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Forward genetic approaches for improved crops

The overall objective of WP4 is to identify associations between traits for drought tolerance and their underlying genes, using forward genetic approaches.   This allelic information will be passed to breeder in the consortium.  The approach we are using differs between the three species because our knowledge of them differs greatly.  We know most about poplar and this allows us to undertake  a complete Genome-Wide Association Analysis (GWAS) since a reference genome and considerable sequence data are already available.  In miscanthus, we focused on genetical genomic analysis using QTL populations that segregate for drought tolerance.  In arundo, we are focused on developing novel resources for harnessing genetic variation by completing a mutagenesis with the development of a new genetic resource that will be assessed for biomass productivity and quality maintenance in droughted conditions.

Five hundred informative clones will be selected from the wide association mapping population grown under two levels of water availability (in WP1) for a Genome Wide Association Study, linking alleles to genes using the sequencing data (from WP 3).  RNA-seq with confirmation using real time qPCR is being used for gene expression in the bulked extreme genotypes.  This enables cis- and trans- eQTL to be elucidated providing powerful insight into the genetic architecture of traits for drought tolerance.

A similar approach will be undertaken in miscanthus, but we are constrained by the fewer knowledge resources available, including no reference genome sequence.  Nevertheless, in such cases RNA sequencing provides powerful gene expression data.  A group of mapping population ‘extremes’ identified from leaf rolling and other drought-associated responses will be identified in the miscanthus mapping population and potential eQTL for drought tolerance identified in this crop.

Our approach to arundo is very different.  Led by the SME Genetilab in Italy, physical mutagenesis of arundo is being developed by testing arundo for radiation dosage and regeneration efficiency.  Different types of tissues (immature inflorescence, young and expanding leaf blade and/or sheat; axillar bud), radiation sources ( -, X- and/or fast neutron), and doses (from 10 Gy- to 100 Gy-equivalent radiation doses) have been tested.  Seedlings have been propagated/transplanted in pots/nursery for a preliminary phenotypic evaluation. Lethality curves and DL50 for each tissue, radiation source and dose, are being established in order to chose the protocol for the large scale mutagenic experiments.  For large-scale mutagenesis, the selected protocol will be applied in order to treat approx. 2,000 samples and obtain approx. 1,000 independent mutagenised regenerants. Following mutagenesis, the samples will be in-vitro and/or in-vivo propagated to obtain approx. 15 clones per mutagenised event.

 

 

For each independent arundo regenerant (approx 1,000), four cloned plants will be transpanted to the field. Plants will be grown following standard agronomic procedures for a total number of 12,000 plants).  During the 4rd and 5th year of the project (2nd and 3rd year of cropping, respectively), productivity and quality traits will be measured.  A parallel field experiment with a sub-selection of the same arundo mutagenised clones will carried out within WP6 in a drought-prone environment.