Greener Journal of Agricultural Sciences
ISSN: 2276-7770; ICV: 6.15
Vol. 4 (2), pp. 039-045, March 2014
Copyright ©2017, the copyright of this article is retained by the author(s)
Research Article (DOI: http://dx.doi.org/10.15580/GJAS.2014.2.021014101)
Determination of Phaseolin Types in Common Bean (Phaseolus vulgaris) Varieties from Turkey
Seher Yıldız Madakbaş1*, Halima Awale2 and James Kelly2
1Ahi Evran University, Agricultural Faculty, Department of Horticulture, Kırşehir-Turkey.
2Michigan State University, Department of Crop, Soil and Microbial Sciences, Michigan- USA.
Article No.: 021014101
The purpose of our study was to determine the phaseolin types of common bean varieties widely consumed in Turkey through protein extraction. Twenty- four bean genotypes, consisting of 22 varieties from Kırsehir province of Turkey and two control varieties (Andean and Mesoamerican) were assessed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed with protein samples extracted from bean genotypes. Gels were stained with Coomassie blue dye R-250 and kept in fixing solution. Our analyses revealed that out of 22 bean samples, 16 contained Andean phaseolin type and 6 contained Mesoamerican phaseolin type. T-test statistical analysis was performed to determine the relationship between seed phaseolin types and seed volumes (length, height and width). The results showed that there was a significant difference between phaseolin and seed volume.
E-mail: seheryldzmadakbas @gmail.com
Phaseolus vulgaris, phaseolin, SDS-PAGE, genotype, Andean, Mesoamerican, seed volume
Common bean (Phaseolus vulgaris L.) is one of the most widely consumed seed based food sources around the world. It is an important legume in Turkey with its pods and seeds being consumed, although the pods are relatively more popular. Fresh common bean production in Turkey is 615,000 tons and dry bean production is 200,000 tons annually (FAO, 2011; FAO, 2012).
Aside from being a perfect nutritional source in terms of some vitamins and minerals, common bean is also rich in proteins, unsaturated fatty acids (linoleic acid) and soluble fiber (Rodiňo et al., 2009). Nutritional contents in bean have therapeutic properties in cardiac and kidney diseases, several cancer types and obesity (De la Fuente et al., 2012).
Common bean contains higher levels of protein (>20%) compared to cereals such as rice and wheat (<15%) (Kaplan, 1965; Brown et al., 1981a; Chandrakanth and Hall, 2008). Phaseolin, a protein mainly stored in seeds, makes up the majority of total seed protein. It has been shown that globulin, which makes up phaseolin, consists of two major parts: vicilin and legumin. Globulin has been utilized in protein studies since total seed protein consists of 35-50% globulins (Ma and Bliss, 1978; Lioi, 1989; Rodiňo et al., 2001a). However, the exact ratio varies between different bean genotypes (Mcleester et al., 1973).
Phaseolin is coded by a single complex loci located in Pv07 linkage group consisting of 6-10 closely tied members of a small gene family (Brown et al., 1981b; Gepts et al., 1986; Koenig et al., 1990; Kami et al., 1995). It has been proven that phaseolin is an important and informative marker in studies on genetic diversity and evolution for both wild and cultivated lines due to its functional and structural properties (de la Fuente et al., 2012). Moreover, these characteristics provide researchers with important opportunities in determining protein compositions of bean seeds qualitatively and quantitatively towards selection for plant breeding (Gepts and Bliss, 1988; Gepts, 1993; Toro et al., 2007; Chandrakanth and Hall, 2008).
It has been shown that phaseolin types vary according to their eco-geographical origin. Two major gene pools were identified. These are Mesoamerican gene pool; from Mexico to Colombia, and Andean gene pool; from south Peru to Nothwestern Argentina. (De la Fuente et al., 2012).Mesoamerican varieties have “S” (Sanilac), “M” (Middle America) or “B” (Boyacá) phaseolin types; Andean varieties have “T” (Tendergreen), “C” (Contender), “H” (Huevo de Huanchaco), “A” (Ayacudo), “J” (Jujuy) and “I” (Inca) phaseolin types. Phaseolin type “C” was reported to be a combination between types “T” and “S” of Mesoamerican and Andean origin respectively. Among cultivated varieties the most popular types are “S” and “T”. Type “B” phaseolin is present only in wild and cultivated varieties of Colombia. Such variety in phaseolin types is thought to have resulted from random outcrosses in nature, contaminations from cultivation areas and multiple cultivation efforts (Brown et al., 1981a; Gepts et al., 1986; Koenig et al., 1990; Kami et al., 1995).
Since the discovery of the Americas, common bean has been traded extensively between the old world and the new world. Common bean came to Europe in 1540s, allowing bean genotypes of Andean and Mesoamerican origin to spread around different regions of the world. It was determined that bean genotypes in Western Europe, the Iberian Peninsula and Africa are mostly of Andean origin since bean varieties in the Iberian Peninsula commonly contains type “C” phaseolin. Bean varieties of Andean origin possessed an advantage over varieties of other origins due to better photoperiodic adaptation. Among varieties of Western Europe, type “T” phaseolin is the most dominant because of favored cultivation due to their green pods (Rodiňo et al., 2001a). Genotypes originating from Andean mountains have adapted to cold European summers better than Mesoamerican genotypes. Thousands of local genotypes of Andean and Mesoamerican gene pools have been cultivated in Europe and preserved in seed banks.
Researchers have utilized molecular markers to determine genetic structures and origins of bean varieties in addition to the seed storage protein “phaseolin” (Piergiovanni et al., 2000; Rodiňo et al., 2003; Logozzo et al., 2007; Limongelli et al., 1996; Perez-Vega et al., 2009; Maras and Sustar-Vozlic, 2013). Polyacrylamide gels have usually yielded the best results in determining phaseolin types. Analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) yields highly stable bands. Thus, types of phaseolin have been identified more easily according to their molecular weight (53kD – 20kD) (Derbyshire et al., 1976; Lioi, 1989).
In this study, the objectives were: 1) pave the way for future studies aiming to determine the structure of phaseolin seed storage protein by determining variability of phaseolin types among common bean genotypes in Turkey, 2) create a preliminary model for phylogenetic studies and 3) assist breeding programs by determining genetic relationships between common bean genotypes.
MATERIALS AND METHODS
Seeds of bean samples
Twenty-two common bean genotypes were provided by the project called: “Characterization of Local Bean Populations in Kırşehir Province” conducted in Kırsehir Ahi Evran University, Faculty of Agriculture, Department of Horticulture in year 2012. Two control genotypes (G 19833-Andean and Dor 364 Mesoamerican) were provided by Michigan State University, Plant Soil and Microbial Sciences Department. Turkish genotypes were G 88-10, G 62, G 68, G 111, G 11, G 28, G 106, G 19, G 70, G 108, G 77, G 105, G 104, G 110, G 16, G 17, G 112, G 64, G 32, G 69, G 35 and G 107.
Protein extraction from seeds
The seed coats from three seeds of each genotype were peeled and the cotyledons were homogenized in 2000 Geno/Grinder (Spex/Ceptiprep) for five minutes at 800 rpm. Fifty milligrams of bean flour was measured and placed into 1.5mLeppendorf tube. 500 µL of Protein Extraction Buffer (0,036 M Tris-HCl pH 8.5) was added and the tubes were gently mixed. Samples were held inside the sonication chamber containing ice and water for one hour and centrifuged (Microcentrifuges Eppendorf Refrigerated 5418R) at 14,000 rpm for 20 minutes. 400 µL of supernatant was transferred into fresh tubes. Cracking buffer with SDS (Tris-HCl 0.625M pH 6.8) was added to the supernatant at 1:1 v/v ratio. Samples were vortexed for two minutes, incubated in 100oC water bath for five minutes and stored at -20oC after being cooled to room temperature for long- term storage (Toro et al., 2007; Ligarreto and Ocampo, 2012).
Electrophoresis was performed after loading samples into NuPAGE 10% Bis-Tris mini gel – NOVEX (provided by Life technologies). Miniprote in Xcell Unit and BIO-RAD SDS-PAGE markers (20kD-103kD) were used. 1µL from samples and 5µL from markers were loaded. Electrophoresis was performed at 110V and 80mA for 80 minutes. Following electrophoresis gels were incubated in Coomassie blue R-250 Buffer at 100 rpm for 1 hour in an orbital shaker. Fixing was performed by incubating the gels in 50ml of fixing solution. The solution was replaced with fresh fixing solution every hour for 3-4 hours (Laemmli, 1970; Bradford, 1976; Ma and Bliss, 1978; Lareo et al., 1993; Rodino et al., 2001b).
Phaseolin types and seed volumes (length, height and width) were determined as performed by Gepts and Bliss (1986). Correlations between phaseolin type and seed volume were determined by Mann-Whitney U Test, the non-parametric equivalent of T-test statistical analysis (Stonehouse and Forrester, 1998).
RESULTS AND DISCUSSION
Twenty-two common bean genotypes and two control varieties were analyzed on SDS-PAGE gels along with a molecular marker (20kD-103kD). Differences in phaseolin types between samples and control varieties are shown in Figure 1 in detail. Band sizes and the number of bands were different between control and sample varieties. Three bands between 49kD and 55kD were observed for Andean genotypes, while for Mesoamerican genotypes two bands between 50kD and 53kD was observed. The number of the bands observed in a specific region of the gel may vary according to the relative weights of the proteins the sample contains. After these bands were highlighted by Coomassie Blue, sharp and proper bands were also obtained along with large and wide bands. This has been reported as a result of different sized seed samples being used in protein extractions (Mcleester et al., 1973), which was confirmed in the gel images we obtained. As can be seen in Figure 1, bean genotypes 14 (G110), 20 (G69), 21 (G35) and 22 (G107) have produced thick bands whereas genotypes 15 (G16) and 16 (G17) have produced proper bands. Such bands were also obtained in other gel images not provided here. Although Phaseolin protein has a narrow range of molecular weight (45-52 kD), phaseolin has been reported to have various molecular weights by different researchers. Mcleester et al. (1973) reported phaseolin having a molecular weight of 53, 47 and 43 kD; Derbyshire et al. (1976) reported 49, 45 and 42 kD; Bollini and Chrispeels(1978) reported 52 and 49 kD(Ahn et al., 1991). The molecular weights of the bands obtained in this study correlates with those reported by other researchers. However the method of extraction and characterization of phaseolin can lead to different results obtained by different laboratories (Derbyshire et al.,1976).SDS-PAGE analysis of phaseolin has been utilized worldwide as an effective tool in analyzing genetic variability, evolutionary relationships and correlation between molecular weight of the band and genotype among bean populations(Brown et al., 1981b; Mcleester et al., 1973; Romero et al., 1975; Gepts, 1998; Singh, 2001; Rodiňo et al., 2001b).We have also observed that SDS-PAGE analysis was useful in tracking the origins of bean genotypes that have been dispersed into various regions because PAGE analyses revealed any changes in genetic variability during the cultivation process. These changes in the Phaseolin locus (Phs) have been shown in Mesoamerican genotypes rather than Andean genotypes (Gepts, 1993; Kami et al., 1995; Chacon et al., 2005).
Phaseolin type, seed volume and color are shown in Table 1. It was determined that out of 22 Turkish bean genotypes, 16 had Andean type phaseolin and 6 had Mesoamerican type phaseolin. Studies on phaseolin and its allozymes have shown that the Andean type phaseolin is the predominant type in worldwide (Singh et al., 1991; Rodiňoet al., 2001; Santalla et al., 2002; Rodiňo et al., 2006). Sustar-Vozlicet al.(2006) have identified Europe as the second center of domestication of the Andean gene pool since the European bean germplasm dates back to 200 years ago. They reported that European accessions have great morphological similarities with the Andean bean genotypes obtained from Chile. Angioi et al. (2010) indicated that Andean genotypes have a greater effect on European accessions compared to Mesoamerican genotypes. European accessions were determined to contain 72% “T”, 21% “S” and 7% “C” type phaseolin and accessions of Iberian Peninsula to contain 43% “C”, 29% “S” and 1% “H” phaseolin. The reason for this is that genotypes of Andean origin are more adapted to ecological conditions compared to Mesoamerican genotypes. Moreover, Andean genotypes containing “T” type phaseolin are preferred in Europe and Turkey because of their green pods which are consumed as fresh bean. It has been reported that among the genotypes consumed as fresh beans, 66% had “T”, 25% had “S” and 9% had “C” type phaseolin (Maras and Sustar-Vozlic, 2013).
In our study we have used T-test statistical analysis to determine the correlation between phaseolin types and seed volumes. Analyses have shown that there is significant difference between phaseolin types in terms of seed volumes (Table 2). Seeds containing Andean type phaseolin were determined to be larger compared to those containing Mesoamerican type phaseolin. Phaseolin alleles were determined to be correlated with seed size and the seed sizes were under control of a quantitative loci (Perez-Vega et al., 2009). Other than phaseolin types, the seed volumes varied among genotypes. Previous studies have shown that “T”, “C”, “H” and “A” (Andean) phaseolin types were associated with larger and wider seeds compared to “S” and “B” (Mesoamerican) genotypes (Gepts et al., 1986; Gepts and Bliss, 1986; Rodiňo et al., 2009).
As shown in Table 1, seeds from Mesoamerican genotypes had dark maroon, dark purple or white color whereas Andean genotypes showed great variability. Morphological analysis of the seeds has shown that small white and red seeds had type “S” phaseolin of Mesoamerican origin, seeds with dark spots on cream background had type “T” phaseolin of Andean origin, seeds with zebra stripes pattern on cream background had type “C” phaseolin and wide kidney shaped seeds with dark maroon-purple and cream seeds containing red veins had both type “T” and “C” phaseolin. White seeds were observed in both Mesoamerican and Andean genotypes, but seeds from the Mesoamerican gene pool showed to be smaller than seeds from the Andean gene pool (Gepts and Bliss, 1988). The analyses we have performed have shown that white seeds of both Andean and Mesoamerican origin had variability in terms of size. The data on seed color with Mesoamerican and Andean origin matches with the provided in the literature. Among these genotypes G 106, G 110 (Andean) and G 11 (Mesoamerican) had an almost black dark purple color. Seeds of control variety DOR-364 (Mesoamerican) were also of the same color. The shared seed color was reported to be resulting from recombination between Andean and Mesoamerican genotypes (Singh et al., 1991; Acquaah and Isleib, 1994; Santalla et al., 2002; Rodino et al., 2006).
Studies on phaseolin type determination aid researchers in understanding wide dispersal of bean genotypes from their original location to their secondary location (De la Fuente et al., 2012). Bean cultivation has been widespread through the Mediterranean region and great variation has occurred. This occurred as a result of different preferences of farmers in various locations.
Phaseolin variability in countries around the Mediterranean region, including Turkey, can be compared to Central and South American countries (Gepts et al., 1986; Lioi, 1989; Chacon et al., 2005). It was reported that 44% of European bean germplasm consists of hybridizations between Andean and Mesoamerican gene pools (Angioi et al., 2010; Santalla et al., 2002). Rodino et al. (2006) have stated that hybrid genotypes have formed a secondary center for genetic variability in bean because wild ancestral genotypes have formed the genetic basis for today’s local and cultivated varieties.
In this study, we have identified phaseolin types in common bean genotypes by protein extraction and SDS-PAGE analysis. However this work helped in the identification of phaseolin types in Turkey in order to reveal the genetic variability in the germplasm since data on current bean collections is limited. In future studies on phaseolin types, the number of seeds has to be high enough to represent the present variability. This study will be a preliminary model for phaseolin studies in Turkey.
This study was conducted in Michigan State University, Plant Soil and Microbial Sciences Department, Bean Genetics Lab in year, 2013. We would like to thank Cesar Umberto Ocampo, Caroline Astudillo-Reyes, Hussein Alameldin, Mete Gurhan Dasci and Asli Akıllı for their assistance in this study.
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Cite this Article: Madakbaş SY, Halima A, James K, 2014. Determination of Phaseolin Types in Common Bean (Phaseolus vulgaris) Varieties from Turkey. Greener Journal of Agricultural Sciences. 4(2):039-045, http://dx.doi.org/10.15580/GJAS.2014.2.021014101.