Investigation of the diversity of effector genes in the banana pathogen, Fusarium oxysporum f. sp. cubense, reveals evidence of horizontal gene transfer.

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana-infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole-genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1-SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF-1α/RPB1/RPB2 (translation elongation factor-1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.

Gene loss in the fungal canola pathogen Leptosphaeria maculans.

Recent comparisons of the increasing number of genome sequences have revealed that variation in gene content is considerably more prevalent than previously thought. This variation is likely to have a pronounced effect on phenotypic diversity and represents a crucial target for the assessment of genomic diversity. Leptosphaeria maculans, a causative agent of phoma stem canker, is the most devastating fungal pathogen of Brassica napus (oilseed rape/canola). A number of L. maculans genes are known to be present in some isolates but lost in the others. We analyse gene content variation within three L. maculans isolates using a hybrid mapping and genome assembly approach and identify genes which are present in one of the isolates but missing in the others. In total, 57 genes are shown to be missing in at least one isolate. The genes encode proteins involved in a range of processes including oxidative processes, DNA maintenance, cell signalling and sexual reproduction. The results demonstrate the effectiveness of the method and provide new insight into genomic diversity in L. maculans.

Identification and characterization of more than 4 million intervarietal SNPs across the group 7 chromosomes of bread wheat.

Despite being a major international crop, our understanding of the wheat genome is relatively poor due to its large size and complexity. To gain a greater understanding of wheat genome diversity, we have identified single nucleotide polymorphisms between 16 Australian bread wheat varieties. Whole-genome shotgun Illumina paired read sequence data were mapped to the draft assemblies of chromosomes 7A, 7B and 7D to identify more than 4 million intervarietal SNPs. SNP density varied between the three genomes, with much greater density observed on the A and B genomes than the D genome. This variation may be a result of substantial gene flow from the tetraploid Triticum turgidum, which possesses A and B genomes, during early co-cultivation of tetraploid and hexaploid wheat. In addition, we examined SNP density variation along the chromosome syntenic builds and identified genes in low-density regions which may have been selected during domestication and breeding. This study highlights the impact of evolution and breeding on the bread wheat genome and provides a substantial resource for trait association and crop improvement. All SNP data are publically available on a generic genome browser GBrowse at www.wheatgenome.info.

Advances in plant genotyping: where the future will take us.

Genetic diversity between individuals can be tracked and monitored using a range of molecular markers. These markers can detect variation ranging in scale from a single base pair up to duplications and translocations of entire chromosomal regions. The genotyping of individuals allows the detection of this variation and it has been successfully applied in plant science for many years. The increasing amounts of sequence data able to be generated using next-generation sequencing (NGS) technologies have produced a vast expansion in the rate of discovery of polymorphisms, with single nucleotide polymorphisms (SNPs) predominating as the marker of choice. This increase in polymorphic marker resources through efficient discovery, coupled with the utility of SNPs, has enabled the shift to high-throughput genotyping assays and these methods are reviewed and discussed here, alongside the recent innovations allowing increased throughput.

New technologies for ultrahigh-throughput genotyping in plant taxonomy.

Molecular genetic markers represent one of the most powerful tools for the analysis of variation between plant genomes. Molecular marker technology has developed rapidly over the last decade, with the introduction of new DNA sequencing methods and the development of high-throughput genotyping methods. Single nucleotide polymorphisms (SNPs) now dominate applications in modern plant genetic analysis. The reducing cost of DNA sequencing and increasing availability of large sequence data sets permit the mining of this data for large numbers of SNPs. These may then be used in applications such as genetic linkage analysis and trait mapping, diversity analysis, association studies, and marker-assisted selection. Here we describe automated methods for the discovery of SNP molecular markers and new technologies for high-throughput, low-cost molecular marker genotyping. Examples include SNP discovery using autoSNPdb and wheatgenome.info as well as SNP genotyping using Illumina's GoldenGate™ and Infinium™ methods.

Next-generation genome sequencing can be used to rapidly characterise sequences flanking T-DNA insertions in random insertional mutants of Leptosphaeria maculans.

BACKGROUND: Banks of mutants with random insertions of T-DNA from Agrobacterium tumefaciens are often used in forward genetics approaches to identify phenotypes of interest. Upon identification of mutants of interest, the flanking sequences of the inserted T-DNA must be identified so that the mutated gene can be characterised. However, for many fungi, this task is not trivial as widely used PCR-based methods such as thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) are not successful. FINDINGS: Next-generation Illumina sequencing was used to locate T-DNA insertion sites in four mutants of Leptosphaeria maculans, a fungal plant pathogen. Sequence reads of up to 150 bp and coverage ranging from 6 to 24 times, were sufficient for identification of insertion sites in all mutants. All T-DNA border sequences were truncated to different extents. Additionally, next-generation sequencing revealed chromosomal rearrangements associated with the insertion in one of the mutants. CONCLUSIONS: Next-generation sequencing is a cost-effective and rapid method of identifying sites of T-DNA insertions, and associated genomic rearrangements in Leptosphaeria maculans and potentially in other fungal species.

Identifying genetic diversity of avirulence genes in Leptosphaeria maculans using whole genome sequencing.

Next generation sequencing technology allows rapid re-sequencing of individuals, as well as the discovery of single nucleotide polymorphisms (SNPs), for genomic diversity and evolutionary analyses. By sequencing two isolates of the fungal plant pathogen Leptosphaeria maculans, the causal agent of blackleg disease in Brassica crops, we have generated a resource of over 76 million sequence reads aligned to the reference genome. We identified over 21,000 SNPs with an overall SNP frequency of one SNP every 2,065 bp. Sequence validation of a selection of these SNPs in additional isolates collected throughout Australia indicates a high degree of polymorphism in the Australian population. In preliminary phylogenetic analysis, isolates from Western Australia clustered together and those collected from Brassica juncea stubble were identical. These SNPs provide a novel marker resource to study the genetic diversity of this pathogen. We demonstrate that re-sequencing provides a method of validating previously characterised SNPs and analysing differences in important genes, such as the disease related avirulence genes of L. maculans. Understanding the genetic characteristics of this devastating pathogen is vital in developing long-term solutions to managing blackleg disease in Brassica crops.

Single nucleotide polymorphism discovery from wheat next-generation sequence data.

Single nucleotide polymorphisms (SNPs) are the most abundant type of molecular genetic marker and can be used for producing high-resolution genetic maps, marker-trait association studies and marker-assisted breeding. Large polyploid genomes such as wheat present a challenge for SNP discovery because of the potential presence of multiple homoeologs for each gene. AutoSNPdb has been successfully applied to identify SNPs from Sanger sequence data for several species, including barley, rice and Brassica, but the volume of data required to accurately call SNPs in the complex genome of wheat has prevented its application to this important crop. DNA sequencing technology has been revolutionized by the introduction of next-generation sequencing, and it is now possible to generate several million sequence reads in a timely and cost-effective manner. We have produced wheat transcriptome sequence data using 454 sequencing technology and applied this for SNP discovery using a modified autoSNPdb method, which integrates SNP and gene annotation information with a graphical viewer. A total of 4,694,141 sequence reads from three bread wheat varieties were assembled to identify a total of 38 928 candidate SNPs. Each SNP is within an assembly complete with annotation, enabling the selection of polymorphism within genes of interest.