Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 97-102, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-218 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Y.-c. Tang, P.-p. Yang, M.-h. Yang, G.-r. He, Y.-w. Cao, L.-f. Xu, J. Ming (2020) Karyotype analysis of Lilium lancifolium and four related cultivars. Caryologia 73(3): 97-102. doi: 10.13128/ caryologia-218 Received: April 13, 2019 Accepted: June 19, 2020 Published: December 31, 2020 Copyright: © 2020 Y.-c. Tang, P.-p. Yang, M.-h. Yang, G.-r. He, Y.-w. Cao, L.-f. Xu, J. Ming. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Karyotype analysis of Lilium lancifolium and four related cultivars Yu-chao Tang1,#, Pan-pan Yang1,#, Mei-hua Yang2, Guo-ren He1, Yu-wei Cao1, Lei-feng Xu1,*, Jun Ming1,* 1 The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Bei- jing 100081, China 2 School of Landscape Architecture, Beijing University of Agriculture, Beijing 100081, China *Corresponding authors. E-mail: xuleifeng@caas.cn (Leifeng Xu); mingjun@caas.cn (Jun Ming) # These authors contribute equally to the article. Abstract. Lilium lancifolium is one of the most important species of genus Lilium. Besides ornamental value, it also has highly edible and medicinal properties. We inves- tigated the karyotypes of L. lancifolium and four related cultivars. The results indicated that the ploidies of four cultivars varied from diploid to tetraploid. Both ‘Flore Pleno’ and ‘Red Velvet’ were triploid (2n=3x=36), consistenting with the wild species L. lan- cifolium. ‘Sweet Surrender’ was diploid (2n=2x=24), and ‘Red Life’ was tetraploid. All karyotypes of candidates belonged to 3B type except ‘Flore Pleno’, which belonged to 3A type. Karyotype symmetry analysis revealed that the wild species L. lancifolium had a middle value of A1, A2, and TF%, which meant that the cultivars related to L. lanci- folium had different tendencies to symmetry compared to L. lancifolium, but whether they were higher or lower was unclear. Keywords: Lilium lancifolium, cultivars, karyotype, chromosome. INTRODUCTION Genus Lilium includes approximately 100-115 wild species (Liang and Tamura, 2000). Lilium lancifolium, the tiger lily, one of the most important species of genus Lilium, is widely distributed in northern and eastern Asia, including Korea, Japan, China and Sakhalin (Feldmaier and McRae, 1982). Besides ornamental value, L. lancifolium is also extensively used as both food and a traditional Chinese medicine for many centuries in China due to its health-promoting properties and treatments of bronchitis, pneumonia, chronic gastritis (Chau and Wu, 2006; Luo et al., 2012; Joung et al., 2007; Kwon et al., 2010; Gao et al., 2015). In addition, L. lancifolium has been used as a modal plant to study the mechanism of bulbil formation in lilies (Yang et al., 2017, 2018; He et al, 2020), because it is one of the four Lilium species which can bear bulbils in leaf axils (McRae, 1998; Liang and Tamura, 2000; Bach and Sochacki, 2012). 98 Yu-chao Tang et al. Karyomorphological investigations are very impor- tant for the views of ta xonomica l, ecologica l and cy tological studies. Although the karyotype features are generally constant in a group of species and even a genus, the variations in structure and/or number can change the number, size, and position of the cen- tromere on chromosomes, causing genetic variation (Ahn et al., 2017). In addition, chromosome number can also complement information of polyploidy and other highly significant genome changes which are invisible by morphological and molecular methods. For more than 40 years, karyotype analysis of many wild Lilium species including L. lancifolium (Gill et al., 1974; Vosa et al., 1976; Gao et al., 2011) and hybrids (Khan et al., 2009; Liu et al., 2011) has been carried out. That provides a lot of significant theoretical basis for cyto- logical and taxonomic studies of Lilium. It is interest- ing that all Lilium species are diploids (2n=2x=24) except L. lancifolium, which consists of ploidy com- plex with diploids, narrowly distributed, and triploids (2n=3x=36), widely distributed in inlands of Chi- na, Korea, Japan and Russia, in nature (Noda 1978, 1986; Kim et al., 2006; Hwang et al., 2011). Because L. lancifolium distributed in China is natural triploid (2n=3x=36) (Noda, 1986), it is highly sterile in cross breeding, neither as male or female parent, which seri- ously stunts the upgrading of its varieties. Recent years, several varieties related to L. lancifo- lium have emerged on market, but the karyotype anal- ysis of L. lancifolium related cultivars have never been reported. Here, we collected four cultivars related to L. lancifolium and investigated the karyotypes of L. lanci- folium and four related cultivars, providing cytological and genetic foundation for the breeding of L. lancifo- lium. MATERIALS AND METHODS Plant materials L. lancifolium and four related cultivars (‘Red Life’, ‘Sweet Surrender’, ‘Red Velvet’, ‘Flore Pleno’) were used as materials in this study (Figure 1). L. lancifolium bulbs were harvested from our farm (Beijing, China: 116.58°E; 40.07°N) in October 2017. Bulbs of L. lancifolium relat- ed cultivars were purchased from Licai Garden Co., Ltd (Zhejiang, China). All bulbs were planted in the matrix of peat, vermiculite, perlite with volume ratio of 5:3:1, in a greenhouse at the Institute of Vegetables and Flow- ers, Chinese Academy of Agricultural Sciences, Beijing, China in March 2018. Root tips were obtained from each population for squashing. Chromsome preparation and observation Actively growing root tips of each populations for the length around 0.5 cm were taken between 9 am and 11 am in a clear day, and pretreated with 0.7% cyclohex- imide solution at 4°C for 10 h, then fixed in Carnoy’s I Fluid (methanol: glacial acetic acid = 3:1) for 24 h at room temperature, and finally kept in 70% alcohal until use. The roots were hydrolyzed in 1 mol/L HCl at 60°C for 10 min, then the chopped root tips were stained in Carbol fuchsin stain for 5~6 min at room tempera- ture. The roots must be rinsed in distilled water for 3 to 5 times before each step. The observations of the best metaphase plates were made using an Olympus CX31 (Olympus light microscope, Tokyo, Japan) equipped with a 100 × /1.25 oil objective and a mounted Canon 550D digital camera (Canon, Japan). Figure 1. The five materials in this study. A: L. lancifolium, B-E: L. lancifolium cultivars: 'Flore Pleno', 'Sweet Surrender', 'Red Life', 'Red Vel- vet', respectively. 99Karyotype analysis of Lilium lancifolium and four related cultivars Karyological analyses The measurement of basic parameters such as long arm (LA), short arm (SA) related to every chromosome was performed utilizing Photoshop V.7. Based on which we calculated total length of genome (TLG), AR (arm ratio), CI (centromeric index), LA% (long arm percent- age), SA% (short arm percentage), TF% (total form per- centage), VRC (value of relative chromatin), A1 (intra- chromosome asymmetry index), A2 (interchromosome asymmetry index) and DI (dispersion index). The Karyo- type formula was calculated according to the definition of metacentric (m), submetacentric (sm), telocentric (t), and sbtelocentric (st), proposed by Levan et al. (1964). The karyotype classification was defined by using the method of Stebbins (1971). Finally, the data analysis was conducted in excel. RESULTS This study revealed detailed pictures of mitotic chro- mosome plates, related karyotypes and karyograms in L. lancifolium and its four cultivars (Figure 2). The related parameters and karyotypic formula were summarized in Table 1 and Table 2. The results showed that, L. lancifolium (Figure 2-A) is triploid, which had a chromosome number of 2n=3x=36; while in the four cultivars, only ‘Flore Pleno’ (Figure 2-B) and ‘Red Velvet’ (Figure 2-E) were trip- loid, with the same chromosome number as L. lanci- folium, 2n=3x=36; ‘Sweet Surrender’ (Figure 2-C) was diploid, 2n=2x=24; and ‘Red life’ (Figure 2-D) was tetra- ploid, 2n=4x=48. In five materials, only L. lancifolium had satellites attached to the first two pairs of chromo- somes, and ‘Flore Pleno’had one group of chromosomes attached with satellites, the other three cultivars had no satellites. According to the karyograms in Figure 2, besides ‘Red Velvet’ had two groups of telocentric chro- mosome, L. lancifolium, ‘Flore Pleno’ and ‘Sweet Surren- der’ had one set telocentric chromosome, and ‘Red life’ has no telocentric chromosome (Figure 2). The length of shortest chromosome in L. lancifoli- um (Table 1-A) is 10.27 μm, which was obviously longer than its four cultivars’. The total genome length ranged from 117.44 to 191.05 μm between tetraploid ‘Red Life’ (Table 1-D) and wild species L. lancifolium in all popu- lations. Even the karyotype formulas of five materials were complicated, at least four different chromosome- types were contained in each species, most of the five materials had the same karyoty pe 3B except ‘Flore Pleno’ (Table 1-B), whose belongs to 3A (Table 1). The Table 1. Karyotype analysis of L. lancifolium and four related cultivars. Sample Chromosome number Chromosome size range (μm) Total genome length (μm) Karyotype Karyotypic formula A 2n=3x=36 10.27-21.15 191.05 3B 3m(2SAT)+3sm(3SAT)+12st+15t+3T B 2n=3x=36 7.57-14.80 127.37 3A 3sm(3SAT)+18st+12t+3T C 2n=2x=24 8.68-19.83 155.29 3B 2m+4sm+4st+12t+2T D 2n=4x=48 6.52-14.15 117.44 3B 4m+8sm+28st+8t E 2n=3x=36 7.25-16.54 153.44 3B 3m+9sm+18st+6T Notes: A: L. lancifolium, B-E: L. lancifolium cultivars: 'Flore Pleno', 'Sweet Surrender', 'Red Life', 'Red Velvet', respectively. Table 2. Mean of parameters of chromosomes analysis of L. lancifolium and four related cultivars. Species TL LA SA AR CI LA% SA% TF% VRC A1 A2 DI A 15.64 12.98 2.66 4.88 0.17 6.79 1.39 16.70 15.92 0.79 0.20 2.47 B 10.45 8.92 1.53 5.83 0.15 7.00 1.20 14.45 10.61 0.81 0.21 3.01 C 12.94 10.49 2.45 4.28 0.19 6.67 1.58 18.95 12.94 0.77 0.26 3.09 D 9.78 7.58 2.20 3.45 0.22 6.45 1.87 22.50 9.79 0.71 0.22 3.44 E 12.79 10.06 2.73 3.68 0.21 6.56 1.78 21.35 12.79 0.70 0.19 3.43 Notes: A: L. lancifolium, B-E: L. lancifolium cultivars: 'Flore Pleno', 'Sweet Surrender', 'Red Life', 'Red Velvet', respectively. TL: total length of chromosome, LA: long arm, SA: short arm, AR: arm ratio, CI: centromeric index, LA%: long arm percentage, SA%: short arm percentage, TF%: total form percentage, VRC: value of relative chromatin, A1: intrachromosome asymmetry index, A2: interchromosome asymmetry index, DI: dispersion index. 100 Yu-chao Tang et al. mean value of the chromosome long arm varied from 7.58 μm to 12.98 μm in ‘Red life’ (Table 2-D) and L. lancifolium (Table 2-A), respectively. The average of short arm lengths ranged between 1.53 μm and 2.66 μm in ‘Flore Pleno’ (Table 2-B) and L. lancifolium. And the average total length of chromosomes varied from 9.78 μm to 15.64 μm in ‘Red life’ and L. lancifolium (Table 2). Figure 2. Mitotic chromosome plates, related karyotypes and karyograms of L. lancifolium and four related cultivars. A: L. lancifolium, B-E: L. lancifolium cultivars: 'Flore Pleno', 'Sweet Surrender', 'Red Life', 'Red Velvet', respectively. 101Karyotype analysis of Lilium lancifolium and four related cultivars To evaluate the symmetry of karyotype, we calcu- lated AR (arm ratio), CI (centromeric index), LA% (long arm percentage), SA%(short arm percentage), TF% (total form percentage), VRC (value of relative chromatin), A1 (intrachromosome asymmetry index), A2 (interchro- mosome asymmetry index) and DI (dispersion index) respectively based on TL (total length of chromosome), LA (long arm) and SA (short arm) (Table 2). The results showed that, among the studied populations, the highest TF% value (22.50) was estimated in ‘Red Life’ and the lowest TF% value (14.45) was estimated in ‘Flore Pleno’, the TF% valve (16.70) of L. lancifolium was the second lowest. The analysis of the intra-chromosome asym- metry (A1) and inter-chromosome asymmetry (A2) revealed that, ‘Red Velvet’ (with mean value of A1=0.70, A2=0.19) presented the smallest asymmetry. In this study, the wild species L. lancifolium had a lowest DI value (2.47), and the DI values four cultivars were much higher (3.01~3.44) (Table 2). DISCUSSION The karyomorphological investigations of Lilium are very important for the views of taxonomical and eco- logical studies (Ahn et al., 2017). In this study, the results showed that, L. lancifolium (A) was triploid, which had a chromosome number of 2n=3x=36, coinciding with pre- vious report (Gao et al., 2011). While the ploidy of the four cultivars related to L. lancifolium (A) varied from diploid to tetraploid. Since the chromosome numbers of F1 hybrids of triploid L. lancifolium × diploid L. leichtli- nii range from 24 to 34 (Suzuki and Yamagishi, 2016), and the chromosome numbers of F1 hybrids of triploid L. lancifolium × tetraploid ‘Brunello’ can reach to 50 (Ma, 2017), these four cultivars we studied might be obtained by hybridization between triploid L. lancifolium and oth- er tetraploid lilies. According to previous studies, all the karyotypes of Lilium belong to 3B and 3A type (Stebbins, 1971; Gao et al., 2011). In our study, only ‘Flore Pleno’ belonged to 3A, the other four belonged to 3B. That cor- roborate that the karyotype of genus Lilium is stable. Both the karyograms and the value of TF% indi- cate that the karyotypes of five materials are very asym- metric. By using the values of A1, A2 (Zarco, 1986) and TF% (Huziwara, 1962), we can evaluate the symmetry of karyotypes among close classes. The present study revealed that the wild species L. lancifolium had a mid- dle value whether of the A1, A2, or TF%, which meant that the cultivars related to L. lancifolium had different tendencies to symmetry compared to L. lancifolium, but whether they were higher or lower was uncertain. DI index plays an important role in arranging the species within the same class of karyotype asymme- try in an advancing order of specialization by permit- ting further gradations, as depicted by species arrange- ment within sections (Lavania and Srivastava, 1992). We found that the wild species L. lancifolium had the lowest DI value compared to its cultivars. This might indicate that the hybrid progenies of L. lancifolium tend to have higher DI values. ACKNOWLEDGEMENTS This work was supported by the National Key R&D Program of China (2019YFD1001002), the National Natural Science Foundation of China (31902043), and the Central Public-interest Scientific Institution Basal Research Fund (IVF-BRF2020019). This research was conducted at the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agri- culture, China. REFERENCES Ahn YJ, Hwang YJ, Younis A, Sung MS, Ramzan F, Kwon MJ, Lim KB. 2017. Investigation of karyotypic com- position and evolution in Lilium species belonging to the section martagon. Plant Biotechnology Reports, 11(6), 407-416. Bach A, Sochacki D. 2012. Propagation of ornamental geophytes: physiology and management systems. In Ornamental Geophytes (pp. 280-305). CRC Press. Chau CF, Wu SH. 2006. The development of regulations of Chinese herbal medicines for both medicinal and food uses. Trends in Food Science & Technology, 17(6), 313-323. Feldmaier C, McRae J. 1982. Lilies. Verlag Eugen Ulmer. Gao J, Zhang T, Jin ZY, Xu XM, Wang JH, Zha XQ, Chen, HQ. 2015. Structural characterisation, physicochemi- cal properties and antioxidant activity of polysaccha- ride from Lilium lancifolium Thunb. Food chemistry, 169, 430-438. Gao YD, Zhou SD, He XJ. 2011. Karyotype studies in thirty‐two species of Lilium (Liliaceae) from China. Nordic Journal of Botany, 29(6): 746-761. Gill BS, Kimber G. 1974. A Giemsa C-banding technique for cereal chromosomes. Cereal Res Commun. 2: 87–94. He G, Yang P, Tang Y, Cao Y, Qi X, Xu L, Ming J. 2020. Mechanism of exogenous cytokinins inducing bulbil formation in Lilium lancifolium in vitro. Plant Cell Reports, 39: 861-872. 102 Yu-chao Tang et al. Huziwara Y. 1962. Karyotype analysis in some genera of Compositae. VIII. Further studies on the chromo- somes of Aster. American Journal of Botany, 49(2): 116-119. Hwang YJ, Kim HH, Kim JB, Lim KB. 2011. Karyotype analysis of Lilium tigrinum by FISH. Horticulture, Environment, and Biotechnology, 52(3): 292-297. Joung YM, Park SJ, Lee KY, Lee JY, Suh JK, Hwang SY, Kang MH 2007. Antioxidative and antimicrobial activities of Lilium species extracts prepared from different aerial parts. Korean Journal of Food Science and Technology, 39(4): 452-457. Khan N, Zhou S, Ramanna MS, Arens P, Herrera J, Visser RG, Van-Tuyl JM. 2009. Potential for analytic breed- ing in allopolyploids: an illustration from Longi- florum× Asiatic hybrid lilies (Lilium). Euphytica, 166(3): 399-409. Kim JH, Kyung HY, Choi YS, Lee JK, Hiramatsu M, Oku- bo H. 2006. Geographic distribution and habitat dif- ferentiation in diploid and triploid Lilium lancifolium of South Korea. Journal of the Faculty of Agriculture, Kyushu University, 51(2): 239-243. Kwon OK, Lee MY, Yuk JE, Oh SR, Chin Y W, Lee HK, Ahn KS. 2010. Anti-inflammatory effects of metha- nol extracts of the root of Lilium lancifolium on LPS- stimulated Raw264. 7 cells. Journal of Ethnopharma- cology, 130(1): 28-34. Lavania UC, Srivastava S. 1992. A simple parameter of dispersion index that serves as an adjunct to karyo- type asymmetry. Journal of Biosciences, 17(2): 179- 182. Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas, 52(2): 201-220. Liang SY, Tamura MN. 2000. Flora of China. Science Press, Beijing. Liu WL, Wu L F, Wu HZ, Zheng SX, Wang JH, Liu FH. 2011. Correlation of saponin content and Fusarium resistance in hybrids from different ploidy levels of Lilium Oriental. Scientia Horticulturae, 129(4): 849- 853. Luo J, Li L, Kong L. 2012. Preparative separation of phe- nylpropenoid glycerides from the bulbs of Lilium lan- cifolium by high-speed counter-current chromatog- raphy and evaluation of their antioxidant activities. Food Chemistry, 131(3): 1056-1062. Ma Bing. 2017. Cross compatibility between interspecific hybrids derived from Lilium lancifolium Thunb. and asia lily cultivers. [M. D. Dissertation]. Shenyang: Shenyang Agriculture University. (in Chinese) McRae EA. 1998. Lilies: a guide for growers and collec- tors. Portland, OR: Timber Press. Noda S. 1978. Chromosomes of diploid and triploid forms found in the natural populations of tiger lily in Tsushima. Journal of Plant Research, 91: 279-283. Noda S. 1986. Cytogenetic behavior, chromosomal dif- ferentiations, and geographic distribution in Lilium lancifolium (Liliaceae). Plant Species Biology, 1(1): 69-78. Stebbins GL. 1971. Chromosomal evolution in higher plants. Chromosomal evolution in higher plants. Edward Arnold. Suzuki T, Yamagishi M. 2016. Aneuploids without bulbils segregated in F1 hybrids derived from triploid Lilium lancifolium and diploid L. leichtlinii crosses. The Hor- ticulture Journal, 85(3): 224-231. Vosa CG. 1976. Chromosome banding patterns in culti- vated and wild barleys (Hordeum spp.). Heredity. 37: 395–403. Yang PP, Xu LF, Xu H, Tang YC, He GR, Cao YW, Ming J. 2017. Histological and transcriptomic analysis dur- ing bulbil formation in Lilium lancifolium. Frontiers in Plant Science, 8: 1508. Yang PP, Xu LF, Xu H, He GR, Feng YY, Cao YW, Ming J. 2018. Morphological and anatomical observation during the formation of bulbils in Lilium lancifolium. Caryologia, 71(2): 146-149. Zarco CR. 1986. A new method for estimating karyotype asymmetry. Taxon, 526-530. Zhang T, Gao J, Jin ZY, Xu XM, Chen HQ. 2014. Protec- tive effects of polysaccharides from Lilium lancifolium on streptozotocin-induced diabetic mice. Interna- tional Journal of Biological Macromolecules, 65: 436- 440.