Introduction
Forage species of Vicia genus have an invaluable importance not only for providing low-cost animal feeds but also they contribute to organic biomass and nitrogen to soil (Yeh et al., Reference Yeh, Yang, Boyle, Ye and Mao1997; Avcioğlu, Reference Avcioğlu2000). Although Vicia species are widespread in the temperate zones of both the hemispheres, it is especially common in the Mediterranean and Middle East (Frediani et al., Reference Frediani, Caputo, Venora, Ravalli, Ambrosio and Cremonini2005; Başbağ et al., Reference Başbağ, Hoşgören and Aydin2013). The centre of diversity and the possible origin for the subgenus Vicia are the Northeastern Mediterranean, including Iraq, Iran, the Southwestern Republics of the former Soviet Union, Syria and Turkey (Maxted, Reference Maxted1995). Turkey, geographically overlapped between the Mediterranean and Near East gene centre, has a very special status for presenting such rich vetch diversity (Harlan, Reference Harlan1971). Therefore, wild and weedy forms of vetch species exist in almost every part of Turkey at altitudes from sea level to 2.200 m (Sabanci, Reference Sabanci, Bennett and Cocks1999). Annual common vetch (Vicia sativa ssp. sativa) is one of the most genetically and phenotypically variable species of Vicia (Davis, Reference Davis1970) and have ability to grow over a wide range of climatic and soil conditions.
Wide range application of polymerase chain reaction (PCR) based on molecular markers is currently the dominant and very powerful tool for genotype characterization and estimation of genetic diversity of crop plants since they are independent of tissue or environment, and allow cultivar identification in the early stages of plant development (Kumar et al., Reference Kumar, Gupta, Misra, Modi and Pandey2009). It had been suggested that diversity estimates based on molecular markers are better suited than pedigree data for parental selection (Tinker et al., Reference Tinker, Fortin and Mather1993). The use of molecular markers for genetic diversity analysis can also serve as a tool to discriminate closely related individuals from different breeding sources (Sun et al., Reference Sun, William, Liu, Kasha and Pauls2001; Tar'an et al., Reference Tar'an, Zhang, Warkenting, Tullu and Vandenberg2005).
In contrast, flow cytometry may successfully be used to determine the genome size and the relative DNA content of unknown samples after a process of comparing the data with the relative fluorescence intensity of nuclei of a reference standard whose genome size has been previously known (Tiryaki and Tuna, Reference Tiryaki and Tuna2012). Genome size has been regarded as a species-specific constant (Greilhuber, Reference Greilhuber1998) and genome size variation of similar species has been called the C-value paradox (Thomas, Reference Thomas1971). The C stands for constancy of DNA amount of unreplicated haploid genome of an individual to indicate genome size variation irrespective of complexity of organisms (Swift, Reference Swift1950). Comparison of C-values of various plant species provides a natural way to explain phylogenetic relationships and systematics of narrow taxonomic groups (Raina, Reference Raina and Kawano1990; Ohri et al., Reference Ohri, Bhargava and Chatterjee2004).
The aims of the present study were to estimate the amount of genetic variability available in various gene pools of common vetch lines collected from natural flora of Turkey or obtained from national or international resources, and to reveal their nuclear DNA contents, and compare the results with three common vetch varieties.
Materials and Methods
Materials
Plant material that presented in Table 1 was either obtained from national or international genetic resources or collected from natural flora of Turkey. Seeds of individual plants of natural flora were collected at locations presented in Table 1. Those seeds were grown and were selfed to propagate enough seeds under the same field conditions for 2 years, during the plant growing seasons of 2008 and 2009. No intra-population diversity was detected for those populations originated from natural flora. The seeds collected from natural flora were also reconfirmed to assure the correct taxonomic classification (V. sativa ssp. sativa) by Biology Department of Kahramanmaraş Sutcu Imam University, Turkey. A total of 27 common vetch lines and three cultivars were used in this study (Table 1).
Source, nuclear DNA content and genome size of 27 lines and three varieties of Vicia sativa used in this study
a 1C nuclear DNA content (mean value ± standard deviation of four samples).
b 1 pg = 978 Mbp (Dolezel et al., Reference Dolezel, Bartos, Voglmayr and Greilhuber2003).
Methods
DNA extraction and quality control
Genomic DNA from young leaves of ten plants of each line or cultivar was bulked and was extracted by using plant genomic DNA extraction mini kit (Favorgen, Pingtung, Taiwan) based on the manufacturer's instruction. The DNA concentrations were estimated by comparing known concentration of λ DNA on 0.8% agarose gel electrophoresis. The DNA samples were diluted to 50 ng/μl using dH2O and stored at − 20°C until used.
Primers and PCR amplification
Details of the primers were summarized in online Supplementary Table S1. Inter-simple sequence repeats (ISSRs) marker analysis was performed according to Zeitkiewicz et al. (Reference Zeitkiewicz, Rafalski and Labuda1994) and was optimized using five ISSR primers. Sequence-related amplified polymorphism (SRAP) marker analysis was done according to Li and Quiros (Reference Li and Quiros2001) and was optimized using 55 primer combinations for 27 common vetch lines and three cultivars. Amplification reactions were carried out in a 25 μl reaction mixture containing 75 mM Tris–HCl, pH 8.8; 20 mM (NH4)2SO4; 2.0 mM MgCl2; 0.2 μM primer; 100 μM each of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP); 1 unit of Taq DNA polymerase; and 10 ng of genomic DNA.
All PCR amplifications were performed in a thermal cycler (Favorgen Gradient PCR, Pingtung, Taiwan). Each primer was optimized to determine the best annealing temperature before used in sample DNA amplification. The PCR reactions were repeated twice to determine the reproducibility of the bands. The ISSR included one cycle of 3 min at 94°C, followed by 49 cycles of 1 min at 94°C, 1 min at 45–51°C (depending upon annealing temperature of the primer presented in Supplementary Table S1, available online), and 2 min at 72°C, followed by a final incubation for 7 min at 72°C. In SRAP, the thermal cycler was programmed to five cycles of 1 min at 94°C, 1 min at 35°C and 1 min at 72°C, for denaturing, annealing and extension, respectively. Then, the annealing temperature was raised to annealing temperature of primers presented in online Supplementary Table S1 for another 35 cycles. ISSR and SRAP amplification products were separated with a 2% (w/v) agarose gel in 1 × TBE buffer at 100 V for 3 h and were stained with ethidium bromide (2 μl/100 ml) before photographed under ultraviolet light.
Nuclear DNA content determination
Nuclear DNA content of four individual plants from each line or cultivar was determined as described previously (Tiryaki and Tuna, Reference Tiryaki and Tuna2012). Briefly, fresh healthy leaf tissues from 3- to 4-week-old seedlings, about 50 mg of target samples and 20 mg of internal standard safflower (Carthamus tinctorius L.) cultivar Dincer were simultaneously excised and placed on ice in a sterile plastic petri dish for the flow cytometer CYTOMICS FC 500 (Beckman Coulter, Inc., Fullerton, CA, USA) analysis. Tissue was chopped into 0.25 to 1 mm segments in 1 ml solution A [24 ml MgSO4 buffer (ice-cold); 25 mg dithiothreitol; 500 μl propidium iodide (PI) stock (5.0 mg PI in 1.0 ml double distilled H2O); 625 μl Triton X-100 stock (1.0 g Triton X-100 in 10 ml double distilled H2O)]. The supernatants were filtered through a 30 μM nylon mesh into a micro-centrifuge tube and centrifuged at high speed (13.000 rpm) for about 20 s. The pellet was resuspended in 400 μl solution B [7.5 ml solution A; 17.5 μl DNase-free RNase] and incubated for 20 min at 37°C before flow cytometric analysis. Samples stained with PI were excited with a 15 mW argon ion laser at 488 nm. Red PI fluorescence area signals (FL2A) from nuclei were collected in the FL2 channel. The absolute DNA amount of each sample was calculated based on the values of the G1 peak means as reported previously (Dolezel and Bartos, Reference Dolezel and Bartos2005).
Data analysis
Amplified DNA fragments were scored as either ‘1’ or ‘0’, representing either the presence or the absence of the bands. Only clear and easily detectable bands were recorded and used for genetic analysis (Supplementary Fig. S1, available online). Genetic distance was calculated by the method of Nei (Reference Nei1972) using PopGen 3.2 program (Yeh et al., Reference Yeh, Yang, Boyle, Ye and Mao1997). Based on the genetic similarity matrix values, the unweighted pair-group method with arithmetic averages (UPGMA) clustering method was used to obtain the dendrogram, depicting genetic relatedness of the lines and cultivars (Mega 4.1 software; Tamura et al., Reference Tamura, Dudley, Nei and Kumar2007).
The polymorphism information content (PIC) of each primer was calculated according to the method by Botstein et al. (Reference Botstein, White, Skolnick and Davis1980) as follows:
$$\begin{eqnarray} PIC = 1 - \sum ( P _{ ij })^{2}, \end{eqnarray}$$
where P ij is the frequency of the ith band revealed by the jth primer. P ij is summed through all the bands revealed by the primers.
The nuclear DNA content data were subjected to analysis of variance using SAS statistical software (SAS, Reference SAS1997), and mean separation was performed by Fisher's least significant difference (LSD) test if F test was significant at P <0.05.
Results
Genetic diversity based on ISSR and SRAP marker analysis
In total, 55 SRAP primer combinations and five ISSR primers produced a total of 229 scorable bands, of which 188 bands (82.1 %) were polymorphic. The number of bands obtained by each primer combination ranged from 1 to 9, with an average of 4.4 bands (Supplementary Table S2, available online). An average polymorphic band produced by primers was 3.62. Me6-Em16 and Me11-Em11 of SRAP primer combinations produced the highest number of bands (nine bands), all of which were polymorphic (Supplementary Table S2, available online); 23 primer combinations showed 100% polymorphism. The primer combinations of Me7-Em10, Me7-Em13, Me8-Em14, Me9-Em12, Me11-Em16, Me13-Em17, Me14-Me15 and Me14-Em17 produced single monomorphic band only (Supplementary Table S2, available online). The PIC values ranged from 0.12 to 0.96, with an average of 0.63. ISSR primer BC-813 and SRAP Me7-Em12 primer combination gave the highest PIC values, 0.96 and 0.95, respectively.
The result from molecular marker-based UPGMA cluster analysis is presented in Fig. 1. The UPGMA trees revealed that 30 vetch genotypes were divided into two main groups as I and II. The first group consisted of eight genotypes. Of them, seven came from natural flora (DV) and were separated as I-A while the line encoded as GB-6 was separated as a single group (I-B). The cluster analysis was able to separate all DV vetch lines from the others and was grouped as subgroup I-A. The second cluster was subdivided into two main groups as II-A and II-B. The line encoded as TA-9 was distinctly separated from the other TA lines. All the lines encoded as CU were clustered together with an exception of CU-5, which showed more similarity to lines encoded as GB-10, IC-6 and IC-7 than the other CU lines. The cultivars encoded as CE-5 and CE-6 were subgrouped together in comparison to the other cultivar (CE-7).
Dendrogram for 27 lines and three varieties of common vetch derived from cluster analysis (UPGMA) based on genetic similarity estimates (Nei, Reference Nei1972) from 55 SRAP and five ISSR marker analysis.
Genetic distance matrix values indicated that lines DV-3 and DV-4 were the closest (0.112), while the highest distinct value (0.627) was determined between lines TA-6 and DV-5 (Table 2).
Nei (Reference Nei1972) genetic distance matrix for 30 common vetch lines and cultivars assessed by SRAP and ISSR markers
Nuclear DNA content
Nuclear DNA content analysis showed no polyploidy within any vetch lines tested (Table 1), while significant (P< 0.0001) nuclear DNA content differences were detected among the lines and cultivars. The mean 2C nuclear DNA content of 30 genotypes was determined as 3.466 pg, which represented an average of 3390.07 Mpb DNA. The highest nuclear DNA content value (3.590 pg) was obtained from line encoded as IC-9, while line encoded as CU-4 had the lowest (3.337 pg). Two cultivars showed a higher 2C content (3.512 pg) than the mean value of 30 genotypes, while lines encoded as DV-1, DV-3 and GB-10 exactly gave the same amount (3.555 pg) of 2C DNA. Lines encoded as CU provided a lower nuclear DNA content than the other genotypes (Table 1).
Discussion
The resolving power of genetic markers is mainly determined by the level of polymorphism detected and microsatellites are presumed to be the fast evolving markers to make genetic analyses within species or among closely related species (Kumar et al., Reference Kumar, Gupta, Misra, Modi and Pandey2009). Previous report has indicated that V. sativa has a complex of well-separated taxa and represents various degrees of phylogenetic divergence (Potokina et al., Reference Potokina, Duncan, Eggi and Tomooka2000). The intra-specific diversity of the widely distributed species of V. sativa was also detected at the DNA level by using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers (Potokina et al., Reference Potokina, Duncan, Eggi and Tomooka2000, Reference Potokina, Blattner, Alexandrova and Bachmann2002). However, it had been suggested that the agronomic performance of V. sativa accessions would still be needed to provide further important information for the utilization of ex situ germplasm collections since AFLP markers produced very similar patterns for those accessions used (Potokina et al., Reference Potokina, Blattner, Alexandrova and Bachmann2002). In addition, they also pointed out the lack of clear intra-specific differentiation within V. sativa, which was attributed to severe reduction of genetic variation and a fast spread of seeds of the cultivated plants. Evaluation of 27 lines and three varieties of common vetch revealed significant intra-specific genetic diversity in the present study. A total of 60 markers (55 SRAP and five ISSR) produced 188 polymorphic bands (82.1 %), and those markers were able to successfully differentiate the lines and cultivars of V. sativa (Fig. 1). Several advantages of the SRAP markers over other marker systems were pronounced, such as simplicity, reveals numerous co-dominant markers, allows easy isolation of bands for sequencing (Robarts and Wolfe, Reference Robarts and Wolfe2014; Li and Quiros, Reference Li and Quiros2001) and they were previously used for a variety of purposes in different crops, including map construction, gene tagging, genomic and complementary DNA (cDNA) fingerprinting, and map-based cloning (Li et al., Reference Li, McVetty, Quiros and Andersen2013). In addition, the SRAP markers preferentially amplify open reading frames, which are expected to be evenly distributed throughout the whole genome (Li and Quiros, Reference Li and Quiros2001). Therefore, the SRAP markers were successfully used to determine the genetic diversity of several other crop species, including Triticum spp. (Fufa et al., Reference Fufa, Baenziger, Beecher, Dweikat, Graybosch and Eskridge2005; Zaefizadeh and Goliev, Reference Zaefizadeh and Goliev2009), Sorghum bicolor (Hussein et al., Reference Hussein, Siddig, Abdalla, Dweikat and Baenziger2014), Allium sativum (Chen et al., Reference Chen, Zhou, Chen, Chang, Du and Meng2013) and Cynodon arcuatus (Huang et al., Reference Huang, Liu, Bai and Wang2013). Detection of relatively high level of polymorphism rate (82.1%) in this study may indicate the presence of variation due to mutation, while genetic similarities among the lines and cultivars might be attributed to cross-pollination within species (Supplementary Table S2, available online). The cDNA-SSR markers were also recently developed for V. sativa subsp. sativa and were tested in 32 accessions (Chung et al., Reference Chung, Kim, Suresh, Lee and Cho2013). An average PIC value of cDNA-SSR markers (0.62) was about the same what we found in this study (0.63) for SRAP and ISSR markers, indicating that SRAP and ISSR DNA markers provided a high level of polymorphism at DNA level for common vetch lines and cultivars. The presence of high genetic diversity within vetch lines and cultivars was also supported by genetic distance matrix data, which ranged from 0.11 to 0.627 (Table 2).
A significant intra-specific nuclear DNA content variation was recently reported for V. sativa by using two different internal standards, namely safflower and barley that has been used to estimate the DNA content of unknown plant material (Tiryaki and Tuna, Reference Tiryaki and Tuna2012). The mean 2C nuclear DNA content was given as 3.481 pg for internal standard of safflower, while about the same mean 2C nuclear DNA content (3.466 pg) of 30 genotypes was determined in this study. Ongoing processes of speciation or genetic divergence are believed to be the main reason for changes in genome size within a narrow group of species (Price, Reference Price1976; Murray, Reference Murray2005). Previous report has pointed out that less than 3% of the variation in genome size has been weakly associated with by variation in the microclimatic conditions (Kalendar et al., Reference Kalendar, Tanskanen, Immonen, Nevo and Schulman2000). However, significant intra-specific genome size variation within V. sativa species determined in this study suggested that this variation might be related to deletions/insertions within V. sativa genome (Petrov, Reference Petrov1997; Gregory, Reference Gregory2003; Bennetzen et al., Reference Bennetzen, Ma and Devos2005) rather than by variation in the microclimatic conditions. Earlier reports also pointed out that the presence of metabolic compounds might be interfering with DNA staining, such as tannins, flavonoids and anthocyanins (Price et al., Reference Price, Hodnett and Johnston2000; Noirot et al., Reference Noirot, Barre, Duperray, Hamon and Kochko2005; Walker et al., Reference Walker, Monino and Correal2006; Bennett et al., Reference Bennett, Price and Johnston2008; Smarda and Bures, Reference Smarda and Bures2010) and may result in very small significant differences between the estimates. Although seeds of common vetch contain antioxidant activity and low-molecular-weight phenolic compounds (Amarowicz et al., Reference Amarowicz, Troszynska and Pegg2008; Pastor-Cavada et al., Reference Pastor-Cavada, Juan, Pastor, Alaiz, Giron-Calle and Vioque2008), the nuclear DNA amount variation of common vetch lines and varieties detected in this study is not likely due to the presence of such metabolic compounds interfering with DNA staining. Intra-specific genome size variation has been also reported for other crops such as soybean (Graham et al., Reference Graham, Nivkell and Rayburn1994; Rayburn et al., Reference Rayburn, Biradar, Bullock, Nelson, Gourmet and Wetzel1997), sunflower (Michaelson et al., Reference Michaelson, Price, Johnston and Ellison1991), pea (Arumuganathan and Earle, Reference Arumuganathan and Earle1991) and maize (Rayburn et al., Reference Rayburn, Auger, Benzinger and Hepburn1989). Our DNA marker data lead to conclusion that differences in genome size within V. sativa is not due to metabolic compounds interfering with DNA staining or microclimatic conditions, but differences in genomic DNA content due to deletion or insertion mutagenesis.
Supplementary material
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/S1479262115000210
Acknowledgements
We are grateful to The Scientific and Technological Research Council of Turkey (TUBITAK, TOVAG Grant no. 107O012) and Kahramanmaraş Sutcu Imam University (KSU, BAP Grant no. 2010/4-6D) for providing financial support. We acknowledge Kahramanmaraş Agricultural Research Institute, Kahramanmaraş, Turkey, for providing field facilities. We thank Metin Tuna for his help on genomic DNA content determination and Ahmet Ilcim for taxonomic classification of the lines collected from natural flora of Turkey. The authors declare that they have no conflict of interest.
