Mapping of the oat crown rust resistance gene Pc91.

Crown rust is an important disease of oat caused by Puccinia coronata Corda f. sp. avenae Eriks. Crown rust is efficiently and effectively managed through the development of resistant oat varieties. Pc91 is a seedling crown rust resistance gene that is highly effective against the current P. coronata population in North America. The primary objective of this study was to develop DNA markers linked to Pc91 for purposes of marker-assisted selection in oat breeding programs. The Pc91 locus was mapped using a population of F7-derived recombinant inbred lines developed from the cross 'CDC Sol-Fi'/'HiFi' made at the Crop Development Centre, University of Saskatchewan. The population was evaluated for reaction to P. coronata in field nurseries in 2008 and 2009. Pc91 mapped to a linkage group consisting of 44 Diversity Array Technology (DArT) markers. DArTs were successfully converted to sequence characterized amplified region (SCAR) markers. Five robust SCARs were developed from three non-redundant DArTs that co-segregated with Pc91. SCAR markers were developed for different assay systems, such that SCARs are available for agarose gel electrophoresis, capillary electrophoresis, and Taqman single nucleotide polymorphism detection. The SCAR markers accurately postulated the Pc91 status of 23 North American oat breeding lines.

Mapping quantitative trait loci associated with barley net blotch resistance.

Net blotch of barley, caused by Pyrenophora teres Drechs., is an important foliar disease worldwide. Deployment of resistant cultivars is the most economic and eco-friendly control method. This report describes mapping of quantitative trait loci (QTL) associated with net blotch resistance in a doubled-haploid (DH) barley population using diversity arrays technology (DArT) markers. One hundred and fifty DH lines from the cross CDC Dolly (susceptible)/TR251 (resistant) were screened as seedlings in controlled environments with net-form net blotch (NFNB) isolates WRS858 and WRS1607 and spot-form net blotch (SFNB) isolate WRS857. The population was also screened at the adult-plant stage for NFNB resistance in the field in 2005 and 2006. A high-density genetic linkage map of 90 DH lines was constructed using 457 DArT and 11 SSR markers. A major NFNB seedling resistance QTL, designated QRpt6, was mapped to chromosome 6H for isolates WRS858 and WRS1607. QRpt6 was associated with adult-plant resistance in the 2005 and 2006 field trials. Additional QTL for NFNB seedling resistance to the more virulent isolate WRS858 were identified on chromosomes 2H, 4H, and 5H. A seedling resistance QTL (QRpts4) for the SFNB isolate WRS857 was detected on chromosome 4H as was a significant QTL (QRpt7) on chromosome 7H. Three QTL (QRpt6, QRpts4, QRpt7) were associated with resistance to both net blotch forms and lines with one or more of these demonstrated improved resistance. Simple sequence repeat (SSR) markers tightly linked to QRpt6 and QRpts4 were identified and validated in an unrelated barley population. The major 6H QTL, QRpt6, may provide adequate NFNB field resistance in western Canada and could be routinely selected for using molecular markers in a practical breeding program.

Targeting the aluminum tolerance gene Alt3 region in rye, using rice/rye micro-colinearity.

Characterization and manipulation of aluminum (Al) tolerance genes offers a solution to Al toxicity problems in crop cultivation on acid soil, which composes approximately 40% of all arable land. By exploiting the rice (Oryza sativa L.)/rye (Secale cereale L.) syntenic relationship, the potential for map-based cloning of genes controlling Al tolerance in rye (the most Al-tolerant cereal) was explored. An attempt to clone an Al tolerance gene (Alt3) from rye was initiated by using DNA markers flanking the rye Alt3 gene, from many cereals. Two rice-derived, PCR-based markers flanking the Alt3 gene, B1 and B4, were used to screen 1,123 plants of a rye F2 population segregating for Alt3. Fifteen recombinant plants were identified. Four additional RFLP markers developed from rice genes/putative genes, spanning 10 kb of a 160-kb rice BAC, were mapped to the Alt3 region. Two rice markers flanked the Alt3 locus at a distance of 0.05 cM, while two others co-segregated with it. The rice/rye micro-colinearity worked very well to delineate and map the Alt3 gene region in rye. A rye fragment suspected to be part of the Alt3 candidate gene was identified, but at this level, the rye/rice microsynteny relationship broke down. Because of sequence differences between rice and rye and the complexity of the rye sequence, we have been unable to clone a full-length candidate gene in rye. Further attempts to clone a full-length rye Alt3 candidate gene will necessitate the creation of a rye large-insert library.

Development of PCR-based codominant markers flanking the Alt3 gene in rye.

Aluminum (Al) toxicity is considered to be a major problem for crop growth and production on acid soils. The ability of crops to overcome Al toxicity varies among crop species and cultivars. Rye (Secale cereale L.) is the most Al-tolerant species among the Triticeae. Our previous study showed that Al tolerance in a rye F6 recombinant inbred line (RIL) population was controlled by a single gene designated as the aluminum tolerance (Alt3) gene on chromosome 4RL. Based on the DNA sequence of a rice (Oryza sativa L.) BAC clone suspected to be syntenic to the Alt3 gene region, we developed two PCR-based codominant markers flanking the gene. These two markers, a sequence-tagged site (STS) marker and a cleaved amplified polymorphic sequence (CAPS) marker, each flanked the Alt3 gene at an approximate distance of 0.4 cM and can be used to facilitate high-resolution mapping of the gene. The markers might also be used for marker-assisted selection in rye or wheat (Triticum aestivum L.) breeding programs to obtain Al-tolerant lines and (or) cultivars.

Inheritance of resistance to covered smut in barley and development of a tightly linked SCAR marker.

Inheritance of resistance to covered smut in the barley line Q21861 was studied using a doubled-haploid population produced by crossing Q21861 with the line SM89010. Based on 3 years of screening in the field and two seasons in the greenhouse, segregation for resistance/susceptibility fits a one-gene ratio, indicating a single major gene for resistance in Q21861. Of 440 random 10-mer primers tested using bulked segregant analysis, one primer (OPJ10) resulted in a reproducible polymorphic band. RAPD marker OPJ10(450) co-segregated in repulsion with the covered smut resistance. This marker was converted to a sequence-characterized amplified region (SCAR) marker linked in coupling (5.5 cM) with the covered smut resistant gene in Q21861. The SCAR marker was amplified in the line TR640 which is also resistant to covered smut, but not in the other resistant lines. The SCAR marker will be useful for marker-assisted selection for covered smut in barley breeding programs.

Studies on rye (Secale cereale L.) lines exhibiting a range of extract viscosities. 1. Composition, molecular weight distribution of water extracts, and biochemical characteristics of purified water-extractable arabinoxylan.

Five rye lines exhibiting a wide range of extract viscosities, along with commercial cultivars of rye and wheat, were compared with respect to their physical and chemical properties. Rye wholemeals contained significantly higher concentrations of total and soluble dietary fiber (TDF and SDF, respectively), total and water-extractable arabinoxylan (TAX and WEAX, respectively), and beta-glucan than did wheat. Significant positive correlations were obtained between rye wholemeal extract viscosity and SDF content (r = 0.90, p < 0.05) and WEAX content (r = 0.89, p < 0.05). Gel permeation chromatography (GPC) of water extracts of rye wholemeals revealed the presence of a high molecular weight fraction (HMWF), which was found in higher concentration in the ryes than in wheat. A significant positive correlation (r = 0.84, p < 0.05) was observed between HMWF content (expressed as a proportion of the total carbohydrate in water extracts) and extract viscosity of rye wholemeals. Treatment of a rye wholemeal extract with xylanase, followed by GPC, indicated that the HMWF consisted primarily of WEAX. Successive treatment of a rye wholemeal extract with alpha-amylase, lichenase, protease, and xylanase confirmed that the viscosity of the extract was primarily related to its content of WEAX. WEAX was isolated from high, intermediate, and low extract viscosity ryes. Structural differences were observed among the three arabinoxylans using H NMR and high-pressure size exclusion chromatography with triple detection. The WEAX from high extract viscosity rye was a higher molecular weight macromolecule exhibiting a higher intrinsic viscosity, a larger radius of gyration, a larger hydrodynamic radius, and a lower degree of branching compared to WEAX from low and intermediate extract viscosity ryes.

Studies on rye (Secale cereale L.) lines exhibiting a range of extract viscosities. 2. Rheological and baking characteristics of rye and rye/wheat blends and feeding value for chicks of wholemeals and breads.

Five rye lines exhibiting a wide range of extract viscosities were evaluated for the rheological and baking properties of their flours, individually and in blends with hard red spring wheat flour. Commercial cultivars of rye and triticale were included in the study as controls. Extract viscosities of rye flours were higher than those of corresponding wholemeals, indicating shifting of water-extractable arabinoxylan into flour during roller milling. Falling numbers of the rye flours correlated positively with their extract viscosities in the presence (r = 0.73, p < 0.05) or absence (r = 0.65, p < 0.05) of an enzyme inhibitor. Farinograms revealed the weakness of rye and triticale flours compared to wheat flour. Extract viscosities of rye flours were negatively correlated (r = -0.65, p < 0.05) with mixing tolerance index and positively correlated (r = 0.64, p < 0.05) with dough stability, suggesting a positive impact of extract viscosity on dough strength. Extract viscosity was negatively correlated (r = -0.74, p < 0.05) with loaf volume and specific volume (r = -0.73, p < 0.05) and positively correlated (r = 0.73, p < 0.05) with loaf weight of rye/wheat bread. Overall, the results indicated that 30% of flour from high or low extract viscosity rye could be incorporated into rye/wheat breads without seriously compromising bread quality. Inclusion of rye, particularly high extract viscosity rye, in chick diets seriously impeded growth performance and feed efficiency. Part of the arabinoxylan survived bread-making and exerted an effect on chicks, although substantially lower digesta viscosities were observed in chicks fed rye bread diets than in those fed rye wholemeals.

Isolation of a cDNA encoding a granule-bound 152-kilodalton starch-branching enzyme in wheat.

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5'-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80-100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum.

Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata.

Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene ß-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30-50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1-2%).

The use of RAPD markers in Hordeum phylogeny.

The phylogenetic relationships among 39 wild Hordeum species, subspecies, and cultivated barley were investigated using RAPD markers as discriminating characters. Seventy-six RAPD fragments were generated using 12 single decameric primers of arbitrary nucleotide sequences. Amplification reactions resulted in fragments ranging in length between 200 and 2000 bp. Clearly resolved bands were scored for their presence or absence in a binary matrix. Amplified products were treated as independent characters to generate a phenogram using the NTSYS-PC package. Tree topology was generally found to be consistent with those based on morphological treatments. However, a few species like H. erectifolium, H. jubatum and, to a lesser extent, H. bulbosum occupied a position different from previous classifications. The results demonstrated that RAPD technology represents a useful and reliable tool for detecting polymorphism for phylogenetic studies. Key words : RAPD analysis, molecular markers, phylogenetic studies, Hordeum species, barley.

Cytological identification of some trisomics of Russian wildrye (Psathyrostachys juncea).

Russian wildrye, Psathyrostachys juncea (Fisch.) Nevski (2n = 2x = 14; NsNs), is an important forage grass and a potential source of germplasm for cereal crop improvement. Because of genetic heterogeneity as a result of its self-incompatibility, it is difficult to identify trisomics of this diploid species based on morphological characters alone. Putative trisomies (2n = 2x + 1 = 15), derived from open pollination of a triploid plant by pollen grains of diploid plants, were characterized by Giemsa C-banding. Based on both karyotypic criteria and C-banding patterns, four of the seven possible primary trisomics, a double-deletion trisomic, and two tertiary trisomics were identified.

Rust resistance in Triticum cylindricum Ces. (4x, CCDD) and its transfer into durum and hexaploid wheats.

In order to counteract the effects of the mutant genes in races of leaf rust (Puccinia recondita f.sp. tritici Rob. ex Desm.) and stem rust (P. graminis f.sp. tritici Eriks. &Henn.) in wheat, exploration of new resistance genes in wheat relatives is necessary. Three accessions of Triticum cylindricum Ces. (4x, CCDD), Acy1, Acy9, and Acy11, were tested with 10 races each of leaf rust and stem rust. They were resistant to all races tested. Viable F1 plants were produced from the crosses of the T. cylindricum accessions as males with susceptible MP and Chinese Spring ph1b hexaploid wheats (T. aestivum, 6x, AABBDD), but not with susceptible Kubanka durum wheat (T. turgidum var. durum, 4x, AABB), even with embryo rescue. In these crosses the D genome of hexaploid wheat may play a critical role in eliminating the barriers for species isolation during hybrid seed development. The T. cylindricum rust resistance was expressed in the F1 hybrids with hexaploid wheat. However, only the cross MP/Acy1 was successfully backcrossed to another susceptible hexaploid wheat, LMPG-6. In the BC2F2 of the cross MP/Acy1//LMPG-6/3/MP, monosomic or disomic addition lines with resistance to either leaf rust race 15 (infection types (IT) 1=, 1, or 1+; addition line 1) or stem rust race 15B-1 (IT 1 or 1+; addition line 2) were selected. Rust tests and examination of chromosome pairing of the F1 hybrids and the progeny of the disomic addition lines confirmed that the genes for rust resistance were located on the added T. cylindricum C-genome chromosomes rather than on the D-genome chromosomes. The T. cylindricum chromosome in addition line 2 was determined to be chromosome 4C through the detection of RFLPs among the genomes using a set of homoeologous group-specific wheat cDNA probes. Addition line 1 was resistant to the 10 races of leaf rust and addition line 2 was resistant to the 10 races of stem rust, as was the T. cylindricum parent. The added C-genome chromosomes occasionally paired with hexaploid wheat chromosomes. Translocation lines with rust resistance (2n = 21 II) may be obtained in the self-pollinated progeny of the addition lines through spontaneous recombination of the C-genome chromosomes and wheat chromosomes. Such translocation lines with resistance against a wide spectrum of rust races should be potentially valuable in breeding wheat for rust resistance.

Transfer of leaf rust and stem rust resistance genes from Triticum triaristatum to durum and bread wheats and their molecular cytogenetic localization.

Six accessions of Triticum triaristatum (Willd) Godr. &Gren. (syn. Aegilops triaristata) (6x, UUMMUnUn), having good resistance to both leaf rust (Puccinia recondita f.sp. tritici Rob. ex Desm) races and stem rust (P. graminis f.sp. tritici Eriks. &Henn.) races, were successfully crossed with both susceptible durum wheats (T. turgidum var. durum L., 2n = 28, AABB) and bread wheats (T. aestivum, 2n = 42, AABBDD). In some crosses, embryo rescue was necessary. The T. triaristatum resistance was expressed in all F1 hybrids. Backcrossing of the F1 hybrids to their wheat parents to produce BC1F1 plants was more difficult (seed set 0-7.14%) than to produce F1 hybrids (seed set 12.50-78.33%). The low female fertility of the F1 hybrids was due to low chromosome pairing. Only gametes with complete or nearly complete genomes from the F1 hybrids were viable. In BC2F4 populations from the cross MP/Ata2//2*MP, monosomic or disomic addition lines (2n = 21 II + 1 I or 22 II) with resistance to leaf rust race 15 (IT 1) were selected. In BC2F2 populations from the crosses CS/Ata4//2*MP and MP/Ata4//2*MP, monosomic or disomic addition lines with resistance to either leaf rust race 15 or stem rust race 15B-1 (both IT 1) were selected. Rust tests and cytology on the progeny of the disomic addition lines confirmed that the genes for rust resistance were located on the added T. triaristatum chromosomes. The homoeologous groups of the T. triaristatum chromosomes in the addition lines from the crosses MP/Ata2//2*MP, CS/Ata4//2*MP, and MP/Ata4//2*MP were determined to be 5, 2, and 7, respectively, through the detecting of RFLPs among genomes using a set of homoeologous group specific wheat cDNA probes. The addition lines with resistance to leaf rust race 15 from the crosses MP/Ata2//2*MP and CS/Ata4//2*MP were resistant to another nine races of leaf rust and the addition line with resistance to stem rust race 15B-1 from the cross MP/Ata4//2*MP was resistant to another nine races of stem rust as were their T. triaristatum parents. Since such genes provide resistance against a wide spectrum of rust races they should be very valuable in wheat breeding for rust resistance.

Variability of the random amplified polymorphic DNA assay among thermal cyclers, and effects of primer and DNA concentration.

The reproducibility of the generation of random amplified polymorphic DNA fragments from three commonly used thermal cyclers was determined using identical assay conditions. In all cases, different results were obtained from the three instruments. Variation in the length of the primer (20 nt or 10 nt) did not have any effect on the reproducibility of the assays from the three machines tested. A DNA concentration of 1 ng generated poorly staining DNA fragments whereas concentrations between 10 ng and 100 ng gave similar banding patterns when using the same thermal cycler. Low concentrations of primer (0.05 microM) did not produce any detectable DNA fragments. Increased primer concentrations of 0.25 microM or higher generated intensely staining DNA fragments, and concentrations above 0.5 microM did not improve the clarity of the banding patterns but did direct the synthesis of increasing amounts of very short DNA fragments. Surprisingly, the 20 nt-long primer was able to direct the synthesis of more DNA fragments than a primer of only 10 nt long.