PUNJAB AGRICULTURAL UNIVERSITY

Biotechnology Centre, Ludhiana -- 141 004, India.

Molecular tagging of stripe rust resistance gene Yr8.

Harjit Singh, and P.J. Sharp, C.R. Wellings, and Sarah Johnston (Plant Breeding Institute, Cobbitty, NSW, Australia).

Isogenic lines carrying four different genes for race specific resistance in the background of susceptible Australian cultivar Avocet S (Table 1) were used to tag genes for resistance to stripe rust. The isogenic lines, donor parents, and Avocet S were tested for their seedling reaction to appropriate avirulent/virulent stripe rust pathotypes to ensure the presence of the respective Yr genes in these lines. Screening of these materials with RAPD markers identified a positive marker associated with the gene Yr8. The lines carrying Yr8 in the Avocet S background and the donor parent Compair consistently had an amplified product of 0.98 kb, obtained with RAPD primer OPV 07. This product was absent in isogenic lines with Yr5,Yr7, or Yr15; their respective donor parents; and in the recurrent parent Avocet S (Fig 1). A product of the same size (0.98 kb) was obtained with the primer OPV 07 in isogenic lines with Yr8 in the background of another stripe rust-susceptible cultivar Harrier S, confirming the of association of this marker with the gene Yr8. Further screening with RAPDs showed that an amplified product of 1.0 kb, obtained with primer OPV06, was absent in the isogenic lines with Yr8 in the backgrounds of Avocet S and the donor parent Compair. This band was present in the isogenic lines with the other Yr genes, in the Avocet S background as well as the cultivar Avocet S. This band may serve as a negative marker for the gene Yr8. The two markers identified in this study may be helpful in pyramiding the Yr8 gene with other stripe rust resistance genes.

Table 1. Materials used for the molecular tagging of the Yr8 stripe rust-resistance gene in Avocet S wheat.

RAPD markers associated with an Aegilops ovata chromosome carrying rust resistance genes.

Harjit Singh and H.S. Dhaliwal.

The evaluation of wild Triticum and Aegilops species at the Punjab Agricultural University, Ludhiana, India, has shown that diploid and polyploid Aegilops species with the C, U, and M genomes are good sources of resistance to P. recondita f. sp. tritici and P. striiformis (Dhaliwal et al. 1993). Crossing of a leaf rust- and stripe rust-resistant accession of Ae. ovata (UUMM) to the rust-susceptible cultivar WL 711, followed by backcrossing to the WL 711 parent has resulted in development of rust-resistant alien substitution lines. C-banding has shown that one of the alien substitution lines possesses a complete chromosome of Ae. ovata with a gene(s) for resistance to leaf rust and stripe rust (Singh and Dhaliwal 1996). The present study was conducted to find RAPD markers that identified the Ae. ovata chromosome with the rust-resistance genes.

An alien substitution line with the Ae. ovata chromosome in a WL 711 background, the donor accession of Ae. ovata(Acc3547), and WL711 were screened with 22 common primers: A09, A10, A17, A18, A24, A25, A45, A47, A62, A70, A83, A86, B13, B27, B30, B33, B37, B39, B71, B73, B79, B86. Amplification with RAPDs was performed in a thermal cycler using 10 ng of template DNA in 25 "WP MathA"Fl of reaction mixture. The amplification mixture was subjected to 35 cycles of denaturation (94"WP MathA"EC for 1 min), annealing (39"WP MathA"EC for 1 min), and extension (72"WP MathA"EC for 2 mins). The amplification products were separated in 1.2 % agarose gels.

An amplification product the size of 0.86 kb was observed in the alien substitution line and the donor line with primer B30. This band was missing in WL711. Similarly, a 2 kb band was obtained with primer A47, and a 60 bp band with primer A86 were present in the alien substitution line and the donor accession of Ae. ovata, but absent in WL 711. The polymorphic bands identified with these three RAPD primers are expected to be associated with the alien chromosome carrying the rust resistance gene(s).

References.

Dhaliwal HS, Singh H, Gill KS, and Randhawa HS. 1993). Evaluation and cataloguing of wheat genetic resources for disease resistance and quality. In: Biodiversity and Wheat Improvement (Damania AB ed). John J. Wiley & Sons, N.Y. and ICARDA, Syria, and Sayce Publishing Co.,U.K. pp123-140.

Singh H and Dhaliwal HS. 1996. Transfer of rust and cereal cyst nematode resistance from Ae. ovata to T. aestivum L. In: Second Intern Crop Sci Congr, New Delhi, India. November, 1996. (In press)

ITEMS FROM ITALY

EXPERIMANTAL INSTITUTE FOR CEREAL RESEARCH

Via Cassia 176, 00191, Rome, Italy.

Black point disease in durum wheat: epidemiological and biochemical aspects.

M. Pasquini, N.E. Pogna, M. Mazza, A. Niglio, L. Sereni, and F. Casini.

Black point of durum wheat can affect grain quality and semolina and pasta products as a result. A survey of several Italian durum wheat cultivars grown in central Italy found the disease to be associated with fungal infection of wheat florets during grain development. Alternaria spp. were the fungi most frequently implicated, and differences in black point incidence were detected among cultivars. Nevertheless, preliminary studies show that a brown-kernel discoloration, similar to that caused by black point, can be induced by simulating extreme environmental conditions.

Biochemically, the brown kernel discoloration seems to be associated with the oxidation of cell-wall phenolic compounds. Therefore, the expression of some cell-wall enzymes involved in these reactions was analyzed. Attention was focused on peroxidases, a class of enzymes often involved in cell-wall response to biotic and abiotic stresses. Peroxidases were extracted from healthy and black-pointed kernels from durum wheat cultivars Grazia, Primadur, Ofanto, and Creso, and analyzed by isoelectrofocusing. Gels were stained in a buffered solution (pH 7.2) of hydrogen peroxide and 4-chloro-naphtol. Preliminary results from the isozyme analyses showed the appearance of a weakly-acid PI band in black-pointed kernels of all the cultivars tested, suggesting the induction of a gene for a peroxidase isozyme. When confirmed, this result may be very useful for breeding for resistance to black point disease.

Innovative uses of cereals.

M. G. D'Egidio.

Cereal crops such as bread and durum wheat, barley, and oats were traditionally used for human grain consumption or animal feed. A new use of these crops may be for biomass production for conversion into products (fructose and fructose polymers) with high added value. In fact, cereals produce and store significant amounts of water-soluble carbohydrates as monosaccharides, sucrose, and fructans; polymers containing several molecules of fructose; and one or more molecules of glucose for much of their growing cycle. Large quantities of fructans are usually stored in the leaves and stems of C₃ cereals when the photosynthetic activities exceed the growth needs. Fructans are then utilized for grain growth and seed metabolism. Previous studies indicated that the maximum accumulation of water-soluble carbohydrates in the stems was at milk phase and reported the influence of environmental conditions. Moreover, production of water-soluble carbohydrates and fructan was found to be maximum for durum wheat (with respect to bread wheat and barley) in different environmental conditions. Previous results on a preliminary characterization of this material show interesting properties of immature wheat grains: a more equilibrated amino acid composition and a high level of fructose polymers. These findings suggest the utilization of this material as functional food.

A bread wheat line lacking the Gli-1/Glu-3 loci on chromosomes 1B and 1D and the Gli-A2 locus on chromosome 6A shows toxicity in coeliac.

A.M. Monari, C. Saponaro, M. De Vincenzi¹, R. Luchetti¹, F. Maialetti¹, and N.E. Pogna.

¹ Istituto Supariore di Sanit, Rome, Italy.

Coeliac disease is a gluten-sensitive enteropathy affecting genetically predisposed individuals. Earlier studies showed that the alcohol-soluble proteins toxic to the coeliac small intestine are able to agglutinate human myelogenous leukemia K562(S) cells, whereas nontoxic proteins are inactive. Peptic-tryptic (PT) digests of alcohol-soluble proteins from bread wheat separate into three fractions, A, B, and C, when separated by affinity chromatography on Sepharose-6B coupled with mannan. Only fraction C peptides are able to agglutinate the K562(S) cells.

Table 1. Peptic-tryptic digest of alcohol-soluble proteins from line TM1, which was active in agglutinating the K596(S) cells.

² Percent of the total amount of peptides loaded onto the Sepharose-6B/mannan column.

A triparental cross was made between the mutant bread wheat genotypes S. Pastore 4A, Alpe 1I, and Reader 2, to determine whether the suppression of certain loci coding for gliadins or LMW-glutenin subunits can affect toxicity of bread wheat in coeliac disease. The latter genotype lacks a-glxxxiadin-encoding locus Gli-A2 on chromosome 6A, whereas S. Pastore 4A and Alpe 1I lack the Gli-B1/Glu-B3 (chromosome 1B) or Gli-D1/Glu-D3 (chromosome 1D) loci, which control the synthesis of some b-, g- and w-gliadins, and also a number of LMW-glutenin subunits. A triple-mutant line named TM1 lacking all the prolamin loci mentioned above was selected among the progeny of this triparental cross. The PT-digest of alcohol-soluble proteins from line TM1 was active in agglutinating the K562(S) cells (Table 1).

However, the minimal agglutinating concentration (MAC) of the PT-digest required to agglutinate 100 % of the cells was almost four times higher in line TM1 than in the normal progeny from the same triparental cross, suggesting that removal of prolamin genes in the FONT SIZE=2 FACE="WP TypographicSymbols"Atriple-nullFONT SIZE=2 FACE="WP TypographicSymbols"@ genotype significantly reduced its toxicity in coeliac disease. The agglutinating activity of line TM1 indicated that prolamins other than chromosome 6A-encoded gliadins exert toxic effects in coeliacs. Separation of PT-digest from line TM1 on a Sepharose-6B mannan column provided the expected three chromatographic fractions. Only peptides in fraction C were found to agglutinate the K562(S) cells at a MAC of 14.8 mg/l as compared to 5.4 mg/l in its normal counterpart (Table 1). Moreover, removal of prolamin genes did not affect the relative amount of fraction C peptides, which represented 1.6 % of the peptides loaded onto the column in both TM1 and its normal sister line.

Introgression of disease-resistance genes from rye into durum wheat.

N.E. Pogna, M. Pasquini, M. Mazza, L. Rovelli, and P. Cacciatori.

The T1BL-1RS wheat-rye translocation has the genes for resistance to leaf rust (Lr26), stem rust (Sr31), and yellow rust (Yr9). The successful transfer of this translocation into tetraploid durum wheats was followed by the induction of allosyndetic recombination between the rye chromosome arm and its wheat homoeologues, in order to overcome the quality defect present in these lines. Low quality is probably due to deleterious rye genes on 1RS and to the absence of the Glu-B3 locus on 1BS that codes for LMW-glutenin subunits.

Phytopathological, molecular, and biochemical analyses on the progeny of crosses between tetraploid wheat lines carrying the T1BL-1RS translocation and the ph1c mutant of the durum wheat Cappelli led to the identification of putative allosyndetic recombinants, which are resistant to leaf rust isolate Pr12, avirulent to Lr26 gene, but lack secalins encoded at the Sec-1 locus on 1RS. A second self-pollinated generation was developed from these putative recombinants. Plants have been obtained that contain the Lr26 and Glu-B3 genes in the absence of Sec-1.

A new locus coding for a 25 kDa rye globulin was found on the translocated 1RS chromosome arm. The high homoeology between chromosomes 1RS and 1BS and the extraction conditions of the protein encoded by this new locus, suggest this globulin to be homoeologous to a 25 kDa bread wheat globulin analyzed by Gomez et al. (Mol Gen Genet 214:541-546, 1988).

Wheat leaf rust in Italy.

M. Pasquini, F. Casulli, L. Sereni, and F. Casini.

Among wheat diseases, leaf rust is widespread under Italian climatic conditions and has been studied intensively over the past 30 years. The virulence spectrum of the pathogen population is different and variable through the years and regions.

Pathogenicity surveys are made yearly in national field trials in critical areas of northern, central, and southern Italy. Data are obtained on the reaction to naturally occurring infections in durum and bread wheat cultivars, of both Italian and foreign origin, new wheat selections, and near isogenic lines with known genes for resistance to P. recondita. Diseased wheat materials are collected from experimental nurseries and commercial fields and tested in the greenhouse.

A decrease in rainfall (from an average of 582.3 mm/year during the 10-year period from 1955-64, to an average of 474.2 mm/year from 1965-94) and a remarkable reduction in the frequency of rainy days (from 98.4 days/year in 1955-64 to 68.6 days/year in 1965-94) have reduced the frequency and severity of leaf rust, especially in southern Italy. In 1995-96, climatic conditions were generally favorable to disease development, and leaf rust was present at high frequency and intensity. The disease also was found in regions where it usually does not appear. Initial leaf rust infections occur mainly through local sources of inoculum present along the southern coasts of the country. The airborne spores from these areas spread inland and to the fields at higher altitudes, to northern Italy, and probably to the surrounding wheat-growing Mediterranean countries.

The data collected in different regions during the last 10 years show that the virulence of P. recondita does not seem to have increased significantly in Italy. Isolates from central and northern Italy with apparently high levels of pathogenicity have been found to be widespread.

A low efficacy has been shown in the field by the near isogenic lines carrying the resistance genes Lr2a, Lr2b, Lr2c, Lr3, Lr3ka, and Lr30. Lines with genes Lr1, Lr9, Lr12, Lr13, Lr15, Lr17, Lr19, Lr22a, Lr24, Lr26, and Lr29 were resistant or moderately resistant to the pathogen in almost all the locations considered.

Many bread and durum wheat cultivars grown in Italy have shown a good field resistance to leaf rust and some of them have maintained the resistance for several years. These cultivars were artificially inoculated in greenhouse with leaf rust isolates and selected for their virulence characteristics or their diffusion in Italian cereal growing areas.

Among durum wheats, Creso, widely cultivated in southern, central, and northern Italy for more than 15 years, still expresses high resistance to leaf rust in both the field and in greenhouse, despite the increasing virulence shown by the pathogen. Creso may have as yet unidentified genes that confer durable resistance. The bread wheat cultivars Fiocco and Maestra, with the T1BL-1RS wheat-rye translocation, have remained resistant for several years to the leaf rust population present in Italy.

Many sources of resistance to the leaf rust pathogen have been identified in durum and bread wheat varieties coming from different origins. Striking disease epidemics and the identification of new virulence gene in the Italian population of P. recondita, have increased efforts to search for new and different resistance sources particularly in wild and related species (T. monococcum, T. dicoccum, T. dicoccoides, Aegilops spp., and S. cereale). There is substantial information on the effectiveness of genes from alien species in conferring resistance in Italy to leaf rust. Transfer to wheat of the several useful traits they contain is being increasingly considered as a breeding strategy that can effectively complement more traditional ones.

Publications.

Alberti I, Casulli F, Pancaldi D, Pasquini M, and Nannetti S. 1996. Indagine epidemiologica sulla ruggine bruna del frumento (Puccinia recondita f.sp. tritici). Emilia-Romagna. Informatore Fitopatologico 10:42-46.

Casulli F, Pasquini M, Pancaldi D, and Grifoni F. 1996. Epidemiological aspects and virulence of Puccinia recondita f.sp.tritici in Italy. Proc 9th Eur and Mediter Cereal Rusts and Mildew Conf, Lunteren, Olanda. 2-6 September, 1996. p. 137.

Ceoloni C, Biagetti M, Ciaffi M, Forte P, and Pasquini M. 1996. Wheat chromosome engineering at the 4x level: the potential of different alien gene transfers into durum wheat. Euphytica 89:87-97.

Cervigni T, D'Egidio MG, Nardi S, and Mariani G. 1992. Fruttani e cereali autunno primaverili. Aspetti biochimici, fisiologici ed agronomici di una destinazione innovativa della coltura. Riv di Agron 26:152-157.

Corazza L, Casulli F, Pasquini M, Balmas V, and Ronga G. 1996. Valutazione di genotipi di frumento duro per resistenza ad alcune importanti malattie fungine. Atti Comvegno Annuale S.I.G.A., Perugia, Italy. 18-21 September, 1996. p. 134.

Corradini C, Canali G, Donini V, and D'Egidio MG. 1995. High-performance chromatographic separation of fructans in a durum wheat. Proc Euro Food Chem VII, Vienna, Austria. pp. 303-306.

Curioni A, Pogna NE, and Dal Belin Peruffo A. 1996. The quantity of bound beta-amylase is related to the size of gluten polymers. In: Gluten 96 (Wrigley CW ed). Cereal Chemistry Division, Royal Australian Chemical Institute, North Melbourne, Sidney. pp 307-311.

Dal Belin PA, Pogna NE, and Curioni A. 1996. Evidence for the presence of disulfide bonds between beta-amylase and low molecular weight glutenin subunits. In: Gluten 96 (Wrigley CW ed). Cereal Chemistry Division, Royal Australian Chemical Institute, North Melbourne, Sidney. pp 312-315.

D'Egidio MG, Cervigni SE., and Cervigni T. 1993. Water soluble carbohydrates in cereal stems at milky phase as raw material for industrial purposes. Proc ICC Intern Symp, Non food uses of cereals, Budapest, Hungary. pp. 73-77.

D'Egidio MG, Cecchini C, Cervigni T, Donini B, and Pignatelli V. 1996. Production of fructose from cereal stems and polyannual cultures of Jerusalem Artichoke. Proc Third Eur Symp Industrial Crops and Products, Reims, France. 22-24 April ( In press).

D'Egidio MG., Cecchini C, Corradini C, Pignatelli V, Donini V, and Cervigni T. 1996. Innovative use of cereals for fructose production. Proc Intern Conf, Cereals: novel uses and processes, Manchester, UK. 4-6 giugno (In press).

De Vincenzi M, Dessi MR, Luchetti R, Pogna NE, Redaelli R, and Galterio. 1996. Toxicity of bread wheat lines lacking prolamins encoded by the Gli-B1/Glu-B3 and Gli-D1/Glu-D3 loci in coeliac disease as determined by their agglutinating activity. ATLA 24:39-48.

Donini B, Pignatelli V, Cervigni T, Cervigni SE, D'Egidio MG, and Mariani G. 1992. Fructans, carbohydrates from crop plants as potential raw material for industry. Proc 7th Intern Conf on Biomass for Energy and Industry. Firenze, Italy. pp. 332-339.

Mazza M, Iori A, Pasquini M, and Pogna NE. 1996. Evidence for a-gliadins encoded by the Gli-B5 locus in durum wheat (Triticum turgidum spp. durum). J Genet Breed 50:in press.

Metakovsky E, Chernacov VM, Upelniek UP, Redaelli R, Dardevet N, Branlard G, and Pogna NE. 1996. Recombination mapping of minor a-gliadin coding loci on chromosome 1A in common wheat: A revision. J Genet Breed. 50:in press.

Pasquini M, Sereni L, Casulli F, Siniscalco A, Lendini M, Mameli L, Gallo G, Monti M, Lo Re L, Coppolino F, Padovan S, Arduini F, and Jenabzadeh P. 1996. Le principali malattie fungine del frumento in Italia nel 1995-96. L'Informatore Agrario. 35:47-54

Pogna NE, Mazza M, Redaelli R, and Ng PKW. 1996. Gluten quality and storage protein composition of durum wheat lines containing the Gli-D1/Glu-D3 loci. In: Gluten 96 (Wrigley CW ed). Cereal Chemistry Division, Royal Australian Chemical Institute, North Melbourne, Sidney. pp 18-22.

Pogna NE, Tusa P, and Boggini G. 1996. Genetic and biochemical aspects of dough quality in wheat. Adv Food Sci 18 (5/6):145-151.

Pogna N, Saponaro C, De Vincenzi M, Luchetti R, and Dessi MR. 1996. Toxicity testing of alcohol-soluble proteins from Triticum monococcum in coeliac disease. Ann Wheat Newslet 42:126.


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