6 th International Symposium on Rose Research and Cultivation, Hannover , 2013

1. Integration of genetic and physical maps of Rosa by means of tyramide-FISH mapping of abiotic stress related genes Ilya Kirov, Katrijn Van Laere, LudmilaKhrustaleva, Ellen De Keyser and Jan De Riek 6th International Symposium on Rose Research and Cultivation, Hannover, 2013 Institute for Agricultural and Fisheries Research Plant Sciences Unit www.ilvo.vlaanderen.be Agriculture and Fisheries Policy Area

2. Joint project Russian State Agrarian University – Moscow Timiryazev Agricultural Academy Institute for Agricultural and Fisheries research (ILVO) – Plant Sciences Unit – Applied Genetics and Breeding

3. Outline Integration of genetic and physical maps of Rosa by means of tyramide-FISH mapping of abiotic stress related genes Genetic map = basedonlinkagebetweentraits = in segregatingpopulations, look forMendel Physicalmaps = from cytogenetics = chromosomepicturesunder microscope = ultimate is the full genome sequence FISH = fluorescent in situhybridisation = a “paintingtool” forgenes

4. Rose population ‘Yesterday’ x R. wichurana (diploid) (diploid) double flower single flower pink colour white colour erect habit prostrate habit F1: segregatingpopulation of 186 genotypes

5. Genetic map of Rosawichurana ‘Yesterday’ R. wichurana 20 AFLP PC; 43 SSR primers QTLs for powdery mildew resistance Flower size and colour Moghaddam et al., Euphytica (2012) R-E R-P

6. Population, offspring of parent ‘Yesterday’ (A) x R. wichurana (B)

7. Linkage map of parent ‘Yesterday’ (total length 536 cM)

8. Linkage map of parent Rosa wichurana(total length 526 cM)

9. common marker: 23 with Spiller et al. 2011 19 with Yan et al. 2005 8 with Debener et al. 2001 a. Dugo et al. 2005 b. Crespel et al. 2002 c. Linde et al. 2006 d. Rajapakse et al. 2001 e. Hibrand-SaintOyant et al., 2008

10. Genetic map of Rosawichurana • Marker assignment to linkage groups is well conserved between different regression maps • Linkage groups are in line with previous maps • Marker order is more variable • Consensus map construction might help • “Graphical” map integration by regression mapping (Spiller et al, 2011, TAG) • Multipoint-likelihood maximization mapping (De Keyser et al, 2010, BMC Molecular Biology) • but is not perfect either • Note: also in genome sequencing, de novo assembly is not always fully conclusive on contig orders

11. Plant Cytogenetics • The oldestform of plant genetics • Karyotyping versus FISH-typeapproaches • Technical focus in FISH on • Probe-sizedetectionlimits (smallest FISH probethatcanbeclearlydiscerned) • Axialresolutionlimits (smallestdistancebetweentwosignalsthatcanberesolvedby a microscope) • Modern applications focus on • Physicalmapping of markers, BACs, contigs • Tool forcomparativegenomics • Observemeiosis live • Breeding tool for interspecific crosses - introgression

12. Cytogenetics in Rosa • Small genome size: 0.83 to 1.30 pg/2C • diploid (2n=2x=14) to octoploid (2n=8x=56): most tetraploid • Small chromosomes • Low mitotic index in shoots and root tips • Thin roots • Notyetanavailable genome sequence: www.rosegenome.org • Sequence homology with Fragaria, Prunus and Malus • Sofar: reports aboutkaryotyping, some FISH

13. Development of a new chromosome preparation method Development of a new chromosome preparation protocol that combines advantages of animal chromosome preparation method and plant drop method of chromosome preparation (Kirov et al. 2013)

14. Karyotyping of R. wichurana

15. Development of a cytogenetic marker system for rose chromosomes To distinguish different chromosomes, accelerate physical mapping on metaphase chromosomes and provide additional source of markers for phylogeny reconstruction Sources of cytogenetic markers Low copy DNA clones Repetitive sequences BAC clones Short-insert small DNA library 454 sequencing analysis by Graph Based Clustering Tool (Novak et al., 2013) NGS data Clustering Repeat identification FISH

16. Mechanism of Tyramide-FISH detection Tyramide-Fluorochrome Chromosomal DNA WASHING STEP H202 О2 HRP Dig labeled DNA Only tyramides coupled to proteins are left on the slide Anti Dig Histones Histones Histones

17. Candidate genes

18. Tyr-FISH mapping of PAL gene Ch7 Relative distance: 77.0 ± 2.1%

19. Tyr-FISH mapping of P5CS gene Ch4 Relative distance: 72.7±3.8%

20. Tyr-FISH mapping of OOMT gene Ch1 Relative distance: 22.6 ± 3.2%

21. Principle of HRM • Specific primers for amplification of EST-SNPs (parents and F1) • PCR amplification in the presence of a saturating fluorescent dye • (the dyefluorescentswhenit is bound with double strand DNA) • Rapid denaturation and renaturation steps • PCR product meltingand detectionof fluorescenceintensity • Segregationpattern of the marker: added to existing map of Rosawichurana (Joinmap 4.0) P: Aa x AA Homozygote (AA) Heterozygote (Aa) F1: 1Aa:1AA

22. Integration of physical position of three genes with genetic linkage map RwLG1 RwLG7 FvChr6 RwLG4 FvChr7 RwChr1 RwChr4 10 Mb RwChr7 10 Mb 20 Mb 30 Mb 20 Mb

23. Conclusions • First time that tyramide FISH was succesful for physical mapping in species with a very small genome and small chromosomes • By HRM it is possible to genetically map DNA fragments even with 1 SNP • Integration between physical and genetic maps: anchoring Rosa linkage groups carrying important genes (e.g. drought stress related) and QTLs (flower size and flower color) • Gaps: low percentage of cells showing signal; abscence of cytogenetic markers of Rosa chromosomes

24. Futureperspectives • Someideas to improve our Tyramide-FISH protocol • Someideas to developchromosome markers to beable to recognise the chromosomes better • Analysis of diploid versus tetraploid/hybrids concerning abiotic stress tolerance • Use our tyramide-FISH protocol for studying the allopolyploidnature of species • Comparativecytogenetics for different important Rosa species

25. Thank you for your attention! Institute for Agricultural and Fisheries Research Plant Sciences Unit www.ilvo.vlaanderen.be Agriculture and Fisheries Policy Area