polymorphic DNAs (RAPDs) between backcross lines and AZ. Among the RAPD fragments, CMNA-38₇₀₀ was linked to the v locus with a recombination frequency of zero, while OPJ-09₈₅₀ and OPP-02₇₀₀ were linked to the v locus at a map distance of 1.4 cM. Thus, the three RAPD markers were clustered around the v locus since the lengths of introgressed chromosomal segments in the L1 and M3-13 lines were no less than 38 cM. The other five RAPD fragments that we identified were not linked to the v locus. Received: 14 January 1997 / Accepted: 14 February 1997