Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.)

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST–RFLP loci in the F1(NA6 × AU6) population. A comprehensive set of EST–SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA6 genetic map contains 88 EST–RFLP and 71 EST–SSR loci with a total map length of 963 cM, while the AU6 genetic map contains 67 EST–RFLP and 58 EST–SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

Genetic Parameters for Production Traits in Queensland Merinos

Genetic and phenotypic parameters are presented for production traits, greasy fleece weight (GFW), yield (YLD), clean fleece weight (CFW), average fibre diameter (DIAM) and liveweight (LWT), in 15 month old medium Peppin Merino sheep at Longreach and Julia Creek, Queensland. Heritabilities for GFW, YLD, CFW, DIAM and LWT were respectively 0.35, 0.62, 0.34, 0.74, and 0.37 for Longreach and 0.23, 0.52, 0.20, 0.67 and 0.56 for Julia Creek. Most estimates were consistent with other reported values. AAABG 13th Conference; Proceedings of the Association for the Advancement of Animal Breeding and Genetics.

Genetic Parameters for Physiological Characters in Merino Rams in Central and North West Queensland

Genetic and phenotypic parameters for respiration rate (RR) and rectal temperature (RT) are presented for weaner and hogget Merino rams, at Longreach and Julia Creek, Queensland. Heritability estimates for RT and RR at both sites and at both ages ranged from moderate to very high. Phenotypic and genetic correlations between these characters are also reported. AAABG 14th Conference; Proceedings of the Association for the Advancement of Animal Breeding and Genetics. AAABG

Comparison of the indirect haemagglutination and gel diffusion test for serotyping Haemophilus parasuis

The aim of this study was to compare the use of indirect haemagglutination (IHA) and gel diffusion (GD) tests for serotyping Haemophilus parasuis by the Kielstein-Rapp-Gabrielson scheme. All 15 serovar reference strains, 72 Australian field isolates, nine Chinese field isolates, and seven isolates from seven experimentally infected pigs were evaluated with both tests. With the IHA test, 14 of the 15 reference strains were correctly serotyped – with serovar 10 failing to give a titre with serovar 10 antiserum. In the GD test, 13 reference strains were correctly serotyped – with antigen from serovars 7 and 8 failing to react with any antiserum. The IHA methodology serotyped a total of 45 of 81 field isolates while the GD methodology serotyped a total of 48 isolates. For 29 isolates, the GD and IHA methods gave discordant results. It was concluded that the IHA is a good additional test for the serotyping of H. parasuis by the KRG scheme if the GD methodology fails to provide a result or shows unusual cross-reactions.

Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: Identification of genetic differences between Haemophilus parasuis isolates

Representational Difference Analysis (RDA) is an established technique used for isolation of specific genetic differences between or within bacterial species. This method was used to investigate the genetic basis of serovar-specificity and the relationship between serovar and virulence in Haemophilus parasuis. An RDA clone library of 96 isolates was constructed using H. parasuis strains H425(P) (serovar 12) and HS1967 (serovar 4). To screen such a large clone library to determine which clones are strain-specific would typically involved separately labelling each clone for use in Southern hybridisation against genomic DNA from each of the strains. In this study, a novel application of reverse Southern hybridisation was used to screen the RDA library: genomic DNA from each strain was labelled and used to probe the library to identify strain-specific clones. This novel approach represents a significant improvement in methodology that is rapid and efficient.

A new data source for fisheries resource assessment: genetic estimates of the effective number of spawners. Final Report to the Fisheries Research and Development Corporation.

The development of innovative methods of stock assessment is a priority for State and Commonwealth fisheries agencies. It is driven by the need to facilitate sustainable exploitation of naturally occurring fisheries resources for the current and future economic, social and environmental well being of Australia. This project was initiated in this context and took advantage of considerable recent achievements in genomics that are shaping our comprehension of the DNA of humans and animals. The basic idea behind this project was that genetic estimates of effective population size, which can be made from empirical measurements of genetic drift, were equivalent to estimates of the successful number of spawners that is an important parameter in process of fisheries stock assessment. The broad objectives of this study were to 1. Critically evaluate a variety of mathematical methods of calculating effective spawner numbers (Ne) by a. conducting comprehensive computer simulations, and by b. analysis of empirical data collected from the Moreton Bay population of tiger prawns (P. esculentus). 2. Lay the groundwork for the application of the technology in the northern prawn fishery (NPF). 3. Produce software for the calculation of Ne, and to make it widely available. The project pulled together a range of mathematical models for estimating current effective population size from diverse sources. Some of them had been recently implemented with the latest statistical methods (eg. Bayesian framework Berthier, Beaumont et al. 2002), while others had lower profiles (eg. Pudovkin, Zaykin et al. 1996; Rousset and Raymond 1995). Computer code and later software with a user-friendly interface (NeEstimator) was produced to implement the methods. This was used as a basis for simulation experiments to evaluate the performance of the methods with an individual-based model of a prawn population. Following the guidelines suggested by computer simulations, the tiger prawn population in Moreton Bay (south-east Queensland) was sampled for genetic analysis with eight microsatellite loci in three successive spring spawning seasons in 2001, 2002 and 2003. As predicted by the simulations, the estimates had non-infinite upper confidence limits, which is a major achievement for the application of the method to a naturally-occurring, short generation, highly fecund invertebrate species. The genetic estimate of the number of successful spawners was around 1000 individuals in two consecutive years. This contrasts with about 500,000 prawns participating in spawning. It is not possible to distinguish successful from non-successful spawners so we suggest a high level of protection for the entire spawning population. We interpret the difference in numbers between successful and non-successful spawners as a large variation in the number of offspring per family that survive – a large number of families have no surviving offspring, while a few have a large number. We explored various ways in which Ne can be useful in fisheries management. It can be a surrogate for spawning population size, assuming the ratio between Ne and spawning population size has been previously calculated for that species. Alternatively, it can be a surrogate for recruitment, again assuming that the ratio between Ne and recruitment has been previously determined. The number of species that can be analysed in this way, however, is likely to be small because of species-specific life history requirements that need to be satisfied for accuracy. The most universal approach would be to integrate Ne with spawning stock-recruitment models, so that these models are more accurate when applied to fisheries populations. A pathway to achieve this was established in this project, which we predict will significantly improve fisheries sustainability in the future. Regardless of the success of integrating Ne into spawning stock-recruitment models, Ne could be used as a fisheries monitoring tool. Declines in spawning stock size or increases in natural or harvest mortality would be reflected by a decline in Ne. This would be good for data-poor fisheries and provides fishery independent information, however, we suggest a species-by-species approach. Some species may be too numerous or experiencing too much migration for the method to work. During the project two important theoretical studies of the simultaneous estimation of effective population size and migration were published (Vitalis and Couvet 2001b; Wang and Whitlock 2003). These methods, combined with collection of preliminary genetic data from the tiger prawn population in southern Gulf of Carpentaria population and a computer simulation study that evaluated the effect of differing reproductive strategies on genetic estimates, suggest that this technology could make an important contribution to the stock assessment process in the northern prawn fishery (NPF). Advances in the genomics world are rapid and already a cheaper, more reliable substitute for microsatellite loci in this technology is available. Digital data from single nucleotide polymorphisms (SNPs) are likely to super cede ‘analogue’ microsatellite data, making it cheaper and easier to apply the method to species with large population sizes.

Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium aphanidermatum populations infecting cucumber in Oman

Seventy three isolates of Pythium aphanidermatum obtained from cucumber from four different regions of Oman and 16 isolates of muskmelon from the Batinah region in Oman were characterized for aggressiveness, sensitivity to metalaxyl and genetic diversity using AFLP fingerprinting. Twenty isolates of P. aphanidermatum from diverse hosts from different countries were also included in the study. Most isolates from Oman were found to be aggressive on cucumber seedlings and all were highly sensitive to metalaxyl (EC50 < 0•80 µg mL−1). Isolates from cucumber and muskmelon were as aggressive as each other on both hosts (P > 0.05), which implies a lack of host specialization in P. aphanidermatum on these two hosts in Oman. AFLP analysis of all isolates using four primer-pair combinations resolved 152 bands, of which 61 (~40%) were polymorphic. Isolates of P. aphanidermatum from Oman and other countries exhibited high genetic similarity (mean = 94.1%) and produced 59 different AFLP profiles. Analysis of molecular variance indicated that most AFLP variation among populations of P. aphanidermatum in Oman was associated with geographical regions (FST = 0.118; P < 0.0001), not hosts (FST = -0.004; P = 0.4323). These data were supported by the high rate of recovery (24%) of identical phenotypes between cucumber and muskmelon fields in the same region as compared to the low recovery (10%) across regions in Oman, which suggests more frequent movement of Pythium inoculum among muskmelon and cucumber fields in the same region compared to movement across geographically separated regions. However, recovering clones among regions and different countries may imply circulation of Pythium inoculum via common sources in Oman and also intercontinental spread of isolates.

Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium spinosum infecting cucumber in Oman

A total of 24 isolates of Pythium spinosum from cucumber obtained from five regions in Oman were characterized for genetic diversity using amplified fragment length polymorphism (AFLP) fingerprinting and three isolates from the Netherlands, South Africa and Japan were included for comparison. Isolates from Oman were also characterized for aggressiveness on cucumber seedlings and sensitivity to metalaxyl. Identity of all isolates was confirmed using sequences of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), which showed more than 99% nucleotide similarity among all isolates. Using six primer-pair combinations, AFLP fingerprinting resolved 295 AFLP markers of which 193 were polymorphic among isolates from other countries and only six were polymorphic among isolates of P. spinosum from Oman. Seven different AFLP phenotypes of P. spinosum were recovered in Oman; two of them were found to contain over 79% of isolates and one was recovered from all regions in Oman. Phenotypes from Oman showed very high (?99%) levels of genetic similarity to each other compared to moderate (mean =53%) levels of genetic similarity with phenotypes from other countries. In addition, all isolates from Oman were found to be highly sensitive to metalaxyl and all were aggressive on cucumber seedlings at 25°C. The high genetic similarity among phenotypes of P. spinosum in Oman as well as recovering two major clones across regions may suggest that P. spinosum has been recently introduced in Oman via a common source.

A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

Assessing for genetic and environmental effects on ruminant feed quality in barley ( Hordeum vulgare )

Grain samples from a combined intermediate and advanced stage barley breeding trial series, grown at two sites in two consecutive years were assessed for detailed grain quality and ruminant feed quality. The results indicated that there were significant genetic and environmental effects for “feed” traits as measured using grain hardness, acid detergent fibre (ADF), starch and in-sacco dry matter digestibility (ISDMD) assays. In addition, there was strong genotypic discrimination for the regressed feed performance traits, namely Net Energy (NE) and Average Daily Gain (ADG). There was considerable variation in genetic correlations for all traits based on variance from the cultivars used, sites or laboratory processing effects. There was a high level of heritability ranging from 89% to 88% for retention, 60% to 80% for protein and 56% to 68% for ADF. However, there were only low to moderate levels of heritability for the feed traits, with starch 30–39%, ISDMD 55–63%, ADF 56–68%, particle size 47–73%, 31–48% NE and ADG 44–51%. These results suggest that there were real differences in the feed performance of barleys and that selection for cattle feed quality is potentially a viable option for breeding programs.

Genetic Impacts of the Hull on Barley Grain Quality

Barley hull plays an important role in malt and feed quality and processing. In this study we measured the variation in hull con-tent along with other grain quality traits namely, kernel discolouration and degree of pre-harvest sprouting, in a single map-ping population. There were significant (p < 0.05) genetic as well as environment effects. In addition, heritability was calculated for hull content to be 29% and 47% for two years’ data. From the analysis, major QTL markers were identified in con-trolling the expression of hull content on chromosomes 2 (2H), and 6 (6H) with significant (P < 0.05) LOD scores of 5.4 and 3.46 respectively. Minor QTLs were identified on 1 (7H), 4 (4H), 5 (1H) and 7 (5H). The region at marker Bmac310 on 4(4H) could be associated with dormancy gene SD4. A number of the QTLs also coincided with regions for either kernel discolouration or preharvest sprouting resistance (dormancy). The results indicate that variation exists for hull content, which is influenced by both growing environment as well as genetically, although the genetic variance explained less than half of the total variance. Further, hull content also impacts on other grain quality attributes including dormancy, sprouting resistance and kernel appearance.

Measurement of genetic and environmental variation in barley (Hordeum vulgare) grain hardness

In this study, we assessed a broad range of barley breeding lines and commercial varieties by three hardness methods (two particle size methods and one crush resistance method (SKCS—Single-Kernel Characterization System), grown at multiple sites to see if there was variation in barley hardness and if that variation was genetic or environmentally controlled. We also developed near-infrared reflectance (NIR) calibrations for these three hardness methods to ascertain if NIR technology was suitable for rapid screening of breeding lines or specific populations. In addition, we used this data to identify genetic regions that may be associated with hardness. There were significant (p<0.05) genetic effects for the three hardness methods. There were also environmental effects, possibly linked to the effect of protein on hardness, i.e. increasing protein resulted in harder grain. Heritability values were calculated at >85% for all methods. The NIR calibrations, with R2 values of >90%, had Standard Error of Prediction values of 0.90, 72 and 4.0, respectively, for the three hardness methods. These equations were used to predict hardness values of a mapping population which resulted in genetic markers being identified on all chromosomes but chromosomes 2H, 3H, 5H, 6H and 7H had markers with significant LOD scores. The two regions on 5H were on the distal end of both the long and short arms. The region that showed significant LOD score was on the long arm. However, the region on the short arm associated with the hardness (hordoindoline) genes did not have significant LOD scores. The results indicate that barley hardness is influenced by both genotype and environment and that the trait is heritable, which would allow breeders to develop very hard or soft varieties if required. In addition, NIR was shown to be a reliable tool for screening for hardness. While the data set used in this study has a relatively low variation in hardness, the tools developed could be applied to breeding populations that have large variation in barley grain hardness.

Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.

The genetic diversity of ampeloviruses in Australian pineapples and their association with mealybug wilt disease

Pineapple mealybug wilt-associated virus 1 (PMWaV-1), 2 (PMWaV-2) and -3 (PMWaV-3) have been detected in Australian commercial pineapple crops, along with a previously undescribed ampelovirus, for which the name Pineapple mealybug wilt-associated virus 5 (PMWaV-5) is proposed. Partial sequences extending from open reading frame 1b through to the heat shock protein homologue were obtained for PMWaV-1, -3 and -5. Phylogenetic analyses of selected regions of these sequences indicated that PMWaV-5 is a distinct species and most closely related to PMWaV-1. The amino acid sequence variation observed in the RNA-dependent RNA polymerase region of PMWaV-1 isolates was 95.8–98.4% and of PMWaV-3 isolates was 92.2–99.5%. In surveys of mealybug wilt disease (MWD) affected crops, none of the four viruses was clearly associated with the disease at all survey sites. A statistically significant association (P < 0.001) between the presence of PMWaV-2 and symptoms was observed at one survey site (site 3), but the virus was at a low incidence at the remaining three survey sites. By contrast, although PMWaV-1 and -3 were equally distributed between symptomless and MWD-affected plants at site 3, there was a statistically significant (P < 0.001) association between each of these two viruses and MWD at sites 1 and 4. At site 2, there was a statistically significant (P < 0.001) association only between PMWaV-3 and MWD. PMWaV-1 was the most commonly found of the four viruses and conversely PMWaV-5 was only occasionally found. Australian isolates of PMWaV-1, -2 and -3 were transmitted by the mealybug species Dysmicoccus brevipes.

The vaccination-challenge trial: the gold standard test to evaluate the protective efficacy of infectious coryza vaccines

Infectious coryza is an upper respiratory tract disease of chickens with the major impact occurring in multi-age flocks. We investigated the relationship between the level of antibodies, as detected by a haemagglutination-inhibition (HI) assay, in infectious coryza-vaccinated chickens and the protection against challenge in those chickens. In one experiment, chickens given a single dose of either of two infectious coryza vaccines lacked a detectable HI response to vaccination but showed significant levels of protection 11 weeks after vaccination. In contrast, in chickens given two doses of an infectious coryza vaccine and challenged 3 weeks after the second vaccine dose, there was a strong serological response with 36/40 birds having a HI titre of 1/20 or greater. In this trial there was an apparent relationship between titre and subsequent protection, with none of the 32 chickens with a titre of 1/40 or 1/80 showing any clinical signs and only one of the same group yielding the challenge organism on culture. In contrast, three of the four vaccinated chickens with a HI titre less than 1/5 developed the typical clinical signs of coryza and yielded the challenge organism on culture. Overall, our results suggest that HI titres cannot be regarded as a definitive predictor of vaccine efficacy. We suggest that the vaccination-challenge trial is the gold standard for the evaluation of the immune response to infectious coryza vaccines.