Examination of Genetic Diversity of Common Bean from the Western Balkans

In this study, genetic diversity of 119 accessions of common bean (Phaseolus vulgaris) from five former Yugoslav republics constituting the western Balkans was assessed by 13 microsatellite markers. This set of markers has proven before to efficiently distinguish between bean genotypes and assign them to either the Andean or the Mesoamerican gene pool of origin. In this study, 118 alleles were detected or 9.1 per locus on average. Four groups (i.e., Slovene, Croatian, Bosnian, and Serbian) showed similarly high levels of genetic diversity as estimated by the number of different alleles, number of effective alleles, Shannon’s information index, and expected heterozygosity. Mildly narrower genetic diversity was identified within a group of Macedonian accessions; however, this germplasm yielded the highest number of private alleles. All five germplasms share a great portion of genetic diversity as indicated by the analysis of molecular variance (AMOVA). On the basis of the scored number of migrants, we concluded that the most intensive gene flow in the region exists in Bosnia and Herzegovina. Cluster analysis based on collected molecular data classified the accessions into two large clusters that corresponded to two gene pools of origin (i.e., Andean and Mesoamerican). We found that Andean genotypes are more prevalent than Mesoamerican in all studied countries, except Macedonia, where the two gene pools are represented evenly. This could indicate that common bean was introduced into the western Balkans mainly from the Mediterranean Basin. Bayesian cluster analysis revealed that in the area studied an additional variation exists which is related to the Andean gene pool. Different scenarios of the origin of this variation are discussed in the article. Common bean (2n = 2x = 22) is the most important edible food legume for direct human consumption in Europe and in the world as it represents a valuable source of proteins, vitamins, fiber, and minerals (Broughton et al., 2003). The Andean region and Mesoamerica are distinguished as the two major centers of origin of this species, according to morphological characters (Singh et al., 1991), seed proteins (Gepts et al., 1986), isozymes (Koenig and Gepts, 1989), DNA markers (Freyre et al., 1996), and sequence data (Schmutz et al., Received for publication 10 Feb. 2015. Accepted for publication 14 Apr. 2015. This work was financially supported by FP7 Project CropSustaIn, grant agreement FP7-REGPOT-CT2012-316205, by grant No. 168/01 from the SEE-ERA.NET.PLUS FP7 Regional Programme and by grant P4-0072 from the Slovenian Research Agency. Accessions in Republic of Srpska, were collected through the National Program for Plant Genetic Resources, with a financial support by Ministry of Science and Technology of the Republic of Srpska.We are thankful toMatej Knapi c fromAgricultural Institute of Slovenia for preparing a geographic map of the western Balkans with collection sites of the studied common bean accessions. Corresponding author. E-mail: marko.maras@kis.si. 308 J. AMER. SOC. HORT. SCI. 140(4):308–316. 2015. 2014). After its domestication in the Americas, common bean promptly spread worldwide (Zeven, 1997). Introduction of this species in Europe dates to the early 16th century when Spanish and Portuguese sailors brought bean specimens to their homelands from both centers of domestication (Gepts and Bliss, 1988). During the last five centuries of cultivation, many landraces and cultivars evolved under diverse environments and farmer preferences in Europe (Zeven, 1997). Though many local cultivars were lost in the last 60 years, there are still many farmers who maintain old local landraces, which are well adapted to the pedoclimatic conditions peculiar to their limited geographical areas, and who have been exchanging their seeds with surrounding areas, mainly in local markets. The pathways of dissemination of the common bean into and across Europe were very complex, with several introductions from America, combined with direct exchanges between European and otherMediterranean countries (Papa et al., 2006). In the past two decades, phaseolin seed protein and other genetic markers have been intensively used to analyze the structure of European common bean populations and distribution of the two gene pools. A prevalence of the Andean ‘‘C’’ and ‘‘T’’ phaseolin types (76%) was first detected by Gepts and Bliss (1988), and was then confirmed by Lioi (1989) in an analysis of a large collection from Italy, Greece, and Cyprus (66% in total), by Logozzo et al. (2007) for a broad European collection (76%), and by others for Portuguese and Spanish genotypes (Rodino et al., 2001, 2003). Similar distribution of Andean and Mesoamerican genotypes has also been observed in phaseolin and molecular marker analyses at a regional scale (Angioi et al., 2009; Limongelli et al., 1996; Piergiovanni et al., 2000; Sicard et al., 2005; Su star-Vozli c et al., 2006). Moreover, several studies have focused on hybridization between the Andean and Mesoamerican gene pools in Europe. This phenomenon was first evidenced in the Iberian Peninsula by analyzing phaseolins, allozymes, and morphological characters (Rodino et al., 2006; Santalla et al., 2002), and later by inter-simple sequence repeat and simple sequence repeat (SSR) markers from both the chloroplast and nuclear genomes of European genotypes (Angioi et al., 2009, 2010; Sicard et al., 2005). Information on genetic diversity of common bean in the western Balkans that encompasses former Yugoslav republics (i.e., Slovenia, Croatia, Bosnia and Herzegovina, Macedonia, and Serbia) is scarce. In this region, common bean represented a very important food in the human diet for centuries. Until World War II, this crop was grown on large areas (>1 million ha) in the field often together with maize (Zea mays). In the second half of the last century new cultivars of both maize and common bean were introduced into crop production, and the old cropping system was abandoned, which subsequently, lead to a great reduction of the areas covered by beans ( 120,000 ha). Different approaches for assessing diversity at the molecular level are presently available. Microsatellites have been considered as the reference markers for cultivar fingerprinting in common bean because they are codominant, widely distributed in the genome, highly polymorphic, and highly repeatable (Powell et al., 1996; Yu et al., 1999). In this study, the genetic diversity of common bean from the western Balkans was assessed by SSR markers. A total of 13 markers that proved in previous studies (Maras et al., 2006, 2013) to be highly polymorphic and as efficient as amplified fragment length polymorphism markers in distinguishing common bean genotypes according to their gene pool of origin (Maras et al., 2008)were employed. The collectedmolecular data allowed us to: 1) examine the relationships among the accessions and the organization of common bean genetic variation in the western Balkans, 2) identify the original gene pool (Andean or Mesoamerican) of the studied plant material, and 3) clarify the bean dissemination process in the western Balkans. Materials and Methods PLANT MATERIAL. A total of 119 common bean landraces from national gene banks of five former Yugoslav republics were used in this study (Table 1; Fig. 1). These included 25 accessions from Bosnia and Herzegovina, 18 from Croatia, 28 from Macedonia [former Yugoslav Republic of Macedonia (FYROM)], 30 from Serbia, and 18 from Slovenia (passport data of the accessions are available upon request). Out of 18 Slovene accessions included, 14 of them have already been assessed for genetic diversity and phaseolin type in our previous studies (Maras et al., 2013; Su star-Vozli c et al., 2006) and were used here as a reference material for the determination of gene pool of origin of the other 105 accessions. DNA EXTRACTION. Total DNA was extracted from bulked leaf material of 10 plants of each accession using BioSprint15 DNA Plant Kit (Qiagen, Germantown, MD) and MagMax Express Magnetic Particle Processor (Life Technologies, Grand Island, NY) following manufacturer’s instructions. Integrity and quality of DNA were evaluated by electrophoresis on 1.0% agarose gels. Concentrations of DNA samples were determined with a fluorometer (DyNA Quant 200; Hoefer, Holliston, MA). MOLECULAR ANALYSES. Thirteen SSR loci developed by Metais et al. (2002) andGaitan-Solis et al. (2002)were employed (Table 2). Amplification reactions were performed with a Veriti Thermal Cycler (Life Technologies) in 10-mL reaction mixtures. Each reaction contained 1 · polymerase chain reaction (PCR) buffer, 2 mM MgCl2, 200 mM nucleoside triphosphates, 0.25 mM unlabeled right primer, 0.25 mM labeled left primer, 0.5 U of Taq DNA Polymerase (Biotools, Madrid, Spain), and 20 ng of genomic DNA. Loci were amplified using a profile of initial denaturation at 95 C for 3 min, followed by 30 cycles of strand denaturation at 94 C for 30 s, primer annealing at 47 to 62 C for 30 s, DNA extension at 72 C for 30 s, and final extension at 72 C for 4min. Fluorescently labeled PCR products were mixed with formamide and internal size standard GeneScan350 ROX (Life Technologies) and genotyped on the 3130xl Genetic Analyzer (Life Technologies). DATA ANALYSES. For each SSR marker, alleles of different sizes were scored. Basic statistics, including observed number of alleles, expected heterozygosity, polymorphic information content (PIC), and probability of identity (PI) were calculated in Identity 1.0 (Wagner and Sefc, 1999) and MicrosatelliteToolkit (Park, 2001). The number of total, effective, and private alleles and alleles with frequency over 5% were calculated for each of the five groups of accessions using GenAlEx 6.1 (Peakall and Smouse, 2006). The same software was used for the estimation of Shannon’s information index and expected heterozygosity of overall loci in single groups of accessions. A