Assessment of internal quality attributes of mandarin fruit. 1. NIR calibration model development

The utility of near infrared spectroscopy as a non-invasive technique for the assessment of internal eating quality parameters of mandarin fruit (Citrus reticulata cv. Imperial) was assessed. The calibration procedure for the attributes of TSS (total soluble solids) and DM (dry matter) was optimised with respect to a reference sampling technique, scan averaging, spectral window, data pre-treatment (in terms of derivative treatment and scatter correction routine) and regression procedure. The recommended procedure involved sampling of an equatorial position on the fruit with 1 scan per spectrum, and modified partial least squares model development on a 720–950-nm window, pre-treated as first derivative absorbance data (gap size of 4 data points) with standard normal variance and detrend scatter correction. Calibration model performance for the attributes of TSS and DM content was encouraging (typical Rc2 of >0.75 and 0.90, respectively; typical root mean squared standard error of calibration of <0.4 and 0.6%, respectively), whereas that for juiciness and total acidity was unacceptable. The robustness of the TSS and DM calibrations across new populations of fruit is documented in a companion study.

Three new Lasiodiplodia spp. from the tropics, recognized based on DNA sequence comparisons and morphology

Botryosphaeria rhodina (anamorph Lasiodiplodia theobromae) is a common endophyte and opportunistic pathogen on more than 500 tree species in the tropics and subtropics. During routine disease surveys of plantations in Australia and Venezuela several isolates differing from L. theobromae were identified and subsequently characterized based upon morphology and ITS and EF1-a nucleotide sequences. These isolates grouped into three strongly supported clades related to but different from the known taxa, B. rhodina and L. gonubiensis, These have been described here as three new species L. venezuelensis sp. nov., L. crassispora sp. nov. and L. rubropurpurea sp. nov. The three could be distinguished easily from each other and the two described species of Lasiodiplodia, thus confirming phylogenetic separations. Furthermore all five Lasiodiplodia spp. now recognized separated from Diplodia spp. and Dothiorella spp. with 100% bootstrap support.

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Identification of small juvenile scombrids from northwest tropical Australia using mitochondrial DNA cytochrome b sequences

Small juveniles of the nine species of scombrids in Australian waters are morphologically similar to one another and, consequently, difficult to identify to species level. We show that the sequence of the mitochondrial DNA cytochrome b gene region is a powerful tool for identification of these young fish. Using this method, we identified 50 juvenile scombrids collected from Exmouth Bay, Western Australia. Six species of scombrids were apparent in this sample of fish: narrow-barred Spanish mackerel (Scomberomorus commerson), Indian mackerel (Rastrelliger kanagurta), frigate tuna (Auxis thazard), bullet tuna (Auxis rochei), leaping bonito (Cybiosarda elegans), and kawakawa (Euthynnus affinis). The presence of Indian mackerel, frigate tuna, leaping bonito, and kawakawa is the first indication that coastal waters may be an important spawning habitat for these species, although offshore spawning may also occur. The occurrence of small juvenile S. commerson was predicted from the known spawning patterns of that species, but other mackerel species (Scomberomorus munroi, Scomberomorus queenslandicus, Scomberomorus semifasiciatus) likely to be spawning during the sampling period were not detected among the 50 small juveniles analyzed here.

Assessment and rationalization of genetic diversity of Papua New Guinea taro (Colocasia esculenta) using SSR DNA fingerprinting

Taro (Colocasia esculenta) accessions were collected from 15 provinces of Papua New Guinea (PNG). The collection, totalling 859 accessions was collated for characterization and a core collection of 81 accessions (10%) was established on the basis of characterization data generated on 30 agro-morphological descriptors, and DNA fingerprinting using seven SSR primers. The selection of accessions was based on cluster analysis of the morphological data enabling initial selection of 20% accessions. The 20% sample was then reduced and rationalized to 10% based on molecular data generated by SSR primers. This represents the first national core collection of any species established in PNG based on molecular markers. The core has been integrated with core from other Pacific Island countries, contributing to a Pacific regional core collection, which is conserved in vitro in the South Pacific Regional Germplasm Centre at Fiji. The core collection is a valuable resource for food security of the South Pacific region and is currently being utilized by the breeding programmes of small Pacific Island countries to broaden the genetic base of the crop.

Effect of DNA extraction on ageing success in coral trout (Plectropomus leopardus) otoliths

To maximize the information commonly collected from otoliths, the effect of DNA extraction on the estimation of age with otoliths was evaluated by comparing sagittal otolith samples from common coral trout (Plectropomus leopardus) for clarity and ageing discrepancies in DNA-extracted and untreated control otoliths. The DNA extraction process had no significant effect, indicating that archived otoliths can be used as a source of DNA while retaining their utility for age estimation.

NIR model development and robustness in prediction of melon fruit total soluble solids

Near infrared spectroscopy (NIRS) can be used for the on-line, non-invasive assessment of fruit for eating quality attributes such as total soluble solids (TSS). The robustness of multivariate calibration models, based on NIRS in a partial transmittance optical geometry, for the assessment of TSS of intact rockmelons (Cucumis melo) was assessed. The mesocarp TSS was highest around the fruit equator and increased towards the seed cavity. Inner mesocarp TSS levels decreased towards both the proximal and distal ends of the fruit, but more so towards the proximal end. The equatorial region of the fruit was chosen as representative of the fruit for near infrared assessment of TSS. The spectral window for model development was optimised at 695-1045 nm, and the data pre-treatment procedure was optimised to second-derivative absorbance without scatter correction. The 'global' modified partial least squares (MPLS) regression modelling procedure of WINISI (ver. 1.04) was found to be superior with respect to root mean squared error of prediction (RMSEP) and bias for model predictions of TSS across seasons, compared with the 'local' MPLS regression procedure. Updating of the model with samples selected randomly from the independent validation population demonstrated improvement in both RMSEP and bias with addition of approximately 15 samples.

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.

DNA amplification fingerprinting analysis of genetic variation within Fusarium oxysporum f.sp zingiberi

Genetic variation among 29 isolates of Fusarium oxysporum f.sp. zingiberi (Foz) collected from diseased ginger rhizome in production regions throughout Queensland was analysed using DNA amplification fingerprinting (DAF). Eight isolates of other Fusarium species and/or formae speciales were included for comparative analysis. Within the Foz isolates, three haplotypes were identified based on 17 polymorphic bands generated with five primers. Two groups showed very little genetic variation (98.6% similarity), whereas the third single isolate was quite distinct in terms of its molecular profile (77.2% similarity). Genetic similarity among the Fusarium solani, F. oxysporum f.sp. lycopersici and F. oxysporum f.sp. cubense races 1, 3 and 4 isolates compared well with the published literature.

The assessment of TGGE for the detection of interspecific and intergeneric DNA-marker polymorphism within Solanaceae

RFLP markers are currently the most appropriate marker system for the identification of uncharacterised polymorphism at the interspecific and intergeneric level. Given the benefits of a PCR-based marker system and the availability of sequence information for many Solanaceous cDNA clones, it is now possible to target conserved fragments, for primer development, that flank sequences possessing interspecific polymorphism. The potential outcome is the development of a suite of markers that amplify widely in Solanaceae. Temperature gradient gel electrophoresis (TGGE) is a relatively inexpensive gel-based system that is suitable for the detection of most single-base changes. TGGE can be used to screen for both known and unknown polymorphisms, and has been assessed here, for the development of PCR-based markers that are useful for the detection of interspecific variation within Solanaceae. Fifteen markers are presented where differences between Lycopersicon esculentum and L. pennellii have been detected by TGGE. The markers were assessed on a wider selection of plant species and found to be potentially useful for the identification of interspecific and intergeneric polymorphism in Solanaceous plants.

An identification tool for the Australian weedy Sporobolus species based on random amplified polymorphic DNA (RAPD) profiles

Based on morphological features alone, there is considerable difficulty in identifying the 5 most economically damaging weed species of Sporobolus [viz. S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur and Schinz, S. fertilis (Steud.) Clayton, S. africanus (Poir.) Robyns and Tourney, and S. jacquemontii Kunth.] found in Australia. A polymerase chain reaction (PCR)-based random amplified polymorphic DNA (RAPD) technique was used to create a series of genetic markers that could positively identify the 5 major weeds from the other less damaging weedy and native Sporobolus species. In the initial RAPD profiling experiment, using arbitrarily selected primers and involving 12 species of Sporobolus, 12 genetic markers were found that, when used in combination, could consistently identify the 5 weedy species from all others. Of these 12 markers, the most diagnostic were UBC51490 for S. pyramidalis and S. natalensis; UBC43310.2000.2100 for S. fertilis and S. africanus; and ORA20850 and UBC43470 for S. jacquemontii. Species-specific markers could be found only for S. jacquemontii. In an effort to understand why there was difficulty in obtaining species-specific markers for some of the weedy species, a RAPD data matrix was created using 40 RAPD products. These 40 products amplified by 6 random primers from 45 individuals belonging to 12 species, were then subjected to numerical taxonomy and multivariate system (NTSYS pc version 1.70) analysis. The RAPD similarity matrix generated from the analysis indicated that S. pyramidalis was genetically more similar to S. natalensis than to other species of the 'S. indicus complex'. Similarly, S. jacquemontii was more similar to S. pyramidalis, and S. fertilis was more similar to S. africanus than to other species of the complex. Sporobolus pyramidalis, S. jacquemontii, S. africanus, and S. creber exhibited a low within-species genetic diversity, whereas high genetic diversity was observed within S. natalensis, S. fertilis, S. sessilis, S. elongates, and S. laxus. Cluster analysis placed all of the introduced species (major and minor weedy species) into one major cluster, with S. pyramidalis and S. natalensis in one distinct subcluster and S. fertilis and S. africanus in another. The native species formed separate clusters in the phenograms. The close genetic similarity of S. pyramidalis to S. natalensis, and S. fertilis to S. africanus may explain the difficulty in obtaining RAPD species-specific markers. The importance of these results will be within the Australian dairy and beef industries and will aid in the development of integrated management strategy for these weeds.

Mitochondrial DNA supports the identification of two endangered river sharks (Glyphis glyphis and Glyphis garricki) across northern Australia

The river sharks (genus Glyphis) are a small group of poorly known sharks occurring in tropical rivers and estuarine waters across northern Australia, south-east Asia and the subcontinent. The taxonomy of the genus has long been unclear due to very few individuals having been caught and examined, resulting in a paucity of data regarding their distribution, biology and ecology. Only recently has attention focussed on the two Australian species, G. glyphis and G. garricki. This study is a result of a rare opportunity to collate the few samples that have been collected from these species and the bull shark Carcharhinus leucas, which shares an overlapping range. These samples were analysed using the DNA barcoding approach (cox1 mitochondrial gene), compared with six other species of carcharhinids and evaluated in light of the current taxonomic classification. Nine species-specific nucleotide differences were found between G. glyphis and G. garricki and no intra-specific variation provides strong support for the separation into distinct species. Significant differences were also observed at the inter-generic level, with Glyphis forming a distinct clade from Carcharhinus. This study provides the basis for future molecular studies required to better address conservation issues confronting G. glyphis and G. garricki in Australia.

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.

DNA bar-coding reveals source and patterns of Thaumastocoris peregrinus invasions in South Africa and South America.

Thaumastocoris peregrinus is a recently introduced invertebrate pest of non-native Eucalyptus plantations in the Southern Hemisphere. It was first reported from South Africa in 2003 and in Argentina in 2005. Since then, populations have grown explosively and it has attained an almost ubiquitous distribution over several regions in South Africa on 26 Eucalyptus species. Here we address three key questions regarding this invasion, namely whether only one species has been introduced, whether there were single or multiple introductions into South Africa and South America and what the source of the introduction might have been. To answer these questions, bar-coding using mitochondrial DNA (COI) sequence diversity was used to characterise the populations of this insect from Australia, Argentina, Brazil, South Africa and Uruguay. Analyses revealed three cryptic species in Australia, of which only T. peregrinus is represented in South Africa and South America. Thaumastocoris peregrinus populations contained eight haplotypes, with a pairwise nucleotide distance of 0.2-0.9% from seventeen locations in Australia. Three of these haplotypes are shared with populations in South America and South Africa, but the latter regions do not share haplotypes. These data, together with the current distribution of the haplotypes and the known direction of original spread in these regions, suggest that at least three distinct introductions of the insect occurred in South Africa and South America before 2005. The two most common haplotypes in Sydney, one of which was also found in Brisbane, are shared with the non-native regions. Sydney populations of T. peregrinus, which have regularly reached outbreak levels in recent years, might thus have served as source of these three distinct introductions into other regions of the Southern Hemisphere.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, interserovar convergence, and evidence of attenuation in Leptospira reference collections.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.