Genetic analysis of Karnal bunt (Neovossia indica) resistance in wheat

Embryos excised from seeds of six generations (P₁, P₂, F₁, BC₁, BC₂ and F₂) of a cross WH 283 x WH 533 were cultured on modified MS medium already inoculated with secondary sporidia ofNeovossia indica. Significant variations for callusing response (CR) (54.55–75.55%) were observed among generations but the presence or absence ofN. indicia did not affect callusing response. A clear inhibition zone (IZ) was formed around each embryo showing callusing. The diameter of IZ varied significantly among generations and was maximum in the resistant genotype, WH 283 (3.60 cm). Fresh weight and dry weight of calli, initiated from embryo cultured and inoculated withN. indica, varied significantly among generations. Coefficient of infection as well as percentage of infection reflected the overdominance of susceptibility. Generation mean analysis showed that the three parameter model was adequate for diameter of IZ only. Six-parameter model showed that additive (in presence ofN. indica), additive and additive x dominance (in absence ofN. indica) effects were also significant. Complementary type of epistasis for fresh weight of calli and dominance, and dominance x dominance effects for dry weight of calli were observed in the presence ofN. indica. Magnitude of additive effects was higher for diameter of IZ in three parameter model. Therefore, selection might assist in improving this trait and thus indirectly help in attaining the resistance towardsN. indica.     

Development of a sequence-characterized amplified region marker for diagnosis of dwarf bunt of wheat and detection of Tilletia controversa Kühn

Aims:  Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is a destructive disease on wheat as well as an important international quarantined disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from amplified fragment length polymorphism (AFLP) for rapid identification of T . controversa .
Methods and Results:  A total of 30 primer combinations were tested by AFLP to detect DNA polymorphisms between T. controversa and related species. The primer combination E08/M02 generated a polymorphic pattern displaying a 451-bp DNA fragment specific for T. controversa . The marker was converted into a sequence-characterized amplified region (SCAR), and specific primers (SC-01₄₉/SC-02₄₁₅), designed for use in PCR detection assays, amplified a unique DNA fragment in all isolates of T. controversa , but not in the related pathogens. The detection limit with the primer set SC-01₄₉/SC-02₄₁₅ was 10 ng of DNA which could be obtained from 11  μ g of teliospores in a 25- μ l PCR reaction.
Conclusions:  An approach to distinguish T. controversa from similar pathogenic fungi has been developed based on the use of a SCAR marker.
Significance and Impact of the Study:  Development of the simple, high throughput assay kit for the rapid diagnosis of dwarf bunt of wheat and detection of T. controversa is anticipated in further studies.     

Dibutyryl c-AMP as an inducer of sporidia formation: Biochemical and antigenic changes during morphological differentiation of Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>) pathogen in axenic culture

Effect of dibutyryl adenosine 3′,5′-cyclic monophosphate (dbc-AMP), an analogue of c-AMP, was investigated on growth and morphological differentiation ofTilletia indica. Exponential growth was observed up to 21 days in both presence and absence of dbc-AMP; however, increasing concentration of dbc-AMP was deleterious to mycelial growth in liquid culture. A slow increase of mycelial biomass up to 21 days and decline at 30 days in the presence of 2.5 mM dbc-AMP was observed, therefore, this concentration was chosen in subsequent investigations. The inhibitory influence of dbc-AMP was further substantiated by decrease in soluble protein. The fungus on exposure to dbc-AMP experienced morphological differentiation from vegetative mycelial phase to sporogenous mycelial phase, and was induced to produce filiform sporidia. Use of quantitative ELISA further suggested that sporidia formation took more than 21 days in the presence of dbc-AMP. Variations of proteins during different stages ofT. indica grown in the presence and absence of dbc-AMP suggested the expression of stage-specific proteins or differential expression of proteins induced by dbc-AMP. The changes in expression of cell surface antigens as evidenced from decrease and increase binding of anti-mycelial and anti-sporidial antibodies in dbc-AMP treated culture by ELISA was further interpreted on the basis of morphological differentiation from mycelial to sporidial phase     

Generation of anti-teliospores antibodies for immunolocalization and characterization of antigenic epitopes of teliospores of Karnal bunt (Tilletia indica) of wheat

Polyclonal antibodies raised against intact teliospores of T. indica in New Zealand albino rabbits were used for the development of indirect immunofluorescence tests. Specificity of anti-teliospore antibodies was evaluated by cross reactivity studies on other bunt, smut and related pathogens. The characteristic reactivity pattern indicated that the antibodies reacted with Tilletia species only. Chemical modifications, heat and enzyme treatments followed by indirect immunofluorescence tests were employed to delineate the molecular nature of the surface antigens. There was partial or no loss in immunoreactivity by methanol, periodate, heat or trypsin treatments. Extensive periodate treatment altered the fluorescence pattern due to changes in configuration of carbohydrate antigen present in episporium. Sequential treatment of periodate and trypsin showed diminished fluorescence due to access of proteolytic enzyme into inner site of episporium thereby cleaving peptide epitope(s) after reorientation of carbohydrate moietiesby periodate treatment. It indicated glycoprotein nature or peptide nature of epitopes on the teliospore surface.     

High resolution mapping and identification of new quantitative trait loci (QTL) affecting susceptibility to Marek's disease

Marek's disease (MD) is a lymphoproliferative disease of chickens that costs the poultry industry approximately $1 billion annually. Genetic resistance to MD is gaining increased attention to augment vaccinal control as disease outbreaks occur more frequently. Previously, analysis of a 272 F2 White Leghorn resource population measured for many MD traits and genotyped for 78 microsatellite markers revealed two and four quantitative trait loci (QTL) with significant and suggestive association, respectively, to one or more MD associated traits. Additional genetic markers have since been scored on the MD resource population to increase QTL resolution and genome coverage. Saturation of four of the QTL regions with 17 markers revealed five new QTL while 32 markers extended the genome coverage by 400 + CM and uncovered three more QTL. QTL analysis by single-point and interval mapping algorithms agreed well when marker saturation was approximately 20 CM or less. Currently 127 genetic markers cover approximately 68% of the genome that contain up to 14 MD QTL associated to one or more MD trait; seven at the significant level and seven at the suggestive level. Individually each QTL accounts for 2-10% of the variation and, in general, resistance was dominant although the resistant allele may come from either parental line. This study suggests that a limited number of genomic regions play a major role in the genetic control of MD resistance. Markers linked to these loci may be useful for selection of MD resistant stock by the poultry industry following verification of the association within their breeding populations.     

The detection and molecular mapping of a major gene for non-specific adult-plant disease resistance against stripe rust (Puccinia striiformis) in wheat

A major gene determining non-specific adult-plant disease resistance against stripe rust (Puccinia striiformis) designated Yrns-B1 was mapped by using a cross between ’Lgst.79–74’ (resistant) and ’Winzi’ (susceptible). Analyzing F₃ lines of two consecutive experimental years contrary modes of inheritance were observed due to the intermediate character of the gene and the difference in the disease pressure during the seasons. Using the disease scoring data of both experimental years independently two maps were constructed detecting Yrns-B1 20.5 and 21.7 cM, respectively, proximal to the wheat microsatellite (WMS) marker Xgwm493 on the short arm of chromosome 3BS. The genetic relationships to other major genes or to quantitative trait loci controlling adult plant disease resistance against rusts in wheat are discussed. Received: 27 May 1999 / Accepted: 28 September 1999     

A new evidence for DNA nicking property of amyloid beta-peptide (1-42): relevance to Alzheimer's disease

Alzheimer's disease (AD) is a complex neurodegenerative disorder with a progressive mental deterioration manifested by memory loss. No definite etiology has been established for AD to date. Amyloid beta (Abeta) protein plays a central role in the pathology of AD through multiple pathways like oxidative stress, apoptosis etc. Recently, our laboratory first time has evidenced localization of Abeta immunoreactivity in apoptotic nuclei of degenerating AD brain hippocampal neurons and also showed that Abeta (1-42) binds and alters the helicity of DNA. The present study provided fundamental data on DNA nicking induced by Abeta. The results showed that Abeta (1-42) has DNA nicking activity similar to nucleases. Further, magnesium ion (1mM) enhanced DNA nicking activity of Abeta. The data on Abeta solution stability on DNA nicking revealed that the oligomers of Abeta (1-42) peptides showed more DNA nicking activity compared to monomers and fibrillar forms. The nuclease specific inhibitor aurintricarboxylic acid prevented the DNA nicking property of Abeta. Transmission electron microscopy (TEM) studies revealed that Abeta causes open circular and linear forms in supercoiled DNA and also clearly evidenced the physical association of protein-DNA complex. The above data indicated that Abeta mimics endonuclease behavior. Our finding of DNA nicking activity of Abeta peptides has biological significance in terms of causing direct DNA damage.     

A mutation at the Ala122 position of acetohydroxyacid synthase (AHAS) located on chromosome 6D of wheat: improved resistance to imidazolinone and a faster assay for marker assisted selection

A new mutation at the acetohydroxyacid synthase (AHAS) locus on chromosome 6D of wheat was analyzed in detail because it conferred an improved resistance to the imidazolinone group of herbicides. Sequence analysis showed that the mutation was at the Ala122 position (A122T), a position in AHAS which has not to date been identified in imidazolinone resistant wheat lines even though the position has been identified in other plants and is associated with resistance. An allele-specific assay for the mutation (in the wheat line Brookton-8) was developed and used in a genetic analysis. Two mapping populations were analysed and the doubled haploid progeny from the cross Brookton-8 × Clearfield STL proved to be most informative. The AHAS^Ala122 mutation (A122T) was allelic to the AHAS^Ser653 mutation (S653N) in Clearfield STL (Imi1, on chromosome 6D) and hence was assigned to the chromosome 6D locus. The analysis of the doubled haploid lines in the mapping population demonstrated the greater resistance conferred by the A122T mutation because lines from the same cross and carrying either the A122T or S653N mutations could be directly compared. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.