Journal of Plant Physiology and Breeding 2021, 11(1): 97-108 ISSN: 2008-5168 Karyological studies and chromosome variation among Iranian endemic Allium species (Amaryllidaceae) Vahid Sayadi1, Ghasem Karimzadeh1*, Mohammad Reza Naghavi2, and Sajad Rashidi Monfared3 Received: February 10, 2018 Accepted: April 15, 2021 1 Department of Plant Genetics and Breeding, College of Agriculture, Tarbiat Modares University, Tehran P. O. Box 14115-336, Iran 2 Agronomy and Plant Breeding Department, Agricultural College, University of Tehran, Karaj, Iran 3 Department of Agricultural Biotechnology, College of Agriculture, Tarbiat Modares University, Tehran P. O. Box 14115-336, Iran * Corresponding author; Email: karimzadeh_g@modares.ac.ir Abstract One of the largest monocotyledonous genus in the Amaryllidaceae family is the Allium genus that includes approximately 900 species. This study aimed to examine the variations and clustering of eight Iranian endemic Allium species based on karyotype features. The species were collected from wild habitats across different geographical areas of Iran. A. sativum, A. stipitatum, A. fistolosum, A. umbellicatum, A. stamineum, A. lenkoranicum, and A. rubellum, were diploids (2n = 2x = 16), but A. atroviolaceum was triploid (2n = 3x = 24). The results represent x = 8 for basic chromosome numbers in all species. Analysis of variance showed significant interspecific variations for all eight chromosomal parameters tested. The mean of chromosome lengths was 11.19 μm, varied from 8.59 μm to 13.81 μm for A. atroviolaceum and A. stipitatum, respectively. In all species, the chromosome types were determined as mostly metacentric (m) and submetacentric (sm), formed five different karyotype formulas of 16m (A. stipitatum, A. fistolosum, A. stamineum), 14m+2sm (A. sativum, A. rubellum), 12m+4sm (A. lenkoranicum), 10m+6sm (A. umbellicatum), and 24m (A. atroviolaceum). According to Stebbins' classification, all karyotypes were grouped in the 1A class and represented the most symmetrical karyotypes. The information obtained from karyotype and chromosome morphology has an appreciable value in understanding the taxon evolution and interrelations. Keywords: Allium L.; Chromosome; Karyotype asymmetry; Ploidy level; Variation Citation: Sayadi V, Karimzadeh G, Naghavi MR, and Rashidi Monfared S, 2021. Karyological studies and chromosome variation among Iranian endemic Allium species (Amaryllidaceae). Journal of Plant Physiology and Breeding 11(1): 97-108. Introduction diseases (Isaacsohn et al. 1998; Su et al. 2006; The genus Allium is one of the largest Londhe et al. 2011; Kim et al. 2012). Central Asia monocotyledonous genera in the Amaryllidaceae is the main center of Allium diversity, while family that includes approximately 900 species western North America is the secondary center of distributed worldwide (Kusterer et al. 2011; distribution (Etoh and Simon 2002; Garcia- Herden et al. 2016; Sayadi et al. 2020). Allium Lampasona et al. 2003; Friesen et al. 2006; Jabbes such as A. cepa, A. fistulosum, and A. sativum has et al. 2011). The current classifications for the been consumed as foods and/or spices around the genus Allium propose 15 subgenera and 56 world. Furthermore, Allium due to organosulfur sections (Friesen et al. 2006), almost 30 Allium composite such as allicin, plays a beneficial role species, containing numerous endemics growing in the prevention and/or treatment of different in Iran (Wendelbo 1971). In the plant systematics, 98 2021, 11(1): 97-108 Sayadi et al. breeding, and genetic studies, karyotypes can removed and pretreated with 0.002 M 8- provide information for the identification of hydroxyquinoline at 25 °C for 4 h in the dark to species and hybrid populations (Anjali and induce cell cycle delay in metaphase. The roots Srivastava 2012). Different ploidy levels have were washed several times in dsH2O and fixed in been reported for Allium as diploid (2n = 2x = 16), 3:1 (v/v) of ethanol and glacial acetic acid triploid (2n = 3x = 24), and tetraploid (2n = 4x = (Carnoy solution) at 4 °C for 24 h. The fixed roots 32). The basic chromosome number of eight (x = were washed in dsH2O, hydrolyzed in 1 M HC1 at 8) is dominant in most subgenera (Baranyi and 60 °C for 14 min in a water bath, and washed in Greilhuber 1999; Guetat et al. 2015). This study water, then stained by aceto-orcein 2% (w/v) at 25 aimed to examine variations and clustering of °C for 2 h in darkness. Finally, for microscopic eight Iranian endemic Allium species based on studies, the karyotype features. individuals were squashed in a drop of 45% (v/v) five root tips from different acetic acid and analyzed per Allium species. Slides Materials and Methods were examined and microscopic photographs Plant materials were taken using a light microscope (Olympus The bulbs of eight different Allium species, BX50; Olympus Optical Co., Ltd., Tokyo, Japan). including A. sativum, A. stipitatum, A. fistolosum, Chromosomes were counted and its parameters A. umbellicatum, A. lenkoranicum, A. rubellum, A. were measured as long arm (L) and short arm (S) stamineum, and A. atroviolaceum, were collected lengths, chromosome length (CL), r-value (S/L), from wild habitats across different geographical arm ratio (AR = L/S), form percentage of areas of Iran. The collected bulbs are being kept in chromosome the Iranian Biological Resource Center (IBRC) chromosome volume (TCV), and centromeric and used in this study. The species codes and index (CI% = S/CL). TCV was measured for each geographical descriptions are presented in Table species, via r2  CL, where "r" is the average 1. radius of (F% the = S/∑CL), chromosome the total cross-section. Karyotype analysis was performed via the use of Chromosome analysis MicroMeasure 3.3 computer program (Reeves Initially, intact bulbs were placed in Petri dishes 2001). The formula of Levan et al. (1964) was and germinated on moist cotton at 20 - 25 °C in used for the karyotypic formula determination. light conditions in a growth chamber. To induce The following parameters were used for the and synchronize cell division, the 0.5 - 1 cm long karyotype symmetrical evolution: TF%: total form root tips of the bulbs were first physically cold percentage; [(ΣS/ΣCL) ×100]; S%: the relative pretreated at 4 °C for 12 h and then were length of the shortest chromosome; RRL: range of maintained at ambient 25 °C for 45 min. For the relative length (RL% cytological preparations, each root tip was dispersion index [the ratio of centromeric gradient max – RL% min); DI: 99 Karyological studies and chromosome variation among Iranian endemic Allium… (ΣS/ΣCL) ×100) concerning the CV of CL]. CL, arm ratio (AR), r-value, F, TCV, and CI Accordingly, coefficient of (Table 2); their means and ranges are presented in chromosome length Stebbins Table 3. The mean value of CL was 11.19 μm and asymmetry categories was used to estimate the varied from 8.59 μm (S8) to 13.81 lm (S2). The karyotype asymmetry. Likewise, Romero-Zarco mean TCV was 25.09 μm3 and ranged from 14.82 (1986) indices: intrachromosomal asymmetry μm3 (S3) to 39.45 μm3 (S2). The mean of CI% index (A1) and interchromosomal asymmetry was 43% and varied from 39% (S7) to 45% (S2, index (A2) were calculated (Romero-Zarco 1986; S3, S8) (Table 3). The karyotype formula and Stebbins 1971; Paszko 2006; Peruzzi et al. 2009; symmetry information for each of the analyzed Zuo and Yuan 2011; Peruzzi and Eroǧlu 2013). species are presented in Table 4. Karyotypes of all of (CVCL) variation and species were classified as class 1A of Stebbins classification Statistical analyses The experiment Karyotype symmetry was presented through TF%, S%, Initially, CVCL, DI, and RRL (Table 4). The maximum assumptions of normality and homoscedasticity TF% value (45.22%) among species was obtained were verified. Then, the analysis of variance in the species S8 and the lowest value (39.43%) in (ANOVA) was performed through the PROC the species S7, which shows S8 and S7 have the GLM of SAS (SAS Institute Inc. 2009), based on maximum and the minimum symmetry of the the cytological data. Fisher's least significant karyotype, respectively. Since a higher S% value differences method at 0.01 probability level was indicates higher symmetry of the karyotype used for mean comparisons. Moreover, the (Gennur et al. 2011), species S4 and species S6 standard errors of the means were calculated. To with S% values of 65.17% and 50.43%, group the Allium species in this study, cluster respectively had the highest symmetric and analysis and principal component analysis (PCA) asymmetric karyotypes. The RRL values showed were performed based on the chromosomal that species S3 with the highest RRL (8.15%) is parameters, using the Minitab 16 software. asymmetric and the species S4 with the lowest randomized out 1971). using a completely was carried (Stebbins design. amount of this index (5.32%) had the highest Results symmetrical karyotypes (Table 4). Among eight Allium species examined, seven Intrachromosomal asymmetry index (A1) revealed were diploid (2n = 2x = 16). Interestingly, S8 with sharp differences among the chromosome arms the chromosome number 2n = 3x = 24 was triploid across different populations. A1 = 0.35 for species (Figure 1). Karyotypes and the ideograms of S7 represented the most asymmetric karyotype studied Allium species are presented in Figure 1 and species S2 and S8 had the most symmetrical and Figure 2, respectively. ANOVA showed karyotype among all species (A1 = 0.18). significant differences among the species for S, L, According to interchromosomal asymmetry (A2), 100 2021, 11(1): 97-108 Sayadi et al. Table 1. Local information of studied Iranian endemic Allium speci Species IBRC* No. Local collection sites - Bahar, Hamadan, Iran A. sativum A. stipitatum P1010429 A. fistulosum - Latitude (N) Longitude (E) 34 55 48 26 34 36 47 46 35 50 51 50 35 50 51 03 35 57 53 08 38 36 45 47 38 36 44 40 35 48 51 47 Mian Mil, Kermanshah, Iran Ghalea Now-e Ghar, Tehran, Iran A. umbilicatum P1009439 Kangelu, Alborz, Iran A. lenkoranikum P1008766 Chashm, Semnan, Iran A. stamineum P1009946 Marand, Western Azarbaijan, Iran A. rubellum P1009972 Qutor, Western Azarbaijan, Iran A. atroviolaceum P1006775 Lavasan, Tehran, Iran Altitude (m) Code in this study 1722 S1 2526 S2 1189 S3 1340 S4 1570 S5 1543 S6 1524 S7 2113 S8 *Iranian Biological Resource Center Table 2. Analysis of variance for chromosomal parameters of studied Iranian endemic Allium species Mean squares S.O.V. df L S CL AR r-value F% TCV Species 7 Error 32 CV% 5.876 ** ** 3.559 ** 17.420 0.078 ** ** 0.0211 ** 0.372 269.30 ** CI% 0.00243** 0.370 0.188 1.058 0.002 0.0004 0.006 1.77 0.00005 9.52 9.03 9.19 2.97 2.61 1.45 5.30 1.63 **p≤ 0.01; MS: mean squares; SOV: source of variation; df: degrees of freedom; L: long arm length; S: short arm length; CL: chromosome length; AR: arm ratio; r-value: S/L; F%: form percentage of chromosome; TCV: the total chromosome volume; CI%: centromeric index Table 3. Mean and range of chromosomal parameters in Allium species Chromosomal parameters S L CL AR r-value F% TCV CI Mean 4.80 6.39 11.19 1.35 0.75 5.37 25.09 0.43 Range (3.88 – 6.23) (4.70 – 7.57) (8.59 – 13.81) (1.22 – 1.55) (0.65 – 0.82) (4.93 – 5.65) (14.82 – 39.45) (0.39 – 0.45) Species that are related to the range* Low High S8 S2 S8 S2 S8 S2 S8 S7 S7 S2, S8 S7 S8 S3 S2 S7 S2, S3, S8 *A. sativum (S1), A. stipitatum (S2), A. fistulosum (S3), A. umbellicatum (S4), A. lenkoranicum (S5), A. stamineum (S6), A. rubellum (S7), and A. atroviolaceum (S8); S: short arm length; L: long arm length; CL: chromosome length; AR: arm ratio; rvalue: S/L; F%: form percentage of chromosome; TCV: the total chromosome volume; CI%: centromeric index 101 Karyological studies and chromosome variation among Iranian endemic Allium… Table 4. Karyotype formulas and symmetry information for each of the analyzed Allium species Species* Karyotype symmetry method (Levan et al. (Stebbins DI TF% (Romero Zarco 1986) 1964) 1971) A1 A2 7m+1sm 1A 0.26 0.20 0.08 42.53 8m 1A 0.18 0.15 0.07 45.05 8m 1A 0.19 0.21 0.09 44.97 5m+3sm 1A 0.31 0.13 0.05 40.41 6m+2sm 1A 0.27 0.16 0.07 42.39 8m 1A 0.23 0.22 0.10 43.62 7m+1sm 1A 0.35 0.19 0.08 39.43 8m 1A 0.18 0.17 0.08 45.22 sativum (S1), A. stipitatum (S2), A. fistulosum (S3), A. umbellicatum (S4), A. S1 S2 S3 S4 S5 S6 S7 S8 * A. S% RRL% 53.94 7.27 63.17 5.73 51.66 8.15 65.17 5.32 60.77 6.11 50.43 8.10 56.58 7.12 59.86 6.36 lenkoranicum (S5), A. CVCL% 19.89 14.92 21.06 12.79 16.18 22.16 19.09 16.81 stamineum (S6), A. rubellum (S7), and A. atroviolaceum (S8); A1: intrachromosomal asymmetry index; A2: interchromosomal asymmetry index; DI: dispersion index; TF%: total form percentage; S%: relative length of the shortest chromosome; RRL: range of relative length; CVCL: coefficient of variation of chromosome length. species S6 and species S4 had the most PCA indicated that the first two principal symmetrical components accounted for 97% of the total and asymmetrical karyotypes, respectively (Table 4). CV% shows the karyotype variation. The symmetry the projected in a 2-dimensional graph (Figure 3A). chromosomes in a species. In a situation where a The first component was highly related to arm karyotype displays the high uniformity of ratio [AR (L/S); - 0.98%] and the second chromosomes or in other words, the karyotype is component was strongly related to the short arm symmetric, the value of this coefficient is low. (S; - 99%). and differences among first two components were The high CV% indicates the size distribution of chromosomes in the karyotype or, in other words, Discussion the karyotype asymmetry (Venora et al. 1991). We studied eight Allium species from Iran in the Species S4 had the highest CVCL% (22.16%) current work. The analyzed Allium species therefore, this species is asymmetric as compared represents x = 8 for basic chromosome numbers, to other species. The lowest CVCL% (12.79%) that had previously been described for this genus belonged to species S6 that represents a (Baranyi and Greilhuber 1999; Guetat et al. 2015; symmetric karyotype among the examined species Salmasi et al. 2019). All examined species, except (Table 4). S8, were diploid (2n = 2x = 16). A. atroviolaseum Cluster analysis represented the presence of (S8) was triploid (2n = 3x = 24). In the present four groups (Figure 3B). The first group included study, a new ploidy level was reported for A. three species (S1, S5, S6); the second group, only atroviolaceum. Besides, we observed 2n = 3x = 24 S2; the third group, two species (S3, S8); the for A. atroviolaseum, where Miryeganeh (2011) fourth group, two species (S4, S7). The results of reported the chromosome number of 2n = 2x = 16. 102 Sayadi et al. 2021, 11(1): 97-108 Figure 1. Somatic chromosome complements of the studied Iranian endemic Allium species [A. sativum (S1), A. stipitatum (S2), A. fistulosum (S3), A. umbellicatum (S4), A. lenkoranicum (S5), A. stamineum (S6), A. rubellum (S7), and A. atroviolaceum (S8)]. Scale bars = 5 μm. Therefore, the present work offers new evidence tetraploid (Maragheh et al. 2019). In Allium for karyotype variation in section Allium; in genera, the diploids are more frequent and our which, variation in ploidy levels occurs (Jiemei et findings are in agreement with previous reports al. 1998; Zhou et al. 2012; Li et al. 2017). (Paknia and Karimzadeh 2011, Salmasi et al. Different ploidy levels have been reported for 2019). In the current work, satellites were not Allium species, for instance, A. sativum as diploid observed in the chromosomes in karyotypes of all (2n = 2x = 16), A. sphaerocephalon as triploid (2n studied species, while it was seen most often in = 3x = 24), and A. porrum (2n = 4x = 32) as other sections of the genus, are not often evident Karyological studies and chromosome variation among Iranian endemic Allium… 103 Figure 2. Idiograms of the studied Iranian endemic Allium species [A. sativum (S1), A. stipitatum (S2), A. fistulosum (S3), A. umbellicatum (S4), A. lenkoranicum (S5), A. stamineum (S6), A. rubellum (S7), and A. atroviolaceum (S8)] in the section Allium (Fritsch and Astanova Advantages that a polyploid gets from genome 1998). Although polyploidy occurs in various doubling, allow those to grow in challenging plants species and plays an essential role in the conditions for the polyploid’s diploid progenitors. evolution of all angiosperms but the evolutionary The roles of triploids in species diversity have success of a species due to the straight result of been indicated in some plant systems (Husband polyploidy is yet obscure (Madlung 2013). 2004; Chester et al. 2012; Miri 2020). This result 104 Sayadi et al. 2021, 11(1): 97-108 Figure 3. A) Dendrogram showing the phonetic relationships among the studied species of Allium. Constructed using the matrix of karyotype similarities and the complete linkage method (Cophenetic correlation r = 0.89). B) Diagram resulting from the principal component analysis (PCA) of the studied Allium species. The first component was highly related to arm ratio [AR (L/S); -0.98%] and the second was strongly related to the short arm (S; -99%). [A. sativum (S1), A. stipitatum (S2), A. fistulosum (S3), A. umbellicatum (S4), A. lenkoranicum (S5), A. stamineum (S6), A. rubellum (S7), and A. atroviolaceum (S8)] 105 Karyological studies and chromosome variation among Iranian endemic Allium… proposes that they can expedite polyploid because they have the highest homology in their speciation. the chromosomal characteristics. Some differences polyploidy-diploidy coexistence or providing to between species about karyotype formula and genetic by neopolyploids. asymmetry indices suggest that structural changes Accordingly, substantial occasion for divergence could have helped to the genus diversity (Seijo among Allium plants can be provided through and Fernández 2003; Karimzadeh et al. 2010, chromosomal variations (Leitch and Leitch 2008; Karimzadeh et al. 2011). Cytogenetic studies as a Soltis et al. 2014). In general, in the current study, valuable tool have been considerably carried out slightly large and medium-sized chromosomes to investigate the phylogenetic relationships were identified in all Allium species examined, among plants, taxonomy, and diversity for many ranging from 8.59 to 13.81 μm. The mechanisms decades. of karyotype differentiation could well be karyotype and chromosome morphology has been described of appreciable value in understanding taxon It occurs variation via via desired habitat promoting variation, vegetative The information obtained from propagation, and polyploidization (Ao 2008). evolution According all although this study provided suitable information, karyotypes were grouped in the 1A class. It is which can be utilized in Allium breeding, genetics, believed that symmetric karyotypes have a lower and evolutionary studies, more studies, e.g. C- grade of development and evolution compared banding with asymmetric karyotypes (Stebbins 1971). hybridization), are still required to clarify the to Stebbins' classification, The PCA grouping was exactly similar to the and and interrelations. FISH In conclusion, (Fluorescence in situ details. results of cluster analysis. 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Plant Systematics and Evolution 297(2): 141-145. ‫‪2021, 11(1): 97-108‬‬ ‫‪Sayadi et al.‬‬ ‫‪108‬‬ ‫مطالعه کاریولوژی و تنوع کروموزومی در گونههای ‪ Allium‬بومی ایران )‪(Amaryllidaceae‬‬ ‫وحید صیادی‪ ،1‬قاسم کریم زاده‪ ،*1‬محمد رضا نقوی‪ 2‬و سجاد رشیدی منفرد‬ ‫‪3‬‬ ‫‪ - 1‬گروه ژنتيك و بهنژادی گياهي‪ ،‬پردیس کشاورزی‪ ،‬دانشگاه تربيت مدرس‪ ،‬تهران ص پ ‪14115-336‬‬ ‫‪ -2‬گروه زراعت و اصالح نباتات‪ ،‬پردیس کشاورزی و منابع طبيعي دانشگاه تهران‪ ،‬کرج‬ ‫‪ -3‬گروه بيوتکنولوژی کشاورزی‪ ،‬پردیس کشاورزی‪ ،‬دانشگاه تربيت مدرس‪ ،‬تهران ص پ ‪14115-336‬‬ ‫*مسئول مکاتبه؛ ‪Email: karimzadeh_g@modares.ac.ir‬‬ ‫چکیده‬ ‫سرده‪ ،Allium ،‬با دارا بودن تقریباً ‪ 900‬گونه یکي از بزرگترین سردههای تك لپهای در خانواده ‪ Amaryllidaceae‬را تشکيل ميدهد‪ .‬هدف از این مطالعه‬ ‫بررسي تنوع و خوشهبندی تعداد هشت گونه ‪ Allium‬بومي ایران بر اساس ویژگيهای کاریوتيپي بود‪ .‬گونههای مورد بررسي از رویشگاههای طبيعي آنان در‬ ‫گونههای‬ ‫که‬ ‫داد‬ ‫نشان‬ ‫نتایج‬ ‫شدند‪.‬‬ ‫آوری‬ ‫جمع‬ ‫ایران‬ ‫مختلف‬ ‫جغرافيایي‬ ‫مکانهای‬ ‫‪ A. lenkoranicum ،A. stamineum ،A. umbellicatum ،A. fistolosum ،A. stipitatum ،A. sativum‬و ‪ A. rubellum‬دیپلوئيد هستند (‪16‬‬ ‫= ‪ ،)2n = 2x‬ولي گونه ‪ A. atroviolaceum‬تریپلوئيد (‪ )2n = 3x = 24‬بود‪ .‬بر اساس نتایج به دست آمده تعداد کروموزوم پایه در همه گونهها برابر با ‪x = 8‬‬ ‫بود‪ .‬نتایج تجزیه واریانس نشان داد که تنوع بين گونهای معنيداری برای هشت ویژگي کروموزومي مورد بررسي وجود دارد‪ .‬ميانگين طول کروموزومها ‪μm‬‬ ‫گونه‬ ‫دو‬ ‫در‬ ‫ترتيب‬ ‫به‬ ‫‪13/81‬‬ ‫‪μm‬‬ ‫تا‬ ‫‪8/59‬‬ ‫‪μm‬‬ ‫از‬ ‫که‬ ‫بود‬ ‫‪11/19‬‬ ‫‪ A. atroviolaceum‬و ‪ A. stipitatum‬متغير بود‪ .‬در همه گونهها‪ ،‬نوع کروموزومها به صورت متاسنتریك (‪ )m‬و ساب متاسنتریك (‪ )sm‬بودند به طوری که‬ ‫تشکيل پنج فرمول مختلف کاریوتيپي شامل‪A. sativum, A. ( 14m+2sm ،)A. stipitatum, A. fistolosum, A. stamineum( 16m :‬‬ ‫‪24sm‬‬ ‫و‬ ‫‪)A.‬‬ ‫(‪umbellicatum‬‬ ‫‪10m+6sm‬‬ ‫‪،)A.‬‬ ‫(‪lenkoranicum‬‬ ‫‪12m+4sm‬‬ ‫‪،)rubellum‬‬ ‫(‪ )A. atroviolaceum‬دادند‪ .‬بر مبنای دستهبندی استبينز‪ ،‬همه کاریوتيپها در کالس ‪ 1A‬گروه بندی شدند که نشان دهنده متقارن ترین کاریوتيپها‬ ‫ميباشد‪ .‬اطالعات به دست آمده از کاریوتيپ و ریختشناسي کروموزوم در درک تکامل تاکسون و روابط متقابل بين آنها از اهميت باالیي برخوردار است‪.‬‬ ‫واژههای کلیدی‪ :‬تقارن کاریوتيپي؛ کروموزوم؛ تنوع؛ سطح پلوئيدی؛ ‪Allium L.‬‬