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Legume Research, volume 44 issue 8 (august 2021) : 888-893

Genetic Relationship in Sainfoin (Onobrychis viciifolia) Landraces Cultivated East Anatolia by using RAPD and ISSR Markers

Mehmet Macit Ertuş1,*
1Department of Plant and Animal Production, Colemerik Vocational School, Hakkari University, Turkey.

  • Submitted04-02-2021|

  • Accepted22-05-2021|

  • First Online 27-07-2021|

  • doi 10.18805/LR-614

Cite article:- Ertuş Macit Mehmet (2021). Genetic Relationship in Sainfoin (Onobrychis viciifolia) Landraces Cultivated East Anatolia by using RAPD and ISSR Markers . Legume Research. 44(8): 888-893. doi: 10.18805/LR-614.

ABSTRACT

Background: Sainfoin (Onobrychis viciifolia) is a forage crop that yields high in arid and calcareous soils and is cultivated in large areas. There aren’t many genetic diversity studies on the varieties of cultured sainfoin. This study was conducted to determine the genetic diversity and the degree of relationship between 23 cultivated landraces and one registered variety. 

Methods: To take samples from the populations, seeds were sown in the field in 2014. Samples were taken from the young leaves of the plants and preserved at -80oC in same year. RAPD and ISSR primers were used in the study. The bands obtained as a result of PCR were recorded and the data of both methods were also evaluated by combining them.

Result: In the study, 5 RAPD and 4 ISSR primers were used and a total of 49 bands were obtained. Of 29 bands obtained using RAPD primers, 20 were found to be polymorphic and of 20 bands obtained using ISSR primers, 15 were found to be polymorphic. It was found that there was a very low correlation between the two methods. Using RAPD and ISSR markers and RAPD + ISSR combination, the similarity index among populations was found to be between 0.25-0.95, 0.5-1.00 and 0.45-0.91, respectively. The Nei’s genetic diversity index was found to be between 0.3365, 0.2656 and 0.3018 with RAPD, ISSR primers and RAPD + ISSR combination, respectively. Based on the dendrograms obtained using RAPD, ISSR primers and RAPD + ISSR combination, the populations under analysis were classified into 3, 3 and 5 groups, respectively. With this study, the closest populations were identified and a significantly high genetic diversity was detected.

INTRODUCTION

Sainfoin (Onobrychis viciifolia) is a forage plant that thrives in arid and poor soils, prevents erosion, is bloat-safe and has high nutritional value and improves soil fertility (Delgado et al., 2008). The cultivated sainfoin is tetraploid genome (2n=4x=28) (Hesamzadeh Hejazi and Ziaei Nasab, 2010; Sepet et al., 2011; Kempf et al., 2016) and is a outcrossing species (Hayot-Carbonero et al., 2011). Sainfoin is known to have been originated from the Middle East and Central Asia (Shen et al., 2019). It is reported that it was first cultivated in settlements around the Rhine Valley at the end of the sixteenth century (Delgado et al., 2008). Although sainfoin has a lifespan of 5-6 years, it dies due to root rot caused by Bembecia scopigera and Sphenoptera carceli following two or three years after it is sown (Avcý et al., 2014). Although it remains on the stand for two years, it is preferred by farmers as it yields higher than alfalfa, especially in unirrigable and calcareous soils. In addition, due to its positive effect on animal health with its tannin content (CT), in recent years it has become the focus of attention as a forage plant (Kempf et al., 2016). Today, sainfoin is cultivated on an area of 153,000 hectares in different regions of Turkey. The sainfoin is an important forage crop in arid and semi-arid regions and is better adapted to areas where alfalfa growth is restricted by environmental conditions (Mohajer et al., 2012). Therefore, it is thought that it will be cultivated even more in areas where there is difficulty accessing irrigation water.
       
Populations that have adapted to the ecological conditions of the region where they are cultivated are referred to as landraces. The widespread use of high-yielding varieties in recent years also entails the protection of landraces facing the danger of extinction. Landraces are important genetic resources for plant breeding and for successful plant breeding, primarily, the genetic diversity of plants must be known (Hejrankesh et al., 2014). However, the identification of existing plant genetic resources has an important place in future genetic studies and the development of approaches (Dos Santos et al., 1994; Demdoum et al., 2012).
       
There are many DNA-based methods used in identifying the genetic variation and in phylogenetic studies. Considering the laboratory facilities, since they involve non-radioactive substances, RAPD and ISSR are methods that can be used to conduct research easily under limited conditions (Kumar et al., 2009). In addition to offering a new and practical approach in taxonomic and phylogenetic comparisons, the ISSR method is also used as a mapping tool for many organisms (Zietkiewicz et al., 1994; Semagn et al., 2006).  RAPD and ISSR methods are used because they exhibit high polymorphism, do not require prior knowledge and are rapid and inexpensive (Bornet et al., 2002; Semagn et al., 2006). Sanghani et al., (2015) used SSR, RAPD and ISSR markers to determine the genetic diversity in Vigna radiata genotypes and found that RAPD markers are effective in determining the genetic relationship between populations. RAPD and ISSR primers are very useful in determining the relationship of the sainfoin and determining its genetic difference (Nosrati et al., 2012; Archer et al., 2013; Rasouli et al., 2013; Hejrankesh et al., 2014; Zarrabian and Majidi, 2015; Sharifi, 2016). Shen et al., (2019) determined the genetic diversity of sainfoin collected from 5 different regions, using EST-SSR markers on 8 plants per region and reported a high level of genetic diversity among individuals. With this study, the aim was to facilitate the selection of parents for breeding programs by identifying the genetic diversity and the degree of relationship among cultivated sainfoin varieties before starting breeding.

MATERIALS AND METHODS

Plant material
 
A total of 23 different sainfoin landraces obtained from 21 different sites at the Eastern Anatolia Region and the Lütfibey variety cultivated by the Eastern Anatolia Agricultural Research Institute were used as the material of this study (Table 1). To take samples from the populations, seeds were sown in the field in 2014. For DNA isolation, thirty plants leaves from each population were taken and stored at -80°C. The laboratory phase of the study, which included DNA extraction and PCR, was carried out in the Laboratory of the Field Crops Department of Van Yüzüncü Yýl University in same year.
 

Table 1: Data on the sainfoin varieties cultivated.


 
Methods
 
Genomic DNA was extracted from young leaves of each genotype following the method by Doyle and Doyle (1987) with minor modifications. DNA concentration was determined by spectrophotometer at 260 nm. The measurement of the OD 280 nm was used to define the content and the ratio OD260/OD280 was between 1.8 and 2.0 (Touil et al., 2008). The 20 μl master mix for the RAPD and ISSR analysis consist of 10x buffer, 0,2 mM of each dNTP, 50mM MgCl2, 5 mM of primer, 1 U Taq polymerase, sterile water and 1 μl (30 ng) of DNA for PCR reaction (Yýldýz et al., 2011).
       
Five RAPD and four ISSR primers were selected from the previous studies on Medicago sativa L. (Denghan-Shoar et al., 1997; Gherardi et al., 1998; Mengoni et al., 2000; Paredes et al., 2002; Touil et al., 2008; Tucak et al., 2008; Petolescu and Nedelea, 2009; Ertus et al., 2014). RAPD amplification was performed in thermo-cycler starting with 4 min. of denaturation at 94°C followed 35 cycle of 1 min at 94°C, 1 min at for annealing (Table 2) and 2 min. at 72°C and final extension of 6 min. at 72°C. The RAPD and ISSR fragments were separated by 1.5% agarose-gel electrophoresis with TAE 1X buffer at 90V for 3 hours and visualized with ethidium bromide. Gels were photographed under UV light to score band were analyzed in binary form for absence (0) or presence (1). Monomorphic bands were excluded from data analysis.
 

Table 2: Characteristic of RAPD primers.


       
Similarity index coefficients of the genetic distance among the genotypes were formed in accordance to the similarity coefficient of Jaccard by SÝMQUAL program and dendrograms with package software NTSYSpc-2.0 according to non-weighted arithmetical average similar group method (UPGMA) (Rohlf, 1997). Nei genetic diversity index were determined using package software POPGENE (Nei, 1973; Yeh et al., 1997; Labate, 2000). The correlation between RAPD and ISSR markers were calculated by means of Mantel test (Mantel, 1967).

RESULTS AND DISCUSSION

RAPD and ISSR amplification
 
Genetic diversity and degree of genetic relationship of 23 landraces collected from 21 different locations in the EasternAnatolia region of Turkey and one registered variety were determined using RAPD and ISSR markers. With the 5 RAPD markers used in the study, a total of 29 bands ranging between 500-1500 bp in size were obtained and 20 of them were found to be polymorphic. All of the 8 bands obtained using RAPD-3 primer, which yielded the highest number of bands, were polymorphic (100%) whereas only one of the three bands obtained using RAPD-1 primer, which yielded the lowest number of bands, was polymorphic (33.3%). Thus, the percentage of polymorphism ranged from 33.3% to 100%. Nosrati et al., (2012) reported the percentage of polymorphism with RAPD markers as 66.67%-84.62%. Hejrankesh et al., (2014) reported a percentage of polymorphism between 75.5%-83.89% with RAPD primers among 10 different sainfoin landraces. Similar to other studies, RAPD primers had a high percentage of polymorphism. A total of 20 bands ranging between 450-1350 bp in size were observed with ISSR primers and 15 of them were found to be polymorphic. Of the bands obtained using ISSR-3 primer, which gave the highest number of bands, 7 were polymorphic (87.5%), whereas only 1 of the bands obtained using ISSR-1 primer, which gave the lowest number of bands, was polymorphic (33.3%) (Table 3). Thus, the percentage of polymorphism was found to be between 33.3%-83.3%.  According to the similarity matrix, there is a low correlation among RAPD and ISSR markers (r=0.063). 
 

Table 3: Characteristic of ISSR primers.


 
Relationship degree and genetic diversity
 
According to the Jaccard similarity coefficient, using RAPD markers, the closest populations were found to be Bozyiðit and Hizan varieties with a similarity coefficient of 0.95 and the most distant populations were found to be Atalan and Lütfibey varieties with a similarity coefficient of 0.25. According to the results obtained with ISSR markers, the highest similarity was between Özalp and Barýþ, Özalp and Muradiye, Barýþ and Muradiye and Dinlence and Yemişlik varieties, all with a similarity coefficient of 1.00. However, the least similarity was between Yukariyaniktas and Erciþ/Merkez varieties, with a coefficient of 0.50. According to the results obtained using the combination of RAPD and ISSR primers, Bozyiðit and Hizan were the closest populations with a similarity coefficient of 0.91, while the most distant were found Atalan and Alacayar and Atalan and Dolutaş, with a similarity coefficient of 0.45. Rasouli et al., (2013) reported that, with RAPD markers, the Nei’s genetic similarity coefficient was between 0.08-0.43 among the landraces. Highly similar populations, which were reported by other researchers, were identified in this study as well.

Genetic diversity among populations was calculated according to Nei’s genetic diversity index. According to the findings, it was determined that Nei’s genetic diversity index was 0.3365, 0.2656 and 0.3018 when RAPD, ISSR and RAPD+ISSR markers were used, respectively. Avcý et al., (2014) reported that the Nei’s genetic diversity index among the different species of Onobrychiswas between 0.2397-0.2916.  Nosrati et al., (2012) reported that the Nei’s genetic diversity index of Onobrychis viciifolia species was between 0.2466-0.3186. In their study using RAPD markers, Hejrankesh et al., (2014), found that Nei’s genetic diversity was between 0.300-0.343 for 10 local varieties of Iran. In a study on 5 populations from Iran, it was reported that Nei’s genetic diversity index was between 0.118 and 0.179 with ISSR markers and was between 0.3640-0.44618 with RAPD markers (Nosrati et al., 2012; Nosrati et al., 2016). Özkan and Bilgen (2018) reported that Nei’s genetic diversity index obtained using 10 SSR markers was 0.210 for 5 different sainfoin populations. Shen et al., (2019) reported that genetic diversity among sainfoin is high. The findings obtained from the study were in concordance with the findings of other researchers and it was determined that sainfoin has high genetic diversity. Özkan and Bilgen (2018) found that the intrapopulation genetic diversity was high, interpopulation genetic diversity was very low in sainfoin populations. These differences were attributed to the number of populations used by the researchers, the collection of these populations from different regions, as well as the use of different markers.
 
Cluster analysis
 
In the dendrogram obtained with RAPD markers, the Atalan variety had a separate branching, Yemişlik and Sirimli were clustered under one group and the other varieties were clustered under two groups (Fig 1). In the dendrogram obtained with ISSR markers, the Yuva population had a separate branching, Sirimli and Yukarikaymaz populations were clustered under one group and the other varieties were clustered under two groups (Fig 2). On the other hand, in the dendrogram obtained with the combination of RAPD and ISSR primers, Yukarikaymaz and Lütfibey varieties were clustered under one group, the other varieties were clustered under three groups, whereas Atalan and Sirimli varieties had their separate branchings (Fig 3). In their study using SSR primers with 32 populations, Kempf et al., (2016) reported that the sainfoin populations were clustered under two main groups. Cluster analysis results in Hejrankesh et al., (2014) showed that 10 different sainfoin varieties had 3 branchings and in Özkan and Bilgen (2019) showed that 5 different populations had three branching.
 

Fig 1: UPGMA dendrogram showing the relationship of O. viciifolia based on RAPD markers.


 

Fig 2: UPGMA dendrogram showing the relationship of O. viciifolia based on ISSR markers.


 

Fig 3: UPGMA dendrogram showing the relationship of O. viciifolia based on primers RAPD + ISSR markers.

CONCLUSION

Identification of the genetic diversity of sainfoin, which is comparable to alfalfa that has been intensively cultivated and yields high in marginal environments, is important. The aim was to identify the genetic diversity and the degree of relationship between 23 sainfoin landraces and one registered sainfoin variety using RAPD and ISSR primers. The high genetic diversity of the cultivated Onobrychis viciifolia was attributed to its ploidy level of 2n=4x=28, chromosome structure and allogamous species. In light of the molecular data, it was found that the cultivated sainfoin landraces were significantly rich genetic resources.               
               
The study also demonstrates that the degree of relationship between sainfoin varieties with high genetic diversity can be distinguished much better using RAPD markers. Based on this information, it can be said that RAPD markers will be helpful when selecting parents in the future breeding of sainfoin landraces.

REFERENCES


  1. Avcı, S., Ilhan, E., Erayman, M., Sancak, C. (2014). Analysis of onobrychis genetic diversity using SSR markers from related legume species. The Journal of Animal and Plant Sciences. 24(2): 556-566.

  2. Demdoum, S., Munoz, F., Delgado, I., Valderrabano, J., Wunsch, A. (2012). EST-SSR cross-amplification and genetic similarity in Onobrychis genus. Genet Resour Crop Evol. 59: 253-260

  3. Dehghan-Shoar, M., Hampton, J.G., Gardiner, S.E. (1997). Genetic analysis among and within populations forming ecotypes and cultivar of alfalfa, Medicago sativa (Leguminosae), using RAPD fragments. Plant Systematics and Evolution, 208: 107-119.

  4. Delgado, I., Salvia, J., Buil, I. andrés, C. (2008). The agronomic variability of a collection of sainfoin accessions. Spanish Journal of Agricultural Research. 6(3): 401-407

  5. Dos Santos, J. B., Nienhuis, J., Skroch, P., Tivang, J., Slocum, M. K. (1994). Comparison of RAPD and RFLP genetic markers in determining genetic similarity among (Brassica oleracea L.) genotypes. Theoretical and Applied Genetics. 87: 909-915.

  6. Doyle, J.J., Doyle, J.L. (1987). A rapid DNA isolation procedure from small quantities of fresh leaf tissues. Phytochem. Bull. 19: 11-15.

  7. Ertus, M.M., Sabancı, C.O., Sensoy, S. (2014). The Determination of Moleculer Diversity Among Some Alfalfa (Medicago sativa L.) Ecotypes Using RAPD Markers. Yyu. J. Agr. Scı. 24(1): 7-15. 

  8. Gherardi, M., Mangin, B., Goffinet, B., Bonnet, D., Huguet, T. (1998). A method to measure genetik distance between allogamous populations of alfalfa (Medicago sativa) using RAPD moleculer markers. Theor. Appl. Genetics. 96: 406-412.

  9. Hesamzadeh Hejazi, S.M., Ziaei Nasab, M. (2010). Cytotaxonomy of some Onobrychis (Fabaceae) species and populations in Iran. Caryologia. 63(1): 18-31.

  10. Hayot Carbonero, C., Mueller-Harvey, I., Brown, T.A., Smith, L. (2011). Sainfoin (Onobrychis viciifolia): A beneficial forage legume. Plant Genetic Resources: Characterization and Utilization. 9(1): 70-85.

  11. Hejrankesh, N., Mousavizadeh, S.A., Haghighi, A.R., Rashidi, V. (2014). Evaluation of genetic diversity of sainfoin (Onobrychis viciifoila scop.) Landraces using rapd markers. Journal of current research in science. 2(6): 739-748.

  12. Kempf, K., Mora-Ortiz, M., Smith, L.M.J., Kölliker, R., Skøt, L. (2016). Characterization of novel SSR markers in diverse sainfoin (Onobrychis viciifolia) germplasm. Genetics. 17: 124.

  13. Kumar, P., Gupta, V.K., Misra, A.K., Modi, D.R., Pandey, B.K. (2009). Potential of Molecular Markers in Plant Biotechnology. Plant Omics Journal. 2(4): 141-162.

  14. Labate, J.A. (2000). Software for population genetic analyses of molecular marker data. Crop. Sci. 40: 1521-1528.

  15. Mantel, N.A. (1967). The detection of disease clustering and a generalized regression approach. Cancer research. 27: 209-220. 

  16. Mengoni, A., Gori, A., Bazzicalupo, M. (2000). Use of RAPD and microsatellite (SSR) variation to ases genetic relationships among populations of tetraploid alfalfa, Medicago sativa. Plant Breeding. 119: 311-317.

  17. Mohajer, S., Jafari, A.A., Taha, R.M., and Ahmad, A.B.A. (2012). Evaluation of yield and morphological traits in 72 genotypes of sainfoin (Onobrychis viciifolia Scop) through factor analysis. Legume Research. 35(2): 132-137.

  18. Nei, M. (1973). Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA. 70: 3321-3323.

  19. Nosrati, H., Feizi, M.A.H., Tarrah, S.S., Haghighi, A.R. (2012). Population GeneticVariation In Sainfoin (Fabaceae) Revealed By Rapd Markers. Analele Universitãþii din Oradea–Fascicula Biologie Tom. XIX. 1: 11-16.

  20. Nosrati, H., Feizi, M.A.H., Latifian, F., Haghighi, A.R. (2016). Eco- Geographical variations of ISSRs among populations of Onobrychis viciifolia (Sainfoin, Fabacae). Analele Universitãþii din Oradea, Fascicula Biologie. 2: 62-66.

  21. Özkan, S., Bilgen, B.B. (2018). Genetic Characterization of Sainfoin (Onobrychis viciifolia) Varieties and Populations Using Microsatellite Markers. Journal of Tekirdag Agricultural Faculty. 16(1). 

  22. Paredes, M., Becerra, V., Rojo, C., Del Pozo, A., Ovalle, C., Aronson, J. (2002). Ecotypic differentiation in Medicago polymorpha L. along an environmental gradient in central Chile. RAPDs studies show little genetic divergence. Euphtica. 123: 431-439.

  23. Petolescu, C., Nedelea, G. (2009). Genetic Diversity Analysis of the In Vitro Regenerated Alfalfa Plants Using Inter Simple Sequence Repeat (ISSR) Markers. Romainan Biotechnological Letters. 14(6): 4882-4886.

  24. Rasouli, M., Jafari, A.A., Tabaei-Aghdaei, S.R., Salehi, P. (2013). Assessment of genetic variability of 36 populations of Sainfoin (Onobrychis sativa) based on RAPD markers. International Journal of Biosciences. 3(10): 15-26.

  25. Rohlf, F.J. (1997). NTSYS-Pc: Numerical Taxonomy and Multivariate Analysis System. Exeter Software, New York.

  26. Sanghani, J., Golakiya, B.A., Dhedhi, K.K. and Patel, S.V. (2015). Molecular characterization of mung bean (Vigna radiata L.) genotypes through RAPD, ISSR and SSR markers. Legume Research. 38: 452-456.

  27. Semagn, K., Bjørnstad, A., Ndjiondjop, M.N. (2006). An overview of molecular marker methods for plants. African Journal of Biotechnology. 5(25): 2540-2568.

  28. Sepet, H., Emre, Ý., Kiran, Y., Kurþat, M., Sahin, A. (2011). Karyological studies on eight species of Onobrychis genus in Turkey. Biologia. 66: 996-1002.

  29. Sharifi, S.M. (2016). Assessment Genetic Diversity Landraces In Onobrychis Sativa Using SSR, RAPD and SRAP Markers. Journal of Agriculture Biotechnology. 1(2): 91-95.

  30. Shen, S., Chai,X., Zhou, Q., Luo, D., Wang, Y., Liu, Z. (2019). Development of polymorphic EST-SSR markers and characterization of the autotetraploid genome of sainfoin (Onobrychis viciifolia). Peer J. 7:e6542. http://doi.org/ 10.7717/peerj.6542.

  31. Touil, L., Guesmi, F., Fares, K., Ferchichi, A. (2008). Genetic diversity of some Mediterranean populations of the cultivated alfalfa (Medicago sativa L.) using ISSR markers. Biotechnology. 7(4): 808-812.

  32. Tucak, M., Popovic, S., Cupic, T., Grljusic, S., Bolaric, S., Kozumplik, V. (2008). Genetic diversity of alfalfa (Medicago spp.) estimated by molecular markers and morphological characters. Periodicum Biologorum. 110(3): 243-249.

  33. Yeh, F.C., Yang, R.C., Boyle, T.B.J., Ye, Z.H., Mao, J.K. (1997). POPGENE, the User Friendly Shareware for Population Genetic Analysis. University of Alberta, Canada. Molecular Biology and Biotechnology Centre.

  34. Yildiz, M., Ekbic, E., Keles, D., Sensoy, S., Abak, K. (2011). Use of ISSR, SRAP and RAPD markers to assess genetic diversity in Turkish melons, Scientia Horticulturae. 130: 349-353.

  35. Zarrabian, M., Majidi, M.M. (2015). Genetic diversity and relationships within and among Onobrychis species using molecular markers. Turk J Bot. 39: 681-692. 
 
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