Discovery of genes associated with fruit ripening in arica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded: chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species.

Host range, symptom expression and RNA 3 sequence analyses of six Australian strains of Cucumber mosaic virus

We have characterised six Australian Cucumber mosaic virus (CMV) strains belonging to different subgroups, determined by the sequence of their complete RNA 3 and by their host range and the symptoms they cause on species in the Solanaceae, Cucurbitaceae and on sweet corn. These data allowed classification of strains into the known three CMV subgroups and identification of plant species able to differentiate the Australian strains by symptoms and host range. Western Australian strains 237 and Twa and Queensland strains 207 and 242 are closely related members of CMV subgroup IA, which cause similar severe symptoms on Nicotiana species. Strains 207 and 237 (subgroup IA) were the only strains tested which systemically infected sweet corn. Strain 243 caused the most severe symptoms of all strains on Nicotiana species, tomato and capsicum and appears to be the first confirmed subgroup IB strain reported in Australia. Based on pair-wise distance analysis and phylogeny of RNA 3, as well as mild disease symptoms on Nicotiana species, CMV 241 was assigned to subgroup II, as the previously described Q-CMV and LY-CMV.

Discovery of genes associated with fruit ripening in Carica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded:chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species.

Phylogeography of the pharaoh cuttle Sepia pharaonis based on partial mitochondrial 16S sequence data

The pharaoh cuttle Sepia pharaonis Ehrenberg, 1831 (Mollusca: Cephalopoda: Sepiida) is a broadly distributed species of substantial fisheries importance found from east Africa to southern Japan. Little is known about S. pharaonis phylogeography, but evidence from morphology and reproductive biology suggests that Sepia pharaonis is actually a complex of at least three species. To evaluate this possibility, we collected tissue samples from Sepia pharaonis from throughout its range. Phylogenetic analyses of partial mitochondrial 16S sequences from these samples reveal five distinct clades: a Gulf of Aden/Red Sea clade, a northern Australia clade, a Persian Gulf/Arabian Sea clade, a western Pacific clade (Gulf of Thailand and Taiwan) and an India/Andaman Sea clade. Phylogenetic analyses including several Sepia species show that S. pharaonis sensu lato may not be monophyletic. We suggest that "S. pharaonis" may consist of up to five species, but additional data will be required to fully clarify relationships within the S. pharaonis complex.

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.

Rice molecular breeding laboratories in the genomics era: Current status and future considerations

Using DNA markers in plant breeding with marker-assisted selection (MAS) could greatly improve the precision and efficiency of selection, leading to the accelerated development of new crop varieties. The numerous examples of MAS in rice have prompted many breeding institutes to establish molecular breeding labs. The last decade has produced an enormous amount of genomics research in rice, including the identification of thousands of QTLs for agronomically important traits, the generation of large amounts of gene expression data, and cloning and characterization of new genes, including the detection of single nucleotide polymorphisms. The pinnacle of genomics research has been the completion and annotation of genome sequences for indica and japonica rice. This information-coupled with the development of new genotyping methodologies and platforms, and the development of bioinformatics databases and software tools-provides even more exciting opportunities for rice molecular breeding in the 21st century. However, the great challenge for molecular breeders is to apply genomics data in actual breeding programs. Here, we review the current status of MAS in rice, current genomics projects and promising new genotyping methodologies, and evaluate the probable impact of genomics research. We also identify critical research areas to "bridge the application gap" between QTL identification and applied breeding that need to be addressed to realize the full potential of MAS, and propose ideas and guidelines for establishing rice molecular breeding labs in the postgenome sequence era to integrate molecular breeding within the context of overall rice breeding and research programs.

Australian and Thai isolates of Burkholderia pseudomallei are distinct by multilocus sequence typing: Revision of a case of mistaken identity

A recent study using multilocus sequence typing (MLST) of Burkholderia pseudomallei isolates found a sequence type (ST60) to be common to both Thailand and Australia, contradicting earlier studies showing complete distinction between isolates from these regions. The ST60 isolates reportedly from Australia had been obtained for MLST from United Kingdom and U.S. collections. We have located and characterized the original Australian isolates; they were collected in 1983, and they are neither ST60 nor B. pseudomallei isolates. The B. pseudomallei MLST database has been corrected, and there is no ST common to isolates verified as obtained from Australia or from Thailand.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Phylogenetic relationships of graminicolous downy mildews based on cox2 sequence data

Graminicolous downy mildews (GDM) are an understudied, yet economically important, group of plant pathogens, which are one of the major constraints to poaceous crops in the tropics and subtropics. Here we present a first molecular phylogeny based on cox2 sequences comprising all genera of the GDM currently accepted, with both lasting (Graminivora, Poakatesthia, and Viennotia) and evanescent (Peronosclerospora, Sclerophthora, and Sclerospora) sporangiophores. In addition, all other downy mildew genera currently accepted, as well as a representative sample of other oomycete taxa, have been included. It was shown that all genera of the GDM have had a long, independent evolutionary history, and that the delineation between Peronosclerospora and Sclerospora is correct. Sclerophthora was found to be a particularly divergent taxon nested within a paraphyletic Phytophthora, but without support. The results confirm that the placement of Peronosclerospora and Sclerospora in the Saprolegniomycetidae is incorrect. Sclerophthora is not closely related to Pachymetra of the family Verrucalvaceae, and also does not belong to the Saprolegniomycetidae, but shows close affinities to the Peronosporaceae. In addition, all GDM are interspersed throughout the Peronosporaceae s lat., suggesting that a separate family for the Sclerosporaceae might not be justified.

Effects of environmental heat on conception rates in lactating dairy cows: Critical periods of exposure

Environmental heat can reduce conception rates (the proportion of services that result in pregnancy) in lactating dairy cows. The study objectives were to identify periods of exposure relative to the service date in which environmental heat is most closely associated with conception rates, and to assess whether the total time cows are exposed to high environmental heat within each 24-h period is more closely associated with conception rates than is the maximum environmental heat for each 24-h period. A retrospective observational study was conducted in 25 predominantly Holstein-Friesian commercial dairy herds located in Australia. Associations between weather and conception rates were assessed using 16,878 services performed over a 21-mo period. Services were classified as successful based on rectal palpation. Two measures of heat load were defined for each 24-h period: the maximum temperature-humidity index (THI) for the period, and the number of hours in the 24-h period when the THI was >72. Conception rates were reduced when cows were exposed to a high heat load from the day of service to 6 d after service, and in wk -1. Heat loads in wk -3 to -5 were also associated with reduced conception rates. Thus, management interventions to ameliorate the effects of heat load on conception rates should be implemented at least 5 wk before anticipated service and should continue until at least 1 wk after service. High autocorrelations existed between successive daily values in both measures, and associations between day of heat load relative to service day and conception rates differed substantially when ridge regression was used to account for this autocorrelation. This indicates that when assessing the effects of heat load on conception rates, the autocorrelation in heat load between days should be accounted for in analyses. The results suggest that either weekly averages or totals summarizing the daily heat load are adequate to describe heat load when assessing effects on conception rates in lactating dairy cows.

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.

Phylogenetic relationships of unclassified, satellitic Pasteurellaceae obtained from different species of birds as demonstrated by 16S rRNA gene sequence comparison

Avian haemophili demonstrating in vitro satellitic growth, also referred to as the V-factor or NAD requirement, have mainly been classified with Avibacterium paragallinarum (Haemophilus paragallinarum), Avibacterium avium (Pasteurella avium), Avibacterium volantium (Pasteurella volantium) and Avibacterium sp. A (Pasteurella species A). The aim of the present study was to assess the taxonomic position of 18 V-factor-requiring isolates of unclassified Haemophilus-like organisms isolated from galliforme, anseriforme, columbiforme and gruiforme birds as well as kestrels and psittacine birds including budgerigars by conventional phenotypic tests and 16S rRNA gene sequencing. All isolates shared phenotypical characteristics which allowed classification with Pasteurellaceae. Haemolysis of bovine red blood cells was negative. Haemin (X-factor) was not required for growth. Maximum-likelihood phylogenetic analysis including bootstrap analysis showed that six isolates were related to the avian 16S rRNA group and were classified as Avibacterium according to 16S rRNA sequence analysis. Surprisingly, the other 12 isolates were unrelated to Avibacterium. Two isolates were unrelated to any of the known 16S rRNA groups of Pasteurellaceae. Two isolates were related to Volucribacter of the avian 16S rRNA group. Seven isolates belonged to the Testudinis 16S rRNA group and out of these, two isolates were closely related to taxa 14 and 32 of Bisgaard, whereas four other isolates were found to form a genus-like group distantly related to taxon 40 and one isolated remained distantly related to other members of the Testudinis group. One isolate was closely related to taxon 26 (a member of Actinobacillus sensu stricto). The study documented major genetic diversity among V-factor-requiring avian isolates beyond the traditional interpretation that they only belong to Avibacterium, underlining the limited value of satellitic growth for identification of avian members of Pasteurellaceae. Our study also emphasized that these organisms will never be isolated without the use of special media satisfying the V-factor requirement.

Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida.

A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes - adk, est, gdh, mdh, pgi, pmi and zwf - and internal fragments of 570-784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

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.

Defining critical soil nutrient concentrations in soils supporting grains and cotton in Northern NSW and Queensland

This project focussed on the phosphorus (P) and potassium (K) status of northern cropping soils. Stores of P and K have been depleted by crop removal and limited fertiliser application, with depletion most significant in the subsoil. Soil testing strategies are confounded by slowly available mineral reserves with uncertain availability. The utility of new soil tests was assessed to measure these reserves, their availability to plants quantified and a regional sampling strategy undertaken to identify areas of greatest P and K deficit. Fertiliser application strategies for P and K have been tested and the interactions between these and other nutrients have been determined in a large field program.