Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle.

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3+, CD4+, CD8+ and [gamma][delta] T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for [gamma][delta] T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.

Characterisation of chicken Campylobacter jejuni isolates using resolution optimised single nucleotide polymorphisms and binary gene markers

The principal objective of this study was to determine if Campylobacter jejuni genotyping methods based upon resolution optimised sets of single nucleotide polymorphisms (SNPs) and binary genetic markers were capable of identifying epidemiologically linked clusters of chicken-derived isolates. Eighty-eight C. jejuni isolates of known flaA RFLP type were included in the study. They encompassed three groups of ten isolates that were obtained at the same time and place and possessed the same flaA type. These were regarded as being epidemiologically linked. Twenty-six unlinked C. jejuni flaA type I isolates were included to test the ability of SNP and binary typing to resolve isolates that were not resolved by flaA RFLP. The remaining isolates were of different flaA types. All isolates were typed by real-time PCR interrogation of the resolution optimised sets of SNPs and binary markers. According to each typing method, the three epidemiologically linked clusters were three different clones that were well resolved from the other isolates. The 26 unlinked C. jejuni flaA type I isolates were resolved into 14 SNP-binary types, indicating that flaA typing can be unreliable for revealing epidemiological linkage. Comparison of the data with data from a fully typed set of isolates associated with human infection revealed that abundant lineages in the chicken isolates that were also found in the human isolates belonged to clonal complex (CC) -21 and CC-353, with the usually rare C-353 member ST-524 being especially abundant in the chicken collection. The chicken isolates selected to be diverse according to flaA were also diverse according to SNP and binary typing. It was observed that CC-48 was absent in the chicken isolates, despite being very common in Australian human infection isolates, indicating that this may be a major cause of human disease that is not chicken associated.

The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley

Using an established genetic map, a single gene conditioning covered smut resistance, Ruh.7H, was mapped to the telomere region of chromosome 7HS in an Alexis/Sloop doubled haploid barley population. The closest marker to Ruh.7H, abg704 was 7.5 cM away. Thirteen loci on the distal end of 7HS with potential to contain single nucleotide polymorphisms (SNPs) were identified by applying a comparative genomics approach using rice sequence data. Of these, one locus produced polymorphic co-dominant bands of different size while two further loci contained SNPs that were identified using the recently developed high resolution melting (HRM) technique. Two of these markers flanked Ruh.7H with the proximal marker located 3.8 cM and the distal marker 2.7 cM away. This is the first report on the application of the HRM technique to SNP detection and to rapid scoring of known cleaved amplified polymorphic sequence (CAPS) markers in plants. This simple, precise post-PCR technique should find widespread use in the fine-mapping of genetic regions of interest in complex cereal and other plant genomes.

Evidence for extensive population structure in the white-spotted eagle ray within the Indo-Pacific inferred from mitochondrial gene sequences

The white-spotted eagle ray Aetobatus narinari is a species complex that occurs circumglobally throughout warm-temperate waters. Aetobatus narinari is semi-pelagic and large (up to 300 cm disc width), suggesting high dispersal capabilities and gene flow on a wide spatial scale. Sequence data from two mitochondrial genes, cytochrome b (cytb) and NADH dehydrogenase subunit 4 (ND4), were used to determine the genetic variability within and among 18 sampling locations in the central Indo-Pacific biogeographical region. Populations in the Indo-Pacific were highly genetically structured with c. 70% of the total genetic variation found among three geographical regions (East China Sea, Southeast Asia and Australia). FST was 0.64 for cytb and 0.53 for ND4, with φST values being even larger, that is, 0.78 for cytb and 0.65 for ND4. This high-level genetic partitioning provides strong evidence against extensive gene flow in A. narinari. The degree of genetic population structuring in the Indo-Pacific was similar to that found on a global scale. Global FST was 0.63 for cytb and 0.57 for ND4, and global φST values were 0.94 for cytb and 0.82 for ND4. This suggests that the A. narinari complex may be more speciose than the two or three species proposed to date. Further sampling and genetic analyses are likely to uncover the ‘evolutionarily significant’ and ‘management’ units that are critical to determine the susceptibilities of individual populations to regional fishing pressures and to provide advice on management options. Network analyses showed a close genetic relationship between haplotypes from the central Indo-Pacific and South Africa, providing support for a proposed dispersal pathway from the possible centre of origin of the A. narinari species complex in the Indo-Pacific into the Atlantic Ocean.

Gene to Phenotype Integrative Science and Modelling Applied to Pioneer elite

This project aims to use simulatiion modelling to improve our understanding of the genetics and physiology of complex traits with a view to increasing the rate of genetic gain in plant breeding programs.

A phylogenetic and taxonomic re-evaluation of the Bipolaris - Cochliobolus - Curvularia Complex

Three genera, Cochliobolus, Bipolaris and Curvularia form a complex that contains many plant pathogens, mostly on grasses (Poaceae) with a worldwide distribution. The taxonomy of this complex is confusing as frequent nomenclatural changes and refinements have occurred. There is no clear morphological boundary between the asexual genera Bipolaris and Curvularia, and some species show intermediate morphology. We investigated this complex based on a set of ex-type cultures and collections from northern Thailand. Combined gene analysis of rDNA ITS (internal transcribed spacer), GPDH (glyceraldehyde 3-phosphate dehydrogenase), LSU (large subunit) and EF1-α (translation elongation factor 1-α) shows that this generic complex divides into two groups. Bipolaris and Cochliobolus species clustered in Group 1 along with their type species, whereas Curvularia species (including species named as Bipolaris, Cochliobolus and Curvularia) clustered in Group 2, with its generic type. The nomenclatural conflict in this complex is resolved giving priority to the more commonly used established generic names Bipolaris and Curvularia. Modern descriptions of the genera Bipolaris and Curvularia are provided and species resolved in this study are transferred to one of these genera based on their phylogeny. © 2012 Mushroom Research Foundation.

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.

Molecular Breeding for Complex Adaptive Traits: How Integrating Crop Ecophysiology and Modelling Can Enhance Efficiency

Progress in crop improvement is limited by the ability to identify favourable combinations of genotypes (G) and management practices (M) in relevant target environments (E) given the resources available to search among the myriad of possible combinations. To underpin yield advance we require prediction of phenotype based on genotype. In plant breeding, traditional phenotypic selection methods have involved measuring phenotypic performance of large segregating populations in multi-environment trials and applying rigorous statistical procedures based on quantitative genetic theory to identify superior individuals. Recent developments in the ability to inexpensively and densely map/sequence genomes have facilitated a shift from the level of the individual (genotype) to the level of the genomic region. Molecular breeding strategies using genome wide prediction and genomic selection approaches have developed rapidly. However, their applicability to complex traits remains constrained by gene-gene and gene-environment interactions, which restrict the predictive power of associations of genomic regions with phenotypic responses. Here it is argued that crop ecophysiology and functional whole plant modelling can provide an effective link between molecular and organism scales and enhance molecular breeding by adding value to genetic prediction approaches. A physiological framework that facilitates dissection and modelling of complex traits can inform phenotyping methods for marker/gene detection and underpin prediction of likely phenotypic consequences of trait and genetic variation in target environments. This approach holds considerable promise for more effectively linking genotype to phenotype for complex adaptive traits. Specific examples focused on drought adaptation are presented to highlight the concepts.