Rebecca Ford

Phylogeny, phylogeography and genetic diversity of the Pisum genus

The tribe Fabeae (formerly Vicieae) contains some of humanitys most important grain legume crops, namely Lathyrus (grass pea/sweet pea/chickling vetches; about 160 species); Lens (lentils; 4 species); Pisum (peas; 3 species); Vicia (vetches; about 140 species); and the monotypic genus Vavilovia. Reconstructing the phylogenetic relationships within this group is essential for understanding the origin and diversification of these crops. Our study, based on molecular data, has positioned Pisum genetically between Vicia and Lathyrus and shows it to be closely allied to Vavilovia. A study of phylogeography, using a combination of plastid and nuclear markers, suggested that wild pea spread from its centre of origin, the Middle East, eastwards to the Caucasus, Iran and Afghanistan, and westwards to the Mediterranean. To allow for direct data comparison, we utilized model-based Bayesian Analysis of Population structure (BAPS) software on 4429 Pisum accessions from three large world germplasm collections that include both wild and domesticated pea analyzed by retrotransposon-based markers. An analysis of genetic diversity identified separate clusters containing wild material, distinguishing Pisum fulvum, P. elatius and P. abyssinicum, supporting the view of separate species or subspecies. Moreover, accessions of domesticated peas of Afghan, Ethiopian and Chinese origin were distinguished. In addition to revealing the genetic relationships, these results also provided insight into geographical and phylogenetic partitioning of genetic diversity. This study provides the framework for defining global Pisum germplasm diversity as well as suggesting a model for the domestication of the cultivated species. These findings, together with gene-based sequence analysis, show that although introgression from wild species has been common throughout pea domestication, much of the diversity still resides in wild material and could be used further in breeding. Moreover, although existing collections contain over 10,000 pea accessions, effort should be directed towards collecting more wild material in order to preserve the genetic diversity of the species.

Abiotic Stress Responses in Plants: Present and Future

Drought, cold, high-salinity and heat are major abiotic stresses that severely reduce the yield of food crops worldwide. Traditional plant breeding approaches to improve abiotic stress tolerance of crops had limited success due to multigenic nature of stress tolerance. In the last decade, molecular techniques have been used to understand the mechanisms by which plants perceive environmental signals and further their transmission to cellular machinery to activate adaptive responses. This knowledge is critical for the development of rational breeding and transgenic strategies to impart stress tolerance in crops. Studies on physiological and molecular mechanisms of abiotic stress tolerance have led to characterisation of a number of genes associated with stress adaptation. Techniques like microarrays have proven to be invaluable in generating a list of stress-related genes. Some of these genes are specific for a particular stress while others are shared between various stresses. Interestingly, a number of genes are shared in abiotic and biotic stress responses. This highlights the complexity of stress response and adaptation in plants. There is a whole cascade of genes involved in abiotic stress tolerance; starting from stress perception to transcriptional activation of downstream genes leading to stress adaptation and tolerance. A number of these genes have been discovered but we still do not have the complete list with all interactions. There is also significant number of genes with unknown functions found to be regulated by abiotic stresses. Understanding the function of these genes and their interaction with other known genes to effect stress adaptation is required.

High-throughput novel microsatellite marker of faba bean via next generation sequencing

BackgroundFaba bean (Vicia faba L.) is an important food legume crop, grown for human consumption globally including in China, Turkey, Egypt and Ethiopia. Although genetic gain has been made through conventional selection and breeding efforts, this could be substantially improved through the application of molecular methods. For this, a set of reliable molecular markers representative of the entire genome is required.ResultsA library with 125,559 putative SSR sequences was constructed and characterized for repeat type and length from a mixed genome of 247 spring and winter sown faba bean genotypes using 454 sequencing. A suit of 28,503 primer pair sequences were designed and 150 were randomly selected for validation. Of these, 94 produced reproducible amplicons that were polymorphic among 32 faba bean genotypes selected from diverse geographical locations. The number of alleles per locus ranged from 2 to 8, the expected heterozygocities ranged from 0.0000 to 1.0000, and the observed heterozygosities ranged from 0.0908 to 0.8410. The validation by UPGMA cluster analysis of 32 genotypes based on Neis genetic distance, showed high quality and effectiveness of those novel SSR markers developed via next generation sequencing technology.ConclusionsLarge scale SSR marker development was successfully achieved using next generation sequencing of the V. faba genome. These novel markers are valuable for constructing genetic linkage maps, future QTL mapping, and marker-assisted trait selection in faba bean breeding efforts.

Stagonosporopsis spp. associated with ray blight disease of Asteraceae

Ray blight disease of pyrethrum (Tanacetum cinerariifolium) is shown to be caused by more than one species of Stagonosporopsis. The Australian pathogen, previously identified as Phoma ligulicola var. inoxydabilis, represents a new species described as Stagonosporopsis tanaceti based on morphological characters and a five-gene phylogeny employing partial sequences of the actin, translation elongation factor 1-alpha, internal transcribed spacers and 5.8S of the nrDNA, 28S large subunit and beta-tubulin 2 gene sequences. Furthermore, the two varieties of Stagonosporopsis ligulicola are elevated to species level as S. chrysanthemi and S. inoxydabilis based on their DNA phylogeny and morphology.

The detection of QTLs in barley associated with endosperm hardness, grain density, grain size and malting quality using rapid phenotyping tools

Using a barley mapping population, ‘Vlamingh’xa0×xa0‘Buloke’ (Vxa0×xa0B), whole grain analyses were undertaken for physical seed traits and malting quality. Grain density and size were predicted by digital image analysis (DIA), while malt extract and protein content were predicted using near infrared (NIR) analysis. Validation of DIA and NIR algorithms confirmed that data for QTL analysis was highly correlated (R2xa0>xa00.82), with high RPD values (the ratio of the standard error of prediction to the standard deviation, 2.31–9.06). Endosperm hardness was measured on this mapping population using the single kernel characterisation system. Grain density and endosperm hardness were significantly inter-correlated in all three environments (rxa0>xa00.22, Pxa0<xa00.001); however, other grain components were found to interact with the traits. QTL for these traits were also found on different genomic regions, for example, grain density QTLs were found on chromosomes 2H and 6H, whereas endosperm hardness QTLs were found on 1H, 5H, and 7H. In this study, the majority of the genomic regions associated with grain texture were also coincident with QTLs for grain size, yield, flowering date and/or plant development genes. This study highlights the complexity of genomic regions associated with the variation of endosperm hardness and grain density, and their relationships with grain size traits, agronomic-related traits, and plant development loci.

Colletotrichum truncatum Pathosystem on Capsicum spp: infection, colonization and defence mechanisms

The disease cycle of the chili (Capsicum annuum) anthracnose fungus Colletotrichum truncatum (formerly C. capsici) was elucidated from a study of infection and colonization of seed, leaves and fruit. Microscopic observations of detached leaves and fruit inoculated with a virulent pathotype (F83B), revealed direct cuticle penetration and, intramural, endophytic and necrotrophic phases of colonization. Seedling and fruit ripening stages were very susceptible to infection with the pathogen causing pre- and post-emergence damage and postharvest fruit rot. Furthermore, a quiescent stage, following leaf infection during the vegetative phase of plant growth served as a potential primary inoculum source for fruit infection. Leaf epidermal cells of the resistant C. chinense PBC932 expressed a strong hypersensitive response 48xa0h after infection (HAI) to both highly virulent (F83B) and less virulent (BRIP 26,974) pathotypes. Infected cells had thickened cell walls, cytoplasm aggregation, and high levels of reactive oxygen species produced 12 HAI. In contrast, the infected epidermal cells of the susceptible C. annuum cultivar Bangchang showed necrosis and rapid cell death after infection by either pathotype. Knowledge of the disease cycle of C. truncatum will be helpful in understanding the behaviour of the pathogen in chili fields which will lead to more efficient application of control measures.

Integration of EST-SSR markers of Medicago truncatula into intraspecific linkage map of lentil and identification of QTL conferring resistance to ascochyta blight at seedling and pod stages

Microsatellite markers have been extensively utilised in the leguminosae for genome mapping and identifying major loci governing traits of interest for eventual marker-assisted selection (MAS). The lack of available lentil-specific microsatellite sequences and gene-based markers instigated the mining and transfer of expressed sequence tag simple sequence repeat (EST-SSR)/SSR sequences from the model genome Medicago truncatula, to enrich an existing intraspecific lentil genetic map. A total of 196 markers, including new 15 M. truncatula EST-SSR/SSR, were mapped using a population of 94 F5 recombinant inbred lines produced from a cross between cv. Northfield (ILL5588)xa0×xa0cv. Digger (ILL5722) and clustered into 11 linkage groups (LG) covering 1156.4xa0cM. Subsequently, the size and effects of quantitative trait loci (QTL) conditioning Ascochyta lentis resistance at seedling and pod/maturity stages were characterised and compared. Three QTL were detected for seedling resistance on LG1 and LG9 and a further three were detected for pod/maturity resistance on LG1, LG4 and LG5. Together, these accounted for 34 and 61% of the total estimated phenotypic variation, respectively, and demonstrated that resistance at the different growth stages is potentially conditioned by different genomic regions. The flanking markers identified may be useful for MAS and for the future pyramiding of potentially different resistance genes into elite backgrounds that are resistant throughout the cropping season.

Expression patterns of C- and N-metabolism related genes in wheat are changed during senescence under elevated CO2 in dry-land agriculture

Projected climatic impacts on crop yield and quality, and increased demands for production, require targeted research to optimise nutrition of crop plants. For wheat, post-anthesis carbon and nitrogen remobilisation from vegetative plant parts and translocation to grains directly affects grain carbon (C), nitrogen (N) and protein levels. We analysed the influence of increased atmospheric CO2 on the expression of genes involved in senescence, leaf carbohydrate and nitrogen metabolism and assimilate transport in wheat under field conditions (Australian Grains Free Air CO2 Enrichment; AGFACE) over a time course from anthesis to maturity, the key period for grain filling. Wheat grown under CO2 enrichment had lower N concentrations and a tendency towards greater C/N ratios. A general acceleration of the senescence process by elevated CO2 was not confirmed. The expression patterns of genes involved in carbohydrate metabolism, nitrate reduction and metabolite transport differed between CO2 treatments, and this CO2 effect was different between pre-senescence and during senescence. The results suggest up-regulation of N remobilisation and down-regulation of C remobilisation during senescence under elevated CO2, which is consistent with greater grain N-sink strength of developing grains.

Genetic transformation of Colletotrichum truncatum associated with anthracnose disease of chili by random insertional mutagenesis

An Agrobacterium tumefaciens ‐mediated transformation (ATMT) system was successfully developed for Colletotrichum truncatum, the causal agent of chili anthracnose. A. tumefaciens carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (gfp) gene was used to transform the conidiospores of two C. truncatum pathotypes F8‐3B and BRIP26974. Optimum transformation efficiency was obtained when equal volumes of A. tumefaciens strain AGL1 carrying either pJF1 or pPK2 binary vector was used to transform C. truncatum conidiospores at 106/ml and co‐cultivated at 24 °C for three days. Southern blot analysis indicated that 87.5% of the transformants contained randomly inserted, single copies of the T‐DNA. Infection and colonisation of chili fruit at the mature red stage with F8‐3B‐GFP and BRIP26974‐GFP confirmed the maintenance of virulence within these transformed pathotypes. In situ studies of infection and colonisation of the susceptible genotype fruit using fluorescent microscopy and transformed isolates of C. truncatum expressing GFP revealed that the pathogen was able to colonise healthy fruit tissue intercellularly in an endophytic manner without producing secondary biotrophic infection structures. The developed transformation system will be used to study the function of pathogenicity genes in C. truncatum using both forward and reverse genetics approaches. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Identification of the MAT1 locus in Stagonosporopsis tanaceti, and exploring its potential for sexual reproduction in Australian pyrethrum fields

Stagonosporopsis chrysanthemi, S. inoxydabilis, and S. tanaceti are closely related Ascomycetes associated with ray blight of the Asteraceae. To date, only S. tanaceti has been identified in Australia, incurring substantial losses to the pyrethrum industry. In contrast to the homothallic S. chrysanthemi and S. inoxydabilis, a sexual state has not been observed for S. tanaceti. The MAT1 locus in S. tanaceti was identified through de novo assembly of shotgun reads, and was further used to develop primers for amplification of the full-length MAT1/2 locus in S. chrysanthemi and S. inoxydabilis. As expected, S. chrysanthemi and S. inoxydabilis possessed a MAT1/2 locus typical of homothallic Dothideomycetes with two adjacent MAT1-1 and MAT1-2 idiomorphs. However, only MAT1-1 could be detected in the assembled genome of S. tanaceti. Although a sexual mode of reproduction cannot be ruled out for S. tanaceti, evidence so far suggests this is absent or occurring at very low frequency in Australian pyrethrum fields.