Grant Daggard

Construction of three linkage maps in bread wheat, Triticum aestivum

Genetic maps were compiled from the analysis of 160-180 doubled haploid lines derived from 3 crosses: Cranbrook × Halberd, CD87 × Katepwa, and Sunco × Tasman. The parental wheat lines covered a wide range of the germplasm used in Australian wheat breeding. The linkage maps were constructed with RFLP, AFLP, microsatellite markers, known genes, and proteins. The numbers of markers placed on each map were 902 for Cranbrook × Halberd, 505 for CD87 × Katepwa, and 355 for Sunco × Tasman. Most of the expected linkage groups could be determined, but 10-20% of markers could not be assigned to a specific linkage group. Homologous chromosomes could be aligned between the populations described here and linkage groups reported in the literature, based around the RFLP, protein, and microsatellite markers. For most chromosomes, colinearity of markers was found for the maps reported here and those recorded on published physical maps of wheat. AFLP markers proved to be effective in filling gaps in the maps. In addition, it was found that many AFLP markers defined specific genetic loci in wheat across all 3 populations. The quality of the maps and the density of markers differs for each population. Some chromosomes, particularly D genome chromosomes, are poorly covered. There was also evidence of segregation distortion in some regions, and the distribution of recombination events was uneven, with substantial numbers of doubled haploid lines in each population displaying one or more parental chromosomes. These features will affect the reliability of the maps in localising loci controlling some traits, particularly complex quantitative traits and traits of low heritability. The parents used to develop the mapping populations were selected based on their quality characteristics and the maps provide a basis for the analysis of the genetic control of components of processing quality. However, the parents also differ in resistance to several important diseases, in a range of physiological traits, and in tolerance to some abiotic stresses.

Late-onset and Recurrent Neonatal Group B Streptococcal Disease Associated with Breast-milk Transmission

The purpose of the study was to determine the epidemiological relationships in three unrelated cases of neonatal late-onset Group B streptococcal (GBS) disease and maternal breast-milk infection with GBS. All deliveries were by cesarean section; case 1 was at term, and cases 2 and 3 were at 32- and 33-wk gestation, respectively. Case 1 relates to a mother with clinical mastitis and recurrent GBS infection in a 20-day-old male infant. Following antibiotic therapy and cessation of breastfeeding, the infant recovered without sequelae. Case 2 refers to a mother with clinical mastitis and the occurrence of late-onset GBS disease in 5-wk-old male twins. Despite intervention, one infant died and the second became ill. Following antibiotic therapy and cessation of breast-feeding, the surviving infant recovered without sequelae. Case 3 refers to a mother with sub-clinical mastitis and late-onset GBS infection occurring in a 6-day-old female twin. Following intervention, the infant recovered but suffered a bilateral thalamic infarction resulting in developmental delay and a severe seizure disorder. Following recovery of GBS from an inapparent mastitis and cessation of breast-feeding, the second infant remained well. Blood cultures from all affected infants and maternal breast milk were positive for GBS. Epidemiological relationships between neonatal- and maternal-derived GBS isolates were confirmed by a random amplified polymorphic DNA polymerase chain reaction assay (RAPD-PCR). This study is significant in that it has demonstrated that maternal milk (in cases of either clinical or sub-clinical mastitis) can be a potential source of infection resulting in either late-onset or recurrent neonatal GBS disease.

Curation of wheat maps to improve map accuracy and QTL detection

Three Australian doubled haploid populations were used to illustrate the importance of map curation in order to improve the quality of linkage maps and quantative trait locus (QTL) detection. The maps were refined and improved by re-examining the order of markers, inspection of the genetic maps in relation to a consensus map, editing the marker data for double crossovers, and determining estimated recombination fractions for all pairs of markers. The re-ordering of markers and replacing genotypes at double crossovers with missing values resulted in an overall decrease in the length of the maps. Fewer apparent genotyping errors, associated with the presence of double recombinants, were identified with restriction fragment length polymorphisms (RFLPs) than with other types of markers used in this study. The complications that translocations may cause in the ordering of markers and subsequent QTL analysis were investigated. QTL analysis using both the original and revised maps indicated that QTL peaks were more sharply located or had improved log-likelihood (LOD) scores in the revised maps. An accurate indication of the QTL peak and a significant LOD score are both essential for the identification of markers suitable for marker-assisted selection. Recommendations are provided for the improvement of the quality of linkage maps.

Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice.

Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.

Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures.

Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 α-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06–0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as −7°C.

Allelopathy, DIMBOA production and genetic variability in accessions of Triticum speltoides

A screening was conducted to study the allelopathic potential of Australian-held accessions of Triticum speltoides. Of 26 accessions, four were found to inhibit root growth in the indicator species, lettuce (Lactuca sativa). The methanol leaf extracts of these accessions significantly reduced the root length of wild oat (Avena spp.). In all but one case, alellopathic accessions contained higher amounts of DIMBOA than did nonallelopathic accessions. Since some variation in allelopathic response was detected within lines, random amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity between and within the allelopathic accessions of Triticum speltoides L. The average genetic similarity between all possible pairs of selected accessions was found to be 55% and ranged from 44% to 88%. Comparison of DNA extracted from different single seedlings within the same accession revealed significant intraaccession genetic diversity (4–24%), although this was much less than that observed between accessions tested. This intraaccession diversity has significant implications for the selection of T. speltoides accessions in breeding or screening programs.

Flour yield QTLs in three Australian doubled haploid wheat populations

Flour yield quantitative trait loci (QTLs) were identified in 3 Australian doubled haploid populations, Sunco × Tasman, CD87 × Katepwa, and Cranbrook × Halberd. Trial data from 3 to 4 sites or years were available for each population. QTLs were identified on chromosomes 2BS, 4B, 5AL, and 6BL in the Sunco × Tasman population, on chromosomes 4B, 5AS, and 6DL in the CD87 × Katepwa population, and on chromosomes 4DS, 5DS, and 7AS in the Cranbrook × Halberd population. In the Sunco × Tasman cross the highest genetic variance was detected with the QTL on chromosome 2B (31.3%), in the CD87 × Katepwa cross with the QTL on chromosome 4B (23.8%), and in the Cranbrook × Halberd cross with the QTL on chromosome 5D (18%). Only one QTL occurred in a similar location in more than one population, indicating the complexity of the flour yield character across different backgrounds.

Combined gene selection methods for microarray data analysis

In recent years, the rapid development of DNA Microarray technology has made it possible for scientists to monitor the expression level of thousands of genes in a single experiment. As a new technology, Microarray data presents some fresh challenges to scientists since Microarray data contains a large number of genes (around tens thousands) with a small number of samples (around hundreds). Both filter and wrapper gene selection methods aim to select the most informative genes among the massive data in order to reduce the size of the expression database. Gene selection methods are used in both data preprocessing and classification stages. We have conducted some experiments on different existing gene selection methods to preprocess Microarray data for classification by benchmark algorithms SVMs and C4.5. The study suggests that the combination of filter and wrapper methods in general improve the accuracy performance of gene expression Microarray data classification. The study also indicates that not all filter gene selection methods help improve the performance of classification. The experimental results show that among tested gene selection methods, Correlation Coefficient is the best gene selection method for improving the classification accuracy on both SVMs and C4.5 classification algorithms.

Targetting AFLP-DNA markers to specific traits and chromosome regions

The amplified fragment length polymorphism (AFLP) technique is widely used in mapping of wheat as high polymorphism rates are obtained and the procedure is relatively simple. In this study the AFLP markers were targetted to wheat chromosome regions of interest, especially those in which random mapping approaches located relatively few markers. Results showed that the combination of bulk segregant analysis and AFLP markers could target new markers to regions of interest in wheat, and as a result, additional markers were identified for the dough mixing time and noodle colour traits. The new markers for noodle colour were closer to this trait than the markers previously identified by random procedures. As a result, their association with the trait was more significant, an aspect that is important for selection efficiency. In order to improve genome coverage, it was found that regions of chromosomes containing telomere sequences could be targetted using AFLPs combined with a telomere sequence anchor primer.

Enhanced shoot regeneration in nine Australian wheat cultivars by spermidine and water stress treatments

The regeneration potential of ageing calli initiated from isolated scutella of immature embryos was increased in nine elite Australian cultivars (QT7208, QT9685, QT7709, Kennedy, Lang, Sunvale, Giles, Petrie and Veery) of wheat (Triticum aestivum L.). Firstly, the effects of 4–32 h of dehydration stress on regeneration of 4- to 20-week old calli were evaluated. Cultivars such as Veery, Kennedy and Sunvale showed significant improvement in regeneration from calli up to 12-weeks old that had undergone 16 h of dehydration stress. Secondly, 4- to 20-week old callus cultures were treated with 0.05–5 mM spermidine to evaluate its effect on regeneration. While spermidine had a negative effect on regeneration from 4-week old calli at all tested concentrations (as compared with untreated controls), there was a 3–50% improvement in the regeneration ability of older calli (16- to 20-week old) of all cultivars. Finally, exogenous application of 1 mM spermidine to 16-week old cultures, in combination with 16 h dehydration stress, improved plant regeneration by 10–65% in all nine cultivars.