Abstract
We investigated the chromosome number and karyotype in seven cultivars of Narcissus spp. using the traditional squash method. The result indicated that all seven cultivars were diploid and the basic chromosome numbers in these seven cultivars were n = 12 and 14. The karyotype was 2B for all seven cultivars. Six of seven cultivars consisted of m and sm chromosome, and only one cultivar contains st chromosome. The detailed karyotypes of the seven cultivars are as follows: Arkle: 2n = 2x = 28 = 3m + 11sm, index of the karyotypic asymmetry (As·k) = 66.57 %; Fortissimo: 2n = 2x = 28 = 4m + 10sm, As·k = 66.60 %; Pink Charm: 2n = 2x = 28 = 3m + 10sm + 1st, As·k = 66.62 %; Mary Bcharmon: 2n = 2x = 24 = 5m + 7sm, As·k = 64.56 %; Barret Browing: 2n = 2x = 24 = 6m + 6sm, As·k = 65.73 %; Romance: 2n = 2x = 24 = 2m + 10sm, As·k = 65.64 %; Dutch Master: 2n = 2x =24 = 3m + 9sm, As·k = 65.95 %. Our results indicated that the karyotype analysis combined with morphological characters could assist to classify the taxonomic relationship of Narcissus spp.
Keywords:
Introduction
Narcissus spp. belongs to Narcissus L. of Amaryllidaceae, which comprises about 25–30 proto-species distributed in Central Europe, the Mediterranean region, West Asia and North Africa (Nuñez et al. Citation2003; Blanchard Citation2004). Because the locations and origin of Narcissus spp. were close, huge hybrid swarms have been consistently formed through natural hybridization for thousands of years. Furthermore, the chromosome number of Narcissus L. species had a great variation ranging from 2n = 14 to 46, and triploid and aneuploidy commonly existed in the natural environment (Brandham Citation1992; González-Aguilera et al. Citation1990). Therefore, the boundaries of Narcissus spp. species and genetic relationships were very complicated and remained unresolved. It is well recognized that many previous cytological studies have been focused on Chinese narcissus（Wang et al. Citation2007; Zhuang et al. Citation1999), but there is a paucity of data for other Narcissus spp.
The classification of Narcissus still remains unclear, and the traditional classification has been mainly based on morphological characteristics, which couldn’t accurately describe the boundaries and the parental relationship between species of narcissus because of high occurrences of natural hybridization. We chose seven cultivars of Narcissus spp. with strong growth and spectacular colors. Moreover, they highly resisted to disease through cultivating for many years. However, it is difficult to recognize them as a distinct species based on morphology. Here, we performed the karyotype analysis for the seven Narcissus spp. to provide their classification and further infer the evolution of the Narcissus. Our experiments suggested that karyotype analysis could provide the basis for understanding of evolution, geographic distribution and morphology differentiation of Narcissus spp. Furthermore, our analysis had important significances to explore the fertility of Narcissus spp. hybrid parents, to guide the genetic breeding work, and especially to clarify the cross combinations between species and the mutation breeding. These analyses will provide an important theoretical basis for the variety breeding and the identification of hybrid offsprings in Narcissus spp.
Materials and methods
We selected and examined seven cultivars of Narcissus spp. that were introduced from Holland. They include Arkle, Fortissimo, Pink Charm, Mary Bcharmon, Barret Browing, Romance and Dutch Master.
Narcissus spp. bulbs were maintained in greenhouse for experiments. One to two cm-long root tips were excised during 8-9 am, and then pretreated with colchicin solution (1 g/L) for 4h at room temperature. The pretreated root tips were fixed using Carnoy’s fixative (anhydrous alcohol: glacial acetic acid = 3:1) for 20 hours. The fixed root tips were then hydrolyzed in HCl (1 mol/L) at 60°C for 10 min, rinsed with distilled water, stained in Card treasure magenta, and squashed to make slides. The slides were examined with a Motic microscope (Motic Instruments Inc., Canada). At least 30 cells of each variety that were in mitotic metaphases were observed and the images with good scattered chromosomes were retained. Chromosome counting and karyotype analysis followed the standard analysis from Li Maoxue (1985). Chromosome type was recorded in accordance with the classified standard from Levan (Citation1964). The karyotype type was adopted on the basis of the classified standard from Stebbins (Citation1971). The karyotype asymmetric coefficient was used to measure the degree of asymmetry (Arano Citation1963).
Results
The karyotype analysis in seven cultivars of Narcissus spp.
The basic chromosome numbers in these seven cultivars were n = 12 and 14. The chromosome karyotype analysis for these seven cultivars is shown in Table .
Table 1. Karyotype analysis in seven cultivars of Narcissus spp. Arm ratio: long/short, chromosome type: m-metacentric, sm-sub metacentric.
Arkle: diploid, and the number of somatic chromosome was 2n = 28. The karyotype formula of Arkle was: 2n = 2x = 28 = 3m + 11sm; the relative length range was 3.74 %–9.46 %; the average arm ratio was 1.99; the proportion of the chromosomes whose arm ratio was greater than 2 was 35.7 % of the total chromosomes; the ratio of the longest chromosome to the shortest one was 2.53; the type of their karyotypes was 2B; the karyotypic asymmetry coefficient (As·k) was 66.57 %; the fifth chromosome, the ninth chromosome and the thirteenth chromosome were m chromosomes, and the remaining chromosomes were sm chromosomes.
Fortissimo: diploids, the number of somatic chromosome was 2n = 28; the karyotype formula was: 2n = 2x = 28 = 4m + 10sm; the relative length range was 4.24 %–9.30 %; the proportion of the chromosomes whose arm ratio was greater than 2 was 50 % of the total chromosomes; the ratio of the longest chromosome to the shortest one was 2.19, the type of their karyotypes was 2B; the karyotypic asymmetry coefficient (As·k) was 66.60 %; the sixth chromosome, the twelfth chromosome, the thirteenth chromosome and the fourteenth chromosome were m chromosomes, and the remaining chromosomes were sm chromosomes.
Pink Charm: diploid, the somatic chromosome number was 2n = 28; the karyotype formula was: 2n = 2x = 28 = 3m + 10sm + 1st; the relative length range was 4.49 %–9.89 %; the proportion of the chromosomes whose arm ratio was greater than 2 was 42.86 % of the total chromosomes; the ratio of the longest chromosome to the shortest one was 2.20, the type of their karyotypes was 2B; the karyotypic asymmetry coefficient (As·k) was 66.62 %; the tenth chromosome was st chromosome, the eighth chromosome and the fourteenth chromosome were m chromosomes, and the remaining chromosomes were sm chromosomes.
Mary Bcharmon: diploid, the somatic chromosome number was 2n = 24; the karyotype formula was: 2n = 2x = 24 = 5m + 7sm; the relative length range was 5.06 %–10.45 %; the proportion of the chromosomes whose arm ratio was greater than 2 was 41.7% of the total chromosomes; the ratio of the longest chromosome to the shortest one was 2.07, the type of their karyotypes was 2B; the karyotypic asymmetry coefficient (As·k) was 64.56 %; the third chromosome, the sixth chromosome, the seventh chromosome, the tenth chromosome and the eleventh chromosome were m chromosomes, and the remaining chromosomes were sm chromosomes.
Barret Browing: diploid, the somatic chromosome number was 2n = 24; the karyotype formula was: 2n = 2x = 24 = 6m + 6sm; the relative length range was 4.57 %–10.14 %; the proportion of the chromosomes whose arm ratio was greater than 2 was 41.7 % of the total chromosomes; the ratio of the longest chromosome to the shortest one was 2.22, the type of their karyotypes was 2B; the karyotypic asymmetry coefficient (As·k) was 65.73 %; the sixth chromosome, the seventh chromosome, the ninth chromosome, the tenth chromosome, the eleventh chromosome and the twelfth chromosome were m chromosomes, and the remaining chromosomes were sm chromosomes.
Romance: diploid, the somatic chromosome number was 2n = 24; the karyotype formula was: 2n = 2x = 24 = 2m + 10sm; the relative length range was 4.49 %–10.55 %; the proportion of the chromosomes whose arm ratio was greater than 2 was 41.7 % of the total chromosomes; the ratio of the longest chromosome to the shortest one was 2.35, the type of their karyotypes was 2B; the karyotypic asymmetry coefficient (As·k) was 65.64 %; the second chromosome and the tenth chromosome were m chromosomes, and the remaining chromosomes were sm chromosomes.
Dutch Master: diploid, the somatic chromosome number was 2n = 24; the karyotype formula was: 2n = 2x = 24 = 3m + 9sm; the relative length range was 4.52 %–10.38 %; the proportion of the chromosomes whose arm ratio was greater than 2 was 50 % of the total chromosomes; the ratio of the longest chromosome to the shortest one was 2.30, the type of their karyotypes was 2B; the karyotypic asymmetry coefficient (As·k) was 65.95 %; the seventh chromosome, the ninth chromosome and the eleventh chromosome were m chromosomes, and the remaining chromosomes were sm chromosomes.
The karyotype comparison in seven cultivars of Narcissus spp.
Three of seven cultivars, Arkle, Fortissimo and Pink Charm had 28 chromosomes, and remaining four cultivars, Mary Bcharmon, Barret Browing and Romance had 24 chromosomes (Fig., Fig.). As shown in Table , the karyotypes for all seven cultivars were 2B. Based on the karyotype formula, six of seven cultivars consisted of m and sm chromosomes, only Pink Charm had a pair of st chromosome. For six cultivars with m and sm chromosome, Barret Browing is the only cultivar that had an equal number of m chromosome and sm chromosome. And the remaining five cultivars have fewer m chromosomes than sm chromosomes. Moreover, the ratios of the longest chromosome to the shortest one were different among seven cultivars, which range around 2.07-2.53 (Table ). The asymmetric coefficient of seven cultivars could be sorted order from high to low as: Pink Charm > Fortissimo > Arkle > Dutch Master > Barret Browing > Romance > Mary Bcharmon. However, we did not observe the secondary constriction and the satellites in these seven cultivars of Narcissus spp.
Fig. 1. Chromosomes of seven cultivars A: Arkle (2n = 28); B: Fortissimo (2n = 28); C: Pink Charm (2n = 28); D: Mary Bcharmon (2n = 24); E: Barret Browing (2n = 24); F: Romance (2n = 24); G: Dutch Master (2n = 24). The label length was 10μm of the lines in the graphs.
Fig. 2. Karyotype of seven cultivars. A: Arkle (2n = 2x = 28 = 3m + 11sm); B: Fortissimo (2n = 2x = 28 = 4m + 10sm); C: Pink Charm (2n = 2x = 28 = 3m + 10sm + 1st); D: Mary Bcharmon (2n = 2x = 24 = 5m + 7sm); E: Barret Browing (2n = 2x = 24 = 6m + 6sm); F: Romance (2n = 2x = 24 = 2m + 10sm); G: Dutch Master (2n = 2x = 24 = 3m + 9sm).
Table 2. The karyotypes comparison in seven cultivars of Narcissus spp.
Discussion
The genetic relationship and classification of Narcissus spp. remained unresolved, which prohibited our further studies of the genetic transformations and their application to cultivation. From our karyotypic analysis, we observed that all seven cultivars were diploid with karyotype 2B. The chromosome has three types as m, sm, and st. sm and m chromosomes appeared in six of seven cultivars and st chromosome only found in cultivar Pink Charm. The length of long arms and the short arms of seven Narcissus spp. were not significantly different and therefore the chromosomes of Narcissus appear almost symmetrical. We observed that three of seven cultivars (Arkle, Fortissimo and Pink Charm) have the same chromosome number as 2n = 28 and the remaining four cultivars (Mary Bcharmon, Barret Browing, Romance and Dutch Master) contain identical chromosome number as 2n = 24. This result indicated that the seven Narcissus spp. cultivars fell into two clusters: the first sister cluster contains Arkle, Fortissimo and Pink Charm; and the second cluster includes Mary Bcharmon, Barret Browing, Romance and Dutch Master. However, the relationship within each cluster remains unclear and needs further investigation. Moreover, the number of chromosomes for these seven cultivars were 28 and 24, which is consistent with previous reports（Brandham Citation1992; González-Aguilera et al. Citation1990). In contrast with previous studies, we did not observe satellites in any of the seven cultivars. This could be explained by the fact that either satellites did not exist in these seven cultivars or they were technically difficult to be detected and need further investigation.
In conclusion, karyotypic analysis combined with morphological enable would enable us to understand better the classification and the taxonomic relationship between Narcissus spp. species. In the meanwhile, the karyotype analysis also provides the basis to select hybrid parents and identify the hybrid offsprings in these seven cultivars of Narcissus spp. for future studies and cultivation.
Acknowledgements
We are grateful to Ms. Feng-xia Luo for providing bulbs of the seven Narcissus spp. species for our experiments. This work was supported by Shenyang City Scientific and Technological project（1071146-3-00）to X.S.
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