Identification of molecular markers for aluminium tolerance in diploid oat through comparative mapping and QTL analysis.

The degree of aluminium tolerance varies widely across cereal species, with oats (Avena spp.) being among the most tolerant. The objective of this study was to identify molecular markers linked to aluminium tolerance in the diploid oat A. strigosa. Restriction fragment length polymorphism markers were tested in regions where comparative mapping indicated the potential for orthologous quantitative trait loci (QTL) for aluminium tolerance in other grass species. Amplified fragment length polymorphism (AFLP) and sequence-characterized amplified region (SCAR) markers were used to provide additional coverage of the genome. Four QTL were identified. The largest QTL explained 39% of the variation and is possibly orthologous to the major gene found in the Triticeae as well as Alm1 in maize and a minor gene in rice. A second QTL may be orthologous to the Alm2 gene in maize. Two other QTL were associated with anonymous markers. Together, these QTL accounted for 55% of the variation. A SCAR marker linked to the major QTL identified in this study could be used to introgress the aluminium tolerance trait from A. strigosa into cultivated oat germplasm.

A molecular linkage map with associated QTLs from a hulless x covered spring oat population.

In spring-type oat ( Avena sativa L.), quantitative trait loci (QTLs) detected in adapted populations may have the greatest potential for improving germplasm via marker-assisted selection. An F(6) recombinant inbred (RI) population was developed from a cross between two Canadian spring oat varieties: 'Terra', a hulless line, and 'Marion', an elite covered-seeded line. A molecular linkage map was generated using 430 AFLP, RFLP, RAPD, SCAR, and phenotypic markers scored on 101 RI lines. This map was refined by selecting a robust set of 124 framework markers that mapped to 35 linkage groups and contained 35 unlinked loci. One hundred one lines grown in up to 13 field environments in Canada and the United States between 1992 and 1997 were evaluated for 16 agronomic, kernel, and chemical composition traits. QTLs were localized using three detection methods with an experiment-wide error rate of approximately 0.05 for each trait. In total, 34 main-effect QTLs affecting the following traits were identified: heading date, plant height, lodging, visual score, grain yield, kernel weight, milling yield, test weight, thin and plump kernels, groat beta-glucan concentration, oil concentration, and protein. Several of these correspond to QTLs in homologous or homoeologous regions reported in other oat QTL studies. Twenty-four QTL-by-environment interactions and three epistatic interactions were also detected. The locus controlling the covered/hulless character ( N1) affected most of the traits measured in this study. Additive QTL models with N1 as a covariate were superior to models based on separate covered and hulless sub-populations. This approach is recommended for other populations segregating for major genes. Marker-trait associations identified in this study have considerable potential for use in marker-assisted selection strategies to improve traits within spring oat breeding programs.

Inheritance and RAPD tagging of multiple genes for resistance to net blotch in barley.

A doubled haploid barley (Hordeum vulgare L.) population that was created from a cross between cultivars 'Léger' and 'CI 9831' was characterized by RAPD (random amplified polymorphic DNA) markers for resistance to isolate WRS857 of Pyrenophora teres Drechs. f. sp. maculata Smedeg., the causal agent of the spot form of net blotch. Resistance, which initially appeared to be conferred by a single gene from the approximate 1:1 (resistant : susceptible) segregation ratio of the doubled-haploid (DH) progeny, was found to be associated with three different genomic regions by RAPD analysis. Of 500 RAPD random primers that were screened against the parents, 195 revealed polymorphic bands, seven showed an association to the resistance in bulks, and these seven markers were mapped to three unlinked genomic regions. Two of these regions, one of which was mapped to chromosome 2, have major resistance genes. The third region has some homology to the chromosome 2 region. This study demonstrates the simultaneous location of markers for more than one gene governing a trait by using RAPD and bulked segregant analysis (BSA).

A molecular linkage map of cultivated oat.

A molecular linkage map of cultivated oat composed of 561 loci has been developed using 71 recombinant inbred lines from a cross between Avena byzantina cv. Kanota and A. sativa cv. Ogle. The loci are mainly restriction fragment length polymorphisms detected by oat cDNA clones from leaf, endosperm, and root tissue, as well as by barley leaf cDNA clones. The loci form 38 linkage groups ranging in size from 0.0 to 122.1 cM (mean, 39 cM) and consist of 2-51 loci each (mean, 14). Twenty-nine loci remain unlinked. The current map size is 1482 cM and the total size, on the basis of the number of unlinked loci, is estimated to be 2932.0 cM. This indicates that this map covers at least 50% of the cultivated oat genome. Comparisons with an A-genome diploid oat map and between linkage groups exhibiting homoeology to each other indicate that several major chromosomal rearrangements exist in cultivated oat. This map provides a tool for marker-assisted selection, quantitative trait loci analyses, and studies of genome organization in oat.

The identification of random amplified polymorphic DNA markers for daylength insensitivity in oat.

Daylength insensitive accessions of Avena sativa L. are being used to develop cultivars that will flower normally when grown under short or long photoperiods. Field data indicate that the insensitivity trait is under the control of a single dominant gene, designated Di1. The random amplified polymorphic DNA (RAPD) technique and bulk segregant analysis of daylength sensitive and insensitive plants were used to find markers for this gene. Five of 200 random decamer primers tested produced polymorphic bands, which were shown to be linked to the trait using 30 homozygous insensitive and 30 homozygous sensitive F3 individuals. Three of the markers produced a band in the presence of the dominant allele, and two in its absence. Segregation analysis showed that markers 221 and 136 could be mapped to within 9.8 +/- 4.6 and 13.9 +/- 5.4 cM of the trait, respectively; that is, close enough to be useful in a breeding program. A study of different cultivars suggested that the band produced by primer 136 is actually the more closely linked marker and the only one present in the original Di1 gene donor CAV2700. The possibility of using both markers in populations derived from different cultivars is discussed.

Identification of an RAPD marker for the crown rust resistance gene Pc68 in oats.

The feasibility of using bulk segregant analysis to identify molecular markers for disease resistance genes in oats was investigated, utilizing random primers in conjunction with polymerase chain reaction technology. Random primers were screened for the amplification of polymorphic DNA fragments on two pools of genomic DNA isolated from plants that were homozygous for the presence and absence of the crown rust resistance gene Pc68. Ten primers were identified that amplified polymorphic DNA fragments. Of these, one was tightly linked, in repulsion, to the target gene, while the other nine were not linked to this trait. The relatively low cost of polymerase chain reaction technology, coupled with rapid leaf disc genomic DNA extraction techniques should result in the effective use of this linked marker in oat breeding selection programs.

Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories.

Random amplified polymorphic DNA (RAPD) analysis appears to offer a cost- and time-effective alternative to restriction fragment-length polymorphism (RFLP) analysis. However, concerns about the ability to compare RAPD results from one laboratory to another have not been addressed effectively. DNA fragments that were amplified by five primers and shown to be reproducibly polymorphic between two oat cultivars (within the Ottawa laboratory) were tested in six other laboratories in North America. Four of the six participants amplified very few or no fragments using the Ottawa protocol. These same participants were able to generate a considerable number of amplified fragments by using their own protocols. The reproducibility of results among laboratories was affected by two factors. First, different laboratories amplified different size ranges of DNA fragments, and, consequently, small and large polymorphic fragments were not always reproduced. Second, although reproducible results were obtained with four of the primers, reproducible results were not obtained with the fifth primer, using the same reaction conditions. It is suggested that if the overall temperature profiles (especially the annealing temperature) inside the tubes are identical among the laboratories, then RAPD fragments are likely to be reproducible.

Identification of a RAPD marker linked to the oat stem rust gene Pg3.

The feasibility of identifying molecular markers linked to disease resistance genes in oats was investigated utilizing random primers in conjunction with polymerase chain reaction technology. A pair of near-isogenic oat lines were screened for polymorphic DNA fragments linked to the stem rust resistance gene Pg3. Two primers were identified which amplified DNA fragments that were polymorphic between the lines analyzed. One primer (ACOpR-2) was shown to be completely linked to the Pg3 locus; the other primer was not linked to either the ACOpR-2 or the Pg3 loci. This type of analysis, combined with rapid leaf disc DNA extraction techniques, offers an effective means of identifying useful molecular markers and of applying them to plant breeding selection strategies.

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species.

Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1-3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A - H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B - H. leporinum; Map C - H. vulgare, H. spontaneum, H. agriocrithon; Map D - H. chilense, H. bogdanii; and Map E - remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.

The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl-tRNA synthetase mutant tsHl and its revertants.

The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5 degrees C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5 degrees C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.

Isolation and characterization of revertants of the mammalian temperature sensitive leucyl-tRNA synthetase mutant tsHl.

Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl-tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl-tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl-tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl-like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl-tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5 degrees C.

An investigation into the potential of a mammalian temperature sensitive leucyl-tRNA synthetase mutant for mutagenesis studies.

Reversion of the mammalian temperature sensitive leucyl-tRNA synthetase mutant CHO tsH1 [33] has been investigated to determine its potential for mutagenesis studies. The protocol for a mutation assay using the new class of temperature sensitive conditional lethal mutants of somatic cells is presented. While it requires careful control of temperature during revertent selection it promises to provide a system complementary to the existing mutation assays. UV and EMS mutagenesis of tsH1 has confirmed that approx. 3TD of expression time are sufficient for complete expression of induced mutants at low mutagen doses (80% survival following UV and 40-50% with EMS). At a higher UV dose resulting in only 10% survival much longer expression times were required which cannot be explained by growth delays alone. While the reason for this is unknown it suggests that care must be taken in studies which require high mutagen doses. Representative revertants which were isolated show a range of phenotypes between those of tsH1 and WT. They appear to be a promising source of extragenic suppressor mutants [33] with alterations in functions affecting protein synthesis.

Effect of leucine on the temperature sensitive phenotype of a mammalian leucyl-tRNA synthetase mutant.

The concentration of leucine in the growth medium has been found to influence the expression of the temperature sensitive phenotype of a mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase. Plating efficiency and growth studies showed that increasing the leucine concentration allows cells to survive at normally non-permissive high temperatures and conversely decreasing the leucine concentration enhances the adverse effectsof high temperature. A similar but smaller effect was noted with isoleucine. It is suggested that this observation may form the basis of a rapid test, useful in directing the investigation of the lesion in similar mutants to pathways involving specific amino acids.