A. De Bustos

Molecular characterisation of the inactive allele of the gene Glu-A1 and the development of a set of AS-PCR markers for HMW glutenins of wheat.

Abstract The present work reports new PCR markers that amplify the complete coding sequence of the specific alleles of the high molecular weight (HMW) glutenin genes. A set of AS-PCR molecular markers was designed which use primers from nucleotide sequences of the Glu-A1 and Glu-D1 genes, making use of the minor diffeences between the sequences of the x1, x2* of Glu-A1, and the x5 and y10 of Glu-D1. These primers were able to distinguish between x2* and the x1 or xNull of Glu-A1. Also x5 was distinguishable from x2, and y10 from y12. The primers amplified the complete coding regions and corresponded to the upstream and downstream flanking positions of Glu-A1 and Glu-D1. Primers designed to amplify the Glu-A1 gene amplified a single product when used with genomic DNA of common wheats and the xNull allele of this gene. This work also describes the cloning and characterisation of the nucleotide sequence of this allele. It possesses the same general structure as x2* and x1 (previously determined) and differs from these alleles in the extension of the coding sequence for a presumptive mature protein with only 384 residues. This is due to the presence of a stop codon (TAA) 1215-bp downstream from the start codon. A further stop codon (TAG), 2280-bp downstream from the starting codon is also found. The open reading frame of xNull and x1 alleles has the same size in bp. Both are larger than x2* which shows two small deletions. The reduced size of the presumptive mature protein encoded by xNull could explain the negative effect of this allele on grain quality.

Physical mapping of repetitive DNA sequences and 5S and 18S-26S rDNA in five wild species of the genus Hordeum.

The genetic relationships between several wild species and subspecies of the genusHordeum were assessed using fluorescencein situ hybridization (FISH). Plant material included natural populations of wild barley growing in Spain of the annual species,H. marinum ssp.marinum (2n=14) andgussoneanum (2n=14), andH. murinum ssp.murinum (2n=28), andleporinum (2n=28) and the perennial speciesH. bulbosum (2n=14) andH. secalinum (2n=28), plus the South American perennial speciesH. chilense (2n=14). FISH was used to locate the chromosomal sites of two rDNA multigene families 5S and 18S–26S (pTa71 and pTa794) and three repetitive DNA sequences (pSc119.2, pAs1 and pHch950) isolated from different species and genera. The seven chromosomes of the diploid species were readily distinguished by their external morphology and hybridization patterns to pTa71, pTa794, pSc119.2 and pAs1. These DNA probes were also useful for the identification of homologous chromosomes and in differentiating these from unidentified chromosomes in the tetraploid taxa. The use of the probe pHch950 permitted intergenomic differentiation in tetraploids and supports the diphyletic origin ofH. murinum andH. secalinum. Thein situ experiments yielded the following conclusions: (1) differences between the subspeciesmarinum andgussoneanum; (2) close relationships between the subspeciesmurinum andLeporinum; and (3) major differences in physical mapping betweenH. bulbosum and the remaining taxa. The genomic and phylogenetic relationships between taxa, as inferred from the results, are discussed.

RAPD variation in wild populations of four species of the genus Hordeum (Poaceae)

Abstract The genetic variation of 102 natural populations of wild barley growing in Spain was assessed using RAPDs (random amplified polymorphic DNA). The plant material included the annual species H. marinum subsp. marinum (22 populations) and subsp. gussoneanum (14), H. murinum subsp. murinum (7) and subsp. leporinum (35), and the perennial species H. bulbosum (17) and H. secalinum (7). Ten of the tested 64 arbitrary 10-mer primers amplified polymorphic DNA in all taxonomic units. Analyses was performed within and between populations, species and subspecies. The primers gave a total of 250 RAPD products. The level of polymorphism varied between taxonomic units depending on the primers employed and the plant reproductive system. In general, the most variable were the allogamous species H. secalinum and H. bulbosum and the autogamous H. marinum subsp. marinum. Among the amplified bands, 69 (27%) were shared by at least two different taxonomic units. The remaining bands were specific. The results demonstrate differences in the degree of similarity between taxonomic units. Jaccard’s similarity coefficients for interval measure within and between populations were used to produce a cluster diagram using the unweighted pair-group method (UPGMA). The different populations of the species and subspecies of Hordeum fell into three groups. The first group contained the populations belonging to both subspecies of H. marinum, plus those of H. secalinum. The populations of H. marinum subsp. gussoneanum were very closely associated. Those of H. marinum subsp. marinum were grouped in a broad cluster. The second group, occupying the innermost position of the tree, was very closely associated with the populations of both subspecies of H. murinum. The third branch segregated H. bulbosum. A series of RAPD markers were investigated by cleaving the amplified products of the same size with restriction endonucleases that recognize targets of 4- or 6-bp. The production of equivalent fragments following cleavage by the same enzyme would seem to demonstrate their homology in samples from different individuals, populations or taxonomic units.

Characterisation and analysis of new HMW-glutenin alleles encoded by the Glu-R1 locus of Secale cereale

This work reports the molecular characterisation of new alleles of the previously reported Glu-R1 locus. Wheat lines carrying the chromosome substitution 1R(1D), rye cultivars and related wild species were analysed. Five new x-type and four y-type Glu-R1 glutenin subunits were isolated and characterised. The coding region of the sequences shows the typical structure of the HMW glutenin genes previously described in wheat, with the N and C-terminal domains flanking the central repetitive region. Tri-, hexa- and nona-peptides found in the central repetitive region of wheat glutenin genes were also present in the rye genes. Duplications and deletions of these motifs are responsible for allelic variation at the Glu-R1 locus. Orthologous genes (from different genomes) were more closely related than paralogous genes (x- and y-type), supporting the hypothesis of gene duplication before Triticeae speciation. Differences in the number and position of cysteine residues identified alleles which in wheat are associated with good dough quality. SDS proteins encoded by some characterised alleles were presumptively identified.

Phylogenetic relationships of the genus Secale based on the characterisation of rDNA ITS sequences

Abstract. Sequence analysis of the internal transcribed spacer of the 18S-5.8S-26S rDNA (ITS-1) region was performed in order to analyse the phylogenetic relationships of eleven taxa of cultivated and wild rye species. The ITS regions were amplified using designed primers. At least ten positive clones of each taxonomic unit were sequenced and compared. Two different ITS sequences were found in three taxa: Secale sylvestre Host, Secale strictum ssp. kuprijanovii Grossh. and Secale strictum ssp. africanum Stapf. Secale sylvestre Host was the species that showed the greatest number of comparative differences in the sequences, and was the most distant of all the taxonomic units analysed. A certain degree of variation was found among all four subspecies of S. strictum analysed. S. strictum Presl ssp. strictum was most closely related to S. strictum ssp. africanum Stapf and S. strictum ssp. kuprijanovii Grossh to S. strictum ssp. anatolicum (Boiss.) Hammer. S. vavilovii showed similarities with this group of subspecies and with the S. cereale group. No differences were found between the weed forms of S. cereale and cultivated rye.

Marker assisted selection to improve HMW-glutenins in wheat

The present work presents the application of new markers based on thePCR technology to amplify the complete coding sequence of the specificalleles of the high molecular weight (HMW) glutenin genes. A set ofAS-PCR molecular markers of the Glu-A1 and Glu-D1 genes wasdesigned, making use of the minor differences between the sequences of thex1, xNull and x2 of Glu-A1, x2, x5, y10 and y12 of Glu-D1. These markers were applied to assist selection inbackcross progenies to improve the glutenin quality in Spanish wheat. Theselection was also assisted using other polymorphic systems (AFLPs) inrecovering the genetic background of the recurrent parent.

Characterisation of two gene subunits on the 1R chromosome of rye as orthologs of each of the Glu-1 genes of hexaploid wheat

Abstract The visco-elastic properties of bread flour are firmly associated with the presence or absence of certain HMW subunits coded by the Glu-1 genes. Identifying allelic specific molecular markers (AS-PCR) associated with the presence of Glu-1 genes can serve as a valuable tool for the selection of useful genotypes. This paper reports the use of primers designed from nucleotide sequences of the Glu-D1 gene of wheat (AS-PCR for Glu-D1y10) that recognise and amplify homologous sequences of the Glu-R1 gene subunits of rye. The primers amplify the complete coding regions and provided two products of different size in rye, in wheats carrying the substitution 1R(1D) and in rye-wheat aneuploid lines carrying the long arm of chromosome 1R. The location, the molecular characterisation of these sequences and their expression during grain ripening seem to demonstrate that the amplification products correspond to structural genes encoding the high-molecular-weight (HMW) glutenins of rye. The homology of the rye gene to subunits encoding HMW glutenins in wheat was confirmed by Southern blots and sequencing. The amplification-products were cloned, sequenced and characterised, and the sequences compared with the main glutenin subunits of wheat and related species. Further, an RT-PCR experiment was performed using primers designed from the sequence of both amplified products. This assay demonstrated that both sequences are expressed in endosperm during grain ripening. The results of these analyses suggest that both gene subunits correspond to x- and y-type genes of the Glu-R1 locus of rye.

Localization of Rad50, a single-copy gene, on group 5 chromosomes of wheat, using a FISH protocol employing tyramide for signal amplification (Tyr-FISH).

An indirect tyramide signal amplification FISH technique (Tyr-FISH) was adapted to detect single-copy genes in plants. In this study, Rad50 loci (responsible for a protein involved in the repair of breaks in double-stranded DNA) were localized on the chromosomes of homologous group 5 in common wheat (Triticum aestivum). Three loci, Rad50A, Rad50B and Rad50D, none of which have been previously mapped using any kind of molecular approach, were localized at similar positions on the short arm of chromosomes 5A, 5B and 5D, respectively. The loci are all proximal to 5S ribosomal gene clusters. The sensitivity and efficiency of the Tyr-FISH technique was tested using 5 differently sized fragments of the gene Rad50D as probes in FISH involving the chromosomes of the diploid wheat Triticum tauschii (the D-genome donor species of the hexaploid wheats). The procedure, which provided specific signal amplification with low level background, detected target DNA sequences as small as 2 kb with a frequency of 37.5%.

AFLP characterization of natural populations of Berberis (Berberidaceae) in Patagonia, Argentina

Abstract. AFLP markers were used to analyse the intra- and interspecific relationships among 22 natural populations of 13 Patagonian species of Berberis and the relationships among the taxa belonging to homoploid and polyploid complexes. Seven primer combinations gave rise to 231 AFLP bands, of which 199 were polymorphic. Correspondence between AFLP data, morphological traits and seed protein bands was also assessed. The dendrogram inferred from AFLP fingerprints showed that, in general, populations of the same species formed closely related groups with high coefficients of similarity. Principal co-ordinates analysis showed two separate subgroups: (i) B. bidentata and their putative ancestors –B. darwinii and B. linearifolia– which form a homogamic group, and (ii) B. buxifolia, B. heterophylla and B. parodii– which could form a polyploid hybrid complex.

The Rad50 genes of diploid and polyploid wheat species. Analysis of homologue and homoeologue expression and interactions with Mre11

The MRN complex plays a central role in the DNA repair pathways of eukaryotic cells and takes part in many other processes, including cell cycle checkpoint signalling, meiosis, DNA replication and telomere maintenance. This complex is formed by the interaction of the products of the Mre11, Rad50 and Nbs1 genes. This paper reports the molecular characterization, expression and interactions of the Rad50 gene in several wheat species with different levels of ploidy. The homoeologous Rad50 wheat genes were found to show a high level of conservation. Most of the RAD50 domains and motifs previously described in other species were also present in wheat RAD50; these proteins are therefore likely to have similar functions. Interactions between the RAD50 wheat proteins and their MRE11 counterparts in the MRN complex were observed. The level of expression of Rad50 in each of the species examined was determined and compared with those previously reported for the Mre11 genes. In some cases similar levels of expression were seen, as expected. The expression of the RAD50 homoeologous genes was assessed in two polyploid wheat species using quantitative PCR. In both cases, an overexpression of the Rad50B gene was detected. Although the results indicate the maintenance of function of these species’ three homoeologous Rad50 genes, the biased expression of Rad50B might indicate ongoing silencing of one or both other homoeologues in polyploid wheat. To assess the consequences of such silencing on the formation of the MRN complex, the interactions between individual homoeologues of Rad50 and their genomic counterpart Mre11 genes were examined. The results indicate the inexistence of genomic specificity in the interactions between these genes. This would guarantee the formation of an MRN complex in wheat.