Ertuğrul TURGUTOĞLU, Görkem SÜLÜ, Aytül KİTAPCI, Yıldız AKA KAÇAR, İlknur POLAT, Özhan ŞİMŞEK, Gül SATAR

Identification of genetic diversity among mutant lemon and mandarin varieties using different molecular markers

Mandarin (Citrus reticulate Blanco) and lemon (C. limon L.) are among the most cultivated Citrus species in the world.Among different approaches, gamma radiation is widely used to induce mutations in citrus breeding studies. Gamma radiation causes DNA damage, randomly inducing several mutations in the genome. Molecular techniquesarewidely used to detect such induced mutations and genetic diversity in plants.In this study, simple sequence repeats (SSR), inter-simple sequence repeat (ISSR) and single-strand conformation polymorphism (SSCP) markers were used to detectthe induced mutations andgenetic diversityin someirradiated mandarin and lemon genotypes in comparison with standard citrus varieties. The irradiated genotypes used in this study derived from populationsthat had been originally obtained with gamma radiation with different gray doses applied on Yerli Mandarin and Antalya YerliYuvarlak lemon varieties. Out of 82 primers (22 SSCP, 30 ISSR and 30 SSR) tested, 55 primers (9 SSCP, 24 ISSR and 22 SSR primers) successfully amplified a total number of 363 amplicons.A single band with an approximate size of 500 nucleotides was determined using the primer ISSR 868 as differentiating between seedy parent and irradiated seedless mandarin genotypes. The polymorphism information content (PIC) values of SSCP, ISSR, and SSR markers ranged from 0.61-0.99, 0.31-0.96, and 0.33-0.96, respectively. Cluster analysis classified lemons and mandarins into 2 subgroups as mutant and commercial lemon genotypes. ISSR markers were found to be more effective to determine the genetic differences among the varieties in comparison to SSCP and SSR markers. The results clearly showed that SSCP, SSR and ISSR markers are important tools to distinguish mutant genotypes andconfirmed their usefulness for phylogenetic studies.

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Agarwal M, Neeta S, Harish P (2008). Advances in molecular marker techniques and their applications in plant sciences. Plant Cell Reports 27: 617-631.
Agisimanto D, Noor NM, Ibrahim R, Mohamad A (2016). Gamma irradiation effect on embryogenic callus growth of Citrus reticulata cv. limaumadu. Sains Malaysiana 45: 329-337.
Aka-Kacar Y, Demirel A, Tuzcu O, Yesiloglu T, Ulas M et al. (2005). Preliminary results on fingerprinting lemon genotypes tolerant to mal secco (Phomatracheiphila Kanc. et Ghik) disease by RAPD markers. Biologia 60: 295-300.
Aka Kacar Y, Simsek O, Donmez D, Boncuk M, Yesiloglu T et al. (2014). Genetic relationships of some Citrus genotypes based on the candidate iron chlorosis genes. Turkish Journal of Agriculture and Forestry 38: 340-347.
Amara MH, Biswas MK, Zhang Z, Guoa WW (2011). Exploitation of SSR, SRAP and CAPS-SNP markers for genetic diversity of Citrus germplasm collection. Scientia Horticulturae 128: 220- 227.
Atak Ç, Alikamanoğlu S, Açık L, Canbolat Y (2004). Induced of plastid mutations in soybean plant (Glycine max L. Merrill) with gamma radiation and determination with RAPD. Mutation Research 556: 35-44.
Barkley NA, Roose ML, Krueger RR, Federici CT (2006). Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). Theoretical and Applied Genetics 112: 1519-1531.
Barkley NA, Krueger RR, Federici CT, Roose ML (2009). What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles. Plant Systematics and Evolution 282: 71-86.
Bermejo A, Pardo J, Cano A (2011). Influence of gamma irradiation on seedless citrus production: pollen germination and fruit quality. Food and Nutrition Sciences 2: 169-180.
Bermejo A, Pardo J, Cano A (2012). Murcottseedless: influence of gamma irradiation on citrus production and fruit quality. Spanish Journal of Agricultural Research 3: 768-777.
Biswas MK, Chai L, Amar MH, Zhang X, Deng XX (2011). Comparative analysis of genetic diversity in Citrus germplasm collection using AFLP, SSAP, SAMPL and SSR markers. Scientia Horticulturae 129: 798-803.
Biswas MK, Xu Q, Mayer C, Deng X (2014). Genome wide characterization of short tandem repeat markers in sweet orange (Citrus sinensis). Plos One 9 (8): e104182. doi: 10.1371/ journal.pone.0104182
Cameron JW, Frost NB (1968). Genetic, breeding and nucellar embryony. In: The Citrus Industry. (Eds.): Reuther W, Batchelor LD and Webber HJ. Berkeley, CA, USA: University California Press, pp. 325-379.
Campos ET, Espinosa MAG,Warburton ML, Varela AS, Monter AV (2005). Characterization of mandarin (Citrus spp.) using morphological and AFLP markers. Interiencia 30: 687-693.
Capparelli R, Viscardi M, Amoroso MG, Blaiotta G, Bianco M (2004). Bianco Inter-simple sequence repeat markers and flow cytometry for the characterization of closely related Citrus limon germplasms. Biotechnology Letters 26: 1295-1299.
Carrillo-Medrano SH, Gutierrez-Espinosa MA, Robles-González MM, Cruz-Izquierdo S (2018). Identification of Mexican lemon hybrids using molecular markers SSR. Revista Mexicana de Ciencias Agrícolas 9: 11-23.
Chang Feng C, Qiao H, Lin H, Qi Gao G, Ju Rong Q et al. (2008). The identification of Citrus sinensis cv. bud mutation by ISSR markers. Southwest China Journal of Agricultural Sciences 21: 1378-1380.
Chapot H (1975). The citrus plant. In: Citrus, CIBA-GEIGY Agrochemicals, Technical Monograph No 4. Basle, Switzerland: CIBA GEIGY Agrochemicals, pp. 6-13.
Chen C, Bowman KD, Choi YA, Dang PM, Rao MN et al. (2008). ESTSSR genetic maps for Citrus sinensis and Poncirus trifoliata. Tree Genetics and Genomes 4: 1-10.
Christov NK, Todorovska EG, Fasoula DA, Ioannides IM, Atanassov AI et al. (2004). Molecular characterization of chemical mutagenesis induced diversity in elite maize germplasm. Genetika 36: 47-60.
Crowley JR (2011). A molecular genetic approach to evaluate a novel seedless phenotype found in Tango, a new variety of mandarin developed from gamma-irradiated W. Murcott. PhD thesis, University of California Riverside, Riverside, CA, USA.
Değirmenci D, Kunter B (2008). SSCP (Single Strand Conformation Polymorphism) markörler aracılığıyla mutasyonların analizi. Mustafa Kemal Üniversitesi Ziraat Fakültesi Dergisi 13: 13-24.
DeğirmenciKarataş D, Kunter B, Coppola G, Velasco R (2010). Analysis of polymorphism based on SSCP markers in gammairradiated (Co60) grape (Vitis vinifera) varieties. Genetics and Molecular Research 9: 2357-2363.
Deng ZN, Gentile A, Nicolosi E, Domina F, Vardi A et al. (1995). Identification of in-vivo and in-vitro lemon mutants by RAPD markers. Journal of Horticultural Sciences 70: 117-125.
Dice LR (1945). Measures of the amount of ecologic association between species. Ecology 26: 297-302.
Doyle JJ, Doyle JL(1990). Isolation of plant DNA fromfresh tissue. Focus 12: 13-15.
Donini B (1982). Mutagenesis applied to improve fruit trees: techniques, methods and evaluation or radiation induced mutations. In: Induced Mutations in Vegetatively Propagated Plants. Vienna, Austria: International Atomic Energy Agency (IAEA), pp. 29-36.
Duran C, Appleby N, Edwards D, Batley J (2009). Molecular genetic markers: discovery, applications, data storage and visualisation. Current Bioinformatics 4: 16-27.
Fang DQ, Roose ML (1997). Identification of closely related citrus cultivars with Inter simple sequence repeat markers. Theoretical and Applied Genetics 95: 408-417.
Federici CT, Fang DQ, Scora RW, Roose ML (1998). Phylogenetic relationships within the genus Citrus (Rutaceae) and related genera as revealed by RFLP and RAPD analysis. Theoretical and Applied Genetics 96: 812-822.
Fu J, Peng ZJ, Cai XD, Guo WW (2011). Regeneration and molecular characterization of interspecific somatic hybrids between Satsuma mandarin and two seedy sweet oranges for scion improvement. Plant Breeding 130 (2): 287-290. doi:10.1111/j.1439-0523
Fujita K, Silver J (1994). Single-strand conformational polymorphism. PCR methods and applications 4: 137-140.
Garcia-Lor A, Luro F, Ollitrault P, Navarro L (2015). Genetic diversity and population structure analysis of mandarin germplasm by nuclear, chloroplastic and mitochondrial markers. Tree Genetics and Genomes 11: 123.
Gentile A, LaMalfa S (2019). New Breeding Techniques for Sustainable Agriculture. In Innovations in Sustainable Agriculture. Cham, Switzerland: Springer International Publishing, pp. 411-437.
Goldenberg L, Yaniv Y, Porat R, Carmi N (2014). Effects of gammairradiation mutagenesis for induction of seedlessness, on the quality of mandarin fruit. Food and Nutrition Sciences 5: 943- 952.
Gülsen O, Roose ML (2001). Lemons: Diversity and relationships with selected citrus genotypes as measured with nuclear genome markers. Journal of American Society for Horticultural Science 126: 309-317.
Gulsen O, Uzun A, Pala H, Canihos E, Kafa G (2007). Development of seedless and mal secco tolerant mutant lemons through budwood irradiation. Scientia Horticulturae112: 184-190.
Gulsen O, Uzun A, Canan I, Seday U, Canihos E (2010). A new citrus linkage map based on SRAP, SSR, ISSR, POGP, RGA and RAPD markers. Euphytica 173: 265-277.
Hearn CJ (1984). Development of seedless orange and grapefruit cultivars through seed irradiation. Journal of American Society for Horticultural Science 109: 270-273.
Hensz RA (1985). “Rio Red” a new grapefruit with a deep-red color. Journal Rio-Grande Valley Horticultural Society 38: 75-76.
Hodgson RW (1967). Horticultural varieties of citrus. In: Reuther W, Webber HJ, Batchelor LD (eds) The Citrus industry. Berkeley, CA, USA: University of California Press, pp. 431-591.
Huang JC, Xiao Y, Zhao CX, Chen ZS, Wang XY (2003). Induction of superior seedless mutation of C. grandis Osbeck cv. Sshatianyou by irradiation. Acta Agriculturae Nucleatae Sinica 17: 171-174.
Huang JH, Wen SX, Zhang YF, Zhong QZ, Yang L et al. (2017). Abnormal megagametogenesis results in seedlessness of a polyembryonic ‘Meiguicheng’ orange (Citrus sinensis) mutant created with gamma-rays. Scientia Horticulturae 217: 73-83.
Hussein EHA, Abd-alla SMM, Awad NA, Hussein MS (2003). Genetic analysis in some Citrus accessions using Microsatellites - and AFLP-based markers. Arabian Journal of Biotechnology 6: 203-222.
Kacar Y, Uzun A, Polat I, Yesiloglu T, Yılmaz B et al. (2013). Molecular characterization and genetic diversity analysis of mandarin genotypes by SSR and SRAP markers. Journal of Food, Agriculture and Environment 11: 516-521.
Khalil SA, Sattar A, Zamir R (2011). Development of sparseseeded mutant kinnow (Citrus reticulata Blanco) through budwood irradiation. African Journal of Biotechnology 10: 65. doi: 10.5897/AJB10.1810
Kajiwara H, Kaneko T, Nishimura M, Ishizaka M (2005). Proteomic identification of RuBisCO/LS gene mutations in radiation mutant of rice by two-dimensional gel electrophorosesis, mass spectrometry, single strand conformation polymorphism and nucleotide sequencing. Rice Genetics Newsletter 20: 1-3.
Konstantinos KV, Panagiotis P, Antonios VT, Agelos P, Argiris NV (2008). PCR–SSCP: A method for the molecular analysis of genetic diseases. Molecular Biotechnology 38: 155-163.
Lamo K, Bhat DJ, Kour K, Solanki SPS (2017). Mutation studies in fruit crops: A Review. International Journal of Current Microbiology and Applied Sciences 6: 3620-3633.
Lombardo G, Schicchi R, Marino P, Palla F (2011). Genetic analysis of Citrus aurantium L. (Rutaceae) cultivars by ISSR molecular markers, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana. doi:10.1080/11263504.2011.557101
Lönnig WE (2005). Mutation breeding, evolution, and the law of recurrent variation. Recent Research Developments in Genetics Breeding 2: 45-70.
Mabberley DJ (2008). Mabberley’s plant-book: a portable dictionary of plants. 3rd ed.. UK: Cambridge University Press.
Mahmoudzadeh H (2013). Bazı Portakal çeşitlerinde genetik farklılıklarını SSCP markırları ile belirlenmesi. Yüksek Lisans Tezi, Çukurova Üniversitesi, Adana, Turkey…
Mallick M, Bharadwaj C, Srivastav M, Sharma N, Awasthi OP (2017). Molecular characterization of Kinnow mandarin clones and mutants using cross genera SSR markers. Indian Journal of Biotechnology 16: 244-249.
Nadeema MA, Nawazb MA, Shahidc MQ, Dogan Y, Comertpay G et al. (2018). DNA molecular markers in plant breeding: current status and recent advancements in genomic selection and genome editing. Biotechnology and Biotechnological Equipment 32: 261-285.
Nicolosi E, Deng ZN, Gentile A, La Malfa S, Continella G et al. (2000). Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theoretical and Applied Genetics 100: 1155-1166.
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989). Detection of polymorphisms of human DNA by gel electrophoresis as single strand conformation polymorphisms. Proceeding National Academy Science 86: 2766-2770.
Pal D, Malik SK, Kumar S, Choudhary R, Sharma KC et al. (2013). Genetic variability and relationship studies of mandarin (Citrus reticulata Blanco) using morphological and molecular markers.Agricultural Research 2: 236-245.
Pestanana RKN, Amorim EP, Ferreira CF, Amorim VBO, Oliveira LS et al. (2011). Agronomic and molecular characterization of gamma ray induced banana (Musa sp.) mutants using a multivariate statistical algorithm. Euphytica 178: 151-158.
Polat I, Turgutoglu E, Kurt S (2015). Determination of genomic diversity within mutant lemon (Citrus Limon L.) and mandarin (Citrus Reticulata) using molecular markers. Pakistan Journal of Botany 47: 1095-1102.
Polat İ (2015). Genetic diversity analysis of Valencia and Navel group sweet orange cultivars by SSR markers. Derim 32: 47-62.
Polat İ (2018). Advanced innovative tools in lemon (Citrus limon L.) Breeding. Advances in Plant Breeding Strategies:Fruits. Chapter 12. Volume 3. Editors: Al-Khayri, Jameel M., Jain, S. Mohan, Johnson, Dennis V. (editors). Berlin, Germany: , Springer, pp. 437-463.
Powell W, Machray GC, Provan J (1996). Polymorphism revealed by simple sequence repeats. Trends in Plant Science 1: 215-222.
Raza H, Khan MM, Khan AA (2003). Review: seedlessness in Citrus. International Journal of Agriculture and Biology 5: 388-391.
Rozen S, Skaletsky HJ (2000). Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (editors). Bioinformatics Methods and Protocols: Methods in Molecular Biology. Totowa, NJ, USA: Humana Press, pp. 365- 386.
Russo F, Donini B,Starrantino S (1981). Mutagenesis applied for citrus improvement. In: Proceedings International Society of Citriculture; Tokyo, Japan. pp. 68-70.
Sankar AA, Moore GA (2001). Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map. Theoretical and Applied Genetics 102: 206-214.
Sato Y, Nishio T (2003). Mutation detection in rice waxy mutants by PCR-RF-SSCP. Theoritical and Applied Genetics 107: 560-567.
Scarano MT, Abbate L, Ferrante S, Lucretti S, Tusa N (2002). ISSRPCR technique: a useful method for characterizing new allotetraploid somatic hybrids of mandarin. Plant Cell Reports 20: 1162-1166.
Shahsavar AR, Izadpanah K, Tafazoli E, Sayed Tabatabaei BE (2007). Characterization of citrus germplasm including unknown variants by inter-simple sequence repeat (ISSR) markers. Scientia Horticulturae112: 310-314.
Smith JSC, Chin ECL, Shu H, Smith OS, Wall SJ, et al. (1997). An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPs and pedigree. Theoretical and Applied Genetics 95: 163-173.
Starrantino A, Russo F, Donini B, Spina P (1988). Lemon mutations obtained by gamma irradiation of the nucellus cultured in vitro. In: 6th International Citrus Congress; Tel Aviv, Israel. pp. 231-235.
Stuber CW, Polacco M, Senior ML (1999). Synergy of empirical breeding, marker assisted selection and genomics to increase crop yield potential. Crop Science 39: 1571-1583.
Sutarto I, Agisimanto D, Supriyanto A (2009). Development of promisingseedless citrus mutants through gamma irradiation. Induced Plant Mutations in the Genomics Era. Rome, Italy: Food and Agriculture Organization of the United Nations, pp. 306-308.
Simsek O, Aka Kacar Y, Yesiloglu T, Olliutrault P (2011). Determination by SSCP markers of the allelic diversity of candidate genes for tolerance to iron chlorosis in citrus germplasm. Proc. 2nd IS on Citrus Biotechnology. Acta Horticulturae 892, ISHS.
Swingle WT, Reece PC (1967). The botany of citrus and its wild relatives. In: Reuther W, Webber HJ, Batchelor LD (editors). The Citrus industry. Berkeley, CA, USA: University of California Press, pp. 389-390.
Tanaka T (1977). Fundamental discussion of Citrus classification. Studia Citrologica 14: 1-6.
Tang XL, Wu ST, Peng CL, Li ZO (1994). Development of seedless citrus cultivars through gamma ray re-irradiation. In: Proceedings series; International Atomic Energy Agency; Vienna, Austria; Food and Agriculture Organization of the United Nations; Rome, Italy. p. 748.
Terol JF, Naranjo MA, Ollitrault P, Talon M (2008). Development of genomic resources for Citrus clementina: Characterization of three deep-coverage BAC libraries and analysis of 46,000 BAC end sequences. BMC Genomics 9: 12
Turgutoğlu E, Demir G, Kurt Ş, Ağsaran B, Polat İ (2014). Çekirdeksiz, dikensiz ve periyodisite göstermeyen mutant Yerli MandarinveAntalya YerliYuvarlak Limon tiplerinin belirlenmesi (Proje ara sonuç raporu). Araştırma Projeleri Faaliyet Raporları. Antalya, Turkey: Batı Akdeniz Tarımsal Araştırma Enstitüsü Müdürlüğü, pp. 34-48.
Usman M, Fatima B, Awais M, Shoukat D, Fatima A et al. (2018). Prospects of mutation breeding in grapefruit (Citrus paradisi Macf.). In: International Horticulture Conference ; Rawalpindi, Pakistan. p. 29.
Uzun B, Lee D, Donini P, Çağırgan I (2003). Identification of a molecular marker linked to the closed capsule mutant trait in sesame using AFLP. Plant Breeding 122: 95-97.
Uzun A, Gulsen O, Yesiloglu T, Aka-Kacar Y, Tuzcu O (2010). Distinguishing grapefruit and pummeloaccessions using ISSR markers. Czech Journal of Genetics Plant Breeding 46 : 170- 177.
Uzun A, Yesiloglu T, Polat I, Aka Kacar Y, Gulsen O et al. (2011). Evaluation of genetic diversity in lemons and some their relatives based on SRAP and SSR markers. Plant Molecular Biology 29: 693-701.
Wang CT, Huang ZJ, He CF, Bi CL, Shen YZ (2001). Detection of the wheat salt-tolerant-mutant using PCR-SSCP combining with direct sequencing. Yi Chuan Xue Bao Journal 28; 852-855 (in Chinese).
Williams TE, Roose ML (2010). Tango-a new, very low-seeded, late-season irradiated selection of ‘W. Murcott’ mandarin from the University of California Riverside. Proceeding of the International Society of Citriculture 1: 202.
Yadav BR, Kale DS (2009). Single strand conformation polymorphism (SSCP) Analysis by Nondenaturing PAGE. Genetics & Genomics: Genotyping. http://www.protocol-online.org.
Yoshida T, Kawase K, Nesumi H (1999). Inheritance of thornlessness in Trifoliate Orange [Poncirus trifoliata (L.) Raf.]. Journal of Japanese Society for Horticultural Science 68: 1104-1110.
Zhang S, Shi Q, Albrecht U, Shatters Jr RG, Stange R et al. (2017). Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants. Horticulture Research 4: 17041. doi: 10.1038/ hortres.2017.41