Karyological diversity of wild Rosa in Yunnan, southwestern China

Hong Ying Jian, Ting Zhang, Qi Gang Wang, Shu Bin Li, Hao Zhang & Kai Xue Tang

Genetic Resources and Crop Evolution An International Journal

ISSN 0925-9864 Volume 60 Number 1

Genet Resour Crop Evol (2013) 60:115-127 DOI 10.1007/s10722-012-9820-z

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Genet Resour Crop Evol (2013) 60:115–127 DOI 10.1007/s10722-012-9820-z

RESEARCH ARTICLE

Karyological diversity of wild Rosa in Yunnan, southwestern China

Hong Ying Jian • Ting Zhang • Qi Gang Wang • Shu Bin Li • Hao Zhang • Kai Xue Tang

Received: 15 October 2011 / Accepted: 18 February 2012 / Published online: 22 March 2012 Ó Springer Science+Business Media Dordrecht 2012

Abstract Wild Rosa resources in Yunnan are very the evolution and speciation in genus Rosa, mainly in abundant and hold many important and useful traits for Sect. Cinnamomeae in alpine areas of northwestern improving modern rose. However, many of them have Yunnan. The cytological data would supply chromo- no cytological data and few were karyologically somal proofs not only for the taxonomy, phylogeny and analysed. In this article, karyological study of 24 wild evolution of genus Rosa in this special geological area, Rosa taxa belonging to different sections was carried but also for the exploitation of useful traits of wild Rosa out by means of traditional squashing and pressing. into the breeding of new cultivars. The chromosome number of 8 taxa and karyotypes of most studied ones were reported for the first time. 22 Keywords Breeding Cytological data Diversity resources were diploids with 14 chromosomes (2n = Polyploidization Rosa 2x = 14), while Rosa moyesii Hemsl. et Wils. and Rosa macrophylla Lindl. from Sect. Cinnamomeae DC. were hexaploids with 42 chromosomes (2n = 6x = 42). Only type 1A and type 2A been found, Introduction karyotypes of genus Rosa in Yunnan were symmetric. Also, the studied taxa differed much from each other in The genus Rosa L. contains about 200 species that are chromosome length, position of centromere, satellite widely distributed throughout the Northern Hemi- position and satellite number, etc. The results implied sphere (Ku and Robertson 2003; Wissemann and Ritz that wild Rosa germplasm in Yunnan were rich in 2005). China is undoubtedly the greatest treasure karyologcal diversity, and polyploidization might have house of Roses and there are about 95 species of wild played and were still playing very important roles in Rosa (Brichet 2003; Ku and Robertson 2003). Yunnan province lies in the southeastern border of China. At the conflux of Sino-Himalayas floristic region, Sino- Japanese floristic region and the Malaysian Forest Flora, with its special geographic environment, unique H. Y. Jian T. Zhang Q. G. Wang S. B. Li weather conditions and ancient geological history, H. Zhang K. X. Tang (&) Yunnan is rich in biodiversity and is a famous plant Yunnan Key Laboratory for Flower Breeding, kingdom (Editor Board of Vegetation of Yunnan Flower Research Institute, Yunnan Academy 1987). As far as genus Rosa was concerned, 58 of Agricultural Sciences, Kunming 650205, Yunnan, People’s Republic of China species/varieties and forms, about half of those in e-mail: [email protected] China and a quarter of those in the world from Sect. 123 Author's personal copy

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Pimpinellifoliae DC., Sect. Cinnamomeae DC., Sect. chromosomes between wild resources and cultivars Chinenses DC., Sect. Synstylae DC., Sect. Banksianae can be used as an important tool for crop genetic and Lindl., Sect. Laevigatae Thory, Sect. Bracteatae Thory breeding (Lespinasse et al. 1976; Schuster 1996). and Sect. Microphylla Cre´p. distribute in Yunnan and Elucidation of the karyotype and the physical structure many of them are endemic to the province. Also, of chromosomes can greatly enhance the exploitation of Yunnan has come into been one of the world’s three genetic variation from wild relatives of crops (Baenziger main cut rose growing areas. With the development of et al. 2006). Wild Rosa species from China had played cut rose industry in Yunnan, studies on the evaluation very important roles in the coming into being of modern of germplasm resources, genetics and breeding of rose roses for providing useful traits especially recurrent arise rapidly in order to improve modern rose varie- flowering and tea scent (Wylie 1954). No fragrance, ties. (Zhang et al. 2006, 2009; Tang et al. 2008; Qiu sensitivity to disease and insects, low tolerance to severe et al. 2009; Bai and Zhang 2008; Yan et al. 2011). climates, etc. are some typical shortcomings of most Rosa is among the first genera of garden flowers to modern cultivars. However, wild Rosa resources in attract the attention of cytologists because of the Yunnan hold many important and useful traits such as importance of cultivated roses as horticultural plants, powdery mildew resistance, large flowers, thornless- in spite of the small size of its chromosomes (Rowley ness, cold resistancy etc. which can be an extended 1967). Cytological reports in rose included chromo- germplasm for breeding improved rose cultivars (Bai some counts, karyotypes and meiotic configuration and Zhang 2008; Zhang et al. 2009;Tangetal.2008). frequencies by means of traditional squashing and Thus, covering the gaps of cytological data of wild Rosa pressing (Liu and Li 1985; Ma and Chen 1991, 1992; species in Yunnan, will supply chromosomal proofs not Ma et al. 1997a; Jian et al. 2010a, b), C-banding (Price only for the taxonomy, phylogeny and evolution of et al. 1981) and FISH (Ma et al. 1997b; Fernandez- genus Rosa in this special geological area, but also for Romero et al. 2001; Akasaka et al. 2002, 2003). Genus the exploitation of useful traits of wild Rosa into the Rosa exhibits a typical polyploidy series with a basic breeding of modern rose cultivars. In this study, the chromosome number of 7. For a very long time, people chromosome number and karyotypes of 24 wild species believed that the number of chromosomes of wild belonging to different sections from different subzones Rosa was euploid predominated with range from of Yunnan were surveyed and reported. 2n = 2x = 14 to 2n = 8x = 56 (Darlington and Wylie 1955; Crane and Byrne 2003). Recently, decaploidy had been observed in Rosa praelucens Materials and methods Byhouwer, an endemic plant to the Zhongdian Plateau, northwestern Yunnan (Jian et al. 2010a). Stems of all the studied species were collected from The largest remaining gaps in the cytological data of their original places and grafted onto R. ‘Natal Briar’ wild Rosa species are in Asiatic members of Sect. during 2003–2008. Detailed collection data were Synstylae and Sect. Pimpinellifoliae (Ma et al. 1997a). shown in Table 2. After the plants survived in For those species in Yunnan, although most of them greenhouse, they were transplanted outside to the rose have had chromosome number information, some taxa germplasm base of FRI, YAAS, which is located in the have no cytological data, especially few were kary- northern suburb of Kunming, Yunnan, China, with an ologically analysed (Table 1). altitude of 1,900 m. Karyotype analysis, the comparison of chromosome All cytological observations were made from shoot measurements, is a traditional cytogenetic step in the tips. Shoot tips were collected from the plants showing comparison of genomes among related species. On one vigorous growth, pretreated in a saturated dichloro- hand, karyotypic changes are frequent in the evolution benzene solution for 2.5 h at 23 °C, then fixed with of higher plants and have frequently served as a basis of Carnoy (1:3 glacial acetic acid/absolute alcohol) at phylogenetic inference (Crane and Byrne 2003), thus 4 °C for 40 min. The fixed tips were hydrolyzed in the results of chromosomal studies may be useful in 1 N HCl at 60 °C for 10 min, stained with carbol plant taxonomy and phylogeny analysis (Singh et al. fuchsin for at least 30 min and squashed on glass 1984;Soodanetal.1988). On the other hand, informa- slides for observation and photos were taken under a tion about difference in number and structure of Nikon E800 microscope (NIKON, JAPAN). 123 ee eorCo vl(03 60:115–127 (2013) Evol Crop Resour Genet Table 1 Rosa species distributed in Yunnan and the related cytological data. All are based on x = 7 Taxa Section Reference 2n Ploidy Karyotype Altitude Habitat

R. omeiensis omeiensis Pimpinellifoliae Ta¨ckholm (1922) 14 2x No Report 2,400–4,000 Slopes, scrub and stream sides Rolfe R. omeiensis glandulosa Pimpinellifoliae No report No Report 2,700–3,800 Slopes and scrubs Yu¨ et Ku R. omeiensis Rolfe f. Pimpinellifoliae No report No Report 2,500–2,800 Slopes pteracantha Rehd. et Wils. R. omeiensis Rolfe f. Pimpinellifoliae No report No Report 2,300–3,400 Sunny slopes paucijuga Yu¨ et Ku R.taronensis Yu¨ et Ku Pimpinellifoliae No report No Report 2,400–3,300 Forest and pastures

R. sericea Lindl. Pimpinellifoliae Ta¨ckholm (1922) 14 2x No Report 2,000–3,600 Sunny slopes Author's R. mairei Le´vl. Pimpinellifoliae No report No Report 1,700–3,200 Sunny slopes and scrubs R. sikangensis Yu¨ et Ku Pimpinellifoliae No report No Report 2,700–3,700 Stream sides, road sides and scrubs R. zhongdianensis Ku Pimpinellifoliae No report No Report 2,600 Unny slopes and scrubs

R. graciliflora Pimpinellifoliae No report No Report 2,700–3,800 Scree Slopes, Scrubs personal Rehd. et Wils. R. farreri Stapf ex Cox Pimpinellifoliae Ta¨ckholm (1922) 14 2x No Report 1,500–3,880 Riverside, Road side and alpine pasture R. lichiangensis Synstylae Song et al. (1989)284x2n= 4x = 28 2,100–2,500 Road sides and scrubs Yu¨ et Ku R. multiflora Thunb. Synstylae Chen et al. (2003)142x2n= 2x = 12 m ? 2sm 2,100–2,500 Road sides and scrubs copy var. multiflora R. multiflora Thunb.var. Synstylae Ma and Chen (1991)213x2n= 3x = 18 m ? 3sm 2,100–2,500 Road sides and scrubs carnea Thory R. multiflora Synstylae No report No Report 2,100–2,500 Road sides and scrubs Thunb.var.cathayensis Rehd. et Wils. R. brunonii Lindl. Synstylae Hurst (1928), 14 2x No Report 1,300–3,000 Forests and scrubs Roberts et al. (2009) R.glomerata Rehd. et Synstylae Roberts et al. (2009) 14 2x No Report 1,200–3,200 Forest edges and scrubs Wils. R. rubus Le´vl. et Vant. Synstylae Ta¨ckholm 1922 14 2x No Report 500–3,400 Forest and road side f.glandulifera Yu¨ et Ku 123 R. filipes Rehd. et Wils. Synstylae Roberts et al. (2009) 14 2x No Report 1,600–1,900 Road side and scrubs 117 118 123 Table 1 continued Taxa Section Reference 2n Ploidy Karyotype Altitude Habitat

R. helenae Rehd. et Synstylae Ta¨ckholm (1922) 14 2x No Report 2,700–3,700 Stream sides, road sides and scrubs Wils. R. longicuspis Bertol. Synstylae Hurst (1928) 14 2x No Report 400–2,900 Forest edges, scrubs and road sides var. longicuspis R. lasiosepala Metc. Synstylae No Report No Report 1,000–1,500 Forest R. soulieana Cre´p. var. Synstylae Hurst (1928) 14 2x No Report 2,000–3,700 Picea forests and stream sides soulieana Cre´p. R. soulieana Cre´p. var Synstylae No Report No Report 2,450–3,000 Slopes, stream sides and pine forest yunnanensis Schneid. R. soulieana Cre´p. var. Synstylae No Report No Report 3,200–3,700 Dry slopes and scrubs microphylla Yu¨ et Ku Author's R. henryi Bouleng. Synstylae Roberts et al. (2009) 14 2x No Report 1,700–2,000 Forest edge, stream sides and scrubs R. weisiensis Yu¨ et Ku Synstylae No Report No Report 1,850–2,300 Scrubs R. prattii Hemsl. Cinnamomeae Darlington and Wylie (1955) 14 2x No Report 1,550–3,800 Slopes, stream sides and scrubs

R. willmottiae Hemsl. Cinnamomeae Ta¨ckholm (1922) 14 2x No Report 3,200–3,300 Slopes and scrubs personal R. forrestiana Bouleng. Cinnamomeae Roberts et al. (2009) 14 2x No Report R. macrophylla Lindl. Cinnamomeae Hurst (1928) 14, 28 2x, 4x No Report 2,700–3,600 Pine forest edges, scrubs and road sides R. sweginzowii Koehne Cinnamomeae Ta¨ckholm (1922), 42 6x No Report 2,500–3,600 Pine forest edges, scrubs and road sides Roberts et al. (2009)

R. sweginzowii Koehne Cinnamomeae No Report No Report 2,800–3,700 Slopes and scrubs copy var. glandulosa Card. R. pseudobanksiae Yu¨ Cinnamomeae No Report No Report 1,700 Road sides

et Ku 60:115–127 (2013) Evol Crop Resour Genet R. sertata Rolfe var. Cinnamomeae Ta¨ckholm (1922), 14, 28 2x, 4x No Report 1,750–3,950 Slopes and sparse forest sertata Rolfe Roberts et al. (2009) R. webbiana Wall. ex Cinnamomeae Hurst (1928), 14, 28 2x, 4x No Report 1,950–3,650 Forest Royle Roberts et al. (2009) R. multibracteata Cinnamomeae Ta¨ckholm (1922), 28,14 4x,2x No Report 2,250–2,800 Forest edge Hemsl. et Wils. Roberts et al. (2009) R. persetosa Rolfe Cinnamomeae Ta¨ckholm 1922 14 2x No Report 3,000–3,200 Slopes R. murielae Rehd. et Cinnamomeae No Report No Report 1,800–2,400 Slopes, stream sides and scrubs Wils. R. moyesii Hemsl.et Cinnamomeae Ta¨ckholm (1922), 42,28 6x,4x No Report 3,000–3,600 Dry slopes pines forest, scrubs Wils. Roberts et al. (2009) ee eorCo vl(03 60:115–127 (2013) Evol Crop Resour Genet Table 1 continued Taxa Section Reference 2n Ploidy Karyotype Altitude Habitat

R. odorata Sweet var. Chinensis Hurst (1928), Liu et al. (2008), 14 2x 2n = 2x = 12 m ? 2sm 700–3,300 Forest edges and road side scrubs odorata Jian et al. (2010b) R. odorata (Andr.) Chinensis Hurst (1928), Ma and 14,21 2x,3x 2n = 3x = 21; 800–2,600 Forest and scrubs Sweet var. gigantea Chen (1992), 2n = 2x = 12 m ? 2sm (Cre´p.) Rehd.et Wils. Jian et al. (2010b) R. odorata (Andr.) Chinensis Jian et al. (2010b)142x2n= 2x = 10 m ? 4sm 1,000–2,500 Slopes and scrubs Sweet var. pseudoindica (Lindl.) Rehd. R. odorata (Andr.) Chinensis Jian et al. (2010b) 14,21 2x,3x 2n = 2x = 12 m ? 2sm; 2,000–2,500 Road sides and scrubs

Sweet var. erubescens 2n = 2x = 10 m ? 4sm; Author's (Focke) Yu¨ et Ku 2n = 3x = 21 m; 2n = 3x = 18 m ? 3sm R. banksiae Ait. var. Banksiae Ma and Chen (1991), 14 2x 2n = 2x = 14 m; 1,500–2,650 Road sides and scrubs banksiae Ait. Liu and Li (1985) 2n = 2x = 12 m ? 2sm personal R. banksiae Ait. var. Banksiae No report No Report 1,600–2,500 Slopes, stream sides and scrubs normalis Regel R. banksiae Ait. f. lutea Banksiae Ta¨ckholm (1922) 14 2x No Report Slopes and scrubs (Lindl.) Rehd. R. cymosa Tratt. var. Banksiae Hurst (1928) 14 2x No Report 200–1,800 Stream sides, road sides and scrubs cymosa copy R. laevigata Michx. Laevigatae Hurst (1928), Liu et al. (2008)14 2x 2n= 2x = 10 m ? 4sm 200–1,400 Slopes, stream sides R. bracteata Wendl. var. Bracteatae Darlington and Wylie (1955)142x2n= 2x = 14 m 200–1,600 Stream sides, road sides and scrubs bracteata R. roxburghii Tratt. Microphyllae Ta¨ckholm (1922), 14 2x 2n = 2x = 14 = 12 m ? 2sm 500–2,500 Road sides and scrubs Song and Li (1989) R. praelucens Microphyllae Jian et al. (2010a) 70 10x 2n = 10x = 70 = 2st ? 2,700–3,300 Sunny slopes and scrubs Byhouwer 6sm ? 62 m 123 119 Author's personal copy

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Table 2 Detailed taxonomic and geological information of the studied species Section Code Species Origin Locality Alt (m)

Pimpinellifoliae 1 R. omeiensis var. omeiensis Lijiang, NW Yunnan 100°1601700E, 27°1200400N 3,150 2 R. sericea Lijiang, NW Yunnan 100°1200100E, 27°0003000N 2,787 3 R. mairei Weixi, NW Yunnan 99°2804100E, 27°1501900N 2,570 4 R. sikangensis Zhongdian, NW Yunnan 103°2707500E, 27°4309100N 3,030 5 R. zhongdianensis Zhongdian,, NW Yunnan 99°3202900E, 28°0004700N 3,003 Cinnamomeae 6 R. moyesii Weixi, NW Yunnan 99°0504200E, 27°3903600N 3,164 7 R. macrophylla Zhongdian, NW Yunnan 99°3703500E, 27°4704800N 3,508 8 R. multibracteata Lijiang, NW Yunnan 100°1101900E, 26°5703000N 2,999 9 R. pseudobanksiae Midu, W Yunnan 25°1606500E, 100°3108900N 1,662 10 R. sertata var. sertata Zhongdian, NW Yunnan 100°0103300E, 32°3505500N 3,035 Synstylae 11 R. multiflora var. multiflora Jinning, C Yunnan 102°2900400E, 24°4005900N 1,920 12 R. brunonii Weixi, NW Yunnan 99°2204300E, 27°0704300N 1,781 13 R. rubus Wenshan, SE Yunnan 105°2502400E, 23°2704100N 1,426 14 R. longicuspis Mile, C Yunnan 103°2302800E, 24°3401200N 1,965 15 R. lasiosepala Xichou, SE Yunnan 105°0605900E, 23°2304400N 1,633 16 R. soulieana var. soulieana Diqin, NW Yunnan 99°1604800E, 28°1404700N 1,951 17 R. weisiensis Weixi, NW Yunnan 99°0205800E, 27°4205400N 1,900 Banksianae 18 R. banksiae var. banksiae Weixi, NW Yunnan 99°0201600E, 27°4705500N 1,987 19 R. banksiae var. normalis Kunming, NW Yunnan 102°4803600E, 25°1201900N 2,078 20 R. banksiae f. lutea Lijiang, NW Yunnan 100°1304900E, 26°5704500N 2,501 21 R. cymosa var. cymosa Wenshan, SE Yunnan 105°0103300E, 23°2204700N 1,428 Laevigatae 22 R. laevigata Funing, SE Yunnan 105°3605000E,3°3500600N 1,000 Microphyllae 23 R. roxburghii Wenshan, SE Yunnan 104°5405900E, 23°2503100N 1,377 Bracteatae 24 R. bracteata Longchuan, W Yunnan 97°4705200E, 24°1104400N 926 NW Yunnan Northwestern Yunnan, W Yunnan Western Yunnan, SE Yunnan Southeastern Yunnan, C Yunnan Center Yunnan

Observations were made on nuclei at the somatic shown in Fig. 1. The Metaphase chromosomes and mitotic interphase and metaphase, and measurements of karyotype of each studied species were shown in chromosome arms were taken from at least ten well- Figs. 2 and 3, respectively. Karyotype comparisons of spread metaphases of five shoot tips. The karyomorpho- the studied species were listed in Table 3. logical classification of the interphase and mitotic prophase chromosomes followed Tanaka (1971, 1977); Nuclei characters of cell mitosis cycle the designation of the centromeric position as median (m), submedian (sm), and subterminal (st) was that of The nuclei characters of interphase nuclei and differ- Levan et al. (1964). The symmetry of karyotype was ent cell mitosis stages of the studied Rosa species were classified according to Stebbins (1971). AS.K% was almost the same as that of Rosa sikangensis Yu¨ et Ku calculated according to that of Arano (1963)andindexof (Fig. 1). The interphase nucleus (Fig. 1a) belonged to relative length (I.R.L) and composing of relative length of the complex chromocenter type (Tanaka 1971, 1977) genome (CRL) was according to that of Kuo et al. (1972). because they exhibited many darkly stained chromo- centers with an irregularly protruding rough surface that gradually transformed into diffuse chromatin. The Results mitotic prophase chromosomes (Fig. 1b), with very distinguishable heterochromatic and euchromatic seg- The nuclei characters of interphase nuclei and differ- ments and the heterochromatic segments distributing ent cell mitosis stages of studied Rosa species were in the interstitial and proximal regions, were of 123 Author's personal copy

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Fig. 1 The interphase nuclei and different cell mitosis stages of R. sikangensis. a Interphase nuclei, b prophase, c metaphase, d anaphase. Bar = 20 lm

interstitial type (Tanaka 1977). In the anaphase and length (CRL) of the studied species in Yunnan varied telophase cells, none chromosomal anomalism such as much. The absolute total length of the longest chromosome doubling, chromosomal bridge and chromosomes were from 1.6 to 3.2 lm, with that of tagged chromosome were observed (Fig. 1d). Rosa cymosa Tratt. var. cymosa being 1.6 lm and that of Rosa soulieana Cre´p. var. soulieana Cre´p. being Chromosome numbers of wild Rosa in Yunnan 3.2 lm. The shortest chromosomes ranged from 0.8 to 2 lm, the seventh pair of chromosome Rosa omeiensis No aneuploidy and no odd ploidy were observed in Rolfe var. omeiensis being 0.8 lm and that of Rosa this study. The chromosome number of Rosa mairie brunonii Lindl., being 1.6 lm. The first pair of Le´vl., R. sikangensis and Rosa zhongdianensis Ku chromosomes from Rosa mairei had the longest from Sect. Pimpinellifoliae, Rosa pseudobanksiae Yu¨ relative total length of 20.15%, while that of Rosa et Ku from Sect. Cinnamomeae, Rosa lasiosepala sericea Lindl. had the shortest relative total length of Metc. and Rosa weisiensis Yu¨ et Ku from Sect. 16.07%. With a relative total length of 12.7%, the last Synstylae, Rosa banksiae Ait. var. normalis Regel and pair of chromosomes from R. multibracteata was the R. banksiae Ait. f. lutea (Lindl.) Rehd., were all longest one and that from R. banksiae var. normalis reported first time. The studied taxa from all sections was the shortest with a relative total length of 9.95% except Sect Cinnamomeae were diploid with 14 among all the studied species. As far as composing of chromosomes (2n = 2x = 14). As for those from relative length were concerned, the genome of Sect. Cinnamomeae, Rosa multibracteata Hemsl. et R. moyesii, R. macrophylla, R. banksiae Ait. var. Wils., R. pseudobanksiae and Rosa sertata Rolfe var. banksiae, R. banksiae var. normalis, R. banksiae f. sertata were diploids with 14 chromosomes (2n = lutea and Rosa bracteata Wendl. var. bracteata 2x = 14), while Rosa moyesii Hemsl. et Wils. and Wendl. were composed with all 4 types of chromo- Rosa macrophylla Lindl. were both hexaploids with somes, long chromosome (L), medium long chromo- 42 chormosomes (2n = 6x = 42) (Fig. 2). some (M1), medium short chromosome (M2) and short chromosome (S). The genomes of Rosa omeien- Karyotype of wild Rosa in Yunnan sis var. omeiensis, Rosa mairei, R. sikangensis and Rosa longicuspis Bertol. var. longicuspis were com- Karyotypes of most studied taxa were reported here posed of 3 types of chromosomes, long chromosome for the first time. All chromosomes of R. pseudobank- (L), medium long chromosome (M1), medium short siae and Rosa multiflora Thunb. var. multiflora were chromosome (M2), without short chromosome. Those metacentric. Genomes of other species were com- of R. pseudobanksiae, R. sertata var. sertata, Rosa posed of metacentric chromosomes (m) and submeta- soulieana var. soulieana and R. weisiensis were centric chromosomes (sm). As Figs. 2, 3, and Table 3 composed of 3 types of chromosomes, medium long showed, the chromosome absolute total length (ATL), chromosome (M1), medium short chromosome (M2) relative total length (TL), and composing of relative and short chromosome (S), without long chromosomes

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Genet Resour Crop Evol (2013) 60:115–127 123 b Fig. 2 Metaphase chromosomes of wild Rosa resources in (L). Genomes of the other 9 species were composed of Yunnan. Note: 1. R. omeiensis var. omeiensis 2. R. sericea 3. medium long chromosome (M1) and medium short R. mairei 4. R. sikangensis 5. R. zhongdianensis 6. R. moyesii 7. R. macrophylla 8. R. multibracteata 9. R. pseudobanksiae 10. chromosome (M2). R. sertata var. sertata 11. R. multiflora var. multiflora 12. R. brunonii The genomes of the studied wild Rosa species 13. R. rubus 14. R. longicuspis var. longicuspis 15. R. lasiosepala 16. differed from each other also in satellite. The fifth pair R. soulieana var. soulieana 17. R. weisiensis 18. R. banksiae var. chromosomes of Rosa sericea, the sixth of Rosa banksiae 19. R. banksiae var. normalis 20. R. banksiae f.lutea21. R. cymosa var. cymosa 22. R. laevigata 23. R. roxburghii 24. mairei, the nineteenth of R. macrophylla, the seventh R. bracteata var. bracteata Bar = 20 lm of R. sertata, Rosa brunnoii and R. banksiae var.

Fig. 3 Karyotype ideogram of wild Rosa resources in Yunnan. 14. R. longicuspis var. longicuspis 15. R. lasiosepala 16. R. Note: 1. R. omeiensis var. omeiensis 2. R. sericea 3. R. mairei 4. R. soulieana var. soulieana 17. R. weisiensis 18. R. banksiae var. sikangensis 5. R. zhongdianensis 6. R. moyesii 7. R. macrophylla banksiae 19. R. banksiae var.normalis20. R. banksiae f.lutea21. 8. R. multibracteata 9. R. pseudobanksiae 10. R. sertata var. R. cymosa var. cymosa 22. R. laevigata 23. R. roxburghii 24. R. sertata 11. R. multiflora var. multiflora 12. R. brunonii 13. R. rubus bracteata Bar = 20 lm 123 Author's personal copy

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Table 3 Karyological characters of wild Rosa resources in Yunnan Taxa Karyotype formula C L/S P As.K% CRL

R. omeiensis var. omeiensis 2n = 2x = 14 = 4sm (1, 6) ? 10 m 1A 1.67 0 58.52 2L ? 4M2 ? 8M1 R. sericea 2n = 2x = 14 = 4sm (1, 6) ? 10 m (2sat, 2A 1.36 0.29 59.52 6M2 ? 8M1 5) R. mairei 2n = 2x = 14 = 2sm (1) ? 12 m (2sat, 6) 1A 1.78 0 57.44 2L ? 4M2 ? 8M1 R. sikangensis 2n = 2x = 14 = 4sm (1, 4) ? 10 m 2A 1.59 0.14 61.15 2L ? 2M2 ? 10M1 R. zhongdianensis 2n = 2x = 14 = 6sm (1, 5 ,6) ? 8 m 1A 1.44 0 61.29 6M2 ? 8M1 R. moyesii 2n = 6x = 42 = 6sm (7, 12, 15) ? 36 m 1A 1.99 0 57.94 4L ? 16M2 ? 20M1 ? 2S R. macrophylla 2n = 6x = 42 = 4sm (7, 13) ? 38 m 1A 1.84 0 57.30 4L ? 10M2 ? 26M1 ? 2S (2sat, 19) R. multibracteata 2n = 2x = 14 = 2sm (4) ? 12 m 1A 1.28 0 56.93 6M2 ? 8M1 R. pseudobanksiae 2n = 2x = 14 = 14 m 1A 1.64 0 55.30 8M2 ? 4M1 ? 2S R. sertata var. sertata 2n = 2x = 14 = 2sm (3) ? 12 m (1sat, 7) 2A 1.41 0.14 58.03 6M2 ? 6M1 ? 2S R. multiflora var. multiflora 2n = 2x = 14 = 14 m 1A 1.66 0 57.83 2L ? 2M2 ? 8M1 R. brunonii 2n = 2x = 14 = 4sm (4, 7, 1sat, 1A 1.47 0 59.06 6M2 ? 8M1 7) ? 10 m R. rubus 2n = 2x = 14 = 2sm (4) ? 12 m 1A 1.58 0 57.29 6M2 ? 8M1 R. lasiosepala 2n = 2x = 14 = 2sm ? 12 m 1A 1.45 0 59.15 8M2 ? 6M1 R. longicuspis var. 2n = 2x = 14 = 2sm (4) ? 12 m 2A 1.51 0.14 59.97 2L ? 2M2 ? 10M1 longicuspis R. soulieana var. soulieana 2n = 2x = 14 = 4sm (1, 5) ? 10 m 1A 1.72 0 59.13 8M2 ? 4M1 ? 2S R. weisiensis 2n = 2x = 14 = 2sm (2) ? 12 m 1A 1.69 0 58.83 6M2 ? 6M1 ? 2S R. banksiae var. banksiae 2n = 2x = 14 = 2sm (3) ? 12 m 2A 1.72 0.14 57.45 2L ? 4M2 ? 6M1 ? 2S R. banksiae var. normalis 2n = 2x = 14 = 2sm (3) ? 12 m (2sat, 7) 2A 1.95 0.14 59.53 2L ? 2M2 ? 8M1 ? 2S R. banksiae f. lutea 2n = 2x = 14 = 4sm (2, 3) ? 10 m 1A 1.77 0 59.28 2L ? 6M2 ? 4M1 ? 2S R. cymosa var. cymosa 2n = 2x = 14 = 4sm (3, 7) ? 10 m 2A 1.45 0.14 58.77 6M2 ? 8M1 R. laevigata 2n = 2x = 14 = 4sm ? 10 m 2A 1.56 0.14 59.74 8M2 ? 6M1 R. roxburghii 2n = 2x = 14 = 2sm ? 12 m 2A 1.51 0.14 58.98 6M2 ? 8M1 R. bracteata 2n = 2x = 14 = 4sm (3, 5) ? 10 m 1A 1.77 0 58.29 2L ? 4M2 ? 6M1 ? 2S C karyotype; L/S ratio of longest chromosome to shortest one; P ratio of chromosome of arm ratio [ 2; As.K% asymmetry index; CRL constitution of relative length normalis had satellites, among which R. sertata var. R. banksiae var. nomalis, Rosa cymosa var. cymosa sertata, R. brunnoii only had one satellite while the and Rosa roxburghii, were 2A, while those of the other 4 species had a pair of satellites. No satellites remained 15 species were 1A. were observed in the other 18 species. Only 1A symmetry and 2A symmetry being found, the karyotypes of the studied species were rather Discussion symmetric. The AS.K% varied from 55.3 to 61.29%, that of R. pseudobanksia was smallest and that of R. The basic chromosome of genus Rosa is proved to be zhongdianensis was the largest. The length ratio of x = 7 by almost all related studies since Ta¨ckholm’s longest chromosome to shortest one (L/S) varied from report in 1920 (Ta¨ckholm 1920). On the basis of both 1.28 (R. multibracteata) to 1.99 (R. moyesii). With one arm ratio and chromosome length, the authors con- or two pairs of chromosomes’ arm ratio (AR) larger cluded that karyotypes of Rosa were largely symmetry than 2, karyotypes of 9 species, namely R. sericea, existed across the genus (Ma and Chen 1991, 1992; R. sikangensis, R. sertata var. sertata, Rosa longicu- Ma et al. 1997a, b; Jian et al. 2010a, b). Our study spis var. longicuspis, R. banksiae var. banksiae, confirmed those previous reports again. The basic

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Genet Resour Crop Evol (2013) 60:115–127 125 chromosome number of wild Rosa in Yunnan is 7 as reported to have high frequencies of polyploidy (Ohba well as in other areas. Also, the karyotypes of almost 1988; Abbott and Brochmann 2003; Brochmann et al. all the studied wild Rosa in Yunnan belonged to 1A or 2004) and it had been known since Ta¨ckholm’s work 2A and were composed with metacentric or metacen- that species or forms in Rosa with the more arctic or tric and submetacentric chromosomes, and thus they alpine distribution were the higher polyploids (Dar- were very symmetric. lington 1942). As for genus Rosa in Yunnan, 22 of the Although wild Rosa species all had a rather studied 24 taxa were diploid, and for all the karyolog- symmetric karyotype, they varied significantly in ically known taxa distributed in Yunnan, totally 25 of chromosome number and karyomorphology. The 36 were diploids. As far as those polyploid taxa were chromosome number of genus Rosa varied from concerned, except the half-cultivated ones, all were 2n = 2x = 14 to 2n = 10x = 70 (Darlington and from the alpine areas of Northwestern Yunnan, which Wylie 1955; Jian et al. 2010a) and many taxa have lies in the south-eastern Himalayan range, now a hot more than one ploidy levels, especially those half- spot both for its biodiversity and for its role in cultivated ones and those from Section Cinnamomeae chromosome evolution (Boufford and Van Dijk 2000; (Ta¨ckholm 1922; Darlington and Wylie 1955; Ma and Nie et al. 2005; Chen et al. 2007). From the natural Chen 1991, 1992; Jian et al. 2010b). On one hand, the distribution point of view, alpine wild rose species existing ploidy levels of wild Rosa in Yunnan included exhibit highly diverse ploidy level from 2x, 4x, 6x and diploidy (2n = 2x = 14) in most reported species, 10x indicating that the extreme environment associ- triploidy (2n = 3x = 21) in Rosa odorata (Andr.) ating with the high altitude promotes the polyploidi- Sweet var. gigantea (Cre´p.) Rehd. et Wils., R. odorata zation in Sect. Cinnamomeae of genus Rosa, as more (Andr.) Sweet var. erubescens (Focke) Yu¨ et Ku and than half of the species from the section were R. multiflora Thunb. var. carnea Thory, tetraploidy polyploidy and many of them were still have more (2n = 4x = 28) in Rosa lichiangensis Yu¨ et Ku,R. than one ploidy levels. In another words, polyploidi- moyesii, R. macrophylla, R. sertata var. sertata, Rosa zation might have played and were still playing very webbiana, R. multibracteata, hexaploidy (2n = important roles in the evolution and speciation in Sect. 6x = 42) in R. moyesii and Rosa sweginzowii Koehne, Cinnamomeae of Genus Rosa in the alpine area of and the newly reported decaploidy (2n = 10x = 70) northwestern Yunnan, as were found by Yuan and in R. praelucens Byhouwer. On the other hand, the Yang (2008), Chen et al. (2007) in the speciation of half-cultivated R. odorata var. erubescens had both Aconitum subgenus Lycoctonum and Buddleja, diploid and triploid individuals. Many Cinnamomeaen respectively. taxa such as R. macrophylla, R. sertata var. sertata, R. webbiana and R. multibracteata were both diploid and Acknowledgments We thank ShuFa Li for collecting and tetraploid, while R. moyesii had both tetraploid and grafting some plant materials, XianQin Qiu, HuiJun Yan and NingNing Zhou for all kinds of helps in daily and lab works, hexaploid populations in previously studies. A new Jihua Wang and Shuping Qu¨ for constructive suggestion. This chromosome number 42 were found in R. macrophylla study was supported by the National Natural Science Foundation which meant that this species had 3 ploidy levels. As of China (Grant No. 31060267), Natural Science Foundation of far as karyomorphology was concerned, different Yunnan Province (2008CD183) and Key Natural Science Foundation of Yunnan Province (2007C0004Z). species were thought to vary much (Ma and Chen 1991, 1992; Ma et al. 1997a, b;Jian et al. 2010b). The taxa in this study also diversified in chromosome length, arm ratio, number and position of satellites, References composing of relative length and karyotype formula, etc. So, wild Rosa species in Yunnan are rich in Abbott RJ, Brochmann C (2003) History and evolution of the arctic flora: in the footsteps of Eric Hulten. Mol Ecol genetic diversity from the karyological point of view. 12:299–313 Polyploidy has long been recognized as a promi- Akasaka M, Ueda Y, Koba T (2002) Karyotype analysis of wild nent force in evolutionary diversification and is one of rose species belonging to septets A by fluorescence in situ hybridization. Chromosom Sci 6:17–26 the most important cytogenetic mechanisms in plant Akasaka M, Ueda Y, Koba T (2003) Karyotype analysis of wild evolution and rapid speciation (Stebbins 1971; Grant rose species belonging to septets B, C, and D by molecular 1981; Levin 2002). Alpine and arctic floras have been cytogenetic method. Breed Sci 53:177–182 123 Author's personal copy

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