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

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

3. Outline 3 Integration of genetic and physical maps of Rosa by means of tyramide-FISH mapping of abiotic stress related genes Genetic map = based on linkage between traits = in segregating populations, look for Mendel Physical maps = from cytogenetics = chromosome pictures under microscope = ultimate is the full genome sequence FISH = fluorescent in situ hybridisation = a “painting tool” for genes

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

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

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. Linkage groups Marker nameSpiller et al., 2011 Yan et al., 2005 Debener et al. 2001 and other authors a,b,c,d,e ‘Yesterday’R. wichurana Rh79 RhAB9-2 RhD201 RhEO506 RhAB15 Rh48 RhB303 Rh50 Rh58 Rh65 RhD221 RhABT12 Rw62C4 Pchgms3 (B6B1) RhAB40 RhP507 Rh93 Rh85 RhAB22 RhE2b Rh72 Rh73 Rw22B6 RhP519 Rw10M24 O1a6 Rh59 Rh77 RhAB38 Rh60 Blfo (NP) Blfa (flower colour) 11122223334444 56 77777 55563211122223334444 56 77777 555632 11 222 33444 44566677 35563211 222 33444 44566677 355632 2 4 4 a, 2 d 4 6 5 a 3, 2 a, 6 b, 3 c, 3 e 2 1 2 3 4 5 6 5 nm 7 2 7 2 6 4 3 2 1 2 4 nm 3 4 5 6 6 nm 7 2 7 2 nm 6 4 Marker not present 2 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-Saint Oyant et al., 2008

10. Genetic map of Rosa wichurana 10 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 11 The oldest form of plant genetics Karyotyping versus FISH-type approaches Technical focus in FISH on Probe-size detection limits (smallest FISH probe that can be clearly discerned) Axial resolution limits (smallest distance between two signals that can be resolved by a microscope) Modern applications focus on Physical mapping of markers, BACs, contigs Tool for comparative genomics Observe meiosis live Breeding tool for interspecific crosses - introgression

12. Cytogenetics in Rosa 12 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 Not yet an available genome sequence: www.rosegenome.org Sequence homology with Fragaria, Prunus and Malus So far: reports about karyotyping, 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 14 Chromosome number Chromosome Length (µm) Relative Length (%) Centromere Index (%) 13.7 ± 0.3017.80 ± 0.2046.00 ± 1.20 23.2 ± 0.6017.00 ± 0.2040.30 ± 1.30 33 ± 0.5015.20 ± 0.2044.30 ± 1.00 42.8 ± 0.4014.00 ± 0.1036.90 ± 0.70 52.6 ± 0.4013.60 ± 0.1041.40 ± 0.70 62.5 ± 0.4012.40 ± 0.2041.80 ± 1.10 72.2 ± 0.50 10.00 ± 0.1023.40 ± 0.90

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

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

17. 17 AbbreviationGene nameFunctionSource of sequence PCR product length PALPhenylalanine ammonia-lyase Principal enzyme of the phenylpropanoid pathway Prunus persica1700 bp OOMTOrcinol o- methyltransferase Involved in synthesis of 3,5-dimethoxytoluene Rosa hybrid sp.1100 bp P5CSDelta-1-pyrroline 5-carboxylate synthetase Key enzyme in proline biosynthesis (osmotic stress-responsive gene) Prunus persica1700 bp Candidate genes

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

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

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

21. Principle of HRM 21 Specific primers for amplification of EST-SNPs (parents and F1) PCR amplification in the presence of a saturating fluorescent dye (the dye fluorescents when it is bound with double strand DNA) Rapid denaturation and renaturation steps PCR product melting and detection of fluorescence intensity Segregation pattern of the marker: added to existing map of Rosa wichurana (Joinmap 4.0) Homozygote (AA) Heterozygote (Aa) P: Aa x AA F1: 1Aa:1AA

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

23. Conclusions 23 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. Future perspectives 24 Some ideas to improve our Tyramide-FISH protocol Some ideas to develop chromosome markers to be able to recognise the chromosomes better Analysis of diploid versus tetraploid/hybrids concerning abiotic stress tolerance Use our tyramide-FISH protocol for studying the allopolyploid nature of species Comparative cytogenetics 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