Structural determination of the K14 capsular polysaccharide from an ST25 Acinetobacter baumannii isolate, D46

The structure of the capsular polysaccharide (CPS) recovered from D46, an extensively antibiotic resistant ST25 Acinetobacter baumannii clinical isolate, was elucidated. The structure was resolved on the basis of NMR spectroscopy and chemical analyses, and was found to contain a branched neutral pentasaccharide with a backbone composed of GalpNAc and Galp residues, all d configured, and a d-Glcp side group. The KL14 gene cluster found in the D46 genome includes genes for four glycosyltransferases but no modules for synthesis of complex sugars, and this is consistent with the structure of K14. The K14 structure and KL14 sequence clarify the relationship between the structure and K locus sequence for A. nosocomialis isolate LUH5541. The identity of the first sugar of the K14 repeat unit (K unit), and the functions of the four encoded glycosyltransferases and Wzy polymerase were predicted.

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

Characterization of 26 new microsatellite loci in the dugong (Dugong dugon)

Twenty-six microsatellite loci have been isolated from a dugong (Dugong dugon). The average heterozygosity was 0.52 with two to 10 alleles per locus surveyed from 50 individuals. The markers are suitable for genetic mark-recapture (PID = 5 × 10-16) in dugongs and they could also be used to quantify physical tag loss, estimate relatedness, assign paternity, elucidate population structure and identify migrants.

New microsatellite loci for Carcharhinid sharks (Carcharhinus tilstoni and C. sorrah) and their cross-amplification in other shark species

Twelve microsatellite DNA markers were isolated in the spot-tail shark (Carcharhinus sorrah) and nine were isolated in Australian black-tip shark (Carcharhinus tilstoni). These loci plus 18 others developed for sharks from the genera Negaprion, Ginglymostoma, Carcharodon and Isurus were tested for amplification success on four species of Carcharhinus (including C. sorrah and C. tilstoni) and four other species representing three diverse families. Cross-amplification was most common within families. Five loci were subsequently tested for polymorphism on 50 C. sorrah and 60 C. tilstoni. The number of alleles per locus was two to 24 and the average heterozygosity was 0.54 (range 0.16-0.87) for C. sorrah and 0.64 (range 0.44-0.78) for C. tilstoni. These loci may be useful tools for genetic analyses of the Carcharhinidae.

Structure of the K12 capsule containing 5,7-di-N-acetylacinetaminic acid from Acinetobacter baumannii isolate D36

The repeat unit of the K12 capsular polysaccharide isolated from the Acinetobacter baumannii global clone 1 clinical isolate, D36, was elucidated by means of chemical and spectroscopical methods. The structure was shown to contain N-acetyl-D-galactosamine (D-GalpNAc), N-acetyl-D-fucosamine and N-acetyl-L-fucosamine linked together in the main chain, with the novel sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-altro-non-2-ulosonic acid (5,7-di-N-acetylacinetaminic acid or Aci5Ac7Ac), attached to D-GalpNAc as a side branch. This matched the sugar composition of the K12 capsule and the genetic content of the KL12 capsule gene cluster reported previously. D-FucpNAc was predicted to be the substrate for the initiating transferase, ItrB3, with the Wzy polymerase making a α-D-FucpNAc-(1 → 3)-D-GalpNAc linkage between the repeat units. The three glycosyltransferases encoded by KL12 are all retaining glycosyltransferases and were predicted to form specific linkages between the sugars in the K12 repeat unit.

Structure of the K6 capsular polysaccharide from Acinetobacter baumannii isolate RBH4

The structure of the capsular polysaccharide (CPS) from an Acinetobacter baumannii global clone 2 (GC2) clinical isolate RBH4 that carries the KL6 gene cluster was elucidated by means of chemical and spectroscopical methods. The repeating unit of K6 CPS is linear and contains N-acetyl-d-galactosamine (d-GalpNAc), two d-galactose (d-Galp) residues and 5,7-di-N-acetylpseudaminic acid (Pse5Ac7Ac). The synthesis of these sugars could be attributed to genes in the KL6 capsule biosynthesis gene cluster, and the formation of the linkages between the sugars were assigned to glycosyltransferases or the Wzy polymerase encoded in KL6.

Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Polymorphic microsatellite loci for the zebra shark Stegostoma fasciatum

We report on the development and characterization of 14 polymorphic microsatellite loci in the zebra shark (Stegostoma fasciatum). Five tetranucleotide and nine dinucleotide loci were polymorphic with heterozygosities ranging from 0.400 to 0.967 and from three to 22 alleles per locus. Cross-species amplification of these zebra shark primers on four other species of orectolobid sharks was not successful.

Genetic population structure of red snappers (Lutjanus malabaricus Bloch & Schneider, 1801 and Lutjanus erythropterus Bloch, 1790) in central and eastern Indonesia and northern Australia

The genetic population structure of red snapper Lutjanus malabaricus and Lutjanus erythropterus in eastern Indonesia and northern Australia was investigated by allozyme electrophoresis and sequence variation in the control region of mtDNA. Samples were collected from eight sites in Indonesia and four sites in northern Australia for both species. A total of 13 allozyme loci were scored. More variable loci were observed in L. malabaricus than in L. erythropterus. Sequence variation in the control region (left domain) of the mitochondrial genome was assessed by RFLP and direct sequencing. MtDNA haplotype diversity was high (L. erythropterus, 0.95 and L. malabaricus, 0.97), as was intraspecific sequence divergence, (L. erythropterus, 0.0-12.5% and L. malabaricus, 0.0-9.5%). The pattern of mtDNA haplotype frequencies grouped both species into two broad fisheries stocks with a genetic boundary either between Kupang and Sape (L. malabaricus) or between Kupang and Australian Timor Sea (L. erythropertus). The allozyme analyses revealed similar boundaries for L. erythropterus. Seven allozymes stocks compared to two mtDNA stocks of L. malabaricus including Ambon, which was not sampled with mtDNA, however, were reported. Possible reasons for differences in discrimination between the methods include: i) increased power of multiple allozyme loci over the single mtDNA locus, ii) insufficient gene sampling in the mtDNA control region and iii) relative evolutionary dynamics of nuclear (allozyme loci) and mitochondrial DNA in these taxa. Allozyme and haplotype data did not distinguish separate stocks among the four Australian locations nor the central Indonesian (Bali and Sape locations) for both L. malabaricus and L. erythropterus.

Gene interactions constrain the course of evolution of phosphine resistance in the lesser grain borer, Rhyzopertha dominica

Phosphine, a widely used fumigant for the protection of stored grain from insect pests, kills organisms indirectly by inducing oxidative stress. High levels of heritable resistance to phosphine in the insect pest of stored grain, Rhyzopertha dominica have been detected in Asia, Australia and South America. In order to understand the evolution of phosphine resistance and to isolate the responsible genes, we have undertaken genetic linkage analysis of fully sensitive (QRD14), moderately resistant (QRD369) and highly resistant (QRD569) strains of R. dominica collected in Australia. We previously determined that two loci, rph1 and rph2, confer high-level resistance on strain QRD569, which was collected in 1997. We have now confirmed that rph1 is responsible for the moderate resistance of strain QRD369, which was collected in 1990, and is shared with a highly resistant strain from the same geographical region, QRD569. In contrast, rph2 by itself confers only very weak resistance, either as a heterozygote or as a homozygote and was not discovered in the field until weak resistance (probably due to rph1) had become ubiquitous. Thus, high-level resistance against phosphine has evolved via stepwise acquisition of resistance alleles, first at rph1 and thereafter at rph2. The semi-dominance of rph2 together with the synergistic interaction between rph1 and rph2 would have led to rapid selection for homozygosity. A lack of visible fitness cost associated with alleles at either locus suggests that the resistance phenotype will persist in the field.

Congruence in QTL for adventitious rooting in Pinus elliottii x Pinus caribaea hybrids resolves between and within-species effects

Targeting between-species effects for improvement in synthetic hybrid populations derived from outcrossing parental tree species may be one way to increase the efficacy and predictability of hybrid breeding. We present a comparative analysis of the quantitative trait loci (QTL) which resolved between from within-species effects for adventitious rooting in two populations of hybrids between Pinus elliottii and P. caribaea, an outbred F1 (n=287) and an inbred-like F2 family (n=357). Most small to moderate effect QTL (each explaining 2-5% of phenotypic variation, PV) were congruent (3 out of 4 QTL in each family) and therefore considered within-species effects as they segregated in both families. A single large effect QTL (40% PV) was detected uniquely in the F2 family and assumed to be due to a between-species effect, resulting from a genetic locus with contrasting alleles in each parental species. Oligogenic as opposed to polygenic architecture was supported in both families (60% and 20% PV explained by 4 QTL in the F 2 and F1 respectively). The importance of adventitious rooting for adaptation to survive water-logged environments was thought in part to explain oligogenic architecture of what is believed to be a complex trait controlled by many hundreds of genes.

Mapping species differences for adventitious rooting in a Corymbia torelliana x Corymbia citriodora subspecies variegata hybrid

Quantitative trait loci (QTL) detection was carried out for adventitious rooting and associated propagation traits in a second-generation outbred Corymbia torelliana x Corymbia citriodora subspecies variegata hybrid family (n=186). The parental species of this cross are divergent in their capacity to develop roots adventitiously on stem cuttings and their propensity to form lignotubers. For the ten traits studied, there was one or two QTL detected, with some QTL explaining large amounts of phenotypic variation (e.g. 66% for one QTL for percentage rooting), suggesting that major effects influence rooting in this cross. Collocation of QTL for many strongly genetically correlated rooting traits to a single region on linkage group 12 suggested pleiotropy. A three locus model was most parsimonious for linkage group 12, however, as differences in QTL position and lower genetic correlations suggested separate loci for each of the traits of shoot production and root initiation. Species differences were thought to be the major source of phenotypic variation for some rooting rate and root quality traits because of the major QTL effects and up to 59-fold larger homospecific deviations (attributed to species differences) relative to heterospecific deviations (attributed to standing variation within species) evident at some QTL for these traits. A large homospecific/heterospecific ratio at major QTL suggested that the gene action evident in one cross may be indicative of gene action more broadly in hybrids between these species for some traits.

The complexities of knowing what it is you are trapping

The effectiveness of any trapping system is highly dependent on the ability to accurately identify the specimens collected. For many fruit fly species, accurate identification (= diagnostics) using morphological or molecular techniques is relatively straightforward and poses few technical challenges. However, nearly all genera of pest tephritids also contain groups of species where single, stand-alone tools are not sufficient for accurate identification: such groups include the Bactrocera dorsalis complex, the Anastrepha fraterculus complex and the Ceratitis FAR complex. Misidentification of high-impact species from such groups can have dramatic consequences and negate the benefits of an otherwise effective trapping program. To help prevent such problems, this chapter defines what is meant by a species complex and describes in detail how the correct identification of species within a complex requires the use of an integrative taxonomic approach. Integrative taxonomy uses multiple, independent lines of evidence to delimit species boundaries, and the underpinnings of this approach from both the theoretical speciation literature and the systematics/taxonomy literature are described. The strength of the integrative approach lies in the explicit testing of hypotheses and the use of multiple, independent species delimitation tools. A case is made for a core set of species delimitation tools (pre- and post-zygotic compatibility tests, multi-locus phylogenetic analysis, chemoecological studies, and morphometric and geometric morphometric analyses) to be adopted as standards by tephritologists aiming to resolve economically important species complexes. In discussing the integrative approach, emphasis is placed on the subtle but important differences between integrative and iterative taxonomy. The chapter finishes with a case study that illustrates how iterative taxonomy applied to the B. dorsalis species complex led to incorrect taxonomic conclusions, which has had major implications for quarantine, trade, and horticultural pest management. In contrast, an integrative approach to the problem has resolved species limits in this taxonomically difficult group, meaning that robust diagnostics are now available.

Population genetic structure of the brown tiger prawn, Penaeus esculentus, in tropical northern Australia

Eight polymorphic microsatellite loci were analysed in six population samples from four locations of the Australian endemic brown tiger prawn, Penaeus esculentus. Tests of Hardy-Weinberg equilibrium were generally in accord with expectations, with only one locus, in two samples, showing significant deviations. Three samples were taken in different years from the Exmouth Gulf. These showed no significant heterogeneity, and it was concluded that they were from a single panmictic population. A sample from Shark Bay, also on the west coast of Australia, showed barely detectable differentiation from Exmouth Gulf (F (ST) = 0 to 0.0014). A northeast sample from the Gulf of Carpentaria showed low (F (ST) = 0.008) but significant differentiation from Moreton Bay, on the east coast. However, Exmouth Gulf/Shark Bay samples were well differentiated from the Gulf of Carpentaria/Moreton Bay (F (ST) = 0.047-0.063). The data do not fit a simple isolation by distance model. It is postulated that the east-west differentiation largely reflects the isolation of east and west coast populations that occurred at the last glacial maximum when there was a land bridge between north-eastern Australia and New Guinea.