Integration of genetic and physical maps of Rosa by means of tyramide-FISH mapping of

abiotic stress related genes

6th 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 ResearchPlant Sciences Unit

www.ilvo.vlaanderen.beAgriculture 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 = 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

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

20 AFLP PC; 43 SSR primersQTLs for powdery mildew resistanceFlower size and colour

Moghaddam et al., Euphytica (2012)

atc+cta-560,0aac+caa-071,4aac+caa-253,6RhD2215,8agg+caa-206,6act+cat-8 act+cat-258,8act+ctg-1915,1agg+caa-816,9aag+cag-1917,1atc+ctc-8118,5atc+ctc-5919,4agg+ctg-3120,1atc+ctc-8021,1Rw62C421,5act+ctg-2322,7acc+cac-923,2act+cat-43 aag+cag-17RhAB12 atc+caa-26aag+ctt-30

23,3

Rw23H523,5RhAB4023,8acc+cat-524,6acc+cat-1725,3aac+cag-625,6atc+caa-2328,7agg+caa-1629,6RhP50730,1act+ctg-26 act+ctg-630,3act+ctg-1630,7atc+caa-7331,8atc+caa-6532,1act+cac-933,2aac+caa-6634,8aac+cag-3636,0aag+ctt-136,6act+cat-4439,7act+ctg-1541,4act+ctg-950,1aag+ctt-551,3atc+cca-1554,5atc+ctc-1154,8Pchgms356,2act+cac-1 aag+cag-4256,9aac+caa-3858,1agg+caa-1858,2act+ctg-14 Rh6058,8agg+cag-4259,1atc+caa-6859,9agg+cag-8762,6atc+ctc-7864,4atc+ctc-7967,5atc+cca-3767,7atc+cca-2368,5Rw5D1172,1aca+cag-1975,3acc+cac-5277,1acc+cac-5077,9acc+cat-679,2acc+cat-3680,0

A4aag+cat-180,0acc+cac-252,2acc+cac-268,8acc+cac-2712,1atc+ctc-913,6aac+cag-316,3act+cat-1817,5atc+cta-2218,9atc+cga-30 atc+cga-2919,5aac+caa-719,7aac+caa-5 act+cat-31act+cat-30 Rw3N1920,1

aag+cat-3720,4atc+cga-3820,9atc+cga-3522,0aac+caa-8922,6act+cac-3823,5agg+caa-3325,1act+cat-3925,9atc+ctc-2226,3aca+caa-1026,8atc+ctc-2127,8atc+ctc-2429,3atc+ctc-2529,5agg+ctg-2 atc+cta-2830,0aag+cag-4430,8aag+cat-2631,9acc+cat-932,7atc+cta-4635,0acc+cat-1235,5acc+cac-8 atc+caa-4535,8aca+cag-1136,8aag+cag-3838,4atc+cta-4539,1acc+cac-1840,1aac+caa-7942,4atc+cta-5042,6atc+caa-245,2Rh9347,4acc+cac-1749,3atc+cga-2649,9atc+ctc-2952,3agg+cag-2757,1aag+cat-3569,0aac+caa-5178,6

A5atc+caa-430,0agg+ctg-273,1agg+cag-64,7aac+cag-156,1atc+caa-5110,4aag+cag-210,5atc+cta-2723,0Rw14H2123,8RhAB3827,1aca+caa-2127,8acc+cat-234,5agg+ctg-2335,7aac+cag-236,9atc+caa-2439,7atc+caa-2242,0aac+caa-4342,7act+cat-342,8Rw18N1943,0aag+ctt-3143,8act+ctg-2443,9atc+cga-3144,0atc+cta-3847,4Rw8B848,1Rw10J1948,9atc+caa-5550,1agg+ctg-2150,5atc+caa-7251,6atc+caa-3552,4agg+ctg-1253,7Rh8556,4agg+caa-3262,6

A6act+ctg-20,0Rw22B62,7atc+cta-44,8atc+caa-599,8atc+caa-2010,8aac+caa-3613,5Rw45E2416,6act+cat-3518,4aac+caa-3520,7atc+caa-3922,9aca+caa-2524,6RhP51925,0atc+caa-1925,3atc+ctc-1226,8aca+caa-2628,5Rh5929,2aag+ctt-1630,1aca+cag-2532,3aac+cag-3235,4atc+cca-2638,7Rw27A11B39,8act+ctg-3940,5Rh7242,0atc+ctc-4342,1Rh7342,5atc+caa-2944,0act+ctg-1347,4aag+cag-5747,5aag+cag-3248,8aag+cat-1349,8atc+caa-4249,9atc+cta-1850,9aag+cag-3153,1atc+ctc-4653,3Rw10M2455,2aac+caa-2655,4aag+cat-6057,8agg+caa-3765,4act+ctg-3874,0

A7

‘Yesterday’

3.4

3.7

2.8

act+cat-220,0RhAB91,4acc+cac-152,1act+ctg-352,7aac+cag-384,3aag+cat-427,3atc+caa-629,5aac+caa-489,7act+ctg-3610,0atc+cca-710,5aac+cag-4211,1aac+cag-4611,2aca+cag-1711,4aag+cat-3911,5aag+cat-31 acc+cat-28aag+ctt-3911,6RhD20112,1acc+cat-812,2aac+cag-4312,3aca+cag-612,6atc+cta-512,9act+cac-813,1act+ctg-3713,6atc+cca-27 act+cac-713,9aag+ctt-1714,7aca+caa-1818,1agg+caa-3 act+cat-3618,7atc+cta-4419,8act+ctg-3419,9atc+caa-3220,9act+ctg-2721,4atc+caa-8123,5atc+cca-2227,7atc+caa-8033,5acc+cac-5133,6aac+caa-9934,8aag+cag-3637,1aac+cag-2345,5aag+cag-2547,6aac+cag-5 atc+ctc-2056,0Rh7958,5aac+caa-2458,6agg+ctg-1760,7atc+cca-1862,4atc+cga-763,7aca+caa-663,9atc+caa-4869,5atc+cta-4871,4

A1acc+cac-470,0acc+cac-486,7Blfa7,4act+cac-409,2Rw29B118,0atc+caa-5818,2aac+cag-3418,7atc+cga-2827,1atc+ctc-827,2act+cac-2429,3aac+caa-9330,7Rh7733,1RhB30335,6atc+cta-4036,7aca+cag-2139,8act+ctg-1841,4act+ctg-1242,6atc+cca-5444,3Rw3k19 act+ctg-30agg+ctg-16 agg+ctg-1844,8

atc+cga-645,4O1a645,6aac+caa-9246,6atc+cca-42 atc+cca-4449,5acc+cac-4650,3atc+cca-1951,8Rh4852,0atc+caa-7153,6atc+cca-5657,4RhAB1557,7atc+cca-2559,5atc+cca-262,1acc+cac-4064,8aac+caa-9666,9RhEO50670,1aca+caa-1772,5atc+ctc-3472,8atc+cga-1973,7atc+cga-1376,9agg+ctg-3678,7aag+cag-4080,0

A2act+cat-230,0aca+caa-166,2atc+caa-7411,1act+cat-913,7agg+caa-3918,8RhM40526,5atc+ctc-3530,4atc+cca-4832,6aca+cag-3433,0acc+cat-4235,8act+cat-437,0atc+caa-5337,7atc+caa-28 act+cat-13act+cat-3837,9agg+caa-2638,2aac+cag-3538,7acc+cac-3838,8aag+ctt-839,2atc+caa-739,4Rh6542,2aag+cag-644,2act+cac-2845,4aca+caa-2446,2agg+cag-2946,6aag+cat-4448,1atc+ctc-3648,9Blfo51,3Rh5057,9atc+ctc-2360,0atc+cca-463,2act+cac-364,3Rh5875,8aac+caa-6376,8atc+ctc-7778,1acc+cat-32 atc+caa-5684,7atc+cta-6088,2act+cac-3189,8

A3

atc+cta-560,0aac+caa-071,4aac+caa-253,6RhD2215,8agg+caa-206,6act+cat-8 act+cat-258,8act+ctg-1915,1agg+caa-816,9aag+cag-1917,1atc+ctc-8118,5atc+ctc-5919,4agg+ctg-3120,1atc+ctc-8021,1Rw62C421,5act+ctg-2322,7acc+cac-923,2act+cat-43 aag+cag-17RhAB12 atc+caa-26aag+ctt-30

23,3

Rw23H523,5RhAB4023,8acc+cat-524,6acc+cat-1725,3aac+cag-625,6atc+caa-2328,7agg+caa-1629,6RhP50730,1act+ctg-26 act+ctg-630,3act+ctg-1630,7atc+caa-7331,8atc+caa-6532,1act+cac-933,2aac+caa-6634,8aac+cag-3636,0aag+ctt-136,6act+cat-4439,7act+ctg-1541,4act+ctg-950,1aag+ctt-551,3atc+cca-1554,5atc+ctc-1154,8Pchgms356,2act+cac-1 aag+cag-4256,9aac+caa-3858,1agg+caa-1858,2act+ctg-14 Rh6058,8agg+cag-4259,1atc+caa-6859,9agg+cag-8762,6atc+ctc-7864,4atc+ctc-7967,5atc+cca-3767,7atc+cca-2368,5Rw5D1172,1aca+cag-1975,3acc+cac-5277,1acc+cac-5077,9acc+cat-679,2acc+cat-3680,0

A4aag+cat-180,0acc+cac-252,2acc+cac-268,8acc+cac-2712,1atc+ctc-913,6aac+cag-316,3act+cat-1817,5atc+cta-2218,9atc+cga-30 atc+cga-2919,5aac+caa-719,7aac+caa-5 act+cat-31act+cat-30 Rw3N1920,1

aag+cat-3720,4atc+cga-3820,9atc+cga-3522,0aac+caa-8922,6act+cac-3823,5agg+caa-3325,1act+cat-3925,9atc+ctc-2226,3aca+caa-1026,8atc+ctc-2127,8atc+ctc-2429,3atc+ctc-2529,5agg+ctg-2 atc+cta-2830,0aag+cag-4430,8aag+cat-2631,9acc+cat-932,7atc+cta-4635,0acc+cat-1235,5acc+cac-8 atc+caa-4535,8aca+cag-1136,8aag+cag-3838,4atc+cta-4539,1acc+cac-1840,1aac+caa-7942,4atc+cta-5042,6atc+caa-245,2Rh9347,4acc+cac-1749,3atc+cga-2649,9atc+ctc-2952,3agg+cag-2757,1aag+cat-3569,0aac+caa-5178,6

A5atc+caa-430,0agg+ctg-273,1agg+cag-64,7aac+cag-156,1atc+caa-5110,4aag+cag-210,5atc+cta-2723,0Rw14H2123,8RhAB3827,1aca+caa-2127,8acc+cat-234,5agg+ctg-2335,7aac+cag-236,9atc+caa-2439,7atc+caa-2242,0aac+caa-4342,7act+cat-342,8Rw18N1943,0aag+ctt-3143,8act+ctg-2443,9atc+cga-3144,0atc+cta-3847,4Rw8B848,1Rw10J1948,9atc+caa-5550,1agg+ctg-2150,5atc+caa-7251,6atc+caa-3552,4agg+ctg-1253,7Rh8556,4agg+caa-3262,6

A6act+ctg-20,0Rw22B62,7atc+cta-44,8atc+caa-599,8atc+caa-2010,8aac+caa-3613,5Rw45E2416,6act+cat-3518,4aac+caa-3520,7atc+caa-3922,9aca+caa-2524,6RhP51925,0atc+caa-1925,3atc+ctc-1226,8aca+caa-2628,5Rh5929,2aag+ctt-1630,1aca+cag-2532,3aac+cag-3235,4atc+cca-2638,7Rw27A11B39,8act+ctg-3940,5Rh7242,0atc+ctc-4342,1Rh7342,5atc+caa-2944,0act+ctg-1347,4aag+cag-5747,5aag+cag-3248,8aag+cat-1349,8atc+caa-4249,9atc+cta-1850,9aag+cag-3153,1atc+ctc-4653,3Rw10M2455,2aac+caa-2655,4aag+cat-6057,8agg+caa-3765,4act+ctg-3874,0

A7

‘Yesterday’

3.4

3.7

2.8

act+cat-220,0RhAB91,4acc+cac-152,1act+ctg-352,7aac+cag-384,3aag+cat-427,3atc+caa-629,5aac+caa-489,7act+ctg-3610,0atc+cca-710,5aac+cag-4211,1aac+cag-4611,2aca+cag-1711,4aag+cat-3911,5aag+cat-31 acc+cat-28aag+ctt-3911,6RhD20112,1acc+cat-812,2aac+cag-4312,3aca+cag-612,6atc+cta-512,9act+cac-813,1act+ctg-3713,6atc+cca-27 act+cac-713,9aag+ctt-1714,7aca+caa-1818,1agg+caa-3 act+cat-3618,7atc+cta-4419,8act+ctg-3419,9atc+caa-3220,9act+ctg-2721,4atc+caa-8123,5atc+cca-2227,7atc+caa-8033,5acc+cac-5133,6aac+caa-9934,8aag+cag-3637,1aac+cag-2345,5aag+cag-2547,6aac+cag-5 atc+ctc-2056,0Rh7958,5aac+caa-2458,6agg+ctg-1760,7atc+cca-1862,4atc+cga-763,7aca+caa-663,9atc+caa-4869,5atc+cta-4871,4

A1acc+cac-470,0acc+cac-486,7Blfa7,4act+cac-409,2Rw29B118,0atc+caa-5818,2aac+cag-3418,7atc+cga-2827,1atc+ctc-827,2act+cac-2429,3aac+caa-9330,7Rh7733,1RhB30335,6atc+cta-4036,7aca+cag-2139,8act+ctg-1841,4act+ctg-1242,6atc+cca-5444,3Rw3k19 act+ctg-30agg+ctg-16 agg+ctg-1844,8

atc+cga-645,4O1a645,6aac+caa-9246,6atc+cca-42 atc+cca-4449,5acc+cac-4650,3atc+cca-1951,8Rh4852,0atc+caa-7153,6atc+cca-5657,4RhAB1557,7atc+cca-2559,5atc+cca-262,1acc+cac-4064,8aac+caa-9666,9RhEO50670,1aca+caa-1772,5atc+ctc-3472,8atc+cga-1973,7atc+cga-1376,9agg+ctg-3678,7aag+cag-4080,0

A2act+cat-230,0aca+caa-166,2atc+caa-7411,1act+cat-913,7agg+caa-3918,8RhM40526,5atc+ctc-3530,4atc+cca-4832,6aca+cag-3433,0acc+cat-4235,8act+cat-437,0atc+caa-5337,7atc+caa-28 act+cat-13act+cat-3837,9agg+caa-2638,2aac+cag-3538,7acc+cac-3838,8aag+ctt-839,2atc+caa-739,4Rh6542,2aag+cag-644,2act+cac-2845,4aca+caa-2446,2agg+cag-2946,6aag+cat-4448,1atc+ctc-3648,9Blfo51,3Rh5057,9atc+ctc-2360,0atc+cca-463,2act+cac-364,3Rh5875,8aac+caa-6376,8atc+ctc-7778,1acc+cat-32 atc+caa-5684,7atc+cta-6088,2act+cac-3189,8

A3

atc+cta-560,0aac+caa-071,4aac+caa-253,6RhD2215,8agg+caa-206,6act+cat-8 act+cat-258,8act+ctg-1915,1agg+caa-816,9aag+cag-1917,1atc+ctc-8118,5atc+ctc-5919,4agg+ctg-3120,1atc+ctc-8021,1Rw62C421,5act+ctg-2322,7acc+cac-923,2act+cat-43 aag+cag-17RhAB12 atc+caa-26aag+ctt-30

23,3

Rw23H523,5RhAB4023,8acc+cat-524,6acc+cat-1725,3aac+cag-625,6atc+caa-2328,7agg+caa-1629,6RhP50730,1act+ctg-26 act+ctg-630,3act+ctg-1630,7atc+caa-7331,8atc+caa-6532,1act+cac-933,2aac+caa-6634,8aac+cag-3636,0aag+ctt-136,6act+cat-4439,7act+ctg-1541,4act+ctg-950,1aag+ctt-551,3atc+cca-1554,5atc+ctc-1154,8Pchgms356,2act+cac-1 aag+cag-4256,9aac+caa-3858,1agg+caa-1858,2act+ctg-14 Rh6058,8agg+cag-4259,1atc+caa-6859,9agg+cag-8762,6atc+ctc-7864,4atc+ctc-7967,5atc+cca-3767,7atc+cca-2368,5Rw5D1172,1aca+cag-1975,3acc+cac-5277,1acc+cac-5077,9acc+cat-679,2acc+cat-3680,0

A4aag+cat-180,0acc+cac-252,2acc+cac-268,8acc+cac-2712,1atc+ctc-913,6aac+cag-316,3act+cat-1817,5atc+cta-2218,9atc+cga-30 atc+cga-2919,5aac+caa-719,7aac+caa-5 act+cat-31act+cat-30 Rw3N1920,1

aag+cat-3720,4atc+cga-3820,9atc+cga-3522,0aac+caa-8922,6act+cac-3823,5agg+caa-3325,1act+cat-3925,9atc+ctc-2226,3aca+caa-1026,8atc+ctc-2127,8atc+ctc-2429,3atc+ctc-2529,5agg+ctg-2 atc+cta-2830,0aag+cag-4430,8aag+cat-2631,9acc+cat-932,7atc+cta-4635,0acc+cat-1235,5acc+cac-8 atc+caa-4535,8aca+cag-1136,8aag+cag-3838,4atc+cta-4539,1acc+cac-1840,1aac+caa-7942,4atc+cta-5042,6atc+caa-245,2Rh9347,4acc+cac-1749,3atc+cga-2649,9atc+ctc-2952,3agg+cag-2757,1aag+cat-3569,0aac+caa-5178,6

A5atc+caa-430,0agg+ctg-273,1agg+cag-64,7aac+cag-156,1atc+caa-5110,4aag+cag-210,5atc+cta-2723,0Rw14H2123,8RhAB3827,1aca+caa-2127,8acc+cat-234,5agg+ctg-2335,7aac+cag-236,9atc+caa-2439,7atc+caa-2242,0aac+caa-4342,7act+cat-342,8Rw18N1943,0aag+ctt-3143,8act+ctg-2443,9atc+cga-3144,0atc+cta-3847,4Rw8B848,1Rw10J1948,9atc+caa-5550,1agg+ctg-2150,5atc+caa-7251,6atc+caa-3552,4agg+ctg-1253,7Rh8556,4agg+caa-3262,6

A6act+ctg-20,0Rw22B62,7atc+cta-44,8atc+caa-599,8atc+caa-2010,8aac+caa-3613,5Rw45E2416,6act+cat-3518,4aac+caa-3520,7atc+caa-3922,9aca+caa-2524,6RhP51925,0atc+caa-1925,3atc+ctc-1226,8aca+caa-2628,5Rh5929,2aag+ctt-1630,1aca+cag-2532,3aac+cag-3235,4atc+cca-2638,7Rw27A11B39,8act+ctg-3940,5Rh7242,0atc+ctc-4342,1Rh7342,5atc+caa-2944,0act+ctg-1347,4aag+cag-5747,5aag+cag-3248,8aag+cat-1349,8atc+caa-4249,9atc+cta-1850,9aag+cag-3153,1atc+ctc-4653,3Rw10M2455,2aac+caa-2655,4aag+cat-6057,8agg+caa-3765,4act+ctg-3874,0

A7

‘Yesterday’

3.4

3.7

2.8

act+cat-220,0RhAB91,4acc+cac-152,1act+ctg-352,7aac+cag-384,3aag+cat-427,3atc+caa-629,5aac+caa-489,7act+ctg-3610,0atc+cca-710,5aac+cag-4211,1aac+cag-4611,2aca+cag-1711,4aag+cat-3911,5aag+cat-31 acc+cat-28aag+ctt-3911,6RhD20112,1acc+cat-812,2aac+cag-4312,3aca+cag-612,6atc+cta-512,9act+cac-813,1act+ctg-3713,6atc+cca-27 act+cac-713,9aag+ctt-1714,7aca+caa-1818,1agg+caa-3 act+cat-3618,7atc+cta-4419,8act+ctg-3419,9atc+caa-3220,9act+ctg-2721,4atc+caa-8123,5atc+cca-2227,7atc+caa-8033,5acc+cac-5133,6aac+caa-9934,8aag+cag-3637,1aac+cag-2345,5aag+cag-2547,6aac+cag-5 atc+ctc-2056,0Rh7958,5aac+caa-2458,6agg+ctg-1760,7atc+cca-1862,4atc+cga-763,7aca+caa-663,9atc+caa-4869,5atc+cta-4871,4

A1acc+cac-470,0acc+cac-486,7Blfa7,4act+cac-409,2Rw29B118,0atc+caa-5818,2aac+cag-3418,7atc+cga-2827,1atc+ctc-827,2act+cac-2429,3aac+caa-9330,7Rh7733,1RhB30335,6atc+cta-4036,7aca+cag-2139,8act+ctg-1841,4act+ctg-1242,6atc+cca-5444,3Rw3k19 act+ctg-30agg+ctg-16 agg+ctg-1844,8

atc+cga-645,4O1a645,6aac+caa-9246,6atc+cca-42 atc+cca-4449,5acc+cac-4650,3atc+cca-1951,8Rh4852,0atc+caa-7153,6atc+cca-5657,4RhAB1557,7atc+cca-2559,5atc+cca-262,1acc+cac-4064,8aac+caa-9666,9RhEO50670,1aca+caa-1772,5atc+ctc-3472,8atc+cga-1973,7atc+cga-1376,9agg+ctg-3678,7aag+cag-4080,0

A2act+cat-230,0aca+caa-166,2atc+caa-7411,1act+cat-913,7agg+caa-3918,8RhM40526,5atc+ctc-3530,4atc+cca-4832,6aca+cag-3433,0acc+cat-4235,8act+cat-437,0atc+caa-5337,7atc+caa-28 act+cat-13act+cat-3837,9agg+caa-2638,2aac+cag-3538,7acc+cac-3838,8aag+ctt-839,2atc+caa-739,4Rh6542,2aag+cag-644,2act+cac-2845,4aca+caa-2446,2agg+cag-2946,6aag+cat-4448,1atc+ctc-3648,9Blfo51,3Rh5057,9atc+ctc-2360,0atc+cca-463,2act+cac-364,3Rh5875,8aac+caa-6376,8atc+ctc-7778,1acc+cat-32 atc+caa-5684,7atc+cta-6088,2act+cac-3189,8

A3

atc+cta-560,0aac+caa-071,4aac+caa-253,6RhD2215,8agg+caa-206,6act+cat-8 act+cat-258,8act+ctg-1915,1agg+caa-816,9aag+cag-1917,1atc+ctc-8118,5atc+ctc-5919,4agg+ctg-3120,1atc+ctc-8021,1Rw62C421,5act+ctg-2322,7acc+cac-923,2act+cat-43 aag+cag-17RhAB12 atc+caa-26aag+ctt-30

23,3

Rw23H523,5RhAB4023,8acc+cat-524,6acc+cat-1725,3aac+cag-625,6atc+caa-2328,7agg+caa-1629,6RhP50730,1act+ctg-26 act+ctg-630,3act+ctg-1630,7atc+caa-7331,8atc+caa-6532,1act+cac-933,2aac+caa-6634,8aac+cag-3636,0aag+ctt-136,6act+cat-4439,7act+ctg-1541,4act+ctg-950,1aag+ctt-551,3atc+cca-1554,5atc+ctc-1154,8Pchgms356,2act+cac-1 aag+cag-4256,9aac+caa-3858,1agg+caa-1858,2act+ctg-14 Rh6058,8agg+cag-4259,1atc+caa-6859,9agg+cag-8762,6atc+ctc-7864,4atc+ctc-7967,5atc+cca-3767,7atc+cca-2368,5Rw5D1172,1aca+cag-1975,3acc+cac-5277,1acc+cac-5077,9acc+cat-679,2acc+cat-3680,0

A4aag+cat-180,0acc+cac-252,2acc+cac-268,8acc+cac-2712,1atc+ctc-913,6aac+cag-316,3act+cat-1817,5atc+cta-2218,9atc+cga-30 atc+cga-2919,5aac+caa-719,7aac+caa-5 act+cat-31act+cat-30 Rw3N1920,1

aag+cat-3720,4atc+cga-3820,9atc+cga-3522,0aac+caa-8922,6act+cac-3823,5agg+caa-3325,1act+cat-3925,9atc+ctc-2226,3aca+caa-1026,8atc+ctc-2127,8atc+ctc-2429,3atc+ctc-2529,5agg+ctg-2 atc+cta-2830,0aag+cag-4430,8aag+cat-2631,9acc+cat-932,7atc+cta-4635,0acc+cat-1235,5acc+cac-8 atc+caa-4535,8aca+cag-1136,8aag+cag-3838,4atc+cta-4539,1acc+cac-1840,1aac+caa-7942,4atc+cta-5042,6atc+caa-245,2Rh9347,4acc+cac-1749,3atc+cga-2649,9atc+ctc-2952,3agg+cag-2757,1aag+cat-3569,0aac+caa-5178,6

A5atc+caa-430,0agg+ctg-273,1agg+cag-64,7aac+cag-156,1atc+caa-5110,4aag+cag-210,5atc+cta-2723,0Rw14H2123,8RhAB3827,1aca+caa-2127,8acc+cat-234,5agg+ctg-2335,7aac+cag-236,9atc+caa-2439,7atc+caa-2242,0aac+caa-4342,7act+cat-342,8Rw18N1943,0aag+ctt-3143,8act+ctg-2443,9atc+cga-3144,0atc+cta-3847,4Rw8B848,1Rw10J1948,9atc+caa-5550,1agg+ctg-2150,5atc+caa-7251,6atc+caa-3552,4agg+ctg-1253,7Rh8556,4agg+caa-3262,6

A6act+ctg-20,0Rw22B62,7atc+cta-44,8atc+caa-599,8atc+caa-2010,8aac+caa-3613,5Rw45E2416,6act+cat-3518,4aac+caa-3520,7atc+caa-3922,9aca+caa-2524,6RhP51925,0atc+caa-1925,3atc+ctc-1226,8aca+caa-2628,5Rh5929,2aag+ctt-1630,1aca+cag-2532,3aac+cag-3235,4atc+cca-2638,7Rw27A11B39,8act+ctg-3940,5Rh7242,0atc+ctc-4342,1Rh7342,5atc+caa-2944,0act+ctg-1347,4aag+cag-5747,5aag+cag-3248,8aag+cat-1349,8atc+caa-4249,9atc+cta-1850,9aag+cag-3153,1atc+ctc-4653,3Rw10M2455,2aac+caa-2655,4aag+cat-6057,8agg+caa-3765,4act+ctg-3874,0

A7

‘Yesterday’

3.4

3.7

2.8

act+cat-220,0RhAB91,4acc+cac-152,1act+ctg-352,7aac+cag-384,3aag+cat-427,3atc+caa-629,5aac+caa-489,7act+ctg-3610,0atc+cca-710,5aac+cag-4211,1aac+cag-4611,2aca+cag-1711,4aag+cat-3911,5aag+cat-31 acc+cat-28aag+ctt-3911,6RhD20112,1acc+cat-812,2aac+cag-4312,3aca+cag-612,6atc+cta-512,9act+cac-813,1act+ctg-3713,6atc+cca-27 act+cac-713,9aag+ctt-1714,7aca+caa-1818,1agg+caa-3 act+cat-3618,7atc+cta-4419,8act+ctg-3419,9atc+caa-3220,9act+ctg-2721,4atc+caa-8123,5atc+cca-2227,7atc+caa-8033,5acc+cac-5133,6aac+caa-9934,8aag+cag-3637,1aac+cag-2345,5aag+cag-2547,6aac+cag-5 atc+ctc-2056,0Rh7958,5aac+caa-2458,6agg+ctg-1760,7atc+cca-1862,4atc+cga-763,7aca+caa-663,9atc+caa-4869,5atc+cta-4871,4

A1acc+cac-470,0acc+cac-486,7Blfa7,4act+cac-409,2Rw29B118,0atc+caa-5818,2aac+cag-3418,7atc+cga-2827,1atc+ctc-827,2act+cac-2429,3aac+caa-9330,7Rh7733,1RhB30335,6atc+cta-4036,7aca+cag-2139,8act+ctg-1841,4act+ctg-1242,6atc+cca-5444,3Rw3k19 act+ctg-30agg+ctg-16 agg+ctg-1844,8

atc+cga-645,4O1a645,6aac+caa-9246,6atc+cca-42 atc+cca-4449,5acc+cac-4650,3atc+cca-1951,8Rh4852,0atc+caa-7153,6atc+cca-5657,4RhAB1557,7atc+cca-2559,5atc+cca-262,1acc+cac-4064,8aac+caa-9666,9RhEO50670,1aca+caa-1772,5atc+ctc-3472,8atc+cga-1973,7atc+cga-1376,9agg+ctg-3678,7aag+cag-4080,0

A2act+cat-230,0aca+caa-166,2atc+caa-7411,1act+cat-913,7agg+caa-3918,8RhM40526,5atc+ctc-3530,4atc+cca-4832,6aca+cag-3433,0acc+cat-4235,8act+cat-437,0atc+caa-5337,7atc+caa-28 act+cat-13act+cat-3837,9agg+caa-2638,2aac+cag-3538,7acc+cac-3838,8aag+ctt-839,2atc+caa-739,4Rh6542,2aag+cag-644,2act+cac-2845,4aca+caa-2446,2agg+cag-2946,6aag+cat-4448,1atc+ctc-3648,9Blfo51,3Rh5057,9atc+ctc-2360,0atc+cca-463,2act+cac-364,3Rh5875,8aac+caa-6376,8atc+ctc-7778,1acc+cat-32 atc+caa-5684,7atc+cta-6088,2act+cac-3189,8

A3

‘Yesterday’

Rw3N190,0acc+cat-444,8aag+ctt-59,0aca+cag-1010,7atc+ctc-2916,3aac+caa-7717,0atc+ctc-917,3aag+cat-317,8aac+caa-2719,2aac+caa-3220,3aac+caa-6722,9atc+cta-43 atc+cta-4224,3agg+caa-3324,9atc+cga-3325,3atc+ctc-2527,9aag+cat-928,0agg+ctg-28 act+cac-2928,3atc+cta-2728,8atc+caa-929,7agg+caa-130,5aac+caa-9833,4atc+caa-3433,9aac+caa-0034,1Rh9334,5agg+cag-637,1aag+cag-239,8aac+caa-7140,1atc+caa-5140,2acc+cat-942,0atc+ctc-2343,9aag+cat-2644,5atc+cta-11 atc+cta-1245,1aca+caa-1046,1aag+cat-3047,8aac+cag-1648,9aac+caa-8855,4aag+cat-1563,6aag+cag-866,8aag+cat-1867,5aac+caa-3070,0aac+caa-7571,2aca+cag-1774,7

B5atc+caa-330,0Rw14H215,4acc+cat-65,9RhAB388,9aag+ctt-3611,4aac+cag-215,2acc+cat-218,6atc+cca-3220,4aag+ctt-3424,0Rw18N1926,6acc+cat-1030,3aac+caa-9932,2Rw8B833,2Rw10J1934,9aac+cag-839,4act+cat-2441,9Rw22A352,6aca+cag-1257,3Rh8558,3atc+cca-5558,9aag+cag-1060,2aac+caa-5461,0act+cac-17 act+cac-21act+cac-2061,1atc+caa-1061,3atc+caa-4363,5act+cat-164,0aag+ctt-464,3atc+cta-1764,9aac+cag-2966,5atc+caa-8267,6RhAB2268,5aca+cag-4 acc+cat-2376,4agg+caa-777,2aac+cag-1381,7acc+cat-3884,4agg+ctg-2489,8

B6atc+caa-180,0aag+ctt-32,6atc+ctc-689,0atc+caa-5410,6Rw22B614,4atc+cca-2920,5Rw45E2420,9aca+caa-2628,0act+ctg-3331,4acc+cat-3931,5aca+caa-2532,0agg+caa-232,5agg+caa-1433,6aac+caa-3534,0atc+caa-1936,1atc+ctc-1436,6Rh5936,8RhP51937,4agg+ctg-2539,8Rw27A11B45,1Rh7246,5Rh7347,2atc+caa-2148,1act+ctg-4048,7atc+ctc-5349,2atc+ctc-5249,8aag+cag-752,2atc+caa-4152,3atc+cta-1853,9atc+ctc-7354,1act+ctg-1354,5aac+caa-7355,4atc+cta-1555,8atc+ctc-7456,3agg+caa-657,7atc+cta-3958,0aag+ctt-2261,1aac+cag-3262,9Rw10M2463,5atc+ctc-7267,7aca+cag-1576,1

B7

acc+cac-510,0aca+caa-44,6aag+ctt-198,3aac+caa-4810,1act+cac-713,6RhAB9-214,1act+cat-1714,9atc+caa-37 aca+cag-7atc+cta-915,6

atc+cta-47 atc+caa-8017,9aac+caa-9020,8aag+cat-3922,7atc+caa-7425,1agg+cag-5626,4aag+cat-3228,0RhD201 act+ctg-529,5aac+caa-4932,8aac+caa-2633,5aca+caa-1834,5atc+ctc-6735,6aca+cag-936,2act+ctg-3537,4aac+cag-4538,3aag+cat-4239,9act+ctg-1 acc+cat-2943,3atc+cca-1344,3acc+cat-1545,2agg+ctg-646,2agg+caa-2349,8acc+cac-5752,2aag+cag-1452,5atc+cca-2252,7atc+caa-1253,7aag+cat-4156,1Rh7960,3

B1

agg+cag-310,0agg+cag-304,7atc+cga-405,2agg+caa-4118,2Blfa18,8agg+cag-7822,1agg+cag-723,6atc+cga-2724,4act+cat-728,9agg+cag-2829,0Rw29B132,7aac+cag-3433,7agg+cag-2633,9act+cat-1636,7agg+ctg-2038,1aac+cag-2440,3atc+ctc-5742,5atc+ctc-6043,4aac+caa-9346,0atc+ctc-848,5aag+cat-1450,0aca+caa-2053,0aac+caa-1654,0agg+ctg-354,5atc+cta-1957,5Rw3K1957,6aag+cat-6359,3atc+cga-1860,9atc+cta-4965,3RhAB1565,8O1a667,5Rh4868,5atc+cga-2070,8aca+caa-1772,3RhEO50677,9aca+caa-3585,3agg+cag-3100,0aca+cag-14101,5

B2

agg+caa-390,0agg+cag-364,5atc+cca-356,7acc+cat-4110,0aag+cat-7013,4aag+ctt-814,4Rh6517,4act+cac-2818,6act+cat-2020,9aac+cag-2221,8atc+ctc-3622,9acc+cat-2524,1agg+caa-2724,7atc+caa-5324,9aca+cag-3427,3atc+cca-2127,8act+ctg-4229,0atc+cga-529,8act+cac-3530,7act+cac-3330,8act+cac-3231,7Rh5031,9RhM40538,6act+ctg-3741,8aag+ctt-1743,1Rh5850,8act+cac-3158,5

B3atc+caa-750,0acc+cat-111,6aac+caa-804,2atc+ctc-474,8act+ctg-45,6aag+ctt-277,9atc+cta-548,3RhD22110,3aag+cat-1612,9act+cat-2614,2atc+cca-1715,7aag+cag-2619,5atc+caa-7922,5atc+cga-3727,0act+cac-1627,9agg+ctg-3529,1atc+cta-5529,2atc+caa-3629,4acc+cac-929,8act+cat-4031,3Rw62C432,1aac+caa-3932,3acc+cat-3332,5acc+cat-17 act+ctg-2333,9RhABT1234,0Rw23H534,5RhAB4035,8act+ctg-2137,0agg+caa-3838,3act+ctg-25 act+ctg-2839,2RhP50739,6agg+caa-4041,4aag+cag-34 acc+cat-149,1aag+cag-3749,8atc+cta-5851,6Rh6055,6acc+cac-2957,2agg+caa-2159,6Pchgms365,8atc+ctc-7666,5

B4

Rosa wichurana

4.54.5

43.5

3.4

Rw3N190,0acc+cat-444,8aag+ctt-59,0aca+cag-1010,7atc+ctc-2916,3aac+caa-7717,0atc+ctc-917,3aag+cat-317,8aac+caa-2719,2aac+caa-3220,3aac+caa-6722,9atc+cta-43 atc+cta-4224,3agg+caa-3324,9atc+cga-3325,3atc+ctc-2527,9aag+cat-928,0agg+ctg-28 act+cac-2928,3atc+cta-2728,8atc+caa-929,7agg+caa-130,5aac+caa-9833,4atc+caa-3433,9aac+caa-0034,1Rh9334,5agg+cag-637,1aag+cag-239,8aac+caa-7140,1atc+caa-5140,2acc+cat-942,0atc+ctc-2343,9aag+cat-2644,5atc+cta-11 atc+cta-1245,1aca+caa-1046,1aag+cat-3047,8aac+cag-1648,9aac+caa-8855,4aag+cat-1563,6aag+cag-866,8aag+cat-1867,5aac+caa-3070,0aac+caa-7571,2aca+cag-1774,7

B5atc+caa-330,0Rw14H215,4acc+cat-65,9RhAB388,9aag+ctt-3611,4aac+cag-215,2acc+cat-218,6atc+cca-3220,4aag+ctt-3424,0Rw18N1926,6acc+cat-1030,3aac+caa-9932,2Rw8B833,2Rw10J1934,9aac+cag-839,4act+cat-2441,9Rw22A352,6aca+cag-1257,3Rh8558,3atc+cca-5558,9aag+cag-1060,2aac+caa-5461,0act+cac-17 act+cac-21act+cac-2061,1atc+caa-1061,3atc+caa-4363,5act+cat-164,0aag+ctt-464,3atc+cta-1764,9aac+cag-2966,5atc+caa-8267,6RhAB2268,5aca+cag-4 acc+cat-2376,4agg+caa-777,2aac+cag-1381,7acc+cat-3884,4agg+ctg-2489,8

B6atc+caa-180,0aag+ctt-32,6atc+ctc-689,0atc+caa-5410,6Rw22B614,4atc+cca-2920,5Rw45E2420,9aca+caa-2628,0act+ctg-3331,4acc+cat-3931,5aca+caa-2532,0agg+caa-232,5agg+caa-1433,6aac+caa-3534,0atc+caa-1936,1atc+ctc-1436,6Rh5936,8RhP51937,4agg+ctg-2539,8Rw27A11B45,1Rh7246,5Rh7347,2atc+caa-2148,1act+ctg-4048,7atc+ctc-5349,2atc+ctc-5249,8aag+cag-752,2atc+caa-4152,3atc+cta-1853,9atc+ctc-7354,1act+ctg-1354,5aac+caa-7355,4atc+cta-1555,8atc+ctc-7456,3agg+caa-657,7atc+cta-3958,0aag+ctt-2261,1aac+cag-3262,9Rw10M2463,5atc+ctc-7267,7aca+cag-1576,1

B7

acc+cac-510,0aca+caa-44,6aag+ctt-198,3aac+caa-4810,1act+cac-713,6RhAB9-214,1act+cat-1714,9atc+caa-37 aca+cag-7atc+cta-915,6

atc+cta-47 atc+caa-8017,9aac+caa-9020,8aag+cat-3922,7atc+caa-7425,1agg+cag-5626,4aag+cat-3228,0RhD201 act+ctg-529,5aac+caa-4932,8aac+caa-2633,5aca+caa-1834,5atc+ctc-6735,6aca+cag-936,2act+ctg-3537,4aac+cag-4538,3aag+cat-4239,9act+ctg-1 acc+cat-2943,3atc+cca-1344,3acc+cat-1545,2agg+ctg-646,2agg+caa-2349,8acc+cac-5752,2aag+cag-1452,5atc+cca-2252,7atc+caa-1253,7aag+cat-4156,1Rh7960,3

B1

agg+cag-310,0agg+cag-304,7atc+cga-405,2agg+caa-4118,2Blfa18,8agg+cag-7822,1agg+cag-723,6atc+cga-2724,4act+cat-728,9agg+cag-2829,0Rw29B132,7aac+cag-3433,7agg+cag-2633,9act+cat-1636,7agg+ctg-2038,1aac+cag-2440,3atc+ctc-5742,5atc+ctc-6043,4aac+caa-9346,0atc+ctc-848,5aag+cat-1450,0aca+caa-2053,0aac+caa-1654,0agg+ctg-354,5atc+cta-1957,5Rw3K1957,6aag+cat-6359,3atc+cga-1860,9atc+cta-4965,3RhAB1565,8O1a667,5Rh4868,5atc+cga-2070,8aca+caa-1772,3RhEO50677,9aca+caa-3585,3agg+cag-3100,0aca+cag-14101,5

B2

agg+caa-390,0agg+cag-364,5atc+cca-356,7acc+cat-4110,0aag+cat-7013,4aag+ctt-814,4Rh6517,4act+cac-2818,6act+cat-2020,9aac+cag-2221,8atc+ctc-3622,9acc+cat-2524,1agg+caa-2724,7atc+caa-5324,9aca+cag-3427,3atc+cca-2127,8act+ctg-4229,0atc+cga-529,8act+cac-3530,7act+cac-3330,8act+cac-3231,7Rh5031,9RhM40538,6act+ctg-3741,8aag+ctt-1743,1Rh5850,8act+cac-3158,5

B3atc+caa-750,0acc+cat-111,6aac+caa-804,2atc+ctc-474,8act+ctg-45,6aag+ctt-277,9atc+cta-548,3RhD22110,3aag+cat-1612,9act+cat-2614,2atc+cca-1715,7aag+cag-2619,5atc+caa-7922,5atc+cga-3727,0act+cac-1627,9agg+ctg-3529,1atc+cta-5529,2atc+caa-3629,4acc+cac-929,8act+cat-4031,3Rw62C432,1aac+caa-3932,3acc+cat-3332,5acc+cat-17 act+ctg-2333,9RhABT1234,0Rw23H534,5RhAB4035,8act+ctg-2137,0agg+caa-3838,3act+ctg-25 act+ctg-2839,2RhP50739,6agg+caa-4041,4aag+cag-34 acc+cat-149,1aag+cag-3749,8atc+cta-5851,6Rh6055,6acc+cac-2957,2agg+caa-2159,6Pchgms365,8atc+ctc-7666,5

B4

Rosa wichurana

4.54.5

43.5

3.4

Rw3N190,0acc+cat-444,8aag+ctt-59,0aca+cag-1010,7atc+ctc-2916,3aac+caa-7717,0atc+ctc-917,3aag+cat-317,8aac+caa-2719,2aac+caa-3220,3aac+caa-6722,9atc+cta-43 atc+cta-4224,3agg+caa-3324,9atc+cga-3325,3atc+ctc-2527,9aag+cat-928,0agg+ctg-28 act+cac-2928,3atc+cta-2728,8atc+caa-929,7agg+caa-130,5aac+caa-9833,4atc+caa-3433,9aac+caa-0034,1Rh9334,5agg+cag-637,1aag+cag-239,8aac+caa-7140,1atc+caa-5140,2acc+cat-942,0atc+ctc-2343,9aag+cat-2644,5atc+cta-11 atc+cta-1245,1aca+caa-1046,1aag+cat-3047,8aac+cag-1648,9aac+caa-8855,4aag+cat-1563,6aag+cag-866,8aag+cat-1867,5aac+caa-3070,0aac+caa-7571,2aca+cag-1774,7

B5atc+caa-330,0Rw14H215,4acc+cat-65,9RhAB388,9aag+ctt-3611,4aac+cag-215,2acc+cat-218,6atc+cca-3220,4aag+ctt-3424,0Rw18N1926,6acc+cat-1030,3aac+caa-9932,2Rw8B833,2Rw10J1934,9aac+cag-839,4act+cat-2441,9Rw22A352,6aca+cag-1257,3Rh8558,3atc+cca-5558,9aag+cag-1060,2aac+caa-5461,0act+cac-17 act+cac-21act+cac-2061,1atc+caa-1061,3atc+caa-4363,5act+cat-164,0aag+ctt-464,3atc+cta-1764,9aac+cag-2966,5atc+caa-8267,6RhAB2268,5aca+cag-4 acc+cat-2376,4agg+caa-777,2aac+cag-1381,7acc+cat-3884,4agg+ctg-2489,8

B6atc+caa-180,0aag+ctt-32,6atc+ctc-689,0atc+caa-5410,6Rw22B614,4atc+cca-2920,5Rw45E2420,9aca+caa-2628,0act+ctg-3331,4acc+cat-3931,5aca+caa-2532,0agg+caa-232,5agg+caa-1433,6aac+caa-3534,0atc+caa-1936,1atc+ctc-1436,6Rh5936,8RhP51937,4agg+ctg-2539,8Rw27A11B45,1Rh7246,5Rh7347,2atc+caa-2148,1act+ctg-4048,7atc+ctc-5349,2atc+ctc-5249,8aag+cag-752,2atc+caa-4152,3atc+cta-1853,9atc+ctc-7354,1act+ctg-1354,5aac+caa-7355,4atc+cta-1555,8atc+ctc-7456,3agg+caa-657,7atc+cta-3958,0aag+ctt-2261,1aac+cag-3262,9Rw10M2463,5atc+ctc-7267,7aca+cag-1576,1

B7

acc+cac-510,0aca+caa-44,6aag+ctt-198,3aac+caa-4810,1act+cac-713,6RhAB9-214,1act+cat-1714,9atc+caa-37 aca+cag-7atc+cta-915,6

atc+cta-47 atc+caa-8017,9aac+caa-9020,8aag+cat-3922,7atc+caa-7425,1agg+cag-5626,4aag+cat-3228,0RhD201 act+ctg-529,5aac+caa-4932,8aac+caa-2633,5aca+caa-1834,5atc+ctc-6735,6aca+cag-936,2act+ctg-3537,4aac+cag-4538,3aag+cat-4239,9act+ctg-1 acc+cat-2943,3atc+cca-1344,3acc+cat-1545,2agg+ctg-646,2agg+caa-2349,8acc+cac-5752,2aag+cag-1452,5atc+cca-2252,7atc+caa-1253,7aag+cat-4156,1Rh7960,3

B1

agg+cag-310,0agg+cag-304,7atc+cga-405,2agg+caa-4118,2Blfa18,8agg+cag-7822,1agg+cag-723,6atc+cga-2724,4act+cat-728,9agg+cag-2829,0Rw29B132,7aac+cag-3433,7agg+cag-2633,9act+cat-1636,7agg+ctg-2038,1aac+cag-2440,3atc+ctc-5742,5atc+ctc-6043,4aac+caa-9346,0atc+ctc-848,5aag+cat-1450,0aca+caa-2053,0aac+caa-1654,0agg+ctg-354,5atc+cta-1957,5Rw3K1957,6aag+cat-6359,3atc+cga-1860,9atc+cta-4965,3RhAB1565,8O1a667,5Rh4868,5atc+cga-2070,8aca+caa-1772,3RhEO50677,9aca+caa-3585,3agg+cag-3100,0aca+cag-14101,5

B2

agg+caa-390,0agg+cag-364,5atc+cca-356,7acc+cat-4110,0aag+cat-7013,4aag+ctt-814,4Rh6517,4act+cac-2818,6act+cat-2020,9aac+cag-2221,8atc+ctc-3622,9acc+cat-2524,1agg+caa-2724,7atc+caa-5324,9aca+cag-3427,3atc+cca-2127,8act+ctg-4229,0atc+cga-529,8act+cac-3530,7act+cac-3330,8act+cac-3231,7Rh5031,9RhM40538,6act+ctg-3741,8aag+ctt-1743,1Rh5850,8act+cac-3158,5

B3atc+caa-750,0acc+cat-111,6aac+caa-804,2atc+ctc-474,8act+ctg-45,6aag+ctt-277,9atc+cta-548,3RhD22110,3aag+cat-1612,9act+cat-2614,2atc+cca-1715,7aag+cag-2619,5atc+caa-7922,5atc+cga-3727,0act+cac-1627,9agg+ctg-3529,1atc+cta-5529,2atc+caa-3629,4acc+cac-929,8act+cat-4031,3Rw62C432,1aac+caa-3932,3acc+cat-3332,5acc+cat-17 act+ctg-2333,9RhABT1234,0Rw23H534,5RhAB4035,8act+ctg-2137,0agg+caa-3838,3act+ctg-25 act+ctg-2839,2RhP50739,6agg+caa-4041,4aag+cag-34 acc+cat-149,1aag+cag-3749,8atc+cta-5851,6Rh6055,6acc+cac-2957,2agg+caa-2159,6Pchgms365,8atc+ctc-7666,5

B4

Rosa wichurana

4.54.5

43.5

3.4Rw3N190,0acc+cat-444,8aag+ctt-59,0aca+cag-1010,7atc+ctc-2916,3aac+caa-7717,0atc+ctc-917,3aag+cat-317,8aac+caa-2719,2aac+caa-3220,3aac+caa-6722,9atc+cta-43 atc+cta-4224,3agg+caa-3324,9atc+cga-3325,3atc+ctc-2527,9aag+cat-928,0agg+ctg-28 act+cac-2928,3atc+cta-2728,8atc+caa-929,7agg+caa-130,5aac+caa-9833,4atc+caa-3433,9aac+caa-0034,1Rh9334,5agg+cag-637,1aag+cag-239,8aac+caa-7140,1atc+caa-5140,2acc+cat-942,0atc+ctc-2343,9aag+cat-2644,5atc+cta-11 atc+cta-1245,1aca+caa-1046,1aag+cat-3047,8aac+cag-1648,9aac+caa-8855,4aag+cat-1563,6aag+cag-866,8aag+cat-1867,5aac+caa-3070,0aac+caa-7571,2aca+cag-1774,7

B5atc+caa-330,0Rw14H215,4acc+cat-65,9RhAB388,9aag+ctt-3611,4aac+cag-215,2acc+cat-218,6atc+cca-3220,4aag+ctt-3424,0Rw18N1926,6acc+cat-1030,3aac+caa-9932,2Rw8B833,2Rw10J1934,9aac+cag-839,4act+cat-2441,9Rw22A352,6aca+cag-1257,3Rh8558,3atc+cca-5558,9aag+cag-1060,2aac+caa-5461,0act+cac-17 act+cac-21act+cac-2061,1atc+caa-1061,3atc+caa-4363,5act+cat-164,0aag+ctt-464,3atc+cta-1764,9aac+cag-2966,5atc+caa-8267,6RhAB2268,5aca+cag-4 acc+cat-2376,4agg+caa-777,2aac+cag-1381,7acc+cat-3884,4agg+ctg-2489,8

B6atc+caa-180,0aag+ctt-32,6atc+ctc-689,0atc+caa-5410,6Rw22B614,4atc+cca-2920,5Rw45E2420,9aca+caa-2628,0act+ctg-3331,4acc+cat-3931,5aca+caa-2532,0agg+caa-232,5agg+caa-1433,6aac+caa-3534,0atc+caa-1936,1atc+ctc-1436,6Rh5936,8RhP51937,4agg+ctg-2539,8Rw27A11B45,1Rh7246,5Rh7347,2atc+caa-2148,1act+ctg-4048,7atc+ctc-5349,2atc+ctc-5249,8aag+cag-752,2atc+caa-4152,3atc+cta-1853,9atc+ctc-7354,1act+ctg-1354,5aac+caa-7355,4atc+cta-1555,8atc+ctc-7456,3agg+caa-657,7atc+cta-3958,0aag+ctt-2261,1aac+cag-3262,9Rw10M2463,5atc+ctc-7267,7aca+cag-1576,1

B7

acc+cac-510,0aca+caa-44,6aag+ctt-198,3aac+caa-4810,1act+cac-713,6RhAB9-214,1act+cat-1714,9atc+caa-37 aca+cag-7atc+cta-915,6

atc+cta-47 atc+caa-8017,9aac+caa-9020,8aag+cat-3922,7atc+caa-7425,1agg+cag-5626,4aag+cat-3228,0RhD201 act+ctg-529,5aac+caa-4932,8aac+caa-2633,5aca+caa-1834,5atc+ctc-6735,6aca+cag-936,2act+ctg-3537,4aac+cag-4538,3aag+cat-4239,9act+ctg-1 acc+cat-2943,3atc+cca-1344,3acc+cat-1545,2agg+ctg-646,2agg+caa-2349,8acc+cac-5752,2aag+cag-1452,5atc+cca-2252,7atc+caa-1253,7aag+cat-4156,1Rh7960,3

B1

agg+cag-310,0agg+cag-304,7atc+cga-405,2agg+caa-4118,2Blfa18,8agg+cag-7822,1agg+cag-723,6atc+cga-2724,4act+cat-728,9agg+cag-2829,0Rw29B132,7aac+cag-3433,7agg+cag-2633,9act+cat-1636,7agg+ctg-2038,1aac+cag-2440,3atc+ctc-5742,5atc+ctc-6043,4aac+caa-9346,0atc+ctc-848,5aag+cat-1450,0aca+caa-2053,0aac+caa-1654,0agg+ctg-354,5atc+cta-1957,5Rw3K1957,6aag+cat-6359,3atc+cga-1860,9atc+cta-4965,3RhAB1565,8O1a667,5Rh4868,5atc+cga-2070,8aca+caa-1772,3RhEO50677,9aca+caa-3585,3agg+cag-3100,0aca+cag-14101,5

B2

agg+caa-390,0agg+cag-364,5atc+cca-356,7acc+cat-4110,0aag+cat-7013,4aag+ctt-814,4Rh6517,4act+cac-2818,6act+cat-2020,9aac+cag-2221,8atc+ctc-3622,9acc+cat-2524,1agg+caa-2724,7atc+caa-5324,9aca+cag-3427,3atc+cca-2127,8act+ctg-4229,0atc+cga-529,8act+cac-3530,7act+cac-3330,8act+cac-3231,7Rh5031,9RhM40538,6act+ctg-3741,8aag+ctt-1743,1Rh5850,8act+cac-3158,5

B3atc+caa-750,0acc+cat-111,6aac+caa-804,2atc+ctc-474,8act+ctg-45,6aag+ctt-277,9atc+cta-548,3RhD22110,3aag+cat-1612,9act+cat-2614,2atc+cca-1715,7aag+cag-2619,5atc+caa-7922,5atc+cga-3727,0act+cac-1627,9agg+ctg-3529,1atc+cta-5529,2atc+caa-3629,4acc+cac-929,8act+cat-4031,3Rw62C432,1aac+caa-3932,3acc+cat-3332,5acc+cat-17 act+ctg-2333,9RhABT1234,0Rw23H534,5RhAB4035,8act+ctg-2137,0agg+caa-3838,3act+ctg-25 act+ctg-2839,2RhP50739,6agg+caa-4041,4aag+cag-34 acc+cat-149,1aag+cag-3749,8atc+cta-5851,6Rh6055,6acc+cac-2957,2agg+caa-2159,6Pchgms365,8atc+ctc-7666,5

B4

Rosa wichurana

4.54.5

43.5

3.4

R. wichurana

R-P R-E

Population, offspring of Parent ‘Yesterday’ (A) and R. wichurana (B)

Dataset AxBLinkage analysis with full

Marker dataset (of both parents)for direct Mapping

Map AB

Sub-Dataset A

Linkage analysis usingonly the marker data-subset present in parent A

Map A

Sub-Dataset B

Linkage analysis usingonly the marker data-subset present in parent B

Map B

Integration of the two parental maps A and B

Map Int. AxB

Population, offspring of Parent ‘Yesterday’ (A) and R. wichurana (B)

Dataset AxBLinkage analysis with full

Marker dataset (of both parents)for direct Mapping

Map AB

Sub-Dataset A

Linkage analysis usingonly the marker data-subset present in parent A

Map A

Sub-Dataset B

Linkage analysis usingonly the marker data-subset present in parent B

Map B

Integration of the two parental maps A and B

Map Int. AxB

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

atc+cta-560,0aac+caa-071,4aac+caa-253,6RhD2215,8agg+caa-206,6act+cat-8 act+cat-258,8act+ctg-1915,1agg+caa-816,9aag+cag-1917,1atc+ctc-8118,5atc+ctc-5919,4agg+ctg-3120,1atc+ctc-8021,1Rw62C421,5act+ctg-2322,7acc+cac-923,2act+cat-43 aag+cag-17RhAB12 atc+caa-26aag+ctt-30

23,3

Rw23H523,5RhAB4023,8acc+cat-524,6acc+cat-1725,3aac+cag-625,6atc+caa-2328,7agg+caa-1629,6RhP50730,1act+ctg-26 act+ctg-630,3act+ctg-1630,7atc+caa-7331,8atc+caa-6532,1act+cac-933,2aac+caa-6634,8aac+cag-3636,0aag+ctt-136,6act+cat-4439,7act+ctg-1541,4act+ctg-950,1aag+ctt-551,3atc+cca-1554,5atc+ctc-1154,8Pchgms356,2act+cac-1 aag+cag-4256,9aac+caa-3858,1agg+caa-1858,2act+ctg-14 Rh6058,8agg+cag-4259,1atc+caa-6859,9agg+cag-8762,6atc+ctc-7864,4atc+ctc-7967,5atc+cca-3767,7atc+cca-2368,5Rw5D1172,1aca+cag-1975,3acc+cac-5277,1acc+cac-5077,9acc+cat-679,2acc+cat-3680,0

A4aag+cat-180,0acc+cac-252,2acc+cac-268,8acc+cac-2712,1atc+ctc-913,6aac+cag-316,3act+cat-1817,5atc+cta-2218,9atc+cga-30 atc+cga-2919,5aac+caa-719,7aac+caa-5 act+cat-31act+cat-30 Rw3N1920,1aag+cat-3720,4atc+cga-3820,9atc+cga-3522,0aac+caa-8922,6act+cac-3823,5agg+caa-3325,1act+cat-3925,9atc+ctc-2226,3aca+caa-1026,8atc+ctc-2127,8atc+ctc-2429,3atc+ctc-2529,5agg+ctg-2 atc+cta-2830,0aag+cag-4430,8aag+cat-2631,9acc+cat-932,7atc+cta-4635,0acc+cat-1235,5acc+cac-8 atc+caa-4535,8aca+cag-1136,8aag+cag-3838,4atc+cta-4539,1acc+cac-1840,1aac+caa-7942,4atc+cta-5042,6atc+caa-245,2Rh9347,4acc+cac-1749,3atc+cga-2649,9atc+ctc-2952,3agg+cag-2757,1aag+cat-3569,0aac+caa-5178,6

A5atc+caa-430,0agg+ctg-273,1agg+cag-64,7aac+cag-156,1atc+caa-5110,4aag+cag-210,5atc+cta-2723,0Rw14H2123,8RhAB3827,1aca+caa-2127,8acc+cat-234,5agg+ctg-2335,7aac+cag-236,9atc+caa-2439,7atc+caa-2242,0aac+caa-4342,7act+cat-342,8Rw18N1943,0aag+ctt-3143,8act+ctg-2443,9atc+cga-3144,0atc+cta-3847,4Rw8B848,1Rw10J1948,9atc+caa-5550,1agg+ctg-2150,5atc+caa-7251,6atc+caa-3552,4agg+ctg-1253,7Rh8556,4agg+caa-3262,6

A6act+ctg-20,0Rw22B62,7atc+cta-44,8atc+caa-599,8atc+caa-2010,8aac+caa-3613,5Rw45E2416,6act+cat-3518,4aac+caa-3520,7atc+caa-3922,9aca+caa-2524,6RhP51925,0atc+caa-1925,3atc+ctc-1226,8aca+caa-2628,5Rh5929,2aag+ctt-1630,1aca+cag-2532,3aac+cag-3235,4atc+cca-2638,7Rw27A11B39,8act+ctg-3940,5Rh7242,0atc+ctc-4342,1Rh7342,5atc+caa-2944,0act+ctg-1347,4aag+cag-5747,5aag+cag-3248,8aag+cat-1349,8atc+caa-4249,9atc+cta-1850,9aag+cag-3153,1atc+ctc-4653,3Rw10M2455,2aac+caa-2655,4aag+cat-6057,8agg+caa-3765,4act+ctg-3874,0

A7

‘Yesterday’

act+cat-220,0RhAB91,4acc+cac-152,1act+ctg-352,7aac+cag-384,3aag+cat-427,3atc+caa-629,5aac+caa-489,7act+ctg-3610,0atc+cca-710,5aac+cag-4211,1aac+cag-4611,2aca+cag-1711,4aag+cat-3911,5aag+cat-31 acc+cat-28aag+ctt-3911,6RhD20112,1acc+cat-812,2aac+cag-4312,3aca+cag-612,6atc+cta-512,9act+cac-813,1act+ctg-3713,6atc+cca-27 act+cac-713,9aag+ctt-1714,7aca+caa-1818,1agg+caa-3 act+cat-3618,7atc+cta-4419,8act+ctg-3419,9atc+caa-3220,9act+ctg-2721,4atc+caa-8123,5atc+cca-2227,7atc+caa-8033,5acc+cac-5133,6aac+caa-9934,8aag+cag-3637,1aac+cag-2345,5aag+cag-2547,6aac+cag-5 atc+ctc-2056,0Rh7958,5aac+caa-2458,6agg+ctg-1760,7atc+cca-1862,4atc+cga-763,7aca+caa-663,9atc+caa-4869,5atc+cta-4871,4

A1acc+cac-470,0acc+cac-486,7Blfa7,4act+cac-409,2Rw29B118,0atc+caa-5818,2aac+cag-3418,7atc+cga-2827,1atc+ctc-827,2act+cac-2429,3aac+caa-9330,7Rh7733,1RhB30335,6atc+cta-4036,7aca+cag-2139,8act+ctg-1841,4act+ctg-1242,6atc+cca-5444,3Rw3k19 act+ctg-30agg+ctg-16 agg+ctg-1844,8atc+cga-645,4O1a645,6aac+caa-9246,6atc+cca-42 atc+cca-4449,5acc+cac-4650,3atc+cca-1951,8Rh4852,0atc+caa-7153,6atc+cca-5657,4RhAB1557,7atc+cca-2559,5atc+cca-262,1acc+cac-4064,8aac+caa-9666,9RhEO50670,1aca+caa-1772,5atc+ctc-3472,8atc+cga-1973,7atc+cga-1376,9agg+ctg-3678,7aag+cag-4080,0

A2act+cat-230,0aca+caa-166,2atc+caa-7411,1act+cat-913,7agg+caa-3918,8RhM40526,5atc+ctc-3530,4atc+cca-4832,6aca+cag-3433,0acc+cat-4235,8act+cat-437,0atc+caa-5337,7atc+caa-28 act+cat-13act+cat-3837,9agg+caa-2638,2aac+cag-3538,7acc+cac-3838,8aag+ctt-839,2atc+caa-739,4Rh6542,2aag+cag-644,2act+cac-2845,4aca+caa-2446,2agg+cag-2946,6aag+cat-4448,1atc+ctc-3648,9Blfo51,3Rh5057,9atc+ctc-2360,0atc+cca-463,2act+cac-364,3Rh5875,8aac+caa-6376,8atc+ctc-7778,1acc+cat-32 atc+caa-5684,7atc+cta-6088,2act+cac-3189,8

A3atc+cta-560,0aac+caa-071,4aac+caa-253,6RhD2215,8agg+caa-206,6act+cat-8 act+cat-258,8act+ctg-1915,1agg+caa-816,9aag+cag-1917,1atc+ctc-8118,5atc+ctc-5919,4agg+ctg-3120,1atc+ctc-8021,1Rw62C421,5act+ctg-2322,7acc+cac-923,2act+cat-43 aag+cag-17RhAB12 atc+caa-26aag+ctt-30

23,3

Rw23H523,5RhAB4023,8acc+cat-524,6acc+cat-1725,3aac+cag-625,6atc+caa-2328,7agg+caa-1629,6RhP50730,1act+ctg-26 act+ctg-630,3act+ctg-1630,7atc+caa-7331,8atc+caa-6532,1act+cac-933,2aac+caa-6634,8aac+cag-3636,0aag+ctt-136,6act+cat-4439,7act+ctg-1541,4act+ctg-950,1aag+ctt-551,3atc+cca-1554,5atc+ctc-1154,8Pchgms356,2act+cac-1 aag+cag-4256,9aac+caa-3858,1agg+caa-1858,2act+ctg-14 Rh6058,8agg+cag-4259,1atc+caa-6859,9agg+cag-8762,6atc+ctc-7864,4atc+ctc-7967,5atc+cca-3767,7atc+cca-2368,5Rw5D1172,1aca+cag-1975,3acc+cac-5277,1acc+cac-5077,9acc+cat-679,2acc+cat-3680,0

A4aag+cat-180,0acc+cac-252,2acc+cac-268,8acc+cac-2712,1atc+ctc-913,6aac+cag-316,3act+cat-1817,5atc+cta-2218,9atc+cga-30 atc+cga-2919,5aac+caa-719,7aac+caa-5 act+cat-31act+cat-30 Rw3N1920,1aag+cat-3720,4atc+cga-3820,9atc+cga-3522,0aac+caa-8922,6act+cac-3823,5agg+caa-3325,1act+cat-3925,9atc+ctc-2226,3aca+caa-1026,8atc+ctc-2127,8atc+ctc-2429,3atc+ctc-2529,5agg+ctg-2 atc+cta-2830,0aag+cag-4430,8aag+cat-2631,9acc+cat-932,7atc+cta-4635,0acc+cat-1235,5acc+cac-8 atc+caa-4535,8aca+cag-1136,8aag+cag-3838,4atc+cta-4539,1acc+cac-1840,1aac+caa-7942,4atc+cta-5042,6atc+caa-245,2Rh9347,4acc+cac-1749,3atc+cga-2649,9atc+ctc-2952,3agg+cag-2757,1aag+cat-3569,0aac+caa-5178,6

A5atc+caa-430,0agg+ctg-273,1agg+cag-64,7aac+cag-156,1atc+caa-5110,4aag+cag-210,5atc+cta-2723,0Rw14H2123,8RhAB3827,1aca+caa-2127,8acc+cat-234,5agg+ctg-2335,7aac+cag-236,9atc+caa-2439,7atc+caa-2242,0aac+caa-4342,7act+cat-342,8Rw18N1943,0aag+ctt-3143,8act+ctg-2443,9atc+cga-3144,0atc+cta-3847,4Rw8B848,1Rw10J1948,9atc+caa-5550,1agg+ctg-2150,5atc+caa-7251,6atc+caa-3552,4agg+ctg-1253,7Rh8556,4agg+caa-3262,6

A6act+ctg-20,0Rw22B62,7atc+cta-44,8atc+caa-599,8atc+caa-2010,8aac+caa-3613,5Rw45E2416,6act+cat-3518,4aac+caa-3520,7atc+caa-3922,9aca+caa-2524,6RhP51925,0atc+caa-1925,3atc+ctc-1226,8aca+caa-2628,5Rh5929,2aag+ctt-1630,1aca+cag-2532,3aac+cag-3235,4atc+cca-2638,7Rw27A11B39,8act+ctg-3940,5Rh7242,0atc+ctc-4342,1Rh7342,5atc+caa-2944,0act+ctg-1347,4aag+cag-5747,5aag+cag-3248,8aag+cat-1349,8atc+caa-4249,9atc+cta-1850,9aag+cag-3153,1atc+ctc-4653,3Rw10M2455,2aac+caa-2655,4aag+cat-6057,8agg+caa-3765,4act+ctg-3874,0

A7

‘Yesterday’

act+cat-220,0RhAB91,4acc+cac-152,1act+ctg-352,7aac+cag-384,3aag+cat-427,3atc+caa-629,5aac+caa-489,7act+ctg-3610,0atc+cca-710,5aac+cag-4211,1aac+cag-4611,2aca+cag-1711,4aag+cat-3911,5aag+cat-31 acc+cat-28aag+ctt-3911,6RhD20112,1acc+cat-812,2aac+cag-4312,3aca+cag-612,6atc+cta-512,9act+cac-813,1act+ctg-3713,6atc+cca-27 act+cac-713,9aag+ctt-1714,7aca+caa-1818,1agg+caa-3 act+cat-3618,7atc+cta-4419,8act+ctg-3419,9atc+caa-3220,9act+ctg-2721,4atc+caa-8123,5atc+cca-2227,7atc+caa-8033,5acc+cac-5133,6aac+caa-9934,8aag+cag-3637,1aac+cag-2345,5aag+cag-2547,6aac+cag-5 atc+ctc-2056,0Rh7958,5aac+caa-2458,6agg+ctg-1760,7atc+cca-1862,4atc+cga-763,7aca+caa-663,9atc+caa-4869,5atc+cta-4871,4

A1acc+cac-470,0acc+cac-486,7Blfa7,4act+cac-409,2Rw29B118,0atc+caa-5818,2aac+cag-3418,7atc+cga-2827,1atc+ctc-827,2act+cac-2429,3aac+caa-9330,7Rh7733,1RhB30335,6atc+cta-4036,7aca+cag-2139,8act+ctg-1841,4act+ctg-1242,6atc+cca-5444,3Rw3k19 act+ctg-30agg+ctg-16 agg+ctg-1844,8atc+cga-645,4O1a645,6aac+caa-9246,6atc+cca-42 atc+cca-4449,5acc+cac-4650,3atc+cca-1951,8Rh4852,0atc+caa-7153,6atc+cca-5657,4RhAB1557,7atc+cca-2559,5atc+cca-262,1acc+cac-4064,8aac+caa-9666,9RhEO50670,1aca+caa-1772,5atc+ctc-3472,8atc+cga-1973,7atc+cga-1376,9agg+ctg-3678,7aag+cag-4080,0

A2act+cat-230,0aca+caa-166,2atc+caa-7411,1act+cat-913,7agg+caa-3918,8RhM40526,5atc+ctc-3530,4atc+cca-4832,6aca+cag-3433,0acc+cat-4235,8act+cat-437,0atc+caa-5337,7atc+caa-28 act+cat-13act+cat-3837,9agg+caa-2638,2aac+cag-3538,7acc+cac-3838,8aag+ctt-839,2atc+caa-739,4Rh6542,2aag+cag-644,2act+cac-2845,4aca+caa-2446,2agg+cag-2946,6aag+cat-4448,1atc+ctc-3648,9Blfo51,3Rh5057,9atc+ctc-2360,0atc+cca-463,2act+cac-364,3Rh5875,8aac+caa-6376,8atc+ctc-7778,1acc+cat-32 atc+caa-5684,7atc+cta-6088,2act+cac-3189,8

A3

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

Rh790,0aag+cat-414,2atc+caa-126,6atc+cca-227,6aag+cag-147,8acc+cac-578,1agg+caa-2310,4agg+ctg-614,0acc+cat-1515,1atc+cca-1315,9acc+cat-29 act+ctg-117,0aag+cat-4220,4aac+cag-4522,0act+ctg-3522,9aca+cag-924,0atc+ctc-6724,6aca+caa-1825,8aac+caa-2626,8aac+caa-4927,5act+ctg-530,7RhD20130,8aag+cat-3232,3agg+cag-5633,9atc+caa-7435,2aag+cat-3937,5aac+caa-9039,5atc+caa-80 atc+cta-4742,4atc+cta-9 aca+cag-744,6atc+caa-3744,7act+cat-1745,4RhAB9-246,2act+cac-746,6aac+caa-4850,2aag+ctt-1952,0aca+caa-455,7acc+cac-5160,3

B1

aca+cag-140,0agg+cag-31,5aca+caa-3516,2RhEO50623,6aca+caa-1729,2atc+cga-2030,7Rh4833,0O1a634,0RhAB1535,7atc+cta-4936,2atc+cga-1840,6aag+cat-6342,2Rw3K1943,9atc+cta-1944,1agg+ctg-347,0aac+caa-1647,5aca+caa-2048,5aag+cat-1451,5atc+ctc-853,0aac+caa-9355,5atc+ctc-6058,1atc+ctc-5759,0aac+cag-2461,2agg+ctg-2063,4act+cat-1664,9agg+cag-2667,6aac+cag-3467,8Rw29B168,9agg+cag-2872,5act+cat-772,6atc+cga-2777,1agg+cag-777,9agg+cag-7879,4Blfa82,7agg+caa-4183,3atc+cga-4096,3agg+cag-3096,8agg+cag-31101,5

B2

agg+caa-390,0agg+cag-364,5atc+cca-356,7acc+cat-4110,0aag+cat-7013,4aag+ctt-814,4Rh6517,4act+cac-2818,6act+cat-2020,9aac+cag-2221,8atc+ctc-3622,9acc+cat-2524,1agg+caa-2724,7atc+caa-5324,9aca+cag-3427,3atc+cca-2127,8act+ctg-4229,0atc+cga-529,8act+cac-3530,7act+cac-3330,8act+cac-3231,7Rh5031,9RhM40538,6act+ctg-3741,8aag+ctt-1743,1Rh5850,8act+cac-3158,5

B3atc+caa-750,0acc+cat-111,6aac+caa-804,2atc+ctc-474,8act+ctg-45,6aag+ctt-277,9atc+cta-548,3RhD22110,3aag+cat-1612,9act+cat-2614,2atc+cca-1715,7aag+cag-2619,5atc+caa-7922,5atc+cga-3727,0act+cac-1627,9agg+ctg-3529,1atc+cta-5529,2atc+caa-3629,4acc+cac-929,8act+cat-4031,3Rw62C432,1aac+caa-3932,3acc+cat-3332,5acc+cat-17 act+ctg-2333,9RhABT1234,0Rw23H534,5RhAB4035,8act+ctg-2137,0agg+caa-3838,3act+ctg-25 act+ctg-2839,2RhP50739,6agg+caa-4041,4aag+cag-34 acc+cat-149,1aag+cag-3749,8atc+cta-5851,6Rh6055,6acc+cac-2957,2agg+caa-2159,6Pchgms365,8atc+ctc-7666,5

B4Rw3N190,0acc+cat-444,8aag+ctt-59,0aca+cag-1010,7atc+ctc-2916,3aac+caa-7717,0atc+ctc-917,3aag+cat-317,8aac+caa-2719,2aac+caa-3220,3aac+caa-6722,9atc+cta-43 atc+cta-4224,3agg+caa-3324,9atc+cga-3325,3atc+ctc-2527,9aag+cat-928,0agg+ctg-28 act+cac-2928,3atc+cta-2728,8atc+caa-929,7agg+caa-130,5aac+caa-9833,4atc+caa-3433,9aac+caa-0034,1Rh9334,5agg+cag-637,1aag+cag-239,8aac+caa-7140,1atc+caa-5140,2acc+cat-942,0atc+ctc-2343,9aag+cat-2644,5atc+cta-11 atc+cta-1245,1aca+caa-1046,1aag+cat-3047,8aac+cag-1648,9aac+caa-8855,4aag+cat-1563,6aag+cag-866,8aag+cat-1867,5aac+caa-3070,0aac+caa-7571,2aca+cag-1774,7

B5

atc+caa-330,0Rw14H215,4acc+cat-65,9RhAB388,9aag+ctt-3611,4aac+cag-215,2acc+cat-218,6atc+cca-3220,4aag+ctt-3424,0Rw18N1926,6acc+cat-1030,3aac+caa-9932,2Rw8B833,2Rw10J1934,9aac+cag-839,4act+cat-2441,9Rw22A352,6aca+cag-1257,3Rh8558,3atc+cca-5558,9aag+cag-1060,2aac+caa-5461,0act+cac-17 act+cac-21act+cac-2061,1atc+caa-1061,3atc+caa-4363,5act+cat-164,0aag+ctt-464,3atc+cta-1764,9aac+cag-2966,5atc+caa-8267,6RhAB2268,5aca+cag-4 acc+cat-2376,4agg+caa-777,2aac+cag-1381,7acc+cat-3884,4agg+ctg-2489,8

B6

aca+cag-150,0atc+ctc-728,4Rw10M2412,6aac+cag-3213,2aag+ctt-2215,0atc+cta-39 agg+caa-618,4atc+ctc-7419,8atc+cta-1520,3aac+caa-7320,7act+ctg-1321,7atc+ctc-7322,0atc+cta-1822,3atc+caa-41 aag+cag-723,9atc+ctc-5226,3atc+ctc-5326,9act+ctg-4027,4atc+caa-2128,0Rh7328,9Rh7229,7Rw27A11B31,0agg+ctg-2536,3RhP51938,7Rh5939,3atc+ctc-1439,6atc+caa-1940,0aac+caa-3542,1agg+caa-1442,6agg+caa-243,6aca+caa-2544,1acc+cat-39 act+ctg-3344,7aca+caa-2648,1Rw45E2455,2atc+cca-2955,6Rw22B661,7atc+caa-5465,5atc+ctc-6867,2aag+ctt-373,5atc+caa-1876,1

B7

R. wichurana

Rh790,0aag+cat-414,2atc+caa-126,6atc+cca-227,6aag+cag-147,8acc+cac-578,1agg+caa-2310,4agg+ctg-614,0acc+cat-1515,1atc+cca-1315,9acc+cat-29 act+ctg-117,0aag+cat-4220,4aac+cag-4522,0act+ctg-3522,9aca+cag-924,0atc+ctc-6724,6aca+caa-1825,8aac+caa-2626,8aac+caa-4927,5act+ctg-530,7RhD20130,8aag+cat-3232,3agg+cag-5633,9atc+caa-7435,2aag+cat-3937,5aac+caa-9039,5atc+caa-80 atc+cta-4742,4atc+cta-9 aca+cag-744,6atc+caa-3744,7act+cat-1745,4RhAB9-246,2act+cac-746,6aac+caa-4850,2aag+ctt-1952,0aca+caa-455,7acc+cac-5160,3

B1

aca+cag-140,0agg+cag-31,5aca+caa-3516,2RhEO50623,6aca+caa-1729,2atc+cga-2030,7Rh4833,0O1a634,0RhAB1535,7atc+cta-4936,2atc+cga-1840,6aag+cat-6342,2Rw3K1943,9atc+cta-1944,1agg+ctg-347,0aac+caa-1647,5aca+caa-2048,5aag+cat-1451,5atc+ctc-853,0aac+caa-9355,5atc+ctc-6058,1atc+ctc-5759,0aac+cag-2461,2agg+ctg-2063,4act+cat-1664,9agg+cag-2667,6aac+cag-3467,8Rw29B168,9agg+cag-2872,5act+cat-772,6atc+cga-2777,1agg+cag-777,9agg+cag-7879,4Blfa82,7agg+caa-4183,3atc+cga-4096,3agg+cag-3096,8agg+cag-31101,5

B2

agg+caa-390,0agg+cag-364,5atc+cca-356,7acc+cat-4110,0aag+cat-7013,4aag+ctt-814,4Rh6517,4act+cac-2818,6act+cat-2020,9aac+cag-2221,8atc+ctc-3622,9acc+cat-2524,1agg+caa-2724,7atc+caa-5324,9aca+cag-3427,3atc+cca-2127,8act+ctg-4229,0atc+cga-529,8act+cac-3530,7act+cac-3330,8act+cac-3231,7Rh5031,9RhM40538,6act+ctg-3741,8aag+ctt-1743,1Rh5850,8act+cac-3158,5

B3atc+caa-750,0acc+cat-111,6aac+caa-804,2atc+ctc-474,8act+ctg-45,6aag+ctt-277,9atc+cta-548,3RhD22110,3aag+cat-1612,9act+cat-2614,2atc+cca-1715,7aag+cag-2619,5atc+caa-7922,5atc+cga-3727,0act+cac-1627,9agg+ctg-3529,1atc+cta-5529,2atc+caa-3629,4acc+cac-929,8act+cat-4031,3Rw62C432,1aac+caa-3932,3acc+cat-3332,5acc+cat-17 act+ctg-2333,9RhABT1234,0Rw23H534,5RhAB4035,8act+ctg-2137,0agg+caa-3838,3act+ctg-25 act+ctg-2839,2RhP50739,6agg+caa-4041,4aag+cag-34 acc+cat-149,1aag+cag-3749,8atc+cta-5851,6Rh6055,6acc+cac-2957,2agg+caa-2159,6Pchgms365,8atc+ctc-7666,5

B4Rw3N190,0acc+cat-444,8aag+ctt-59,0aca+cag-1010,7atc+ctc-2916,3aac+caa-7717,0atc+ctc-917,3aag+cat-317,8aac+caa-2719,2aac+caa-3220,3aac+caa-6722,9atc+cta-43 atc+cta-4224,3agg+caa-3324,9atc+cga-3325,3atc+ctc-2527,9aag+cat-928,0agg+ctg-28 act+cac-2928,3atc+cta-2728,8atc+caa-929,7agg+caa-130,5aac+caa-9833,4atc+caa-3433,9aac+caa-0034,1Rh9334,5agg+cag-637,1aag+cag-239,8aac+caa-7140,1atc+caa-5140,2acc+cat-942,0atc+ctc-2343,9aag+cat-2644,5atc+cta-11 atc+cta-1245,1aca+caa-1046,1aag+cat-3047,8aac+cag-1648,9aac+caa-8855,4aag+cat-1563,6aag+cag-866,8aag+cat-1867,5aac+caa-3070,0aac+caa-7571,2aca+cag-1774,7

B5

atc+caa-330,0Rw14H215,4acc+cat-65,9RhAB388,9aag+ctt-3611,4aac+cag-215,2acc+cat-218,6atc+cca-3220,4aag+ctt-3424,0Rw18N1926,6acc+cat-1030,3aac+caa-9932,2Rw8B833,2Rw10J1934,9aac+cag-839,4act+cat-2441,9Rw22A352,6aca+cag-1257,3Rh8558,3atc+cca-5558,9aag+cag-1060,2aac+caa-5461,0act+cac-17 act+cac-21act+cac-2061,1atc+caa-1061,3atc+caa-4363,5act+cat-164,0aag+ctt-464,3atc+cta-1764,9aac+cag-2966,5atc+caa-8267,6RhAB2268,5aca+cag-4 acc+cat-2376,4agg+caa-777,2aac+cag-1381,7acc+cat-3884,4agg+ctg-2489,8

B6

aca+cag-150,0atc+ctc-728,4Rw10M2412,6aac+cag-3213,2aag+ctt-2215,0atc+cta-39 agg+caa-618,4atc+ctc-7419,8atc+cta-1520,3aac+caa-7320,7act+ctg-1321,7atc+ctc-7322,0atc+cta-1822,3atc+caa-41 aag+cag-723,9atc+ctc-5226,3atc+ctc-5326,9act+ctg-4027,4atc+caa-2128,0Rh7328,9Rh7229,7Rw27A11B31,0agg+ctg-2536,3RhP51938,7Rh5939,3atc+ctc-1439,6atc+caa-1940,0aac+caa-3542,1agg+caa-1442,6agg+caa-243,6aca+caa-2544,1acc+cat-39 act+ctg-3344,7aca+caa-2648,1Rw45E2455,2atc+cca-2955,6Rw22B661,7atc+caa-5465,5atc+ctc-6867,2aag+ctt-373,5atc+caa-1876,1

B7

R. wichurana

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

      Linkage groups    

Marker name Spiller et al., 2011

Yan et al., 2005

Debener et al. 2001 and other authorsa,b,c,d,e

‘Yesterday’ R. wichurana

Rh79RhAB9-2RhD201RhEO506RhAB15Rh48RhB303Rh50Rh58Rh65RhD221RhABT12Rw62C4Pchgms3 (B6B1)RhAB40 RhP507Rh93Rh85RhAB22RhE2bRh72Rh73Rw22B6RhP519Rw10M24O1a6Rh59Rh77RhAB38Rh60Blfo (NP)Blfa (flower colour)

11122223334444  56  77777 555632

11 222 33444  44566677    355632

   22     4  4a, 2d

44  6      5a

    3, 2a, 6b, 3c, 3e

2

1112 2223334444445665 nm7777727 26432

1112224 nm3334444445666 nm77777272 nm64Marker not present2

common marker:23 with Spiller et al. 201119 with Yan et al. 2005 8 with Debener et al. 2001

a. Dugo et al. 2005 b. Crespel et al. 2002c. Linde et al. 2006d. Rajapakse et al. 2001e. Hibrand-Saint Oyant et al., 2008

10

Genetic map of Rosa wichurana

• 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 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

• 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

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

Chromosomenumber

ChromosomeLength (µm)

RelativeLength (%)

CentromereIndex (%)

1 3.7 ± 0.30 17.80 ± 0.20 46.00 ± 1.202 3.2 ± 0.60 17.00 ± 0.20 40.30 ± 1.303 3 ± 0.50 15.20 ± 0.20 44.30 ± 1.004 2.8 ± 0.40 14.00 ± 0.10 36.90 ± 0.705 2.6 ± 0.40 13.60 ± 0.10 41.40 ± 0.706 2.5 ± 0.40 12.40 ± 0.20 41.80 ± 1.107 2.2 ± 0.50 10.00 ± 0.10 23.40 ± 0.90

Development of a cytogenetic marker system for rose chromosomes

Short-insert small DNA library

454 sequencing analysisby 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 clonesBAC clones

Repetitive sequences

Sources of cytogenetic markers

Chromosomal DNA

HRP

Tyramide-Fluorochrome

О2

AntiDig

Dig labeled DNA

HistonesHistonesHistones

Mechanism of Tyramide-FISHdetection

Only tyramides coupled to

proteins are left on the slide

H202

WASHING STEP

17

Abbreviation Gene name Function Source of sequence

PCR product length

PAL Phenylalanine ammonia-lyase

Principal enzyme of the phenylpropanoid pathway

Prunus persica 1700 bp

OOMT Orcinol o-methyltransferase

Involved in synthesis of 3,5-dimethoxytoluene

Rosa hybrid sp. 1100 bp

P5CS Delta-1-pyrroline 5-carboxylate synthetase

Key enzyme in proline biosynthesis (osmotic stress-responsive gene)

Prunus persica 1700 bp

Candidate genes

18

Tyr-FISH mapping of PAL gene

Relative distance: 77.0 ± 2.1%

Ch7

19

Tyr-FISH mapping of P5CS gene

Relative distance: 72.7±3.8%

Ch4

20

Tyr-FISH mapping of OOMT gene

Relative distance: 22.6 ± 3.2%

Ch1

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 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

Integration of physical position of three genes with genetic linkage map

RwLG4aac-caa-01acc-cac-51AAC-CAA-345atc-cca-27act-cac-8rhd201acc-cat-8aca-cag-17aag-ctt-40acc-cat-28atc-cca-7ATC-CAA-325act-ctg-36atc-caa-62aag-cat-39aag-ctt-17AAC-CAA-276act-cat-36agg-caa-3atc-cta-44act-ctg-34aac-caa-49atc-caa-32atc-caa-81ATC-CAA-105act-ctg-35acc-cat-29act-ctg-1AAC-CAA-416atc-cca-13AGG-CAG-380atc-ctc-67aag-cag-36aac-cag-45aca-cag-9AAC-CAA-314agg-caa-23aag-cag-14ATC-CTC-338P5CSaag-cag-25agg-cag-85AGG-CAG-386rh79atc-cta-47act-cat-17atc-caa-37aca-cag-7atc-cca-18atc-cga-7AAC-CAA-138atc-caa-48

acc-cac-57

1

agg-ctg-36

atc-cga-13

atc-ctc-34ATC-CTC-182rheo506

aac-caa-96

acc-cac-40

atc-cca-25rhab15-1AAC-CAA-94ATC-CAA-418atc-cca-19rh48acc-cac-46atc-cca-42AAC-CAA-341AAC-CAA-419ssr8-2act-ctg-30ssr8-1OOMTagg-ctg-16atc-cca-54atc-cga-6act-ctg-18atc-cta-40rhb303act-cac-36

aac-caa-93atc-cga-28

Flower_coloraac-cag-33act-cat-33

atc-cta-51agg-cag-30

aag-cat-63

2

aca-caa-16

aag-ctt-10

rhm405acc-cat-41AAC-CAA-58(2)atc-ctc-35AGG-CAG-161acc-cat-42act-cat-4aac-cag-44aag-ctt-9aac-cag-35agg-caa-26atc-caa-28act-cat-38act-cat-13aag-ctt-8acc-cac-38atc-caa-7rh65-1rh65-2atc-cca-48aag-cag-6aag-cat-44atc-ctc-36Flower_sizePALrh50-1atc-cca-4act-cac-3

rh58-2rh58-1aac-caa-63atc-ctc-77

acc-cat-32atc-caa-56atc-cta-60

atc-caa-76

agg-cag-50

3

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

RwChr1

RwChr4

RwChr710 Mb

20 Mb

30 Mb

FvChr6

10 Mb

20 Mb

FvChr7aac-caa-01acc-cac-51AAC-CAA-345atc-cca-27act-cac-8rhd201acc-cat-8aca-cag-17aag-ctt-40acc-cat-28atc-cca-7ATC-CAA-325act-ctg-36atc-caa-62aag-cat-39aag-ctt-17AAC-CAA-276act-cat-36agg-caa-3atc-cta-44act-ctg-34aac-caa-49atc-caa-32atc-caa-81ATC-CAA-105act-ctg-35acc-cat-29act-ctg-1AAC-CAA-416atc-cca-13AGG-CAG-380atc-ctc-67aag-cag-36aac-cag-45aca-cag-9AAC-CAA-314agg-caa-23aag-cag-14ATC-CTC-338P5CSaag-cag-25agg-cag-85AGG-CAG-386rh79atc-cta-47act-cat-17atc-caa-37aca-cag-7atc-cca-18atc-cga-7AAC-CAA-138atc-caa-48

acc-cac-57

1

agg-ctg-36

atc-cga-13

atc-ctc-34ATC-CTC-182rheo506

aac-caa-96

acc-cac-40

atc-cca-25rhab15-1AAC-CAA-94ATC-CAA-418atc-cca-19rh48acc-cac-46atc-cca-42AAC-CAA-341AAC-CAA-419ssr8-2act-ctg-30ssr8-1OOMTagg-ctg-16atc-cca-54atc-cga-6act-ctg-18atc-cta-40rhb303act-cac-36

aac-caa-93atc-cga-28

Flower_coloraac-cag-33act-cat-33

atc-cta-51agg-cag-30

aag-cat-63

2

aca-caa-16

aag-ctt-10

rhm405acc-cat-41AAC-CAA-58(2)atc-ctc-35AGG-CAG-161acc-cat-42act-cat-4aac-cag-44aag-ctt-9aac-cag-35agg-caa-26atc-caa-28act-cat-38act-cat-13aag-ctt-8acc-cac-38atc-caa-7rh65-1rh65-2atc-cca-48aag-cag-6aag-cat-44atc-ctc-36Flower_sizePALrh50-1atc-cca-4act-cac-3

rh58-2rh58-1aac-caa-63atc-ctc-77

acc-cat-32atc-caa-56atc-cta-60

atc-caa-76

agg-cag-50

3

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

aac-caa-01acc-cac-51AAC-CAA-345atc-cca-27act-cac-8rhd201acc-cat-8aca-cag-17aag-ctt-40acc-cat-28atc-cca-7ATC-CAA-325act-ctg-36atc-caa-62aag-cat-39aag-ctt-17AAC-CAA-276act-cat-36agg-caa-3atc-cta-44act-ctg-34aac-caa-49atc-caa-32atc-caa-81ATC-CAA-105act-ctg-35acc-cat-29act-ctg-1AAC-CAA-416atc-cca-13AGG-CAG-380atc-ctc-67aag-cag-36aac-cag-45aca-cag-9AAC-CAA-314agg-caa-23aag-cag-14ATC-CTC-338P5CSaag-cag-25agg-cag-85AGG-CAG-386rh79atc-cta-47act-cat-17atc-caa-37aca-cag-7atc-cca-18atc-cga-7AAC-CAA-138atc-caa-48

acc-cac-57

1

agg-ctg-36

atc-cga-13

atc-ctc-34ATC-CTC-182rheo506

aac-caa-96

acc-cac-40

atc-cca-25rhab15-1AAC-CAA-94ATC-CAA-418atc-cca-19rh48acc-cac-46atc-cca-42AAC-CAA-341AAC-CAA-419ssr8-2act-ctg-30ssr8-1OOMTagg-ctg-16atc-cca-54atc-cga-6act-ctg-18atc-cta-40rhb303act-cac-36

aac-caa-93atc-cga-28

Flower_coloraac-cag-33act-cat-33

atc-cta-51agg-cag-30

aag-cat-63

2

aca-caa-16

aag-ctt-10

rhm405acc-cat-41AAC-CAA-58(2)atc-ctc-35AGG-CAG-161acc-cat-42act-cat-4aac-cag-44aag-ctt-9aac-cag-35agg-caa-26atc-caa-28act-cat-38act-cat-13aag-ctt-8acc-cac-38atc-caa-7rh65-1rh65-2atc-cca-48aag-cag-6aag-cat-44atc-ctc-36Flower_sizePALrh50-1atc-cca-4act-cac-3

rh58-2rh58-1aac-caa-63atc-ctc-77

acc-cat-32atc-caa-56atc-cta-60

atc-caa-76

agg-cag-50

3

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

RwLG1 RwLG7

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

Future perspectives

• 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

Thank you for your attention!

Institute for Agricultural and Fisheries ResearchPlant Sciences Unit

www.ilvo.vlaanderen.beAgriculture and Fisheries Policy Area