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Genetic Diversity of Pinto and Fresh Bean (Phaseolus Vulgaris

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Genetic Diversity of Pinto and Fresh Bean (Phaseolus Vulgaris Turkish Journal of Agriculture and Forestry Turk J Agric For (2020) 44: 417-427 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-2002-9 Genetic diversity of pinto and fresh bean (Phaseolus vulgaris L.) germplasm collected from Erzincan province of Turkey by inter-primer binding site (iPBS) retrotransposon markers 1, 2 3,4 3 Halil İbrahim ÖZTÜRK *, Atilla DURSUN , Arash HOSSEINPOUR , Kamil HALİLOĞLU 1 Health Services Vocational School, Erzincan Binali Yıldırım University, Erzincan, Turkey 2 Department of Horticulture, Faculty of Agriculture, Ataturk University, Erzurum, Turkey 2 Department of Field Crops, Faculty of Agriculture, Ataturk University, Erzurum, Turkey 4 Crop and Horticultural Science Research Department, Ardabil Agricultural and Natural Resources Research and Education Center, AREEO Ardabil (Moghan), Iran Received: 03.02.2020 Accepted/Published Online: 26.06.2020 Final Version: 02.08.2020 Abstract: In this study, genetic diversity among 71 bean genotypes (41 pinto beans and 30 fresh bean genotypes) and 4 commercial cultivars (Aleyna, Gina, Perolar, and Serra) commonly grown at Erzincan province in Turkey were investigated. The iPBS (inter-primer binding site) markers were used to investigate the genetic diversity of the genotypes. Polymorphism ratio was identified as 100% in all primers. Twenty-seven primers were studied and number of alleles in primers varied between 13–69 (with an average value of 37.14). According to cluster analysis based on iPBS data, genotypes were divided into 2 groups. Polymorphic information content (PIC) values varied between 0.19 (iPBS 2077) and 0.42 (iPBS 2381, iPBS 2385, and iPBS 2389) with an average PIC value of 0.33. According to genetic structure analysis, genotypes were divided into 2 subpopulations. Majority of the genotypes of the regions from where beans were collected were placed in the same groups. Key words: Genetic variation, molecular marker, Phaseolus vulgaris, population structure 1. Introduction Repeat (ISSR) (Dagnew et al., 2014), Simple Sequence Beans (Phaseolus vulgaris L, 2n = 2x = 22), produced over Repeats (SSR) (Sarıkamış et al., 2009; Bilir et al., 2019), the large fields both worldwide and in Turkey, are significant Sequence-Related Amplified Polymorphism (SRAP) self-pollinating species belonging to Leguminosae family (Ceylan et al., 2014), and Expressed Sequence Tag (EST) (Dursun et al., 2010; Öztürk and Dursun, 2018; Yeken et (Garcia et al., 2011), all to assess genetic diversity and al., 2018). They have a quite large genetic diversity and relationships among several Phaseolus vulgaris species. this diversity needs genetic characterization. The primary Inter-primer binding site (iPBS) is a universal method for objective in characterization of plant genetic sources is DNA fingerprinting and retrotransposon isolation. It is to put forth the genetic variations among seed samples an amplification technique and do not require sequence or populations, and then use these samples to determine data. The iPBS technique has successfully been used for the quantity and distribution of genetic variation among assessment of genetic diversity in common beans (Nemli the populations (Piergiovanni et al., 2006; Akbulut et al., 2015; Aydın and Baloch, 2019) quinoa (Hossein- et al., 2013). Genetic assessments through screening Pour et al., 2019a), adonis (Hossein-Pour, 2019b) and plant genetic sources with the molecular markers and saffron (Gedik et al., 2017). However, there are very few identification of genetic relationships and similarities studies about the use of iPBS-retrotransposon markers among the genotypes will construct the bases for further for assessment of genetic diversity in pinto and fresh bean breeding studies (Sarıkamış, 2014). Various methods have genotypes. Also, inexistence of a genetic identification used molecular markers including Amplified Fragment research on fresh bean with the aid of iPBS markers Length Polymorphisms (AFLP) (Sustar-Vozlic et al., 2006), have made the present study unique in this sense. In Random Amplified Polymorphic DNA (RAPD) (Erdinc this study, some local pinto and fresh bean genotypes et al., 2017), Sequence Characterized Amplified Region commonly grown in Erzincan (Turkey) district were (SCAR) (Madakbaş et al., 2016), Inter Simple Sequence collected and genetic differences among the genotypes * Correspondence: [email protected] 417 This work is licensed under a Creative Commons Attribution 4.0 International License. ÖZTÜRK et al. / Turk J Agric For were put forth with the aid of iPBS marker method. LTR- iPBS primers previously developed by Kalendar et al. retrotransposons have primer binding sites (PBS) around (2010). Among these 40 iPBS primers, 27 of them with 5’ LTR. The 3’ tip of cellular tRNA is bound to PBS and good/excellent PCR products were selected and were initiates mRNA synthesis of retrotransposon (Kumar and used for genotyping the whole set of Phaseolus vulgaris L. Bennetzen, 1999). In iPBS method, the regions so close genotypes. as to have implications between 2 LTR retrotransposons 2.4. Data analysis are amplified in reverse directions. Primers are developed TotalLab TL120 software was used for analysis of iPBS band from the preserved regions of PBS. iPBS produces 15–50 patterns. The iPBS amplification products were scored as bands with a length of between 100–5000 bp. Just because present (1) or absent (0). Only the clear and strong bands of high number of copies in investigated genomes of iPBS were scored and were used in further analyses. Numerical sequences, iPBS produces several numbers of net bands Taxonomy and Multiware Analysis System (NTSYSpc and it is easy to score them in standard agarose gels stained version 2.0) was used based on Dice similarity matrix with ethidium bromide. iPBS method yields polymorphic (Dice, 1945) to assess the genetic similarities among fingerprints behaving as dominant marker (Kalendar et the genotypes. UPGMA (unweighted pair-group mean al., 2010). Present findings will reveal genetic variations average) tree was constructed with the aid of the same among the genotypes and generate the bases for the software and a PCA (principle component analysis) was further breeding studies. Such findings will also provide conducted (Rohlf, 2000). PIC (polymorphism information an integrity in genetic identification studies on pinto and content) (PIC=1−Σpi2, where Pij is the frequency of the fresh bean and will also reduce workloads and costs of pattern j for each marker i) was used to assess the diversity breeders. The aim of the present study was to evaluate the of each iPBS marker (Anderson et al., 1993). POPGEN1.32 population structure and genetic diversity of pinto and was used to determine genetic parameters, effective fresh bean genotypes collected from Erzincan province number of alleles (ne), Nei’s genetic diversity (h), and using iPBS markers. Shannon’s information index (I) (Yeh et al., 1997). Model- based cluster analysis was used with the aid of Structure 2. Materials and methods v. 2.2 software to determine genetic structure of the This study was carried out in the Molecular Biology and genotypes (Pritchard et al., 2000a; Pritchard et al., 2000b). Genetics Laboratory of the Field Crops Department of Number of populations (K) expected to present in each Atatürk University in 2017. 10 runs (generally varied between 2–10) characterized 2.1. Collection of plant material by a set of distinctive allele frequencies at each locus and In this study, 71 local bean genotypes (41 pinto beans and the individuals were sited in K clusters. MCMC (Markov 30 fresh beans) commonly produced in Erzincan province Chain Monte Carlo) posterior probabilities were estimated. were collected. Besides, 4 commercial cultivars (1 pinto The most expected value of K was predicted with the aid of bean and 3 fresh bean) in widely grown in the province Evanno’s ∆K method (Evanno et al., 2005) and Structure were used to compare with local genotypes (Table 1). Harvester (Earl, 2012). 2.2. Genomic DNA isolation Sample plants were grown in a greenhouse. Bulk DNA 3. Results and discussion of 71 individuals per accession was prepared from young 3.1. Polymorphism revealed by iPBS primers leaves of 2-week-old plants in Laboratory of Molecular Of 40 iPBS primers used in this study, 27 yielded Biology and Genetics, Department of Field Crops, sufficiently clear and scorable bands. With these 27 Agriculture Faculty, Ataturk University, Turkey in 2017. primers, 1003 visible and scorable bands were generated Genomic DNA extractions were performed as described by (Table 2). In similar studies conducted on beans, Nemli Zeinalzadehtabrizi et al. (2015) with slight modifications. et al. (2015) screened 83 iPBS primers and reported that DNA quality was affirmed through electrophoresis in 47 iPBS primers (56%) yielded visible and scorable bands. 0.8% agarose gel. The NanoDrop ND-1000 UV/Vis In another study, average number of polymorphic iPBS- spectrophotometer (Thermo Fisher Scientific, USA) was retrotransposon bands was reported as 3.8 and number of used to determine DNA concentrations. For iPBS analysis, bands per primer was reported as 25 (Gedik et al., 2017). final DNA concentration was adjusted to 50 µg/mL. Diluted Additionally, iPBS-retrotransposon primers yielded DNA samples were stored at –20 °C for Polymerase chain greater data than IRAP (Boronnikova and Kalendar, 2010), reaction (PCR) reactions. RAPD (Bukhari et al., 2015) and AFLP (Sustar-Vozlic et 2.3. iPBS marker
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