Regular Article
Mixed-ploidy in Iranian endemic Cymbopogon olivieri (Boiss.) Bor: A chromosomal and holoploid genome size study
2024 Volume 89 Issue 2 Pages 127-131
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2024 Volume 89 Issue 2 Pages 127-131
Cymbopogon olivieri is one of the important species belonging to the family Poaceae. The natural habitat of this plant is tropical regions. In particular, it is distributed in tropical and subtropical regions of Africa, Asia, and America. Two species, C. olivieri and C. parkeri have been reported in Iran. The genus has been proven to possess antioxidant, antimicrobial, and antibacterial properties. Taxonomic identification and species classifying of Lemongrass is very difficult due to the ease of cross-breeding and the existence of a large number of species and races. This study aimed to study variations and clustering of 10 Iranian endemic populations of C. olivieri (Boiss.) Bor based on chromosomal and holoploid genome size features. The results of karyotype analysis clearly showed the mixed-ploidy. Among which, six populations were diploids (2n=2x=20), three tetraploids (2n=4x=40), and a hexaploid (2n=6x=60). The mean chromosome length (CL) was determined as 1.87 µm for diploids, 1.73 µm for tetraploids, and 1.25 µm for a hexaploid. The average holoploid genome sizes of the diploids, tetraploids, and a hexaploid were 4.42, 4.64, and 4.91 pg, respectively. These results may provide relevant information for Cymbopogon breeding studies.
Cymbopogon olivieri is one of the important plant species belonging to the family Poaceae with stiff, upright stem, and short rhizomes (Ganjewala and Gupta 2013). The natural habitat of this plant is tropical regions. In particular, it is distributed in tropical and subtropical regions of Africa, Asia, and America. In sources, up to 56 species and 120 varieties for the Cymbopogon genus have been revealed (Mohammadi and Mansouri 2011). In some reports, up to 120 species were reported (Akhila 2009). Two species, C. olivieri and C. parkeri were reported in Iran (Bor 1970; Weiss 1997). Taxonomic identification and species classifying of C. olivieri is very difficult due to the ease of cross-breeding and the existence of a large number of species and races (Bor 1960). Piperitone was the major composition of the essential oil Cymbopogon olivieri (Sonboli et al. 2010; Mahboubi and Kazempour 2012). In traditional medicine, the leaves and roots of Cymbopogon olivieri are widely used as antiseptics and to treat stomach diseases (Zargari 1995). Furthermore, this genus has been proven to possess antioxidant, antimicrobial, and antibacterial properties (Chalabian and Moosavi 2003; Kazempour 2012). Data obtained from the karyotypes can be used for many purposes in plant breeding such as the identification of species and the population study resulting from a cross between individuals (Anjali and Srivastava 2012). Moreover, one of the reliable techniques to screen DNA content in plant is the use of flow cytometry (FCM) (e.g., Karimzadeh et al. 2010, 2011; Tarkesh Esfahani et al. 2020; Abbasi-Karin et al. 2022; Firoozi et al. 2022; Mehravi et al. 2022; Zarabizadeh et al. 2022; Sayadi et al. 2022; Khakshour et al. 2024). In a study on Cymbopogon, it was found that these genera have different ploidy levels, ranging from diploid (2n=20) to tetraploid (2n=40), and hexaploid (2n=60) (Babu 1936; Bor 1953). Thakur et al. (2021) reported that the four varieties of Cymbopogon they studied were diploid with a chromosome number (2n=20). For C. flexuosus, despite the similarity in the ploidy level, FCM studies showed differences in genome size between the studied genotypes (2.77 bp and 2.26 bp; Jagadeeshan et al. 2022). In plant breeding programs, knowledge of genetic diversity and the management of genetic resources are essential (Lee et al. 2021). This study aimed to study the possible mixed-ploidy and clustering of 10 Iranian endemic populations of C. olivieri based on chromosome length and holoploid genome size features.
Ten populations of Cymbopogon olivieri (Boiss.) Bor were collected from the natural habitats in various geographic locations of Iran as described in Table 1. For chromosome analysis, pretreatment, fixation, and microscopy were performed according to Sayadi et al. (2021). The fixed roots were hydrolyzed in 1 M HC1 at 60°C for 12 min in a water bath, followed by staining by 4% (w/v) hematoxylin for 4 h in darkness at room temperature. Following chromosome counting, the measurement of chromosome length (CL) was conducted using a MicroMeasure 3.3 software (Reeves 2002).
| Population codes | Local collection locations | Latitude (N) | Longitude (E) | Altitude (m) |
|---|---|---|---|---|
| P01 | Hormozgan, Bandar Abbas | 27°26′11.9″ | 56°59′26.8″ | 180 |
| P02 | Hormozgan, Bandar Abbas | 27°29′36.8″ | 56°56′02.9″ | 153 |
| P03 | Hormozgan, Bandar Abbas | 27°28′02.2″ | 56°21′06.3″ | 126 |
| P04 | Hormozgan, Bandar Abbas | 27°29′52.6″ | 56°15′51.6″ | 200 |
| P05 | Hormozgan, Bandar Abbas | 27°49′26.3″ | 56°26′43.1″ | 1,080 |
| P06 | Hormozgan, Bandar Abbas | 27°46′44.8″ | 56°24′56.0″ | 746 |
| P07 | Hormozgan, Bandar Abbas | 27°41′13.1″ | 56°14′34.1″ | 463 |
| P08 | Hormozgan, Bandar Abbas | 27°46′44.0″ | 56°04′04.2″ | 688 |
| P09 | Hormozgan, Bandar Abbas | 28°00′25.5″ | 55°54′23.6″ | 764 |
| P10 | Hormozgan, Bandar Abbas | 28°12′42.5″ | 55°52′37.7″ | 868 |
FCM analysis was performed according to Sayadi et al. (2022). Glycine max cv. Polanka (2C DNA=2.50 pg) was used as an internal reference standard plant. To estimate the holoploid genome size, a flow cytometer BD FACSCantoTM-KE (BD Biosciences, Bedford, MA, USA) with an argon ion laser (488 nm) was employed. A BD FACSDiva™ software was utilized for the analysis of the raw data (Mehravi et al. 2022) and the gating of histograms was carried out, using a Partec FloMax ver. 2.4e. (Partec, Münster, Germany). Statistical analyses were conducted using a completely randomized design with 5 replications for chromosome length data and 3 replications for holoploid genome size. Values of the G1 peak means represent the absolute DNA amount of a sample (Doležel et al. 2003). Normality tests of data and analysis of variance (ANOVA) were conducted via the PROC GLM of SAS (SAS Institute Inc 2009). Mean comparisons were conducted using Fisher’s least significant differences (LSD).
The results of ANOVA showed significant differences in chromosome length and holoploid 2C DNA within each ploidy level (Table 2). The obtained results from the karyotype analysis of the 10 populations studied (C. olivieri) showed clear mixed-ploidy. Among those, 6 populations were diploids (2n=2x=20), 3 tetraploids (2n=4x=40), and 1 hexaploid (2n=6x=60; Fig. 1). The ploidy levels in Cymbopogon are different, such that diploid (2n=20) to tetraploid (2n=40), and hexaploid (2n=60) have been found in this genus (Babu 1936; Bor 1953; Thakur et al. 2021). The obtained results in the present study agree with those reported previously. The mean value of CL was determined as 1.87 µm for diploids, varying from 1.71 µm (P04) to 2.06 µm (P06); 1.73 µm for tetraploids, ranging from 1.68 µm (P09) to 1.81 µm (P07); and 1.25 µm for a hexaploid (P10; Table 3). It can be asserted that the diversity within the genus may have arisen due to structural changes (Seijo and Fernández 2003). The comprehension of taxon evolution and interrelations is enhanced via the information obtained from karyomorphology (Furo et al. 2020; Sayadi et al. 2021). The obtained results of the means comparisons within each ploidy level confirmed clear mixed ploidy and high intraspecific variation in this parameter among C. olivieri populations (Table 2). The mean holoploid genome size of the diploids was 4.42 pg, varying from 3.43 pg (P02) to 5.02 pg (P05). That of tetraploids was 4.64 pg, ranging from 5.41 pg (P07) to 3.54 pg (P09), and that of a hexaploid was 4.91 pg (P10; Table 4). It was reported that 2C DNA average for a tetraploid population of C. flexuosus was 2.77 pg, while that of another population with the same ploidy level (tetraploid) was 2.26 pg (Jagadeeshan et al. 2022). Cymbopogon is a genus containing profound genetic diversity, such that 2C DNA content for C. martini (diploid) was 1.86 pg, C. flexuosus (tetraploid) was 3.28 pg, and C. citratus (tetraploid) was 3.51 pg (Jagadeeshan et al. 2021). Germplasm diversity is essential for plant conservation and improvement (Jagadeeshan et al. 2021; Lee et al. 2021). The genome size can undergo alterations in response to environmental stress, implying that quick processes exist that can change the genome in a few generations even between very closely related organisms (Arnault and Dufournel 1994; Capy et al. 2000; Karimzadeh et al. 2010, 2011; Fablet and Vieira 2011; Abbasi-Karin et al. 2022; Firoozi et al. 2022; Mehravi et al. 2022; Sayadi et al. 2022; Zarabizadeh et al. 2022; Khakshour et al. 2024). The results of the current report may provide relevant information for C. olivieri breeding studies. To increase the potential applicability of this genus, this plant still needs more investigation on the genetic variability, expanding the range of research on its genetic characteristics as well as developing breeding methods.
| S.O.V. | Df | MS | CV% | |
|---|---|---|---|---|
| CL | Population (2x) | 5 | 0.12994** | 10.86 |
| Error | 294 | 0.02174 | ||
| Population (4x) | 2 | 0.06638* | 10.97 | |
| Error | 297 | 0.02063 | ||
| 2C DNA | Population (2x) | 5 | 1.72581** | 3.52 |
| Error | 13 | 0.02446 | ||
| Population (4x) | 2 | 3.41877** | 3.19 | |
| Error | 7 | 0.02092 |
*, ** Significant differences at 5% and 1% probability levels, respectively. S.O.V.: sources of variation, Df: degrees of freedom, MS: mean squares, CV: coefficient of variation.
Specimens were stained in aceto-orcein 2% (w/v) at 25°C for 2 h in darkness. Scale bars=5 µm.
| Population | Ploidy level | 2n | CL (µm) |
|---|---|---|---|
| P01 | 2x | 20 | 1.772b±0.052 |
| P02 | 2x | 20 | 1.774b±0.059 |
| P03 | 2x | 20 | 2.004a±0.057 |
| P04 | 2x | 20 | 1.714b±0.057 |
| P05 | 2x | 20 | 1.889ab±0.052 |
| P06 | 2x | 20 | 2.060a±0.056 |
| P07 | 4x | 40 | 1.818a±0.043 |
| P08 | 4x | 40 | 1.698b±0.037 |
| P09 | 4x | 40 | 1.688b±0.034 |
| Average | 2x | 20 | 1.87±0.024 |
| 4x | 40 | 1.73±0.022 | |
| 6x | 60 | 1.25±0.018 | |
| Total | — | 1.69±0.016 |
Means with the same symbol letters (a and b) within each ploidy level are not significantly different at p＜0.01 (2x) and p＜0.05 (4x), using LSD.
| Populations* | Ploidy level | Holoploid genome size | Monoploid genome size | ||
|---|---|---|---|---|---|
| 2C DNA Mean (pg)±Se | 1C DNA (pg) | 1Cx DNA (pg) | 1Cx DNA (Mbp) | ||
| P01 | 2x | 3.496b±0.009 | 2.42 | 2.42 | 2366.76 |
| P02 | 2x | 3.436b±0.067 | 2.42 | 2.42 | 2366.76 |
| P03 | 2x | 4.853a±0.039 | 2.45 | 2.45 | 2396.10 |
| P04 | 2x | 4.833a±0.197 | 2.68 | 2.68 | 2621.04 |
| P05 | 2x | 5.017a±0.019 | 2.49 | 2.49 | 2435.22 |
| P06 | 2x | 4.896a±0.029 | 1.77 | 1.77 | 1731.06 |
| Average | 4.421 | 2.37 | 2.37 | 2319.49 | |
| P07 | 4x | 5.416a±0.002 | 1.75 | 0.87 | 850.86 |
| P08 | 4x | 4.980b±0.092 | 1.72 | 0.86 | 841.08 |
| P09 | 4x | 3.545c±0.138 | 2.51 | 1.25 | 1222.50 |
| Average | 4.647 | 1.99 | 0.99 | 971.48 | |
| P10 | 6x | 4.910±0.064 | 2.45 | 0.82 | 801.96 |
| LSD1% | 0.0165 | ||||
In “2C DNA” column, means with the same symbol letters (a, b, and c) within each ploidy level are not significantly different at p＜0.01, using LSD.
The authors gratefully acknowledge the support provided for this survey by the Tarbiat Modares University, Tehran, Iran.
Ghasem Karimzadeh verified the methods and supervised the study. Abbas Yari carried out the experiment. Sajad Rashidi Monfared contributed to the implementation of the research, and the analysis of the data. Vahid Sayadi verified the data and wrote the manuscript. All authors discussed the results and contributed to the final manuscript.
