Studies on genetic divergence in pomegranate (Punica granatum L.) using SRAP markers C. Kanupriya, D. Manmohan Kumar, P. Nischita, M. Gayathri, K.V. Ravishankar and P. Sampath Kumar1 Division of Biotechnology ICAR-Indian Institute of Horticultural Research Hesaraghatta Lake Post, Bengaluru, 560089, India E-mail: kanu@iihr.ernet.in ABSTRACT Pomegranate genotypes have been characterized mainly on the basis of morphological traits; but, these traits are affected to a large extent by environmental and cultivation conditions, resulting in their ambiguous discrimination. Molecular markers are more suited for accurate discrimination of genotypes and cultivars. Sequence-Related Amplified Polymorphism (SRAP) markers were used in the present study to analyze polymorphism among the important pomegranate genotypes grown in India. The total number of bands generated by 30 SRAP primers for 12 genotypes was 1448, with an average of 48.3 bands per primer. Polymorphism varied from 2.7 to 73.9, with an average of 40.95%. Similarity-value based on Jaccard’s Coefficient ranged from 0.63 (between cvs. Naina and Amlidana) to 0.95 (between cvs. Kabul Yellow and Jalore Seedless). UPGMA (un-weighted pair group method with arithmetic mean) analysis was performed and a dendrogram was constructed using Jaccard’s similarity matrix. The 12 genotypes used grouped into 5 clusters. SRAP markers were found suitable for determining variability among the pomegranate genotypes studied. Key words: Pomegranate, molecular markers, SRAP, genetic diversity J. Hortl. Sci. Vol. 10(2):125-129, 2015 INTRODUCTION Pomegranate (Punica granatum L.) is commercially cultivated in Iran, Afghanistan, India and the Mediterranean countries. Iran is believed to be its primary centre of origin. In India, pomegranate occurs naturally just in the Western Himalayan regions of the states of Jammu and Kashmir, Himachal Pradesh and Uttarakhand. In recent years, the area under this crop has increased substantially, mainly because of its versatility, adaptability, drought resistance, low maintenance-costs and a steady high yield (Narzary et al, 2010). In the global food industry, pomegranate figures among a novel category of exotic plant sources termed as ‘super fruit’. Extracts from this plant (juices, seed-oil and peel) have been reported as exhibiting a strong antioxidant activity helpful in preventing cancer and cardiovascular diseases (Shishodia et al, 2006). Therefore, breeding for useful traits in this crop has gained importance. To do this, determination of genetic relationships and precise identification of the genotypes (to conserve genetic diversity) is required. Pomegranate genotypes have been mainly evaluated in the past based on morphological characters; but, these traits are affected by the environment and cultivation conditions, and do not result in a clear discrimination (Kumar, 1999). Molecular or DNA-based markers are more suitable for accurate discrimination between genotypes and cultivars. As per literature, most of the studies on pomegranate diversity have been made using molecular markers such as randomly amplified polymorphic DNA (RAPD) (Sarkhosh et al, 2009; Hasnaoui et al, 2010), amplified fragment length polymorphism (AFLP) (Jbir et al, 2008, 2009), and, simple sequence repeat microsatellites (SSR) (Pirseyedi et al, 2010; Ebrahimi et al, 2010), but not using SRAP. SRAP (Sequence-Related Amplified Polymorphism) is a PCR-based marker developed by Li and Qurios (2001). The technique of SRAP consists of preferential amplification of open reading frames (ORFs) using PCR. For this purpose, a combination of two types of primers is employed. The first type (forward primer) is 17 bp long, and consists of a fixed sequence of 14 nucleotides, rich in C and G, with three selective bases at the 3' end. 1Division of Fruit Crops, ICAR-Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Bengaluru, 560089, Karnataka, India 126 This primer preferentially amplifies exonic regions (which are generally rich in these two nucleotides). The second type of primer (reverse) of 18 bp contains a sequence of 15 nucleotides rich in A and T, and three selective bases at the 3' end. This primer preferentially amplifies intronic regions and the regions with promoters (as these are rich in the nucleotides A and T) (Ferriol et al, 2003). Therefore, this method is widely used for assessing genetic diversity and species diversity (Budak et al, 2004). With this unique primer design, SRAP markers are known to be more reproducible, stable and less-complex than the other molecular markers. We used SRAP markers in our study to analyze the diversity in 12 important pomegranate genotypes found in India. MATERIAL AND METHODS The study was conducted at ICAR-Indian Institute of Horticultural Research, Bengaluru, India. Twelve cultivars/ hybrids were selected for this study from the available pomegranate germplasm collection at the institute. The plant material used was young leaves, sampled from adult trees. Total DNA was extracted from the leaf using a modified Cetyl Trimethyl Ammonium Bromide (CTAB) method (Kanupriya et al, 2013). Integrity and quality of the DNA so isolated was determined by agarose gel electrophoresis (0.8%). DNA quantification was done using GeneQuant UVSpectrophotometer (GE Healthcare BioSciences Ltd., England), and diluted as per standard procedure. A set of 30 SRAP primer combinations was used for amplifying DNA from 12 genotypes (Table 1) of pomegranate. Additional information on primers is presented in Table 2. PCR was carried out with an initial denaturing temperature of 94°C for 5min, followed by 5 cycles at 94°C of 1min each, 35°C for 1min, 72°C for 1min, followed by 35 cycles at 94°C for 1min, 50°C for 1min, 72°C for 1min; and, a final extension at 72°C for 5min (Li and Quiros, 2001). PCR products were separated on 2% agarose gel. The size of all the amplification products was estimated from comparison with a standard molecular-weight DNA ladder (1kb). Bands were scored as discrete variables, using “1” to indicate presence and “0” to indicate absence of any particular band. The data was then statistically analyzed by Winboot software to obtain Jaccard’s similarity matrix, and, the corresponding dendrogram was drawn for obtaining clusters using NTSYS PC 2.11 software (Rohlf, 2000) RESULTS AND DISCUSSION The total number of bands generated using 30 SRAP primers for the 12 genotypes was 1448, with an average of Table 1. Twelve genotypes of pomegranate used in this study and their description Sl. Genotype Description No. 1 Amlidana F1 hybrid of a cross (Ganesh x Nana); grows well under tropical climate; with quality fruit attributes, is superior to the sour variety Daru, whose trees come up naturally in the temperate regions of North India; fruits provide a more acidic (16.18%) anardana and higher fruit yield/tree; short-statured trees suitable for high-density planting, increased fruit yield/unit area 2 Ruby Early maturing, thin skin, red aril, sweet, soft-seeded; selection resulting from progeny of <(Ganesh x Kabul) x Yercaud-F2 x (Ganesh x Gul Shah Red)- F2> 3 Bhagwa Larger fruit size; sweet, bold and attractive aril; glossy, very attractive; saffron-coloured, thick skin makes it suitable for distant markets; less susceptible to fruit spot or thrips compared to other pomegranate varieties; considering all these attributes, ‘Bhagawa’ is recommended for cultivation in pomegranate-growing regions of Maharashtra. 4 Ganesh A selection from ‘Alandi’, developed by Dr. Cheema at Pune; soft seeded with pinkish flesh and juice of agreeable taste; heavy bearer. 5 Jodhpur Red Medium-size fruit, with hard rind; fleshy aril, light pink, sweet, juicy; seed moderately-hard; a favourite cultivar in Rajasthan, the plant is erect, bearing medium-size fruits (150g to 170g each); seeds hard, red in colour; juice slightly acidic with TSS 14 to 16%; reported as highly susceptible to cracking 6 Jalore Seedless Known cultivar from Rajasthan and Gujarat; bears small-sized (90g to 110g), round fruits; seeds normally small and soft; juice agreeable in taste and red in colour. 7 Kabul Yellow This plant has yellow pigmentation in petiole base, leaf margins, flower buds and fruit rind 8 Naina This is an exotic variety from Sri Lanka 9 Daya This is an exotic variety from Sri Lanka. 10 Daru It is a sour pomegranate, which grows wild on Himalayan foothills. Though its sugar content is over 10 per cent, yet it tastes sour due to high acid content. Its seeds are sundried to make ANARDANA. It can withstand cold and dry conditions and is considered to be important source of resistance to diseases and pests 11 Muscat This is a favourite variety of kohlar (Maharashtra) dueto its large and sweet fruits with soft seeds. It is highly suitable for Pune and Sholapur areas of Maharashtra and some parts of Karnataka. 12 Nana This is miniature plant and is grown for ornamental purposes. It is believed to be drought tolerant and does not yield fruits/ flowers. Kanupriya et al J. Hortl. Sci. Vol. 10(2):125-129, 2015 127 Fig. 1. Gel profile showing PCR products obtained from the SRAP primer combination Me7 + Em7 [1-12 serially represents pomegranate varieties (genotypes) Amlidana, Bhagwa, Daru, Daya, Ganesh, Jodhpur Red, Jalore Seedless, Kabul Yellow, Muscat, Nana, Naina and Ruby, respectively; L is 1kb ladder] 48.3 bands per primer. The total number of SRAP loci (Table 2) are arranged in ascending order (percentage polymorphism) from the primer pair Me1+Em9 to the primer pair Me1+Em3. Highest number of bands (92) was observed in the primer pair Me7+Em7 (Fig. 1). Polymorphism per cent varied from 2.7 to 73.9, with an average of 40.95%. Similarity-value, based on Jaccard’s coefficient, ranged from 0.63 (between cvs. Naina and Amlidana) to 0.95 (between cvs. Kabul Yellow and Jalore Seedless) (Table 3). UPGMA (un-weighted pair group method with arithmetic mean) analysis was made, and a dendrogram was constructed using Jaccard’s similarity matrix involving data generated from 30 SRAP primers on 12 genotypes of pomegranate (Fig. 2). These genotypes grouped into four clusters. ‘Amlidana’ and ‘Bhagwa’ were placed in one cluster. ‘Amlidana’ is the progeny of ‘Ganesh’ and ‘Nana’, while, ‘Bhagwa’ is a selection of unknown parentage. It is likely that these share some common ancestors. ‘Daru’, found growing wild in the Himalayan foothills, separated itself from the rest of the genotypes. It is a sour pomegranate growing in the wild in Himalayan foothills. It is commercially an important genotype, since, its seeds are sun-dried to turn into anardana. This genotype withstands cold and dry conditions, and is considered an important source of resistance to diseases and pests. The third cluster was made up of seven phenotypically divergent genotypes, viz., Daya, Muscat, Jalore Seedless, Kabul Yellow, Nana, Ganesh and Jodhpur Red, while, the fourth and last cluster consisted of cvs. Naina and Ruby. All these genotypes are phenotypically divergent; cvs. Muscat, Jalore Seedless and Kabul Yellow are grown in dry regions of the North-West of India, while, cv. Daya is originally from Sri Lanka. ‘Nana’ is an ornamental pomegranate variety of very short stature. Parentage of ‘Naina’ is not known, and, it is likely that it shares with Ruby common ancestors. Similarity-value based on Jaccard’s coefficient clearly demonstrates that the genotype, Naina, is divergent from Amlidana; while, the genotypes Kabul Yellow and Jalore Seedless are greatly similar to each other. Overall similarity between genotypes was high, reflecting weak genetic differentiation even among genotypes belonging to various geographical regions. Apart from the genotype, the nature of the markers used in a study also has an influence on the picture emerging on genetic diversity, since, the preferential amplification-region differs with the marker used. Aradhya et al (2006) used AFLP analysis to assess genetic diversity within a pomegranate collection maintained at National Clonal Germplasm Repository (NCGR), Davis, California, Table 2. Primer combinations in SRAP markers used in the study, showing their amplification and polymorphism Sl. Primer Total number Polymorphism Percentage No. of SRAP loci 1 Me1+Em9 46 34 73.9 2 Me4+Em9 43 39 72.0 3 Me7+Em9 39 27 69.2 4 Me4+Em2 67 13 64.2 5 Me3+Em3 33 21 63.6 6 Me2+Em7 34 21 61.8 7 Me9+Em9 57 33 57.9 8 Me8+Em3 28 15 53.5 9 Me8+Em6 55 29 52.7 10 Me8+Em5 72 36 50.0 11 Me1+Em8 47 23 48.9 12 Me10+Em5 45 21 46.6 13 Me9+Em3 45 21 46.6 14 Me7+Em4 45 20 44.4 15 Me1+Em1 21 9 42.8 16 Me8+Em7 62 26 41.9 17 Me2+Em5 41 17 41.4 18 Me4+Em3 58 22 37.9 19 Me8+Em9 19 7 36.8 20 Me8+Em8 55 19 34.5 21 Me7+Em6 35 11 31.4 22 Me4+Em4 51 15 29.4 23 Me2+Em4 31 7 22.5 24 Me7+Em7 92 20 21.7 25 Me10+Em3 61 13 21.3 26 Me9+Em7 60 12 20.0 27 Me10+Em8 24 4 16.6 28 Me8+Em4 43 6 13.9 29 Me1+Em5 54 6 11.1 30 Me1+Em3 37 1 2.7 and reported that despite the different origin of individual accessions, diversity was very low. Accessions from the same place were analyzed recently for evaluating diversity, by Parvaresh et al (2012) using microsatellite markers. They Genetic divergence studies in pomegranate using SRAP markers J. Hortl. Sci. Vol. 10(2):125-129, 2015 128 Table 3. Pair-wise genetic distance calculated using Jaccard’s similarity matrix between 12 genotypes of pomegranate using SRAP markers Genotype Amlidana Bhagwa Daru Daya Ganesh Jodhpur Jalore Kabul Muscat Nana Naina Ruby Red Seedless Yellow Amlidana 1.00 Bhagwa 0.89 1.00 Daru 0.70 0.76 1.00 Daya 0.77 0.81 0.79 1.00 Ganesh 0.75 0.75 0.82 0.86 1.00 Jodhpur Red 0.74 0.76 0.74 0.85 0.87 1.00 Jalore Seedless 0.74 0.78 0.76 0.88 0.89 0.87 1.00 Kabul Yellow 0.75 0.76 0.74 0.86 0.89 0.87 0.95 1.00 Muscat 0.77 0.80 0.75 0.89 0.86 0.86 0.92 0.93 1.00 Nana 0.74 0.77 0.76 0.85 0.85 0.83 0.92 0.89 0.90 1.00 Naina 0.63 0.67 0.76 0.75 0.76 0.75 0.77 0.77 0.78 0.81 1.00 Ruby 0.65 0.70 0.79 0.77 0.80 0.76 0.80 0.81 0.81 0.82 0.90 1.00 fruit crop. This is corroborated by molecular analysis carried out in various countries by researchers quoted above, and by our present work using SRAP markers. However, it may be noted that the material analyzed in this study represents a fraction of the germplasm conserved worldwide. Future studies need to explore germplasm available elsewhere and attempt to relate the diversity observed to other traits, for identifying parents for breeding programmes. These can range from agronomic and quality-related traits (such as antioxidant content), to novel uses of pomegranate, such as in aesthetics. Generally, our study confirmed the narrow genetic base reported in P. granatum, and emphasizes a need to widen the existing genetic diversity through further exploration. Although SRAP markers in our study were able Kanupriya et al J. Hortl. Sci. Vol. 10(2):125-129, 2015 Fig. 2. Dendrogram based on UPGMA analysis by Jaccard’s similarity matrix using data generated from 30 SRAP primers for 12 genotypes of pomegranate reported a high level of genetic diversity within a group and a low level among groups. Moslemi et al (2010) and Yuan et al (2007) also reported a low diversity in Iranian and Chinese genotypes, using AFLP markers. On the other hand, Narzary et al (2010) reported a high genetic diversity across natural populations of the Western Himalayan region of India, based on inter-simple sequence repeat (ISSR) markers. As seen in Fig. 1, SRAP markers showed good stability, repeatability and also clear bands, which facilitated easy scoring. SRAP markers preferentially amplify the ORF regions of DNA, and have been demonstrated to be more powerful at revealing genetic diversity among closely-related cultivars than are SSR, ISSR or RAPD markers as such in other crops like buffalo grass (Budak et al, 2004), okra germplasm (Gulsen et al, 2007), Cucurbit pepo germplasm (Ferriol et al, 2003) and Brassica (Li and Quires, 2001). The weak genetic differentiation seen between genotypes of pomegranate is attributed to an inherently narrow genetic base. The family Punicaceae is monogeneric, comprising just two species, P. granatum L. and P. protopunica L., the latter restricted to Island of Socotra. This narrow genetic base, along with domestication of the desirable genotypes, human selection and clonal propagation, has led to high levels of genetic uniformity in this ancient 129 to determine variability among the pomegranate accessions tested, a combination of multiple molecular techniques (AFLP, ISSR and SSRs) may lead to a more accurate estimation of genetic diversity, and, relate the diversity observed to qualitative traits in future studies. REFERENCES Aradhya, M. 2006. http://www.ars-grin.gov/npgs/ cgc_reports/woody2006/ncgrdavis2006.html Budak, H., Shearman, R.C., Parmaksiz, I., Gaussoin, R.E., Riordan T.P. and Dweikat, I. 2004. Molecular characterization of buffalo grass germplasm using sequence-related amplified polymorphism markers. Theor. Appl. Genet., 108:328-334 Ebrahimi, S., Sayed Tabatabaei, B.E. and Sharifnabi, B. 2010. Microsatellite isolation and characterization in pomegranate (Punica granatum L.). Iranian J. Biotech., 8:156-163 Ferriol, M., Pico, B. and Nuez, F. 2003. Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP Markers. Theor. Appl. Genet., 107:271-282 Gulsen, O., Karagul, S. and Abak, K. 2007. Diversity and relationships among Turkish okra germplasm by SRAP and phenotypic marker polymorphism. Biologia Bratislava, 62:41-45 Hasnaoui, N., Mars, M., Chibani, J. and Trifi, M. 2010. Molecular polymorphism in Tunisian pomegranate (Punica granatum L.) as revealed by RAPD fingerprints. Diversity, 2:107-113 Jbir, R., Hasnaoui, N., Mars, M., Marrakchi, M. and Trifi, M. 2008. Characterization of Tunisian pomegranate (Punica granatum L.) cultivars using amplified fragment length polymorphism analysis. Sci. Hortic., 115:231-237 Kanupriya, C., Nischita, P. and Ravishankar, K.V. 2013. An efficient method of genomic DNA isolation from pomegranate. Indian J. Hort., 70:584-586 Kumar, L.S. 1999. DNA markers in plant improvement. Biotech. Adv., 17:143-183 Li, G. and Quiros, C.F. 2001. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: Its application to mapping and gene tagging in Brassica. Theor. Appl. Genet. 103:455-461 Moslemi, M., Zahravi, M. and Bakhshi Khaniki, G. 2010. Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers. Sci. Hortic., 126:441-447 Narzary, D., Rana, T.S. and Ranade, S.A. 2010. Genetic diversity in inter-simple sequence repeat profiles across natural populations of Indian pomegranate (Punica granatum L.). Pl. Biol., 12:806-813 Parvaresh, M., Talebi, M., Ebrahim, B. and Tabatabaei, S. 2012. Molecular diversity and genetic relationship of pomegranate (Punica granatum L.) genotypes using microsatellite markers. Sci. Hortic., 138:244-252 Pirseyedi, S.M., Valizadehghan, S., Mardi, M., Ghaffari, M.R., Mahmoodi, M., Zahravi, M., Zeinalabedini, M. and Khayam Nekoui, S.M. 2010. Isolation and characterization of novel microsatellite markers in pomegranate (Punica granatum L.). Int’l. J. Mol. Sci., 11:2010-2016 Rohlf, F.J. 2000. NTSYS-pc: Numerical Taxonomy and Multivariate Analysis System, Version 2.2. Exeter Software. Setauket, New York, USA Sarkhosh, A., Zamani, Z., Fatahi, R. and Ranjbar, H. 2009. Evaluation of genetic diversity among Iranian soft- seed pomegranate accessions by fruit characteristics and RAPD markers. Sci. Hortic., 121:313-319 Shishodia, S., Adams, L., Bhatt, I.D. and Aggarwal, B.B. 2006. Anticancer potential of pomegranate. In: Pomegranates. Ancient Roots to Modern Medicine, 1st edn. Taylor and Francis Group, Boca Raton, FL, USA, pp. 107-116 Yuan, H., Yin, Y., Qu, J., Zhu, L. and Li, Y. 2007. Population genetic diversity in Chinese pomegranate (Punica granatum L.) cultivars revealed by fluorescent-AFLP markers. J. Genet. Genom., 34:1061-1071 (MS Received 24 March 2015, Revised 15 September 2015, Accepted 28 September 2015) Genetic divergence studies in pomegranate using SRAP markers J. Hortl. Sci. Vol. 10(2):125-129, 2015