Confirmation of QTL mapping and marker validation for partial seedling resistance to crown rot in wheat line '2-49'

We have tested the efficacy of putative microsatellite single sequence repeat (SSR) markers, previously identified in a 2-49 (Gluyas Early/Gala) × Janz doubled haploid wheat (Triticum aestivum) population, as being linked to partial seedling resistance to crown rot disease caused by Fusarium pseudograminearum. The quantitative trait loci (QTLs) delineated by these markers have been tested for linkage to resistance in an independent Gluyas Early × Janz doubled haploid population. The presence of a major QTL on chromosome 1DL (QCr.usq-1D1) and a minor QTL on chromosome 2BS (QCr.usq-2B1) was confirmed. However, a putative minor QTL on chromosome 2A was not confirmed. The QTL on 1D was inherited from Gluyas Early, a direct parent of 2-49, whereas the 2B QTL was inherited from Janz. Three other putative QTLs identified in 2-49 × Janz (on 1AL, 4BL, and 7BS) were inherited by 2-49 from Gala and were not able to be confirmed in this study. The screening of SSR markers on a small sample of elite wheat genotypes indicated that not all of the most tightly linked SSR markers flanking the major QTLs on 1D and 1A were polymorphic in all backgrounds, indicating the need for additional flanking markers when backcrossing into some elite pedigrees. Comparison of SSR haplotypes with those of other genotypes exhibiting partial crown rot resistance suggests that additional, novel sources of crown rot resistance are available.

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.

DArT markers: Diversity analyses and mapping in Sorghum bicolor

The sequential nature of gel-based marker systems entails low throughput and high costs per assay. Commonly used marker systems such as SSR and SNP are also dependent on sequence information. These limitations result in high cost per data point and significantly limit the capacity of breeding programs to obtain sufficient return on investment to justify the routine use of marker-assisted breeding for many traits and particularly quantitative traits. Diversity Arrays Technology (DArT™) is a cost effective hybridisation-based marker technology that offers a high multiplexing level while being independent of sequence information. This technology offers sorghum breeding programs an alternative approach to whole-genome profiling. We report on the development, application, mapping and utility of DArT™ markers for sorghum germplasm. Results: A genotyping array was developed representing approximately 12,000 genomic clones using PstI+BanII complexity with a subset of clones obtained through the suppression subtractive hybridisation (SSH) method. The genotyping array was used to analyse a diverse set of sorghum genotypes and screening a Recombinant Inbred Lines (RIL) mapping population. Over 500 markers detected variation among 90 accessions used in a diversity analysis. Cluster analysis discriminated well between all 90 genotypes. To confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525 integrating DArT and other marker types. In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM. The genetic linkage map had an average marker density of 1/2.39 cM, with an average DArT marker density of 1/3.9 cM. Conclusion: We have successfully developed DArT markers for Sorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications.

DNA markers linked to yield, yield components, and morphological traits in autotetraploid lucerne (Medicago sativa L.)

We have mapped and identified DNA markers linked to morphology, yield, and yield components of lucerne, using a backcross population derived from winter-active parents. The high-yielding and recurrent parent, D, produced individual markers that accounted for up to 18% of total yield over 6 harvests, at Gatton, south-eastern Queensland. The same marker, AC/TT8, was consistently identified at each individual harvest, and in individual harvests accounted for up to 26% of the phenotypic variation for yield. This marker was located in linkage group 2 of the D map, and several other markers positively associated with yield were consistently identified in this linkage group. Similarly, markers negatively associated with yield were consistently identified in the W116 map, W116 being the low-yielding parent. Highly significant positive correlations were observed between total yield and yield for harvests 1-6, and between total yield and stem length, tiller number, leaf yield/plant, leaf yield/5 stems, stem yield/plant, and stem yield/5 stems. Highly significant QTL were located for all these characters as well as for leaf shape and pubescence.

Simple sequence repeat markers associated with three quantitative trait loci for black point resistance can be used to enrich selection populations in bread wheat

Black point in wheat has the potential to cost the Australian industry $A30.4 million a year. It is difficult and expensive to screen for resistance, so the aim of this study was to validate 3 previously identified quantitative trait loci (QTLs) for black point resistance on chromosomes 2B, 4A, and 3D of the wheat variety Sunco. Black point resistance data and simple sequence repeat (SSR) markers, linked to the resistance QTLs and suited to high-throughput assay, were analysed in the doubled haploid population, Batavia (susceptible) × Pelsart (resistant). Sunco and Pelsart both have Cook in their pedigree and both have the Triticum timopheevii translocation on 2B. SSR markers identified for the 3 genetic regions were gwm319 (2B, T. timopheevii translocation), wmc048 (4AS), and gwm341 (3DS). Gwm319 and wmc048 were associated with black point resistance in the validation population. Gwm341 may have an epistatic influence on the trait because when resistance alleles were present at both gwm319 and wmc048, the Batavia-derived allele at gwm341 was associated with a higher proportion of resistant lines. Data are presented showing the level of enrichment achieved for black point resistance, using 1, 2, or 3 of these molecular markers, and the number of associated discarded resistant lines. The level of population enrichment was found to be 1.83-fold with 6 of 17 resistant lines discarded when gwm319 and wmc048 were both used for selection. Interactions among the 3 QTLs appear complex and other genetic and epigenetic factors influence susceptibility to black point. Polymorphism was assessed for these markers within potential breeding material. This indicated that alternative markers to wmc048 may be required for some parental combinations. Based on these results, marker-assisted selection for the major black point resistance QTLs can increase the rate of genetic gain by improving the selection efficiency and may facilitate stacking of black point resistances from different sources.

Recovery of intravenously infused chromium EDTA and lithium sulphate in the urine of cattle and their use as markers to measure urine volume

A series of metabolism experiments investigated the recovery of continuous-, intravenously infused chromium complexed with ethylenediamine tetra-acetic acid (CrEDTA) and lithium sulphate in the urine of cattle with a view to using the markers to estimate urine and metabolite output in grazing cattle. The recovery of Cr in urine from these infusions was similar (90%) in metabolism trials when cattle consumed three very contrasting diets: high-grain formulated pellet, lucerne hay (Medicago sativa) or low-quality native grass hay (predominantly Heteropogon contortus). By contrast, Li recovery in urine averaged 46.3 +/- 0.40% and 72.6 +/- 0.43% for native pasture and lucerne hays, respectively, but was not constant across days. There was negligible transfer of Cr from CrEDTA in blood serum to the rumen or faeces, whereas appreciable quantities of infused Li were found in both. The ratio of urine volume estimated by spot samples and marker dilution of Cr, to urine volume measured gravimetrically, was 1.05. In grazing studies using rumen-fistulated (RF) steers grazing seven different tropical and temperate grass and legume pastures, the ratio of concentrations of purine derivatives (PD) to Cr in spot samples of urine was shown to vary diurnally in the range of 49% to 157% of the average 24 h value. This finding indicated the need for regular sampling of urine to achieve an accurate average value for the PD: Cr ratio in urine for use in estimating urinary PD excretion and hence microbial protein production in the rumen. It was concluded that continuous, intravenous infusion of CrEDTA resulted in a constant recovery of Cr in the urine of cattle across diets and, provided an intensive sampling regime was followed to account for diurnal variation, it would be suitable as a marker to estimate urine volume and urinary output of PD in grazing cattle.

A consensus genetic map of sorghum that integrates multiple component maps and high-throughput Diversity Array Technology (DArT) markers

Background: Sorghum genome mapping based on DNA markers began in the early 1990s and numerous genetic linkage maps of sorghum have been published in the last decade, based initially on RFLP markers with more recent maps including AFLPs and SSRs and very recently, Diversity Array Technology (DArT) markers. It is essential to integrate the rapidly growing body of genetic linkage data produced through DArT with the multiple genetic linkage maps for sorghum generated through other marker technologies. Here, we report on the colinearity of six independent sorghum component maps and on the integration of these component maps into a single reference resource that contains commonly utilized SSRs, AFLPs, and high-throughput DArT markers. Results: The six component maps were constructed using the MultiPoint software. The lengths of the resulting maps varied between 910 and 1528 cM. The order of the 498 markers that segregated in more than one population was highly consistent between the six individual mapping data sets. The framework consensus map was constructed using a "Neighbours" approach and contained 251 integrated bridge markers on the 10 sorghum chromosomes spanning 1355.4 cM with an average density of one marker every 5.4 cM, and were used for the projection of the remaining markers. In total, the sorghum consensus map consisted of a total of 1997 markers mapped to 2029 unique loci ( 1190 DArT loci and 839 other loci) spanning 1603.5 cM and with an average marker density of 1 marker/0.79 cM. In addition, 35 multicopy markers were identified. On average, each chromosome on the consensus map contained 203 markers of which 58.6% were DArT markers. Non-random patterns of DNA marker distribution were observed, with some clear marker-dense regions and some marker-rare regions. Conclusion: The final consensus map has allowed us to map a larger number of markers than possible in any individual map, to obtain a more complete coverage of the sorghum genome and to fill a number of gaps on individual maps. In addition to overall general consistency of marker order across individual component maps, good agreement in overall distances between common marker pairs across the component maps used in this study was determined, using a difference ratio calculation. The obtained consensus map can be used as a reference resource for genetic studies in different genetic backgrounds, in addition to providing a framework for transferring genetic information between different marker technologies and for integrating DArT markers with other genomic resources. DArT markers represent an affordable, high throughput marker system with great utility in molecular breeding programs, especially in crops such as sorghum where SNP arrays are not publicly available.

High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira.

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars. These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potentia.

Stem Cell Markers in Cancer: Do They Have Clinical Significance?

In cancer, a subpopulation of malignant cells expresses markers of normal stem cells. These cells have the potential of initiating tumor growth and therefore also tumor recurrence. Thus, these cells are called cancer stem cells. A myriad of markers have been applied to identify these cells, but no single marker can be found exclusively in cancer stem cells. In many types of cancer, clinical recurrence and tumor progression are the main causes of mortality, despite intense oncological treatment. It has been proposed that the presence of cancer stem cells causes this resistance to therapy. The scope of this thesis is to investigate the role of stem cell markers and genes in the clinical setting. Especially, the aim was to elucidate the clinical significance of stem cell markers as novel prognostic and diagnostic tools in cancer. Tumor biopsy material from central nervous system tumors (oligodendroglioma, astrocytoma and glioblatoma), neural crest derived tumors (pheochromocytomas) and oral carcinoma was screened for stem cell markers. Initially, 15 stem cell markers were screened in a test series of gliomas. The markers applied for expanded tumor analyses (in 305 cases of glioma, 42 cases of pheochromocytoma, and 73 cases of oral carcinoma) were BMI-1, Snail, p16, mdm2, and c-Myc. Data on marker expression was compared with clinical and pathological parameters. In gliomas, BMI-1 expression was found in nearly all tumors analyzed, but the frequency of BMI-1 expressing cells was highly variable, ranging from 1 to 100%. In oligodendroglioma, BMI-1 expression was identified as a prognostic marker independent of tumor grade and clinical parameters. In pheochromocytoma, Snail expression was shown to distinguish between the metastatic and non-metastatic forms of the tumor. Snail expression was seen only in metastatic tumors, whereas non-metastatic tumors did not commonly express Snail. Finally, in oral carcinoma, BMI-1 expression was seen in roughly 80% of tumors, and Snail expression was high or very high in all cases. The lack of BMI-1 expression was associated with early relapse in oral carcinoma.

Negligible evidence for regional genetic population structure for two shark species Rhizoprionodon acutus (Rüppell, 1837) and Sphyrna lewini (Griffith & Smith, 1834) with contrasting biology

Biodiversity of sharks in the tropical Indo-Pacific is high, but species-specific information to assist sustainable resource exploitation is scarce. The null hypothesis of population genetic homogeneity was tested for scalloped hammerhead shark (Sphyrna lewini, n = 237) and the milk shark (Rhizoprionodon acutus, n = 207) from northern and eastern Australia, using nuclear (S. lewini, eight microsatellite loci; R. acutus, six loci) and mitochondrial gene markers (873 base pairs of NADH dehydrogenase subunit 4). We were unable to reject genetic homogeneity for S. lewini, which was as expected based on previous studies of this species. Less expected were similar results for R. acutus, which is more benthic and less vagile than S. lewini. These features are probably driving the genetic break found between Australian and central Indonesian R. acutus (F-statistics; mtDNA, 0.751–0.903, respectively; microsatellite loci, 0.038–0.047 respectively). Our results support the spatially homogeneous monitoring and management plan for shark species in Queensland, Australia.

The extent of population genetic subdivision differs among four co-distributed shark species in the Indo-Australian archipelago

Background: The territorial fishing zones of Australia and Indonesia are contiguous to the north of Australia in the Timor and Arafura Seas and in the Indian Ocean to the north of Christmas Island. The area surrounding the shared boundary consists of a variety of bio-diverse marine habitats including shallow continental shelf waters, oceanic trenches and numerous offshore islands. Both countries exploit a variety of fisheries species, including whaler (Carcharhinus spp.) and hammerhead sharks (Sphyrna spp.). Despite their differences in social and financial arrangements, the two countries are motivated to develop complementary co-management practices to achieve resource sustainability. An essential starting point is knowledge of the degree of population subdivision, and hence fisheries stock status, in exploited species. Results: Populations of four commercially harvested shark species (Carcharhinus obscurus, Carcharhinus sorrah, Prionace glauca, Sphyrna lewini) were sampled from northern Australia and central Indonesia. Neutral genetic markers (mitochondrial DNA control region sequence and allelic variation at co-dominant microsatellite loci) revealed genetic subdivision between Australian and Indonesian populations of C. sorrah. Further research is needed to address the possibility of genetic subdivision among C. obscurus populations. There was no evidence of genetic subdivision for P. glauca and S. lewini populations, but the sampling represented a relatively small part of their distributional range. For these species, more detailed analyses of population genetic structure is recommended in the future. Conclusion: Cooperative management between Australia and Indonesia is the best option at present for P. glauca and S. lewini, while C. sorrah and C. obscurus should be managed independently. On-going research on these and other exploited shark and ray species is strongly recommended. Biological and ecological similarity between species may not be a predictor of population genetic structure, so species-specific studies are recommended to provide new data to assist with sustainable fisheries management.

Fifteen microsatellite loci for the jungle perch, Kuhlia rupestris

We developed and optimized 15 polymorphic microsatellite loci in the jungle perch, Kuhlia rupestris. Loci were screened in a single population (n = 24) from Fraser Island, Queensland, Australia. Number of alleles per locus ranged from 3 to 19 and observed heterozygosity from 0.25 to 1. No significant linkage disequilibrium was detected between any pair of loci. Genotype proportions for these loci in the population sampled were in Hardy–Weinberg equilibrium.

Determination of management units for grey mackerel fisheries in northern Australia.

The requirement for Queensland, Northern Territory and Western Australian jurisdictions to ensure sustainable harvest of fish resources and their optimal use relies on robust information on the resource status. For grey mackerel (Scomberomorus semifasciatus) fisheries, each of these jurisdictions has their own management regime in their corresponding waters. The lack of information on stock structure of grey mackerel, however, means that the appropriate spatial scale of management is not known. As well, fishers require assurance of future sustainability to encourage investment and long-term involvement in a fishery that supplies lucrative overseas markets. These management and fisher-unfriendly circumstances must be viewed in the context of recent 3-fold increases in catches of grey mackerel along the Queensland east coast, combined with significant and increasing catches in other parts of the species' northern Australian range. Establishing the stock structure of grey mackerel would also immensely improve the relevance of resource assessments for fishery management of grey mackerel across northern Australia. This highlighted the urgent need for stock structure information for this species. The impetus for this project came from the strategic recommendations of the FRDC review by Ward and Rogers (2003), "Northern mackerel (Scombridae: Scomberomorus): current and future research needs" (Project No. 2002/096), which promoted the urgency for information on the stock structure of grey mackerel. In following these recommendations this project adopted a multi-technique and phased sampling approach as carried out by Buckworth et al (2007), who examined the stock structure of Spanish mackerel, Scomberomorus commerson, across northern Australia. The project objectives were to determine the stock structure of grey mackerel across their northern Australian range, and use this information to define management units and their appropriate spatial scales. We used multiple techniques concurrently to determine the stock structure of grey mackerel. These techniques were: genetic analyses (mitochondrial DNA and microsatellite DNA), otolith (ear bones) isotope ratios, parasite abundances, and growth parameters. The advantage of using this type of multi-technique approach was that each of the different methods is informative about the fish’s life history at different spatial and temporal scales. Genetics can inform about the evolutionary patterns as well as rates of mixing of fish from adjacent areas, while parasites and otolith microchemistry are directly influenced by the environment and so will inform about the patterns of movement during the fishes lifetime. Growth patterns are influenced by both genetic and environmental factors. Due to these differences the use of these techniques concurrently increases the likelihood of detecting different stocks where they exist. We adopted a phased sampling approach whereby sampling was carried out at broad spatial scales in the first year: east coast, eastern Gulf of Carpentaria (GoC), western GoC, and the NW Northern Territory (NW NT). By comparing the fish samples from each of these locations, and using each of the techniques, we tested the null hypothesis that grey mackerel were comprised of a single homogeneous population across northern Australia. Having rejected the null hypothesis we re-sampled the 1st year locations to test for temporal stability in stock structure, and to assess stock structure at finer spatial scales. This included increased spatial coverage on the east coast, the GoC, and WA. From genetic approaches we determined that there at least four genetic stocks of grey mackerel across northern Australia: WA, NW NT (Timor/Arafura), the GoC and the east Grey mackerel management units in northern Australia ix coast. All markers revealed concordant patterns showing WA and NW NT to be clearly divergent stocks. The mtDNA D-loop fragment appeared to have more power to resolve stock boundaries because it was able to show that the GoC and east coast QLD stocks were genetically differentiated. Patterns of stock structure on a finer scale, or where stock boundaries are located, were less clear. From otolith stable isotope analyses four major groups of S. semifasciatus were identified: WA, NT/GoC, northern east coast and central east coast. Differences in the isotopic composition of whole otoliths indicate that these groups must have spent their life history in different locations. The magnitude of the difference between the groups suggests a prolonged separation period at least equal to the fish’s life span. The parasite abundance analyses, although did not include samples from WA, suggest the existence of at least four stocks of grey mackerel in northern Australia: NW NT, the GoC, northern east coast and central east coast. Grey mackerel parasite fauna on the east coast suggests a separation somewhere between Townsville and Mackay. The NW NT region also appears to comprise a separate stock while within the GoC there exists a high degree of variability in parasite faunas among the regions sampled. This may be due to 1. natural variation within the GoC and there is one grey mackerel stock, or 2. the existence of multiple localised adult sub-stocks (metapopulations) within the GoC. Growth parameter comparisons were only possible from four major locations and identified the NW NT, the GoC, and the east coast as having different population growth characteristics. Through the use of multiple techniques, and by integrating the results from each, we were able to determine that there exist at least five stocks of grey mackerel across northern Australia, with some likelihood of additional stock structuring within the GoC. The major management units determined from this study therefore were Western Australia, NW Northern Territory (Timor/Arafura), the Gulf of Carpentaria, northern east Queensland coast and central east Queensland coast. The management implications of these results indicate the possible need for management of grey mackerel fisheries in Australia to be carried out on regional scales finer than are currently in place. In some regions the spatial scales of management might continue as is currently (e.g. WA), while in other regions, such as the GoC and the east coast, managers should at least monitor fisheries on a more local scale dictated by fishing effort and assess accordingly. Stock assessments should also consider the stock divisions identified, particularly on the east coast and for the GoC, and use life history parameters particular to each stock. We also emphasise that where we have not identified different stocks does not preclude the possibility of the occurrence of further stock division. Further, this study did not, nor did it set out to, assess the status of each of the stocks identified. This we identify as a high priority action for research and development of grey mackerel fisheries, as well as a management strategy evaluation that incorporates the conclusions of this work. Until such time that these priorities are addressed, management of grey mackerel fisheries should be cognisant of these uncertainties, particularly for the GoC and the Queensland east coast.