6. Molecular and Cytological Characterisation (part of WP-05)

The objective is to provide molecular and molecular cytogenetics markers for the characterisation of Crocus germplasm and application in selection of improved cultivars for saffron production. Thus, the molecular characterization of Crocus will allow:

-To identify clearly the off types in some collections

-To appreciated the extend of diversity of various geographic or genetic groups cultivated and wild.

-To clarify the genetic relationships between these groups (heterozygosity of varieties). Indeed, an important genetic missing has occurred over the past, because the ornamental value of Crocus , with transfer and hybridisations made between cultivated species

A range of appropriate molecular marker are available for such purposes: Microsatellites (SSRs), inter-SSR and inter-retroelement methods, and gene based markers (PCR and hybridization based) will be used. A wide range of accessions of both cultivated Saffron and wild species will be used for characterization, and one or two outgroups will be included in analysis (Romulea and Iris) to allow robust rooting of phylogenies.

Standard primers for phylogenetic analysis will be used to amplify gene segments from nuclear, chloroplast and mitochondrial genes. Molecular-based trees will be constructed to infer relationships between groups and accessions. Molecular-based analysis will show how much diversity is present in the genus; this data will be in a form that it can be used to develop conservation policy.

In addition to estimating genetic diversity, these tools can be utilized to establish molecular markers for phenotypic traits of interest. Genes which are widely conserved between species, with a focus on those associated with biotic and abiotic stress tolerance, will be amplified and sequenced from a range of accessions, using conserved sets of PCR primers. Genes associated with corm development and dormancy will be isolated and characterized from a range of accessions. Gene expression analyses, will allow to found more efficient alleles. Genes associated to flavour and other associated quality traits important in Crocus commercialization. Several genes directly involved in flavour have been identified in C. sativus , and several biochemical markers are used to determine the quality of flavour and aroma in saffron, we would like to correlated molecular and biochemical flavour traits.