High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

Background: Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results: We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions: Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.

Behavioural responses of the seven-spot ladybird Coccinella septempunctata to plant headspace chemicals collected from four crop Brassicas and Arabidopsis thaliana, infested with Myzus persicae

1 Insects using olfactory stimuli to forage for prey/hosts are proposed to encounter a ‘reliability–detectability problem’, where the usability of a stimulus depends on its reliability as an indicator of herbivore presence and its detectability. 2 We investigated this theory using the responses of female seven-spot ladybirds Coccinella septempunctata (Coleoptera: Coccinellidae) to plant headspace chemicals collected from the peach-potato aphid Myzus persicae and four commercially available Brassica cultivars; Brassica rapa L. cultivar ‘turnip purple top’, Brassica juncea L. cultivar ‘red giant mustard’, Brassica napus L. cultivar ‘Apex’, Brassica napus L. cultivar ‘Courage’ and Arabidopsis thaliana. For each cultivar/species, responses to plants that were undamaged, previously infested by M. persicae and infested with M. persicae, were investigated using dual-choice Petri dish bioassays and circular arenas. 3 There was no evidence that ladybirds responded to headspace chemicals from aphids alone. Ladybirds significantly preferred headspace chemicals from B. napus cv. Apex that were undamaged compared with those from plants infested with aphids. For the other four species/cultivars, there was a consistent trend of the predators being recorded more often in the half of the Petri dish containing plant headspace chemicals from previously damaged and infested plants compared with those from undamaged ones. Furthermore, the mean distance ladybirds walked to reach aphid-infested A. thaliana was significantly shorter than to reach undamaged plants. These results suggest that aphid-induced plant chemicals could act as an arrestment or possibly an attractant stimulus to C. septempunctata. However, it is also possible that C. septempunctata could have been responding to aphid products, such as honeydew, transferred to the previously damaged and infested plants. 4 The results provide evidence to support the ‘reliability–detectability’ theory and suggest that the effectiveness of C. septempunctata as a natural enemy of aphids may be strongly affected by which species and cultivar of Brassica are being grown.

Foraging strategies of natural enemies of the peach-potato aphid Myzus persicae

The foraging strategies of two natural enemies of the peach-potato aphid Myzus persicae: the seven-spot ladybird Coccinella septempunctata and the parasitoid wasp Diaeretiella rapae, were investigated. Specifically the roles of plant semiochemicals in the location of plants infested with M. persicae by these natural enemies were examined. I investigated the olfactory responses of female C. septempunctata to volatiles collected from M. persicae and four Brassica cultivars; Brassica rapa, B. juncea, B. napus cultivar ‘Apex’ and B. napus cultivar ‘Courage’ and wild-type Arabidopsis thaliana that were: undamaged, previously infested by M. persicae and infested with M. persicae. C. septempunctata showed no attraction to volatiles from M. persicae alone. C. septempunctata significantly changed its searching behaviour in response to plant volatiles from B. rapa, B. napus cv. ‘Apex’ and Arabidopsis infested with M. persicae. C. septempunctata was also found to display a significant turning bias when foraging on a branching horizontal wire stem. A model was developed to investigate how turning biases affect the foraging efficiency of C. septempunctata in dichotomous branched environments. Simulations using this model indicated that turning biases could potentially increase searching efficiency. D. rapae showed a significant preference for volatiles from M. persicae infested wild-type Arabidopsis but no preference to volatiles from M. persicae alone or M. persicae honeydew. Volatile emissions by Arabidopsis were shown to be localised to the area of aphid-infestation rather than systemic. Using gas chromatography plants infested with M. persicae were shown to emit a quantitatively different volatile blend than undamaged plants. In experiments with jasmonate mutants of Arabidopsis the jasmonate (octadecanoid) wound response pathway was implicated as being important for the production of M. persicae induced volatiles, attractive to D. rapae. Other wound response pathways were also found to be involved in the production of the full blend of M. persicae induced volatiles.

A simple and versatile 2-dimensional platform to study plant germination and growth under controlled humidity

We describe a simple, inexpensive, but remarkably versatile and controlled growth environment for the observation of plant germination and seedling root growth on a flat, horizontal surface over periods of weeks. The setup provides to each plant a controlled humidity (between 56% and 91% RH), and contact with both nutrients and atmosphere. The flat and horizontal geometry of the surface supporting the roots eliminates the gravitropic bias on their development and facilitates the imaging of the entire root system. Experiments can be setup under sterile conditions and then transferred to a non-sterile environment. The system can be assembled in 1-2 minutes, costs approximately 8.78$ per plant, is almost entirely reusable (0.43$ per experiment in disposables), and is easily scalable to a variety of plants. We demonstrate the performance of the system by germinating, growing, and imaging Wheat (Triticum aestivum), Corn (Zea mays), and Wisconsin Fast Plants (Brassica rapa). Germination rates were close to those expected for optimal conditions.

Hybridization between Brassica napus and B-rapa on a national scale in the United Kingdom

Mike J. Wilkinson, Luisa J. Elliott, Jo?l Allainguillaume, Michael W. Shaw, Carol Norris, Ruth Welters, Matthew Alexander, Jeremy Sweet, David C. Mason (2003). Hybridization between Brassica napus and B-rapa on a national scale in the United Kingdom, Science, 302 (5644), 457-459. RAE2008

Hybridization between Brassica napus and B. rapa on a national scale in the United Kingdom

Measures blocking hybridization would prevent or reduce biotic or environmental change caused by gene flow from genetically modified (GM) crops to wild relatives. The efficacy of any such measure depends on hybrid numbers within the legislative region over the life-span of the GM cultivar. We present a national assessment of hybridization between rapeseed (Brassica napus) and B. rapa from a combination of sources, including population surveys, remote sensing, pollen dispersal profiles, herbarium data, local Floras, and other floristic databases. Across the United Kingdom, we estimate that 32,000 hybrids form annually in waterside B. rapa populations, whereas the less abundant weedy populations contain 17,000 hybrids. These findings set targets for strategies to eliminate hybridization and represent the first step toward quantitative risk assessment on a national scale.

Thermal and pressure stability of myrosinase enzymes from black mustard (Brassica nigra L. W.D.J Koch. var. nigra), brown mustard (Brassica juncea L. Czern. var. juncea) and yellow mustard (Sinapsis alba L. Subsp Maire) seeds

This study investigates the effects of temperature and pressure on inactivation of myrosinase extracted from black, brown and yellow mustard seeds. Brown mustard had higher myrosinase activity (2.75 un/mL) than black (1.50 un/mL) and yellow mustard (0.63 un/mL). The extent of enzyme inactivation increased with pressure (600-800 MPa) and temperature (30-70 °C) for all the mustard seeds. However, at combinations of lower pressures (200-400 MPa) and high temperatures (60-80 °C), there was less inactivation. For example, application of 300 MPa and 70 °C for 10 minutes retained 20%, 80% and 65% activity in yellow, black and brown mustard, respectively, whereas the corresponding activity retentions when applying only heat (70 °C, 10min) were 0%, 59% and 35%. Thus, application of moderate pressures (200-400 MPa) can potentially be used to retain myrosinase activity needed for subsequent glucosinolate hydrolysis.

Simple sequence repeats reveal uneven distribution of genetic diversity in chloroplast genomes of Brassica oleracea L. and (n=9) wild relatives

Diversity in the chloroplast genome of 171 accessions representing the Brassica 'C' (n = 9) genome, including domesticated and wild B. oleracea and nine inter-fertile related wild species, was investigated using six chloroplast SSR (microsatellite) markers. The lack of diversity detected among 105 cultivated and wild accessions of B. oleracea contrasted starkly with that found within its wild relatives. The vast majority of B. oleracea accessions shared a single haplotype, whereas as many as six haplotypes were detected in two wild species, B. villosa Biv. and B. cretica Lam.. The SSRs proved to be highly polymorphic across haplotypes, with calculated genetic diversity values (H) of 0.23-0.87. In total, 23 different haplotypes were detected in C genome species, with an additional five haplotypes detected in B. rapa L. (A genome n = 10) and another in B. nigra L. (B genome, n = 8). The low chloroplast diversity of B. oleracea is not suggestive of multiple domestication events. The predominant B. oleracea haplotype was also common in B. incana Ten. and present in low frequencies in B. villosa, B. macrocarpa Guss, B. rupestris Raf. and B. cretica. The chloroplast SSRs reveal a wealth of diversity within wild Brassica species that will facilitate further evolutionary and phylogeographic studies of this important crop genus.

Cloning and characterization of microRNAs from Brassica napus

A library containing approximately 40,000 small RNA sequences was constructed for Brassica napus. Analysis of 3025 sequences obtained from this library resulted in the identification of 11 conserved miRNA families, which were validated by secondary structure prediction using surrounding sequences in the Brassica genome. Two 21 nt small RNA sequences reside within the arm of a pre-miRNA like stem-loop structure, making them likely candidates for novel non-conserved miRNAs in B. napus. Most of the conserved miRNAs were expressed at similar levels in a F1 hybrid B. napus line and its four double haploid progeny that showed marked variations in phenotypes, but many were differentially expressed between B. napus and Arabidopsis. The miR169 family was expressed at high levels in young leaves and stems, but was undetectable in roots and mature leaves, suggesting that miR169 expression is developmentally regulated in B. napus. © 2007 Federation of European Biochemical Societies.

DrsG from Streptococcus dysgalactiae subsp equisimilis inhibits the antimicrobial peptide LL- 37

SIC and DRS are related proteins present in only four of the more than 200 Streptococcus pyogenes emm-types. These proteins inhibit complement mediated lysis and/or the activity of certain antimicrobial peptides. A gene encoding a homologue of these proteins, herein called DrsG, has been identified in the related bacterium Streptococcus dysgalactiae subsp equisimilis (SDSE). Here we show that geographically dispersed isolates representing 14 of 50 emm-types examined possess variants of drsG. However not all isolates within the drsG-positive emm-types possess the gene. Sequence comparisons also reveal a high degree of conservation in different SDSE emm-types. To examine the biological activity of DrsG, recombinant versions of two major DrsG variants, DrsGS and DrsGL, were expressed and purified. Western blot analysis using antisera raised to these proteins demonstrated both variants to be expressed and secreted into culture supernatant. Unlike SIC, but similar to DRS, DrsG does not inhibit complement mediated lysis. However, like both SIC and DRS, DrsG is a ligand of the cathelcidin LL-37 and is inhibitory to its bactericidal activity in in vitro assays. The greatest similarity between DrsG and DRS/SIC is found in the signal sequence at the amino terminus and proline rich domains in the C-terminal half of the protein. Conservation of prolines in this latter region also suggests these residues are important in the biology of this family of proteins. This is the first report demonstrating the activity of an AMP inhibitory protein in SDSE. These results also suggest that inhibition of AMP activity is the primary function of this family of proteins. The acquisition of complement inhibitory activity of SIC may reflect its continuing evolution.

Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Recent advances in the molecular biology of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease

Twelve years ago our understanding of ratoon stunting disease (RSD) was confined almost exclusively to diagnosis of the disease and control via farm hygiene, with little understanding of the biology of the interaction between the causal agent (Leifsonia xyli subsp. xyli) and the host plant sugarcane (Saccharum spp. hybrids). Since then, research has focused on developing the molecular tools to dissect L. xyli subsp. xyli, so that better control strategies can be developed to prevent losses from RSD. Within this review, we give a brief overview of the progression in research on L. xyli subsp. xyli and highlight future challenges. After a brief historical background on RSD, we discuss the development of molecular tools such as transformation and transposon mutagenesis and discuss the apparent lack of genetic diversity within the L. xyli subsp. xyli world population. We go on to discuss the sequencing of the genome of L. xyli subsp. xyli, describe the key findings and suggest some future research based on known deficiencies that will capitalise on this tremendous knowledge base to which we now have access.

Comparison of Culture and a Novel 5' Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.

The host range and biology of Cometaster pyrula; a biocontrol agent for Acacia nilotica subsp. indica in Australia

Prickly acacia, Acacia nilotica subsp. indica (Benth.) Brenan, a major weed of the Mitchell Grass Downs of northern Queensland, Australia, has been the target of biological control projects since the 1980s. The leaf-feeding caterpillar Cometaster pyrula (Hopffer) was collected from Acacia nilotica subsp. kraussiana (Benth.) Brenan during surveys in South Africa to find suitable biological control agents, recognised as a potential agent, and shipped into a quarantine facility in Australia. Cometaster pyrula has a life cycle of approximately 2 months during which time the larvae feed voraciously and reach 6 cm in length. Female moths oviposit a mean of 339 eggs. When presented with cut foliage of 77 plant species, unfed neonates survived for 7 days on only Acacia nilotica subsp. indica and Acacia nilotica subsp. kraussiana. When unfed neonates were placed on potted plants of 14 plant species, all larvae except those on Acacia nilotica subsp. indica and Acacia nilotica subsp. kraussiana died within 10 days of placement. Cometaster pyrula was considered to be highly host specific and safe to release in Australia. Permission to release C. pyrula in Australia was obtained and the insect was first released in north Queensland in October 2004. The ecoclimatic model CLIMEX indicated that coastal Queensland was climatically suitable for this insect but that inland areas were only marginally suitable.