Karyotype variation and biochemical analysis of five Vicia species

Fabaceae is considered as the third largest family, which includes more than 727 genera and 20,000 species. The genus Vicia has from 180 to 210 species. Vicia species have a great economical and agricultural importance. Karyotype study of chromosomes and SDS-PAGE for seed storage proteins (soluble and non-soluble proteins) were carried out on five Vicia species (Vicia macrocarpa, Vicia sativa, Vicia narbonensis, Vicia ervilia) collected from IPK, Germany, and Vicia faba from Agriculture Research Centre, Giza, Egypt, to find out the phylogenetic relationships among these species. From karyotype of studied Vicia species chromosomes, it was found that V. macrocarpa, V. sativa, and V. faba had six pairs of chromosome (2n = 12) while V. narbonensis and V. ervilia had seven pairs of chromosome (2n = 14). The most related species was found between V. ervilia and V. narbonensis (77.8%) depending on seed soluble protein similarity level, but between V. narbonensis and V. macrocarpa was 70.0% depending on seed non-soluble protein similarity level, while between V. ervilia and V. narbonensis, the most related species was 69.0% depending on collective data of both soluble and non-soluble seed storage protein. The phylogenetic relationships between the studied species depending on collective data of protein markers and karyotype characteristic were as follows: V. ervilia is closely related to V. narbonensis, while V. narbonensis is related to V. macrocarpa and V. ervilia, but the degree of relation between V. narbonensis and V. macrocarpa is less than the relation between V. narbonensis and V. ervilia. Equally, while V. sativa is closely related to V. macrocarpa, but V. faba is distant from all other studied species.


Introduction
Fabaceae is considered the second family after cereal crops in agricultural importance based on area harvested and total production; this family contains more than 727 genera and 20,000 species (Gepts et al. 2005). The species in genus Vicia (180 to 210 species) are widely distributed throughout the world. This genus has two subgenera, Vicia and Vicilla, and the subgenus Vicilla is considered more primitive and diverse than the subgenus Vicia (Hanelt and Mettin 1989;Maxted 1993).
The subgenus Vicilla is divided into 17 sections including forage species. Kupicha (1976) suggested that the subgenus Vicia is smaller and coherent, containing 38 species divided into 5 sections. This subgenus contains the more agriculturally important species of V. faba (section Faba), V. sativa (section Vicia), and V. narbonensis (section Narbonensis).
Karyological studies had an important role in improvement and solving taxonomic problems between the related species (Lavia et al. 2009;Murti et al. 2012). The cytogenetic comparisons based on chromosome size, centromeric index, and banding patterns between related species occurred by staining chromosomes with different dyes such as feulgen, orcein, or carmine (Cremonini 1992;Galasso et al. 1994;Cremonini et al. 1998;Fuchs et al. 1998).
Until recently, cytotaxonomic relationships between species were performed using conventional staining methods to visualize the chromosomes. The development and application of banding techniques for plants have proved to be a practical tool for identifying chromosomes as well as providing much information regarding species relationships. The most popular staining procedures include Q, G, C, R, and silver stain banding which have been developed for brightfield microscopy (Casperson et al. 1970;Howell et al. 1975;Fominaya et al. 1988;Jellen et al. 1993;Jellen and Ladizinsky 2000).
Polyacrylamide gel electrophoresis (PAGE) plays an important role in the analysis of protein profile. PAGE is considered the most widespread technique. Seed storage proteins were used in investigating genetic diversity and evaluation of taxonomic and genetic associations in Vicia (Emre et al. 2010).
The aim of our research is to find out the phylogenetic relationships between five Vicia species (V. macrocarpa (Moris) Betol., V. sativa (L.) ssp. Sativa convar var. sativa, V. narbonensis (L.) var. narbonensis, V. ervilia (L.) Willd, and V. faba var. sakha 3) by studing the karyotype of chromosomes and seed storage protein (soluble and non-soluble proteins) profile of the studied species.

Materials and methods
All the laboratory experiments were carried out in the laboratories of Genetics and Cytology Department, Genetic Engineering and Biotechnology Research Division, National Research Centre, Giza, Egypt.

Chromosome preparation
The seeds of Vicia species were germinated on moist filter paper at 20°C. Root tips of about 1-2 cm length were excised. The roots were treated with ice-cold water for 20-22 h to arrest the chromosome at metaphase then fixed in Carnoyʼs solution I (3:1 v/v) absolute ethanol to glacial acetic acid for 24 h at 4°C, then stored in a refrigerator in 70% ethanol; after that, the roots were incubated in 1% cellulose and 1% pectinase (v/v) which were dissolved in 0.01 M citrate buffer pH 4.8 at 37°C for 1 h. Root tips were squashed on slides in a drop of 45% acetic acid, frozen on liquid nitrogen to remove the coverslips. After that, slides were washed with Carnoyʼs fixative solution and air dried. The slides were stained with DAPI; after that, they were examined under florescence microscope.

Karyotype characteristics
After photos were captured with a camera connected to a computer, the chromosomes of each cell were arranged using the Adobe Photoshop 6.0 software. After finishing the arrangement of chromosomes of one species, a computer program (Micromeasure 3.3) was used to measure the total length of each chromosome, length of the short arm, length of the long arm, arm ratio (long/short), centromeric index [short/(long + short)], and the relative length (RL) for each chromosome (percentage of total length of haploid complement). The ideograms for the 5 species were drawn in Corel-Draw program.
The procedure of Bebeli and Kaltsikes (1985) was followed to describe the chromosome types in the five species according to the location of the centromere, i.e., metacentric to cover the M-chromosomes with an arm ratio (S:L) between 1:1.35, submetacentric to cover the Sm chromosomes with an arm ratio between 1.36 and 1.75, and subtelocentric to cover the St-chromosomes with an arm ratio greater than 1.76.

Seed storage protein profiles using SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method proposed by Laemmli (1970), as modified by Studier (1973). Water-soluble proteins (WSP) and water-non-soluble proteins (WNSP) were extracted from the seed of five Vicia species.

Karyotype characteristics
The somatic chromosome number of two species (V. narbonensis and V. ervilia) is 2n = 14, while in the other three species (V. sativa, V. macrocarpa, and V. faba) is 2n = 12. All of the examined species have one secondary constriction except for V. sativa which has two. For karyotypic analysis, chromosomes were captured by a cooled CCD camera and analyzed on a computer with image analysis software (Photoshop 0.6). Chromosomes were randomly numbered, and the total length and lengths of the short arm (S) and long arm (L) were measured for each chromosome. Using chromosomemeasuring software, the short and long arms of the homologous chromosome pairs were measured and identification based on chromosome arm ratio (L/S). In the karyogram construction, chromosome pairs were ordered from longest to shortest based on the relative length of each pair of chromosomes. At least 10 wellspread chromosome preparations of each species were analyzed to validate the karyogram construction for each species. The total length and the length of the short arm, long arm, arm ratio, the relative length of each chromosome, and the chromosome types of the five Vicia species are depicted in Table 1. The karyotype of the five Vicia species and their ideograms are shown in Fig. 1.

Seed storage protein profiles using SDS-PAGE
Seed storage proteins (soluble and non-soluble proteins) fractions were used to find out the relationships between the studied five Vicia species (Fig. 2).
The studied Vicia species were examined for seed storage protein profile, and the data was subjected to unweighted pair-group method with arithmetical average (UPGMA) to find the phylogenetic relationships among the species. Table 2 and Fig. 3 represented the relationship between the studied Vicia species depending on seed soluble protein similarity level, and it was found as follows: V. ervilia and V. narbonensis are the most related species (77.8%), then comes V. macrocarpa which is most related to V. narbonensis (64.5%), V. sativa which is most related to V. macrocarpa (60.0%), and peripheral position comes V. faba which has almost the same relationship with the other studied species, while Table 3 and Fig. 4 showed the relationship between the studied five species depending on seed non-soluble protein similarity level, and it was found as follows: V. narbonensis and V.
macrocarpa are the most related species (70.0%), then comes V. sativa, V. ervilia, and V. faba. The collective data of both soluble and non-soluble seed proteins were analyzed using UPGMA (Table 4 and Fig. 5); the relationship between the studied five species was as follows: V. ervilia and V. narbonensis were the most related species (69.0%), then V. narbonensis and V. macrocarpa (67.2%), V. macrocarpa and V. sativa (60.7%), and V. faba at a peripheral position.

Discussion
The karyotypes of several species have been established based on chromosome size and centromeric index in addition to the traditional process for karyotyping by adding a dye to metaphase chromosomes. Different dyes that affect different areas of the chromosomes are used for a range of identification purposes. One common dye used is Giemsa; this dye is effective because it markedly stains the bands on a chromosome; each chromosome can then be identified by its banding patterns (Cremonini 1992; Galasso et al. 1994;Cremonini et al. 1998;Fuchs et al. 1998); however, this approach is limited by the similar morphology of chromosomes in many species.
In our results, V. ervilia L. (Willd) had seven pairs of chromosomes (2n = 14): three metacentric, one submetacentric, and three subtelocentric chromosomes, while V. faba (var. sakha 3) had six pairs of chromosomes (2n = 12): one metacentric and five subtelocentric chromosomes which disagree with Hizume et al. (1980) in chromosome type; they studied the C-banding patterns on V. faba using Giemsa stain and found that the number of chromosomes 2n = 12 (one metacentric, three subtelocentric, and two telocentric); the metacentric chromosome associated with secondary constriction on short arm. SDS-PAGE considered a genetic markers in analyses of genetic distances between species to determine the taxonomic relationship (Tamkoc and Arslan 2011).
In our study, the five Vicia species were examined for protein profile levels and the data was subjected to unweighted pair-group method with arithmetical average (UPGMA) to find the phylogenetic relationships among the species. First, the relationship between the studied five Vicia species depending on seed soluble protein  similarity level was as the follows: V. ervilia and V. narbonensis are the most related species (77.8%), then comes V. macrocarpa which is most related to V. narbonensis (64.5%), and V. sativa which is most related to V. macrocarpa (60.0%), and peripheral position comes V. faba which has almost the same relationship with the other studied species (Table 2 and Fig. 3). Second, the relationship between the studied five species depending on seed non-soluble protein similarity level was as the follows: V. narbonensis and V. macrocarpa are the most related species (70.0%), then comes V. sativa, V. ervilia, and V. faba (Table 3 and Fig. 4), while the relationship between the studied five species depending on the collective data of both soluble and non-soluble proteins using UPGMA was as the follows: V. ervilia and V. narbonensis were the most related species (69.0%), then comes V. narbonensis and V. macrocarpa (67.2%), then comes V. macrocarpa and V. sativa (60.7%), and V. faba at a peripheral position (Table 4 and Fig. 5).
There are a few studies made to determine the genetic diversity between faba bean and its related Vicia species (Haider et al. 2001). In that study, 13 taxa representing 6 Vicia species (V. sativa, V. villosa, V. monantha, V. narbonensis, and V. cinerea, in addition to V. faba) were collected from the north-west coastal region of Egypt. SDS-PAGE indicated clear differences between different Vicia species as well as within the taxa of the same species. The results indicated that V. monantha and V.
villosa are most closely related to V. cinerea, V. faba, and V. narbonensis, which are completely distant. The marked differences between V. faba and other wild species indicated that none of the latter can be considered as the wild progenitor of V. faba, while Kahraman et al. (2016) implicated that protein banding patterns for investigating 22 bean genotypes provided a clear classification by view of selection criteria. Similarity dendrogram presented two main groups that showed ranges nearly 20-75% and 50-90%, respectively.

Conclusion
The phylogenetic relationships among the studied species depending on the seed storage proteins profile (soluble and non-soluble proteins) using SDS-PAGE and karyotype characteristic were as follows: V. ervilia was closely related to V. narbonensis, and V. narbonensis was related to V. macrocarpa, but the degree of relation between V. narbonensis and V. macrocarpa was less than the relation between V. narbonensis and V. ervilia which were the most related species, while V. sativa was closely related to V. macrocarpa, but V. faba was distant from all other studied species. This study helps the breeder to perform the breeding program without consumption of a long duration for doing several crossing over for doing a hybridization between different species.