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Adult Plant Resistance (APR) genes are broad-spectrum, partial-resistance genes that have the potential to contribute to sustainable control of wheat rust diseases. However, their isolation and characterization are complicated by the lack of precise molecular markers required for their identification, and therefore their use in plant breeding programs has been limited. Recent developments including the falling cost of sequencing and the increasing use of sequence capture methods to reduce genome complexity have enabled previously intractable methods such as mutational genomics to clone genes in wheat. Despite their increasing ease of use, many of these approaches require prior knowledge of the gene space and, in some cases, the gene family of the target gene to be cloned. As the APRs cloned so far do not belong to any common gene family, it is not possible to use general features of these identified APRs to conduct biased searches for novel APRs. This project aims to use an unbiased gene isolation technique called MutChromSeq, which combines chromosome flow-sorting and mutational genomics, and is independent of fine mapping, to rapidly clone the recently discovered APR gene Lr68 (Leaf Rust 68). Cloning APRs allows breeders to trace genes cheaply and quickly using gene-specific markers, enabling them to build effective and durable resistance gene pyramids. It also allows us to elucidate any common mechanism of action they have, helping researchers and breeders understand better the basis of their durable resistance. At the same time, the generation time of wheat has become one of the major limiting factors for the response time of breeders to rust epidemics. Thus, this project also aims to combine marker-assisted selection with accelerated generation advancement ('speed breeding') for rapid germplasm structuring and field performance evaluation.
The identification of R-genes using traditional map-based approaches is a long, laborious process, not to mention the time required for subsequent development of cultivars incorporating the new resistances. Breeders seek to reduce the length of breeding cycles, and researchers require new tools to accelerate discovery and understanding of mechanisms associated with durable resistance, especially adult plant resistance (APR). A new method for rapid generation advancement, known as ‘speed breeding’, significantly reduces the length of breeding cycles, provide increased recombination during line development and enable selection in early generations. The speed breeding protocol uses controlled temperature regimes and 24h light to accelerate plant growth and development. Phenotyping methods adapted for use in the speed breeding system permit year-round evaluation of APR to rust pathogens within 5 weeks from time of sowing. RNA sequencing (RNA-Seq) technology has revolutionized gene expression profiling in plants. We previously used RNAseq to identify novel transcripts and miRNAs associated with seedling resistance (Lr28) leading to identification of transcription factors and miRNA families (e.g. miR36, miR37 and miR39) involved in signalling and defense response (Kumar et al. J. Nuc. Acids 2014:570176). In this study we report the application of speed breeding and RNAseq technologies for the purpose of rapidly identifying transcripts and miRNA associated with APR. Wheat landraces harbouring novel sources of resistance were grown under speed breeding conditions and sampled for RNA at key growth stages, before and after inoculation, which enabled discovery of differentially expressed miRNAs. Our next steps are aimed at validating these genetic factors associated with APR in order to better understand the signalling pathways and deliver tools to assist the assembly of robust wheat cultivars for the future.