Lilium rosthornii is a perennial herb native to China. This Lilium species is an attractive ornamental due to its orange or yellowish flower color, extended floral longevity, curved flower shape, and purple spots in the perianth. With all these desirable flower traits, it is a superior parent for Lilium breeding. Moreover, L. rosthornii is important in traditional Chinese medicine (Long et al., 2004).
Lilium rosthornii was originally primarily distributed in valleys and alongside rivers in provinces such as Sichuan, Chongqing, Hubei, and Guizhou. In recent years, most wild populations of L. rosthornii have been reduced due to habitat destruction and loss, and the natural distribution of this species is also shrinking gradually, such that L. rosthornii is becoming endangered (Wang, 2009). However, little is known about the biology and cytology of this species. He (2006) reported on pollen viability and identified the optimal conditions for pollen storage, providing some reference for cross breeding. Zhao (2012) studied the micropropagation and rapid propagation of L. rosthornii in an attempt to establish the best system for regenerating L. rosthornii.
Nonetheless, cytological studies of L. rosthornii remain scarce. Although Qing (2011) and Liu et al. (2010) both analyzed the karyotype of L. rosthornii, their results were markedly different. Qing proposed that the karyotype of L. rosthornii was type 3A [length ratio of the longest chromosome to the shortest chromosome (Lt/St) < 2:1; percentage of chromosomes with an arm ratio of more than 2:1 between 51% and 99%] and that chromosomes 1, 7, and 9 were satellite chromosomes, whereas Liu et al. (2010) considered the karyotype of L. rosthornii to be type 3B (Lt/St between 2:1 and 4:1; percentage of chromosomes with an arm ratio of more than 2:1 between 51% and 99%), with chromosomes 1 and 6 constituting satellite chromosomes. Both of those studies used L. rosthornii samples collected from the Jinfoshan area of Nanchuan, Chongqing. The inconsistency between these results complicates the collection, identification, and use of L. rosthornii, as researchers and readers do not know whether this disagreement is caused by differences in the materials themselves or data interpretation. Chromosomal karyotype analysis is mainly based on chromosome number, the arm length ratio, and the locations of secondary constrictions. However, different chromosome preparation methods often cause deviations in the determination of the arm length ratio and location of secondary constrictions (particularly the former). Further studies of this species will provide valuable information for the conservation and utilization of L. rosthornii resources.
Giemsa C-banding and fluorescent in situ hybridization (FISH) techniques are both traditional karyotyping methods. In C-banding analysis, chromosomes are first treated with hydrochloric acid (HCl) and barium hydroxide [Ba(OH)2] and then painted with Giemsa stain (Sigma-Aldrich, St. Louis, MO). Different chromosomes typically show different banding patterns that can be used in chromosome identification. In FISH analysis, specific DNA sequences such as ribosomal RNA genes are first labeled with fluorescein, biotin, or digoxin, which can emit visible fluorescence or be detected by visible fluorescence when induced by ultraviolet light. These RNA probes are then hybridized with chromosomes fixed on slides or nylon membranes. The application of FISH to plant chromosome characterization using rDNAs began in the late 1980s and has become a powerful tool for localizing specific DNA sequences on plant chromosomes (Schwarzacher et al., 1989). The rDNAs (45S and 5S), which are repeated sequences in the genome, are commonly used for physical mapping of plant chromosomes due to their universal occurrence and redundancy (Hasterok et al., 2001). Simultaneous FISH of 45S and 5S rDNAs has been approved as a useful tool for the identification of various plants. Taketa et al. (1999) compared the locations of 5S and 18S-25S rDNA in nine Hordeum L. species and concluded that rDNA markers are useful for the investigation of chromosome evolution and phylogeny. Mizuochi et al. (2007) first cloned 5S and 45S rDNA from Tulipa fosteriana Hoog and then determined the locations of these two types of rDNA in Tulipa gesneriana L. and T. fosteriana. 5S and 45S rDNA FISH has also been used to characterize chromosomes from Brassica L. (Hasterok et al., 2001), Cucumis sativus L. cv. Winter Long (Koo et al., 2002), and Nicotiana L. (Lim et al., 2005). Compared with the traditional karyotype analysis, C-banding and FISH provide more uniform and reproducible results. Giemsa C-banding and FISH techniques have been used in the study of Lilium chromosomes (Lee et al., 2014; Sultana et al., 2011). However, to our knowledge, no molecular cytogenetic studies have been performed to date in L. rosthornii.
The PI banding technique was first described by Hanson et al. (1995), who used it with bacterial artificial chromosomes. Peterson et al. (1999) used this technique to determine the location of single-copy and low-copy sequences in tomato (Solanum lycopersicum L). Their results showed that the observation of PI fluorescence revealed bright red fluorescent bands in the nucleolus organizer region (NOR) and in pericentromeric heterochromatin in many chromosomes. More recently, PI fluorochromes have been used in genome mapping using FISH (Nath et al., 2015). In this study, we combined the C-banding, PI-banding, and 45S rDNA FISH methods to characterize the chromosomes from L. rosthornii, which was collected in 2004 from Nanchuan, Chongqing Province, China.
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