Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(4): 111-121, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1721 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Narges Firoozi, Ghasem Karimzadeh, Mohammad Sadegh Sabet, Vahid Sayadi (2022). Intraspecific karyomorphological and genome size variations of in vitro embryo derived Iranian endemic Asafoetida (Ferula assa-foetida L., Apiaceae). Caryologia 75(4): 111-121. doi: 10.36253/caryolo- gia-1721 Received: June 29, 2022 Accepted: December 06, 2022 Published: April 28, 2023 Copyright: © 2022 Narges Firoozi, Ghasem Karimzadeh, Mohammad Sadegh Sabet, Vahid Sayadi. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits 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. Intraspecific karyomorphological and genome size variations of in vitro embryo derived Iranian endemic Asafoetida (Ferula assa-foetida L., Apiaceae) Narges Firoozi, Ghasem Karimzadeh*, Mohammad Sadegh Sabet, Vahid Sayadi Department of Plant Genetics and Breeding, College of Agriculture, Tarbiat Modares Uni- versity, Tehran P. O. Box 14115-336, Iran *Corresponding author. E-mail: karimzadeh_g@modares.ac.ir Abstract. Asafoetida (Ferula assa-foetida L.) is one of the endemic medicinal plants in Iran. Analysis of karyomorphology and 2Cx DNA measurements (monoploid genome size) of 18 Iranian endemic Ferula assa-foetida populations were performed. The in vitro embryo-derived root tips were examined for karyological studies, via technique of squash and stain with 2% (w/v) aceto-orcein. Seeds of the Ferula samples and leaves of Solanum lycopersicum as standard reference (2C DNA = 1.96 pg) were stained with propidium iodide (PI), using flow cytometric (FCM) technique. All the studied popu- lations were diploids (2n = 2x = 22) with mean chromosome length (CL) of 3.95 μm, varied from 3.05 μm (P7) to 4.94 μm (P18). The mean total chromosome volume (TCV) was 0.98 μm3, ranged from 0.47 μm3 (P7) to 1.57 μm3 (P3). Two-typed chromo- somes (“m”, “sm”) formed three classes of karyotype formula. Karyotypes were mostly symmetrical and fell in 1A and 2A Stebbins category. The monoploid genome size of Iranian endemic Ferula assa-foetida populations is being stated for the first time; its mean value was 4.51 pg, ranged from 4.09 (P4) to 4.69 pg (P16). Intraspecific kary- omorphological and genome size variations were clearly confirmed in studied Ferula assa-foetida. Keywords: Chromosome, Ferula assa-foetida, Flow cytometry, Karyotype, 2Cx DNA. 1. INTRODUCTION Ferula assa-foetida belongs to Apiaceae family that grows in Iran, Kash- mir in Pakistan, and Afghanistan. Asafoetida production from Ferula assa- foetida as a source is confined to southern Iran (Farhadi et al. 2019; Barzegar et al. 2020). Iranian flora consists of 30 species of Ferula, most of which are endemic (Khajeh et al. 2005; Farhadi et al. 2019). It is herbal and permanent and enlarges to 2 cm high (Khajeh et al. 2005). Apiaceae family had very low germination owing to seed dormancy (Nadjafi et al. 2006). Hence, the ger- mination of Ferula’s seeds was complicated. To accelerate the breakage of 112 Narges Firoozi et al. its seed dormancy, various methods were applied such as soaking with running water and treating with either chilling temperature or GA3 (Keshtkar et al. 2008; Has- sani et al. 2009; Zare et al. 2011). The oleo-gum-resin has got from taking away of the stems or cut off the roots that have a sulfurous smell and bitter taste. Ferula spe- cies, due to its chemical compounds, play a useful role in the treatment of various diseases. The oleo-gum-resin is antiseptic, antifungal, antibiotic, laxative, indiges- tion, antiviral, antidiabetic whooping cough, cramp, infertilitypain, and cancer chemopreventive (Aruna and Sivaramakrishnan 1992; Dehpour et al. 2009; Lee et al. 2009). The attribute properties of these plants have ses- quiterpene coumarins and e few monoterpenes (El- Razek et al. 2001). Terpene coumarins have anti-HIV activity (Zhou et al. 2000). In plant sciences, for a large number of plants in order to DNA content’s screening is used of flow cytometry (FCM) as a powerful and reli- Figure 1. Geographic distribution of sampled Ferula assa-feotida L. on the map of Iran, using ArcGIS 113Intraspecific karyomorphological and genome size variations of in vitro embryo derived Iranian endemic Asafoetida able technique (e.g., Loureiro et al. 2005; Mahdavi and Karimzadeh 2010; Karimzadeh et al. 2010, 2011; Abedi et al. 2015; Tarkesh Esfahani et al. 2020; Zarabizadeh et al., 2022). It mainly focused on cell cycle analysis, meas- urement of nuclear DNA content, and determination of ploidy level. It can be used to determine monoploid and holoploid genome size (Doležel et al. 2003, 2007; Greil- huber et al. 2005(. Furthermore, in plant systematics and plant breeding, karyotypes can make available evi- dence and data for species identification and the study of populations resulting from a cross between individu- als (Anjali and Srivastava 2012). In a study on Ferula assa-foetida, it was found that this plant is diploid with a chromosome number 22, grouping in 2A class accord- ing to Stebbins classification (Zhao et al. 2006). Like- wise, the same chromosome number of 22 was reported by El-Alaoui-Faris et al. (2006) in five different species of Ferula; F. gouliminensis, F. cossoniana, F. tingitana, F. sauvagei, and F. atlantica. Furthermore, in a study conducted in China on two species of F. liacentiana and F. bungeana, the 22-chromosome number was also reported (Qixin and Menglan, 1993). It is significant to note that some studies and reports on F. assa-foetida in outside of its native range have mistakenly identified the species. In other words, some species that produce asa- foetida are often misidentified as F. assa-foetida (Cham- berlain, 1977). Awareness of genetic diversity and the management of genetic resources are considered as the main parts of plant breeding programs. The first step in plant breeding is to understand the genome structure and the germplasm collection (Lee et al. 2021). Taken together, these situations indicate the need for basic investigations especially cytogenetic studies. The key aim of the current survey was to study intraspecific genome size and karyo-morphological variations among 18 F. assa-foetida populations of Iran. 2. MATERIALS AND METHODS Seeds of 18 Iranian endemic populations of Feru- la assa-foetida L. were used for this study. The germ- plasm collection of the Iranian Biological and Resource Center (IBRC), Tehran, Iran from where the seeds were obtained. Geographical description, climatic informa- tion, the population codes used in this study and the gene bank codes are present in Table 1. Since previously reported methods, including chilling temperature and treating GA3, was not satisfactory to achieve good germi- nation, hence, in vitro embryo culture was the best, and the most suitable technique (Zare et al. 2011; Suran et al. 2016). 2.1. In vitro embryo culture At first, seeds were soaked in running water for 24 h sterilized as follows: Table 1. Local information of the collected Iranian endemic Ferula assa-foetida L. Mean rainfall (mm) Mean Temp (ºC) Altitude (m) Longitude (E) Latitude (N) Local collection locations Population code 13.66 20.20 1817 52º 33’ 00.0’’ 33º 27’ 54.0’’ Esfahan, Iran P1 60.52 15.25 2750 51º 26’ 55.3’’ 30º 55’ 49.9’’ Kohkiloyeh Boyerahmad, Iran P2 15.87 20.82 1795 54º 20’ 00.0’’ 29º 12’ 00.0’’ Fars, Iran P3 5.54 22.91 669 56º 55’ 50.6’’ 33º 35’ 39.1’’ Khorasan, Iran P4 4.32 20.42 2158 54º 38’ 23.8’’ 32º 06’ 43.4’’ Yazd, Iran P5 5.08 20.54 1720 54º 09’ 53.6’’ 31º 38’ 13.4’’ Yazd, Iran P6 5.08 20.54 831 55º 38’ 20.4” 33º 06’ 43.2” Yazd, Iran P7 4.32 20.42 2158 54º 38’ 23.6’’ 32º 06’ 43.4’’ Yazd, Iran P8 5.08 20.54 1950 54º 14’ 42.5’’ 31º 38’ 41.7’’ Yazd, Iran P9 5.08 20.54 2160 54º 09’ 53.6’’ 31º 38’ 13.5’’ Yazd, Iran P10 5.08 20.54 3279 54º 05’ 27.0’’ 31º 37’ 41.2’’ Yazd, Iran P11 9.04 20.00 2164 57º 54’ 50.4” 29º 18’ 10.8” Kerman, Iran P12 12.05 17.07 2000 56º 45’ 00.0’’ 30º 48’ 00.0’’ Kerman, Iran P13 9.04 20.00 2200 56º 25’ 00.0’’ 31º 08’ 00.0’’ Kerman, Iran P14 12.05 17.07 1900 57º 07’ 00.0’’ 30º 17’ 00.0’’ Kerman, Iran P15 4.05 21.05 2600 57º 18’ 00.0’’ 30º 30’ 00.0’’ Kerman, Iran P16 11.49 16.67 1850 55º 07’ 57.0’’ 30º 17’ 40.0’’ Kerman, Iran P17 7.04 19.23 2335 56º 60’ 00.0’’ 30º 20’ 20.4’’ Kerman, Iran P18 114 Narges Firoozi et al. · Wash the seeds with five drops of washing liquid for 2 min. · Seeds placement under running water for 5 min. · Seeds placement in ethanol 70% for 2 min. · Wash seeds with distilled water for 5 min. · Seeds placement in sodium hypochlorite 5.25 (v/v) for 20 min. · Wash seeds with dsH2O three times and each time for 2 min in laminar airflow. After this step, the embryos were excised by push the bottom of seeds and were transferred to Murashige and Skoog medium (Murashige and Skoog 1962). The embryos in Petri dishes were placed in an incubator at photoperiod with a 16 h light/8 h dark and 25 °C. It was germinated after three to four days. 2.2. Karyomorphological analysis For the preparations of karyology, actively around one cm-long in vitro growing roots were removed and pre-treated in colchicine (0.05% (w/v)) for 4 h in the dark at 4 °C to impel metaphase arrest. After that, the roots were subsequently fixed in freshly Carnoy’s fixa- tive (3 absolute ethanol: 1 glacial acetic acid (v/v) ratio) at 4 °C for 24 h (Karimzadeh et al. 2010, 2011). Using distilled water, the fixed roots were washed then in a water bath its hydrolyzed in 1M Hydrochloric acid (HCl) for 10 min at 60 °C, after these steps, staining was per- formed for 3 h with aceto-orcein (2% (w/v)) at 25 °C (Karimzadeh et al. 2011). The five well-expand monolay- er metaphase plates from various individuals were exam- ined per Ferula assa-foetida populations. Photographs were taken in high resolution via a light microscope (BX50 model, Olympus Optical Co., Ltd., Tokyo, Japan) armed with an digital camera (DP12, Olympus Optical Co., Tokyo, Japan). Eight chromosomal parameters were either inves- tigated as short arm (S) and long arm (L) lengths, or measured as chromosome length (CL = L + S), r-value (S/L), arm ratio (AR; L/S), total chromosome volume (TCV = πr2 CL, where r = average chromosome radius), form percentage (F% = S/SCL), and centromeric index (CI = S/CL). Chromosome types were determined, via Levan et al. (1964) formula and Idiograms were drawn from the mean values. The following parameters were also assessed for analysis karyotype asymmetry: the difference of range relative length (RL%Max - RL%Min), karyotype total form percentage (TF% = ΣS/ΣCL × 100), dispersion index (DI = (Mean CI × CVCL) /100), mean centromeric asymmetry (MCA), coefficient of variation of chromosome length (CVCL) (Paszko 2006; Peruzzi et al. 2009), Romero-Zarco (1986) intrachromosomal (A1) and interchromosomal (A2) asymmetry indices; and Stebbins asymmetry categories for the investigation of karyotype asymmetry were assessed (Stebbins 1971). 2.3. Flow cytometric analysis For each Asafetida population, the monoploid 2Cx- value was calculated through Flow cytometric analy- sis. Hence, to prepare nuclear suspensions, four seeds (Jedrzejczyk and Sliwinska, 2010) of each Asafetida sam- ple along with the healthy fresh young leaves of Solanum lycopersicum cv. Stupicke (2C DNA = 1.96 pg; Doležel et al., 2007) as the internal reference standard plant were chopped with a sharp razor blade in ice-cold woody plant buffer (WPB, Loureiro et al., 2007). Flow cytomet- ric analysis was carried out via PI (Propidium Iodide) staining method. The nuclei suspension was examined via the f low cytometer (BD FACSCanto II, BD Bio- sciences, Bedford, MA, USA), through BD FACSDivaTM Software. The gained data were transferred to Flowing Software (ver. 2.5.0, Cell Imaging Core, Turku Centre for Biotechnology) to be editable in Partec FloMax software (ver. 2.4e, Partec, Münster, Germany). The range of gat- ing zone was calculated on obtained histograms from FCM, via the FloMax. Healthy fresh young leaves of the Asafetida sample’s seeds and the standard reference were chopped, using a sharp razor blade. this action performed in ice-cold nuclear extraction buffer WPB (Woody Plant Buffer, Loureiro et al. 2007). The chopped seeds and leaves were filtered via a 30 μm green nylon mesh (Partec, Münster, Germany). One ml of staining buffer, 50 µg ml-1 of PI (Fluka) solution and 50 µg ml-1 of RNase (Sigma-Aldrich Corporation, MO, USA) stock solution were added to each sample. For the stained nuclei, the relative fluorescence intensity was calculated via FCM on a linear scale. For each sample, flow cyto- metrically minimum 5000 nuclei were evaluated. The values of the means of G1 peak were used to estimate the absolute DNA amount of the sample (Doležel et al. 2003, 2007; Greilhuber et al. 2005; Doležel and Bartoš 2005; Mahdavi and Karimzadeh 2010; Karimzadeh et al. 2011) as follows: Sample 2CX DNA (pg) = (Sample G1 peak mean/Stand- ard G1 peak mean) × Standard 2C DNA (pg). Monoploid genome size was estimated based on above converting formula in the form of base pair (Doležel et al. 2003). It should be noted that one pg of DNA equivalent to 978 mega base pairs (Mbp) were con- sidered (Doležel et al. 2003). 115Intraspecifi c karyomorphological and genome size variations of in vitro embryo derived Iranian endemic Asafoetida 2.4. Statistical analysis In First normality test was carried out for the obtained data from karyotypic and f low cytometric studies and then were investigated for karyological data in fi ve replications and for nuclear DNA content three replications based on a completely randomized design (CRD). Differences between means were measured through the least signifi cant diff erence (LSD). Minitab 17 soft ware package was used for multivariate statistical analysis. principal components analysis (PCA) was then carried out based on data matrix to evaluate the contri- bution of each karyotypic parameter to the ordination of populations. A cluster analysis on chromosomal param- eters was carried out, through the Ward’s method and the Euclidean distance to assess similarities and varia- tions amid the populations. 3. RESULTS All 18 Iranian endemic populations of Ferula assa- foetida were diploid (2n = 2x = 22). Obtained karyotypes from somatic complement and the haploid comple- ment’s idiograms of studies F. assa-foetida populations are demonstrated in fi gures 2 and 3, respectively. Based on the ANOVA results, among populations for either all chromosomal parameters, or monoploid genome size were signifi cant diff erences (P < 0.01; 2Cx DNA; Table 2). Th e mean chromosome length (CL) was determined as 3.95 μm, varied from 3.05 μm (P7) to 4.94 μm (P18). Th e mean CI of the complement, varied from 41.15% (P18) to 45.57% (P1). Th e mean TCV was 0.98 μm3, ranged from 0.47 μm3 (P7) to 1.57 μm3 (P3). According to numerous karyotypic symmetrical indices tested, F. assa-foetida populations indicated various symmetrical clusters. Th e maximum value of TF% was recognized in P1 (45.3%) and the lowest value was identifi ed in P14 (41.3%). Th e uppermost and the lowermost values of the various range of relative length (DRL) were detected in P8 (4.79%; the most asymmetric) and P1 (2.25%; the most symmetric), respectively. Th e highest and lowest values of CVCL %were identifi ed in P8 (15.53%; the maxi- mum asymmetric) and P1 (7.17%; the maximum sym- metric), respectively. Similar to the results of DRL and P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 Figure 2. Karyotypes of somatic chromosome complement of 18 Iranian endemic Ferula assa-foetida L. (n = 2x = 22) populations. Scale bars = 5 µm. Ch r omosome leng th (µ Chr oosome leng th (µ P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 om P18 Figure 3. Idiograms of haploid chromosome complement of 18 Ira- nian endemic Ferula assa-foetida L. (2n = 2x = 22) populations. 116 Narges Firoozi et al. CVCL%, the highest value of dispersion index (DI) was detected in P8 (0.07; the most asymmetric), while P1 dis- played the lowest (0.03; the most symmetric). In conclu- sion, three (DRL, CVCL% and DI) among five karyotypic symmetrical indices examined, confirmed that all 18 F. assa-foetida populations examined P8 and P1 performed to have the most asymmetrical and symmetrical karyo- types, respectively. The highest value of MCA was identi- fied in P14 (18.75 %) while P1 demonstrated the lowest value (9.1 %). Two-typed chromosomes were recognized in all populations by using Levan et al. (1964) chromo- some nomenclature: “m” (centromere at medium region) and “sm” (centromere at sub medium region); formed 3 different karyotypic formulas as follows: 22m (nine populations), 20m+2sm (six populations) and 18m+4sm (three populations; Table 3). Karyotypes of all popula- tions were ordered in the 1A and 2A classes of Stebbins classification (Stebbins 1971). For further detailed stud- ies of asymmetry, A1 and A2 indices were also calculated (Romero-Zarco, 1986). The highest A1 = 0.30 was found in P14, showing the highest inter chromosomal differ- ence, resulted in asymmetric karyotype and the lowest A1 = 0.16 is related to the P1 which demonstrations the highest symmetry. The highest A2 = 0.16 was related to Table 2. ANOVA of chromosomal parameters (a) and monoploid genome size (2Cx DNA; b) of Ferula assa-foetida L. S.O.V. Df MS S L CL AR r-value F% TCV CI a) Chromosomal parameters Population 17 12.58** 0.19** 0.18** 2.35** 2.42** 1.85** 20.12** 2.32** Error 972 0.79 0.005 0.005 0.97 0.97 0.98 0.66 0.97 b) Monoploid genome size (2Cx DNA) S.O.V. Df 2Cx DNA Population 17 0.066** Error 36 0.004 ** Significant difference (P < 0.01). Table 3. Karyotypic parameters of Ferula assa-foetida L. (2n = 2x = 22). Populations Stebbins’ category Karyotype formula CVCL% DI DRL% TF% MCA Asymmetry Indices (Romero-Zarco, 1986) A2 A1 P1 1A 22m 7.17 0.03 2.25 45.30 9.00 0.07 0.16 P2 1A 20m+2sm 13.78 0.06 4.21 43.54 14.11 0.14 0.23 P3 1A 22m 14.45 0.06 4.40 43.34 14.26 0.14 0.24 P4 1A 20m+2sm 10.88 0.05 3.27 43.26 14.24 0.11 0.24 P5 1A 22m 13.16 0.06 3.89 43.21 13.48 0.13 0.23 P6 1A 22m 12.79 0.05 4.03 42.61 15.58 0.13 0.26 P7 1A 22m 14.01 0.06 4.29 42.97 14.92 0.14 0.24 P8 1A 22m 15.53 0.07 4.79 42.95 14.34 0.16 0.24 P9 1A 22m 13.71 0.06 4.22 43.05 14.05 0.14 0.23 P10 1A 22m 11.65 0.05 3.65 42.67 14.89 0.12 0.25 P11 1A 20m+2sm 12.86 0.05 3.99 43.06 15.31 0.13 0.25 P12 1A 20m+2sm 14.94 0.06 4.48 41.78 17.14 0.15 0.27 P13 2A 18m+4sm 13.78 0.06 4.24 41.50 17.59 0.14 0.28 P14 1A 18m+4sm 13.73 0.06 4.07 41.31 18.75 0.14 0.30 P15 1A 22m 14.94 0.06 4.62 42.67 13.03 0.15 0.22 P16 2A 20m+2sm 11.67 0.05 3.43 42.58 16.06 0.12 0.26 P17 1A 20m+2sm 13.37 0.06 4.12 41.39 17.25 0.13 0.28 P18 1A 18m+4sm 13.92 0.06 4.10 41.67 17.69 0.14 0.29 117Intraspecifi c karyomorphological and genome size variations of in vitro embryo derived Iranian endemic Asafoetida P8; they have the highest chromosomal asymmetry and the lowest A2 = 0.07 was related to P1, having the highest intra chromosomal symmetry (Table 3). The scatter diagram of these indices (Figure 4) shows fi ve groups of populations. Th e principal compo- nent analysis of the karyotypic parameters was carried out to estimate total variation and its parameters quota in populations. Th e PCA representing that the fi rst three principal components account for 99% of the cumula- tive variation, and they were plotted in a 2-dimensional graphic (Figure 5). Th e Ward (Khodadadi et al. 2014) phenogram constructed based on karyotype similarities (Figure 6) shows six major clusters. Th e principal com- ponent analysis resulting populations arrangement from this exam entirely fi ts with that obtained with the Ward grouping analysis. Th us, the obtained results suggested that populations within one cluster, having the high homology in chromosomal diff erences. The result of FCM data was confirmed for nor- mality and analyzed based on completely randomized design (CRD) with three replicate cells. ANOVA showed among- populations high signifi cant diff erence (P < 0.01) for monoploid genome size (2Cx DNA content; Table 2). Th e mean value was 4.51 pg (Table 4), varied from 4.09 pg in P4 to 4.69 pg in P16. Th e histograms obtained for 27 A 2 A1 Figure 4. Scatter plot of intrachromosomal (A1) and interchromo- somal (A2) asymmetries of 18 Iranian Ferula assa-foetida L. popu- lations 28 First component S ec on d c om p on en t Figure 5. Diagram resulting from principal components analysis (PCA) of Ferula assa-foetida L. populations. 29 Observations D is ta n ce Figure 6. Dendrogram showing the phenetic relationships among the studied populations of Ferula assa-foetida L. populations. Table 4. Mean (± Se) comparisons of monoploid genome size (2Cx DNA) of Iranian endemic Ferula assa-foetida L. (2n = 2x = 22). 1Cx DNA (Mbp) 1Cx DNA (pg) Mean 2Cx DNA (pg) ± Se Population 2166.27 2.215 4.43 ± 0.075efg P1 2146.71 2.195 4.39 ± 0.047fg P2 2122.26 2.170 4.34 ± 0.006g P3 2000.01 2.045 4.09 ± 0.042h P4 2210.28 2.260 4.52 ± 0.036bcde P5 2185.83 2.235 4.47 ± 0.010def P6 2259.18 2.310 4.62 ± 0.035ab P7 2176.05 2.225 4.45 ± 0.049defg P8 2244.51 2.295 4.59 ± 0.012abc P9 2273.85 2.325 4.65 ± 0.015a P10 2190.72 2.240 4.48 ± 0.060cdef P11 2176.05 2.225 4.45 ± 0.025defg P12 2234.73 2.285 4.57 ± 0.019abcd P13 2229.40 2.280 4.60 ± 0.038abc P14 2244.51 2.295 4.59 ± 0.026abc P15 2293.41 2.345 4.69 ± 0.030a P16 2283.63 2.335 4.67 ± 0.019a P17 2268.96 2.320 4.64 ± 0.056ab P18 2000.01-2293.41 2.045-2.345 4.09-4.69 Range Means with the same symbol letter in a “Mean 2Cx DNA (pg)” col- umn are not signifi cantly diff erent (P > 0.01), using LSD test. 118 Narges Firoozi et al. analyzing nuclear DNA amount included two peaks (Figure 7): the left peaks refer to the G1 of Solanum lycopersicum cv. Stupicke as reference standard plant (Doležel et al. 2007) and the right peaks to the G1 of F. assa-foetida samples. In other words, using 1Cx DNA monoploid genome size in Mbp, the mean value of all populations was 2205.39 Mbp (Table 4). 4. DISCUSSION Ferula assa-foetida belongs to Apiaceae fam- ily includes about 170 identifi ed taxa, of which 30 spe- cies have been detected in different phytogeographi- cal regions in Iran (Zomorodian et al., 2018). It is an important perennial herb with medicinal benefi ts which is native to central Asia and Iran (Farhadi et al. 2019). Many pharmaceutical properties and medical benefi ts had been reported for Ferula (Dehpour et al. 2009; Lee et al. 2009). For increasing the potential applicability of this genus, this perennial plant still needs more investi- gation on the genetic variability, expanding the range of research on its genetic characteristics as well as develop breeding methods. In the current study, we studied the Iranian endem- ic Ferula assa-foetida populations. Eighteen Iranian endemic populations of Asafoetida (F. assa-foetida L.) were cytogenetically assessed on the basis of karyo- morphology and genome size in the current study; they were all diploids (2n = 2x = 22). Mostly, in Ferula genera the diploids are more frequent and obtained results in the present study are in agreement with those reported on Ferula assa-foetida (Zhao et al. 2006) and on other species of Ferula (Wanscher 1931; Qixin and Menglan 1993; Ghaff ari et al. 2005; El-Alaoui-Faris et al. 2006; Sağiroğlu and Duman 2006; Bernard et al. 2007). Th e obtained results of ANOVA show a signifi cant diff erence in terms of all chromosomal traits. (Table 2), confi rm- ing intraspecifi c karyomorphological diversity in the studied Iranian endemic Asafoetida populations, which would be benefi cial for the success of the breeding pro- grams. The mean chromosome length (CL) was 3.95 μm, varied from 3.05 μm (P7) to 4.94 μm (P18). In other words, a remarkable 1.89 μm variation in chromosome size was detectable among such a number of examined Iranian endemic Asafoetida. In the present investiga- tion two-typed chromosomes (“m”, “sm”) formed three classes of karyotype formula, comprised: 22m for nine populations (P1, P3, P5- P10, P15), 20m+2sm for six populations (P2, P4, P11, P12, P16, P17(, and 18m+4sm for three populations (P13, P14, P18). Zhao et al. (2006), studying on Ferula fukanensis, similarly reported the two-typed chromosomes of “m” and “sm”. Th e studied karyotypes were grouped in the 1A and 2A classes based on classifi cation of Stebbins. In a study on Ferula fukan- ensis, karyotypes were classifi ed as 2A (Zhao et al. 2006), which was consistent with the results of the current study in the P13 and P16 populations. It has been alleged that symmetric karyotypes have a lower grade of evolu- tion in comparison with asymmetric karyotypes (Steb- bins 1971). Understanding taxon evolution, and interre- lations are facilitated through the information obtained from karyotype, and chromosome morphology (Furo et al. 2020; Sayadi et al. 2021). In the current investiga- tion, the scatter diagram shown the populations in fi ve groups that exactly fi ts with the principal component analysis resulting species arrangement. Furthermore, the results noted that populations inside a cluster have the maximum homology of chromosomes. According to Number of n uclei P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 Relative nuclear DNA content (a. u.) Figure 7. Flow cytometric histograms of monoploid genome size (2Cx DNA (of 18 Iranian endemic Ferula assa-foetida L. popula- tions. Th e left peaks refer to G1 of the Solanum lycopersicum cv. Stupicke (2C DNA = 1.96 pg) reference standard and the right peaks refer to G1 of Ferula assa-foetida L. samples. 119Intraspecific karyomorphological and genome size variations of in vitro embryo derived Iranian endemic Asafoetida karyotypes studies, the most fertile offspring can be pro- duced by crossing the populations having the maximum chromosomal homology. The results of principal compo- nent analysis, and cluster analysis for the chromosomal traits, it is possible to introduce populations in groups 2 and 3 due to the shortest distance, to intersect and pro- duce maximum fertile offspring. Accordingly, crossing between populations in a cluster is recommended, for instance between P13 with P14, P16, or P17, and also P12 with P18. The karyotypes in the primitive species are usually highly symmetrical, but that is not necessarily the case. In other words, a distinct and more evidence is ever required to evaluate the direction in changes of karyotype (Peruzzi and Eroǧlu 2013). It has been noted that the evolutionary relationships via asymmetry indi- ces usage for the establishment may not be straightfor- ward. It can be stated that the genus diversity may have resulted from the structural changes. Some differences in asymmetry indices and karyotype formula between species may have contributed to this diversity (Seijo and Fernandez 2003). As stated in the Stebbins (1971) classification, the karyotypes were mostly symmetric (Table 3). In the pre- sent study, to achieve greater measurement accuracy, additional parameters were also assessed in addition to Stebbins asymmetry categories, including TF%, DI, CVCL, MCA, A1, and A2 asymmetry indices for karyotype asymmetry analysis (Table 3). Some have argued and suggested that Stebbins’ (1971) classification as a qualita- tive method is not so strong and lower flexible regarding the types of conclusions it can provide (Paszko, 2006). The average of DI% was 5.6% (from 3% in P1 to 7% in P7). All of the populations were symmetric based on TF% parameter, that the mean value was 42.71%, rang- ing from 41.3% (P14) to 45.3% (P1). Some chromosomal disorders are probably a factor of gradual changes in the amounts of TF%. The appearance changes in the chro- mosomes morphology were happens due to the various causes such as translocated or duplicated chromosomes (Das et al. 1998). When the populations are similar in Stebbins classification, estimates A1 and A2 parameters of Romero-Zarco (1986) to determine the further asym- metric karyotype are necessary. As A1 index gets lower in P1 represents karyotype symmetry and a higher value in P14 assessed greater asymmetry. The A2 parameter was 13.22%, ranging from 7% (P1) to 16% (P8). Prevent- ing interspecific cross accomplishment and offspring infertility may have ensued from the difference in result- ing karyotype symmetry. Obvious intraspecific diversity was also detected in terms of monoploid genome size (2Cx DNA) among examined Iranian endemic Asafoetida populations. Hence, on the other hand, the 2Cx DNA amounts of 18 Iranian endemic F. assa-foetida L. populations are being reported for the first time, having the mean value of 4.51 pg, varied from 4.09 (P4) to 4.69 pg (P16). In other words, in our knowledge, few reports were found in the literature for the genome size estimation even in other Ferula species. For example, 2.90 pg was reported for 2C DNA amount of F. communis by Olmedilla et al. )1985) and 4.92 pg in F. heuffelii by Siljak-Yakovlev et al. (2010). Changes in the genome size (increases or decreases) may have participated in the genus diversity and evolution (Seijo and Fernandez 2003). 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