EU-COST Action SaffronomicsWG1 meeting

The roadmap to unveil the genomic sequence of saffronHotel Savoia, Procida, May 16-17, 2014

Friday, May 16

9.15 Welcome (G. Giuliano)

1. Josè-Antonio Fernandez-Perez: The CrocusbankcollectionCrocusbank is a large collection, composed of accessions from across the world with manyof Spanish varieties. Different accessions show morphological/agronomic differences despite saffron of clonal origin, possibly due to epigenetic variations. Saffron industry undergoes crisis due to adulteration and WG1 Saffronomics (including WG1) should provide tools for fraud/adulteration tracing; some tools already exist (DNA sequences and/or metabolites discriminating btw saffron and other adulterants)

2. Matteo Busconi: Epigeneticvariation in saffron12 different genotypes identified on the basis of AFLPs, withlargest variation observed among Iranian accessions. Methylation-sensitive AFLPsdetected larger variation (28 different methyl-genotypes); variations associate withedaphoclimaticvariability (origin).

3. Giovanni Giuliano: Sequencing of complexgenomesCompleted genome sequencing projects underlined the complexities of sequencing polyploid genomes Development of novel sequencing protocols (longer reads: MiSeq, PacBio) makes separation between haplotypes at the assembly step more feasible. Alternatively, chromosomes from allopolyploid genomes can be separated through flow sorting techniques.

4. StephaneRombauts: Genome annotation and optical mappingOptical mapping as a tool to drive superscaffolding and scaffold orientation. BioNano technology is the best but at least 100kb scaffolds needed for optimal performance. High Molecular Weight DNA (> 500kb) needed, not easy.Genome annotation: Eugene software, integrates extrinsic data (a priori informed system): > 40 different tracks can be loaded for prediction, including RNAseq data. Manual curation necessary; VIB-Ghent has a portal (Orcae) for that.

Lunch

5. Gabino Sanchez Perez: Biodiversity sequencing in Solanum85 tomato species sequenced, among which 3 outside the tomato clade that evidenced, beyond small translocations and inversions, also the presence of “new” genomic regions. The identification of such regions has been made possible also due to the improvements of the PacBioread length, increased to an average of 8-10 kb (up to 20 kb reads can be obtained). This eases the obtainment of longer contigs but also requires particularly careful library preparation coupled with a strict size selection to obtain fragments long enough to exploit the power of the sequencer.

6. Pat Heslop-Harrison: Cytogenetics of CrocusFluorescent labelling of chromosomes does not improve the discrimination between paralogs and different genomes that has proven difficult; only for one chromosome such differentiation has given results. No variation in saffron by IRAPs, but huge inter- and intra-specific variation in other species. Looking at single genes in a saffron EST database (D’Agostinoet al.), has evidenced that in ca 70 cases 3 alleles could be identified with a putative ratio 2:1, thereby suggesting the emergence of C. sativus from the conjunction of a non-reduced gamete with a reduced one from another species (not sure which one).Most likely progenitors from IRAP, in situ hybridization and sequence analysis: close species to C.cartwrightianus and C. pallasii ssp. pallasii

7. Thomas Schmidt: Progress in saffron chromosome identification using FISH18S-5.8S-25S rRNA, 5S rRNA genes and two satellite repeats were applied as probes for chromosome identification using FISH. These experiments resulted in unambiguous identification of 7 out of 8 chromosomes of each haploid supplement. However, homeologous chromosomes of the three subgenomes were not distinguished. More satellite repeats have been identified and are currently tested for chromosome specificity under investigation to establish a FISH karyotype. No single probe is available able to distinguish the chromosomes of the three subgenomes(max 2).

8. Sergio Lucretti: The FISHIS technique for separating complex genomesThis technique is a combination of FISH and flow sorting, useful for separating genomes and discriminate allopolyploids; it relies on probes able to discriminate the genomes. Already shown to work for Triticaceae. Beyond specific probes, actively dividing and easily synchronizable cells are needed. The technique could be applied to saffron, if corms in

hydroponicculturecould generate enough roots and specific probes are found able to discriminate between homeologous chromosomes. If 2 out of 3 chromosomes derive from the same species, they will probably difficult to discriminate but the technique will still diminish overall complexity. The technique can sort DNA fragments 40-100kb long, useful for generating fosmid libraries and PacBio sequencing.

9. BahattinTanyolac: Chloroplast genome sequencingThe Crocus chloroplast sequence is very conserved with respect to those of other plant species. Chloroplast sequences from several different Crocus species including putative ancestors of saffron, have been amplified and sent for sequencing. Results will be available in June. In collaboration with J. Dolezel, a bilateral Turkish-Czech project was presented for the shotgun sequencing of a single flow-sorted chromosome.

10.Carsten Pedersen: Wild crocus species: genetic resources of Saffron crocus?Not possible to discriminate between species using Structure; more markers needed, perhaps by GBS. Use of wild to increase yield: build a new triploid (or higher ploidy): no idea how long it will take to obtain seeds.

17.30 General discussion

Saturday, May 17

11.Silvia Fluch: Use of methylation-based genome filtration to identify transcribed regions of the genome of SaffronGene filtration testedin rice using methylation sensitive enzymes results in > 90% genes recovered. The same system has been used as a proof-of-concept in Norway spruce (large genome) and is currently being applied in saffron. Library preparation is ongoing and MiSeq sequencing will be performed.

12.Marco Pietrella: The saffron stigma transcriptomeSaffron stigma transcriptome at 6 different developmental stages has been sequenced and assembled. Significant differences between early- and late-stage maturation: ca 1,000 genes differentially expressed among which Transcription factors. Presumably within the transcriptome several genes putatively involved in pathogen response and in the biosynthesis of secondary metabolites of potential interest are present and can be fished for searching for other interesting enzymes. Analysis evidenced >1,000

putative SSR that could be used for genotyping. Trancriptome data will be used to assess the genomic origin of the plant.

13.Giovanni Giuliano: Cloning the enzyme catalyzing the first step in stigma apocarotenoid biosynthesisUsing transcriptome data, a number of candidates for the cleavage of zeaxanthin could be identified and, using in vitro and in vivo assays, an enzyme was isolated as the responsible of the cleavage.

Coffee break

14.Andreas Voloudakis: dsRNA application for plant virus control and endogenous plant gene expression regulation. A new functional genomics tool?dsRNA has been used successfully for “plant vaccination” against plant viruses. Initial experiments using specific siRNAs aggravate symptoms of virus infection.The method is systemic and nucleotide-specific. The same approach works in endogenous gene expression control, but only locally. dsRNA can be used for functional genomics analysis in saffron. Alternatively, virus induced gene silencing (VIGS) could be employed, but a suitable virus-vector is needed.

General discussion

Lunch

15.Marta Roldan-Medina: concluding remarksGrant Holder Manager, Marta Roldán, informed about the presentation of SO IoannaStravidou “COST: 2014 year of transition” in the APC 2014, highlighting that the transition requires from us an adaptive effort to accomplish the new COST rules.

16.White paper writing session Draft is available, will be circulated beginning of June after a

second revision. Missing sections identified that should be included in the whitepaper;responsible partners identified and tasks were assigned

Paper will be addressed to people willing to join and fund the project

Beneficiaries and impact: food and agriculture vs scientific community (Text needs to be generated) (Pat)

Genotyping text expanded by Silvia Cytogenetics part to be revised From the whitepaper we will try to go for joint publication

18:30 Dinner

Summary of discussion:

Arguments for sequencing the genome:1. Understanding of the botanical position of saffron and of its parents.2. Use for better classification of Crocusbankmaterial through barcoding3. Answer biological questions related to:

a. epigenomics, b. origin, c. medical applicationsd. proteome/metabolome analysis, e. food adulteration,f. functional genomics

At present, the main limiting factor is the availability of corms of a reference accession. Due to the lack of sufficient amount of material from Crocusbank, commercial material will be used.Options: Kozani, LaMancha.

Supply of material from Crocusbank: Material requests from different partners (Thomas; Matteo, Giovanni, Marian, Jaroslav, Sergio, Bahattin; request for corms, leafs, DNA, stigmas) on Saffron accessions as well as wild species is not easy to satisfy due to i) ‘exotic material’ i.e. wild species do not grow very well under Spanish conditions and thus material is limited and ii) management of the Crocusbank is currently difficult due to lack of funding.

Agreement reached:Jose Antonio will provideapprox1,000 corms of aLaMancha commercial variety (present in Crocusbank) ––first estimates200 for Turkey, 100 Germany, 200 for ENEA, 100 for Silvia, 200 for Leicester. Silvia will send out a list to all WG1 partners to get their exact requirements.

Technology:There are 2 options i) whole genome shotgun ii) chromosome sorting; parallel approach will be followed.Shotgun sequencing: 10-20x coverage in 500 bpMiSeqpseudoreads, then assembly using Newbler (Rombauts). Libraries made in Rome/Wageningen, runs distributed among partners owning a Miseq. Giovanni/Gabino will circulate costs. Each run should provide 15 Gb, ie 1.5x coverage of the genome. Contributing partners: Giovanni; Silvia, Gabino, Matteo, Jose Antonio, Bahattin. More sought within Saffronomics. Andreas Voloudakismetioned that he will investigate for possible financial participation from Greece. Aim to complete the work in October and submit a first abstract to the Plant and Animal Genome meeting.

HMW DNA: Necessary for mate pairs and BAC/fosmid library construction. ENEA/AIT will work on developing appropriate protocols.Scaffolding: Wait for Lucigen Kit for chimera-freelong-jump (20-40 Kb) mate pairs (due in June).BACs/Fosmids: Fosmid library will be outsourced if enough funding is raised. Dolezel will be asked for his availability to prepare a BAC library.Transcriptome sequencing: 6 stigma developmental stages are done (ENEA) and will be publicly available as soon as paper is accepted - ENEA has funding for 15 more (different tissues + stigmas/anthers/tepals from wild species).Material made available by Crocusbank (???) or by other partners (Pedersen).ENEA could do metabolomic profiling on the same samples. Test foractive transposons in the transcriptome. Schmidt will send TE sequences to ENEA that will return contigs showing homology. Other species:C cartriwghtianus: sequenced by F. Blattner((IPK Gatersleben - pers. comm. of german Colleagues). Silvia Fluch(as WG1 leader) will contact him to invite him to join the COST action and to find out more about the status of the genome.Epigenetic differences: Bisulfite sequencing of different accessions as soon as first genome draft will be available

Project financing:A funding goal of 5,000.- EUR per partner is reasonable. Giovanni will contact potential partners as soon as a first draft of the white paper is finished. Inkind contributions to the project will bealso taken into account.