Cytogenetic Diversity and Characterization of Vicia sativa Subspecies

This research was carried out at the Ankara University Faculty of Agriculture, Biotechnology Laboratory between 2016-2017. The seeds of the accessions used in this research were kindly provided by Esvet Acikgoz (Uludag University, Bursa, Turkey). The research material consisted of multiple entries collected from the five subspecies given in Table 1.
 

 
The seeds of all accessions were germinated in petri dishes at room temperature (25°C) for two or three days until a root was obtained at a length of around 3 cm. All cytological observations were then made from samples collected from root tips at 2-3 days old. The root tips were pre-treated in 6% a-monobromonaphtalane at 4°C for 7-8 h and then fixed in glacial acetic acid for 30 minutes and transferred to 70% ethanol for long term storage. For analysis, the root tips were hydrolyzed with 1 N HCl for 12-18 minutes at room temperature (25°C). After hydrolyzing, the root tips were stained with 2% aceto-orcein in absolute darkness for 2.5 h. Finally, samples were crushed in 45% acetic acid, as described by Martin et al., (2018). The chromosomes were observed during the mitotic metaphase using an Olympus BX-51 (Olympus Corp., Japan) microscope. The chromosome number and karyotype measurements were recorded for at least ten cells per accession. The selected cells were photographed using the Olympus BX-51 camera at room temperature (25°C), at an 8000x magnification and used for the measurements. Six chromosomal parameters, chromosome length (c), relative length (RL), long arm length (L), short arm length (S), arm ratio (AR: L/S) and centromeric index (S/C), were measured using the Micro Measure 3.3 program (Reeves 2001).  Ideograms were drawn based on the ratio of long arm length to short arm length. Karyotype formulas of all Vicia sativa genotypes were determined using the method described by Levan et al., (1964). Five parameters were used in order to estimate the karyotype asymmetry. The difference in relative length (DRL:MaxRL – MinRL) (Zarco 1986) and total form percentage (TF, 100 × ΣS/CL) were calculated (Huziwara 1962). The intrachromosomal asymmetry index A₁ = [1 – (Σ (short arm/long arm)/n)] and the interchromosomal asymmetry index; A₂ = [standard deviation (S)/mean length (X)] calculated by Zarco 1986. The mean centromeric asymmetry (M_CA) parameter was used to calculate the asymmetry of each chromosome (Peruzzi and Eroglu, 2013).

In this study, the chromosome numbers and detailed chromosome measurements for the Vicia sativa subspecies were determined. All analyzed accessions of the Vicia sativa complex were diploid. The subsp. angustifolia, macrocarpa, sativa and segetalis were found to have 2n=2x=12 chromosomes (Fig 1 and 2), whereas the subsp. cordata and nigra  were found to have 2n = 2x = 10 (Fig 1 and 2).
 

 

       
The results also confirmed that the basic chromosome numbers were x = 5 and 6, as had been previously reported. Additionally, the karyotype formulas of Vicia sativa subspecies with the same basic chromosome numbers were found to have higher similarity rates. Ideograms (Fig 2) were generated to using the mean values of chromosomes. Karyotype formulas and quantitative analyses of the Vicia sativa subspecies differed among cultivars as well as subspecies of different origins. However, the karyotypical differences were found to be much higher among subspecies of different origins. The chromosomes observed in the study were mainly submetacentric or subtelocentric types.
 
Cv. Antalya and subsp. macrocarpa “Ericek” were shown to have the same karyotype formula of 2n = 12 = 6m + 6sm. Following this, subsp. angustifolia (2 m + 2 sm + 8 st) and cv. Soner (4 sm + 8st) karyotypes had four pairs of subtelocentric chromosomes. It was determined that four pairs of chromosomes were also submetacentric in cv. Beyaz (4 m + 8 sm) and subsp. macrosperma “5283” (8 sm + 4 st). The karyotype formulas of subsp. cordata and subsp. nigra, with the chromosome number 2n = 10, were determined to be 2 sm + 8 st and 2 m + 4 sm + 4 st, respectively (Table 2).
 

 
When the average long arm length of Vicia sativa subspecies was analyzed, subsp. segetalis was observed to be the longest. It was followed by subsp. cordata and subsp. angustifolia. It was also determined that cv. Antalya had the shortest average long arm length among the subspecies. Additionally, chromosome no 1 of subsp. segetalis had the highest long arm length (7.038 µm) out of all the genotypes (Table 3). The average chromosome lengths of the accessions varied between 2.64 - 5.65 µm, as indicated in Table 2. When the length of the longest chromosome of the accessions was compared, the range was found to be 3.251 µm and 9.036 µm in Antalya and subsp. angustifolia, respectively. On the other hand, the shortest chromosome length range was also observed in cv. Antalya with 1.686 µm and subsp. cordata with 4.477 µm (Table 3).
 

 
The range of the total haploid chromosome length in the accessions was determined to be between 15.863 µm (cv. Antalya) and 33.882 µm (subsp. segetalis). Furthermore, cv. Soner (22.208 µm) and subsp. macrosperma “5283” (21.934 µm) are the genotypes with the most similar haploid chromosome lengths. This information has given insight into the chromatin content of the analyzed genotypes (Konischenko et al., 2014).
 
The results of the karyotype asymmetry index analysis showed that intrachromosomal asymmetry was higher than interchromosomal asymmetry in the karyotypes of Vicia sativa subspecies (Table 4). Subsp. segetalis had the highest A₁ (0.72) and M_CA (55.87) value and the lowest TF value (21.22%). Thus, subsp. segetalis was determined to be the most asymmetric karyotype. On the contrary, cv. Antalya was the most symmetric karyotype among the genotypes with A₁ (0.41) and M_CA (26.99) values and with the highest TF (35.54%) value. When the interchromosomal asymmetry results were analyzed (Table 4), subsp. angustifolia was shown to have the highest A₂ (0.52) and DRL (23.68) values and it was therefore determined to be the most asymmetric karyotype among the genotypes. Subsp. nigra had the lowest A₂ (0.06) and DRL (3.10) values.
 

 
In close agreement with this study, the same chromosome numbers were reported in earlier studies for the Vicia sativa subspecies analyzed in this paper. However, the subspecies may contain different chromosome numbers within itself. For example, the accessions of subsp. sativa showed 2n= 10, 12 and 14, subsp. angustifolia, cordata and nigra showed 2n= 10 and 12 and subsp. macrocarpa showed 2n= 12 and 14, in the mixed Vicia sativa populations (Yamamoto 1968; Hollings and Stace, 1974; Ladizinsky 1978; Ladizinsky and Temkin, 1978; Zohary and Plitmann, 1979; Kamari et al., 1994; Weber and Schifino-Wittmann, 1999; Arslan et al., 2012; Martin et al., 2018).
 
Analyzing karyotype asymmetry provides a good point of reference for the general morphology of the plant karyotypes (Zarco 1986; Zuo and Yuan 2011). Asymmetry is defined by the dominance of almost the same size m and sm chromosomes (Zuo and Yuan 2011; El-Bok et al., 2014). Changes in chromosome morphology have often been associated with evolution in higher plants. In this study, in order to determine the asymmetric values in the karyotypes of genotypes, the intrachromosomal (A₁) and interchromosomal (A₂) asymmetric indexes of Zarco (1986) were used. The intrachromosomal asymmetry index (A₁) indicated the arm ratio of each pair of homologous chromosomes, where the interchromosomal asymmetry index (A₂) was applied to variation coefficient. The most asymmetric values in the genotypes were therefore determined by the indices A₁ and A₂.

Theoretically, differences observed in length and the structure of chromosome morphology could be explained away as gradual alterations, which occurred through the evolution of the karyotype during natural or manual selection. 
 
Detailed chromosome measurements and karyotype asymmetry degrees of all Vicia sativa subspecies are not available in the published literature. However, throughout this experiment, the cytological and karyological characteristics of Vicia sativa subspecies have been identified. Our findings could play a significant role in gaining an understanding of their genetic stability and potential for utilization in breeding programs. The gene pool that can be used in plant breeding programs is divided into three groups, according to hybridizability and proximity relationships (Harlan and de Wet, 1971; Von Bothmer et al., 2003; Kanwar et al., 2020). The primary gene pool group consists of modern (elite) varieties, old varieties, landraces and close relative species. Subspecies, which are compatible and similar in terms of cytological characteristics, are more likely to succeed in the transfer of desirable features during cross-breeding and/or self-fertilization studies (Devi et al., 2020). Chromosome numbers are 2n=2x=10, subsp. cordata and subsp. nigra to each other, are thought to give fertile progeny when crossed obtained. It can be speculated that fertile offspring can be obtained from the hybrids of other subspecies and cultivars with a chromosome number of 2n=2x=12. Research has accelerated putting superior features from close species and subspecies into cultivated types with the techniques and technologies developed in recent years. In this study, the potential of the hybridization of subspecies with cultivated types and possible genetic barriers among subspecies have been revealed at the cytological level.