Journal of Applied Botany and Food Quality 88, 222 - 227 (2015), DOI:10.5073/JABFQ.2015.088.032 1Alata Horticultural Research Station, Erdemli, Mersin-Turkey 2Department of Field Crops, Faculty of Agriculture, Namik Kemal University, Tekirdag-Turkey 3Department of Horticulture, Faculty of Agriculture, Ataturk University, Erzurum-Turkey Genetic characterization of banana clones grown in Turkey based on nuclear DNA content and SRAP markers Hasan Pinar1, Mustafa Unlu1, Mustafa Bircan1, Filiz Baysal1, Gulsemin Savas Tuna2, Metin Tuna2, Sezai Ercisli3* (Received March 24, 2015) Summary Amplified Polymorphic DNA (RAPD) (DAS et al., 2009), Restriction Fragment Length Polymorphism (RFLP) (HIPPOLYTE et al., 2010), This study was conducted to investigate the genetic relationships Amplified Fragment Length Polymorphism (AFLP) (OPARA et al., among banana clones grown in Turkey based on their nuclear DNA 2010), Inter Simple Sequence Repeats (ISSR) (LU et al., 2011), Se- contents and SRAP markers. Four banana clones including ‘Dwarf quence Related Amplified Polymorphism (SRAP) (PHOTHIPAN et al., Cavendish’, ‘Grand Nain’, ‘Azman’ and local ‘Erdemli’ were used 2005) and Simple Sequence Repeats (SSR) markers (MILLER et al., as plant material. Nuclear DNA content of the banana cultivars 2010). Sequence Related Amplified Polymorphism (SRAP) was first was estimated by flow cytometer and varied between 1.766 pg (‘Er- used in Brassicaceae for marker development and mapping (LI and demli’) and 2.028 pg (‘Grand Nain’). ‘Azman’ contained 1.973 pg QUIROS, 2001). Subsequently this system has been used in many fruit and ‘Dwarf Cavendish’ 1.936 pg nuclear DNA. Genetic similarities crops such as citrus (UZUN et al., 2009), apricot (UZUN et al., 2010) of 4 banana clones were between 0.63-0.91 based on SRAP molecu- etc. for genetic diversity and fingerprinting studies because it is a lar markers. The local ‘Erdemli’ banana clone was the most distinct simple and also efficient marker system that can be adapted for a va- from the others. In conclusion, there is a high level of genetic varia- riety of purposes in different crops, including map construction, gene tion among Erdemli and other banana clones grown in Turkey and tagging, genomic and cDNA fingerprinting and map based cloning the local clone ‘Erdemli’ is the most distinct one. This study showed (LI and QUIROS, 2001). that nuclear DNA content analysis together with molecular markers Flow cytometry is among the most common methods used to assess could be useful to assess the relationships among banana clones. numerous cellular characteristics and to analyze cells individually (LOUREIRO et al., 2006). The method was considered as the fastest Introduction and the most reliable technique (SUDA and TRAVNICEK, 2006). The method was initially developed for human cells but then was adapted Fruits have long been valued as part of a nutritious and tasty diet. for plant cells as a reliable tool for estimation of nuclear DNA con- The flavours provided by different fruit species are among the most tent and ploidy level constitutions in plants (DOLEZEL et al., 1994; preferred in the world, and it is increasingly evident that fruits not TUNA et al., 2006; OZKAN et al., 2010; TIRYAKI and TUNA, 2012). only taste good, but also good for human health (BACVONKRALJ et al., DOLEZEL et al. (1994) modified the technique for analysis of nuclear 2014; ROP et al., 2014). genomes of bananas and plantains. The method has been employed Bananas and plantains belong to the genus Musa and they are the in finding out the DNA nuclear contents of various banana varieties most significant food and cash crops worldwide, especially in tropi- since the early 1980s (DOLEZEL, 1991). cal and sub-tropical regions. World total annual banana produc- D’HONT et al. (1999) carried out a study with flow cytometry method tion is around 102 million tons (FAO, 2013). The genus Musa is to assess genetic differences between Musa genomes and reported divided into two sections: Musa and Callimusa; the Musa section significant differences between the sizes of Musa A, B, S and T ge- includes 33 species (HAKKINEN, 2013). The section contains tri- nomes. He was able to indicate that S and T genomes were larger ploid varieties evolved from two wild diploids (2n = 2x = 22) as of than A and B genomes and T genome was the largest one. The infor- seminiferous species, M. acuminata Colla (genomes AA) and M. mation provided by the researchers was then employed by the other balbisiana (genomes BB). In general, bananas consist of two major researchers as a significant diagnostic tool to assess the differences categories as of those consumed as dessert (constituting about 43% between Musa genomes and it was reported in previous studies that of world production) and cooking bananas (constituting the remain- DNA content of AA genome accessions varied between 1.22-1.27 pg ing 57% of world production) (JONES, 2000). (LYSAK, 1999) or between 1.20-1.33 pg (KAMATE, 2001). ARUMU- Some banana clones can be cultivated in subtropical regions between GANATHAN and EARLE (1991) also reported the similar DNA content 20° and 30° north and south of the equator. Several subtropical re- estimations for triploid accessions with the estimations of LYSAK gions protected from cold weather, such as the Mediterranean coast- (1999) and KAMATE (2001). LYSAK et al. (1999) determined DNA at line, are also suitable for growing bananas. Banana has been grown a level of 1.81 pg in a triploid accession. in some subtropical parts of Turkey since 1934 (GUBBUK et al., 2004) In this study, nuclear DNA contents of ‘Dwarf Cavendsish’, ‘Grand and annual banana production is increasing each year and has reached Nain’ and local clones ‘Azman’ and ‘Erdemli’, grown in Turkey were 210.000 tons in Turkey (FAO, 2013). determined for the first time through flow cytometry and also genetic Several direct or indirect methods have been used to identify the relationships among these cultivars were assessed by SRAP marker. genomes in Musa. These include molecular markers, isozymes, re- trotransposons, in situ hybridization and flow cytometry. A number of techniques have been used to evaluate the level of ge- Materials and methods netic variability in Musa via molecular markers, including Random Plant material International ‘Dwarf Cavendish’ and ‘Grand Nain’, local ‘Azman’ * Corresponding author and ‘Erdemli’ banana clones were used as the plant material of the Determine of genetic relationships among banana clones by SRAP markers 223 present study. All plants were grown in protected areas of open fields ing 0.5 ml extraction buffer. The crude nuclear suspension was then and selected from a commercial orchard in Erdemli, Mersin. Some transferred to a labelled test tube through 50-micron nylon mesh. yield and leaf characteristics such as finger length (cm), finger weight 1.5 ml of staining buffer was then added to the test tube and the (g), plant height (cm), number of hands (bunch-1), number of fingers samples were stored at 4 °C for approximately 60 min in dark. PI (hand-1), bunch weight (kg/plant), leaf petiole length (cm) and leaf fluorescence emitted from nuclei was collected through a 560 nm petiole width (cm) were measured by using ten plants from each long-pass filter and a 630 nm band-pass filter. For each sample, fluo- clone as shown in Tab. 1. Data were analyzed statistically via JMP rescence data were acquired from at least 3000 nuclei. Three inde- 7.0 (Copyright © 2007 SAS Institute Inc.). pendent replications were performed and histograms with coefficient of variation (CV) above 5.75% were rejected. The formula used for converting fluorescence values to DNA content was: Nuclear DNA DNA isolation content = (mean position of unknown peak)/(mean position of known) Total genomic DNA was extracted from fresh leaf tissues of four X DNA content of known standard (Rice, Osmancik cultivar). banana clones for molecular analysis by using the CTAB procedure as described by DOYLE and DOYLE (1990). DNA concentration was measured with a microplate spectrophotometer (BioTek Instruments, Results and discussion Inc., Vinooski, USA), and 10 ng/mL DNA templates were made using TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) buffer. Yield and leaf characteristics Yield is the most important criterion for banana production. Some yield and leaf characteristics of 4 banana clones grown in Turkey are SRAP analysis provided in Tab. 1. ‘Grand Nain’ had the highest values with regard A total of 19 SRAP primer combinations were used for all banana to finger length, finger weight, hand number, and bunch weight; ‘Er- clones (Tab. 2). PCR reaction components and PCR cycling para- demli’ had the lowest value with regard to all these characteristics. meters were as described by UZUN et al. (2009). PCR products ‘Azman’ and ‘Grand Nain’ clones were similar to each other, but were separated on 2% agarose gels in 1X TBE buffer (89 mM Tris, ‘Dwarf Cavendish’ clones had lower values when compared with 89 mM Boric acid, 2 mM EDTA) at 115 volts for 3 h. The fragment ‘Azman’ and ‘Grand Nain’ with regard to plant height and the other patterns were photographed under UV light for further analysis. A horticultural characteristics (Tab. 1). 100 bp standard DNA ladder was used to confirm the appropriate markers for SRAP analysis. Three replications were used to confirm markers. Molecular analysis was carried out as follows: each band SRAP analysis was scored as present (1) or absent (0) and data were analyzed with The number of morphological markers for identifying the clones of the Numerical Taxonomy Multivariate Analysis System (NTSYS-pc) banana is limited and they do not often reflect genetic relationships software (ROHLF, 2000). A similarity matrix was constructed using because of interaction with the environment, epistasis and the largely SRAP data based on Dice’s coefficient.