Genetic Diversity and Population Structure of Iranian tulips revealed by EST-SSR and NBS-LRR Markers

Document Type: Research paper


1 Ph.D. Student, Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran.

2 Department of Horticulture, Faculty of Agriculture, Shiraz University, Shiraz, IR IRAN

3 Department of plant breeding-Wageningen University

4 Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran.

5 Department of Crop Production and Plant Breeding, College of Agriculture, Shiraz University, Shiraz, Iran

6 Institute of Biotechnology, Shiraz University, Shiraz, Iran.

7 Department of Plant Protection, College of Agriculture, Shiraz University, Shiraz, Iran.

8 Department of Plant Breeding, Wageningen University, Wageningen, the Netherlands.


The genus Tulipa L. (Liliaceae) comprises about 100 species and Iran is considered as one of the main origins of tulips. In this research, genetic diversity and population structure of 27 wild populations of tulips collected from Iran were studied by 15 highly polymorphic and reproducible expressed sequenced tag-simple sequence repeat (EST-SSR) markers and 8 nucleotide binding site (NBS)-enzyme combinations. According to EST-SSR genotyping, the average of gene diversity (GD) and polymorphism information content (PIC) were 0.66 and 0.62, respectively. However, the values of GD and PIC were equal for each NBS primer–enzyme combination which ranged between 0.85-0.95 with a mean value of 0.91. The mean value of resolving power (EST-SSR = 1.93; NBS-LRR = 17.39) indicated that the NBS markers had higher discriminatory power compared to the EST-SSR markers. UPGMA clustering confirmed the results of PCA which was further confirmed by Bayesian model-based STRUCTURE analysis. Population structure analysis detected 3 and 4 gene pools for 27 wild tulip germplasms with EST-SSR genotyping and NBS-LRR profiling, respectively. The AMOVA results indicated that molecular variation among populations (ΦPT = inter-population variation) was 82% and 93% of the total variation for EST-SSR and NBS-LRR markers, respectively. The results of this study will help the conservation and phylogenetic studies of tulips.


Amos B, Pemberton J. 1992. DNA fingerprinting in non-human populations. Current Opinion in Genetics and Development 2, 857-860.

Ballesteros-Mejia L, Lima NE, Lima-Ribeiro MS, Collevatti RG. 2016. Pollination mode and mating system explain patterns in genetic differentiation in Neotropical plants. PLoS One 11:e0158660. doi:10.1371/journal.pone.0158660

Brownstein MJ, Carpten JD, Smith JR. 1996. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques 1004-6, 1008-10

Caser M, Scariot V, Arens P. 2010. Consequences of geographical habitats on population structure and genetic diversity in Campanula spp. International Journal of Plant Biology 1, 22-29.

Chistiakov DA, Hellemans B, Volckaert FAM. 2006. Microsatellites and their genomic distribution, evolution, function and applications: A review with special reference to fish genetics. Aquaculture 255, 1–29.

Christenhusz MJ, Govaerts R, David JC, Hall T, Borland K, Roberts PS, Tuomisto A, Buerki S, Chas MW, Fay MF. 2013. Tiptoe through the tulips–cultural history, molecular phylogenetics and classification of Tulipa (Liliaceae). Botanical Journal of the Linnean Society 172, 280-328.

Ellis JR, Burke JM. 2007. EST-SSRs as a resource for population genetic analyses. Heredity 99, 125-132.

Eujayl I, Sledge MK, Wang L, May GD, Chekhovskiy K, Zwonitzer JC, Mian MA. 2004. Medicago truncatula EST-SSRs reveal cross-species genetic markers for Medicago spp. Theoretical and Applied Genetics 108, 414-422.

Evanno G, Regnaut S, Goudet J. 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology 14, 2611-2620.

Fulton T, Chunwongse J, Tanksley S. 1995. Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Molecular Biology Report 13, 207-209.

Hildebrand CE, David C, Torney C, Wagner P. 1994. Informativeness of polymorphic DNA markers. The human genome project: deciphering the blueprint of heredity. University Science Books, Sausalito, 100-102.

Kiani M, Memariani F, Zarghami H. 2012. Molecular analysis of species of Tulipa L. from Iran based on ISSR markers. Plant Systematics and Evolution 298, 1515-1522. 

Kimura M, Crow JF. 1964. The number of alleles that can be maintained in a finite population. Genetics 49, 725-738.

Kutlunina NA, Polezhaeva MA, Permyakova MV. 2013. Morphologic and AFLP analysis of relationships between tulip species Tulipa biebersteiniana (Liliaceae).  Russian Journal of Genetics 49, 401-410.

Leigh F, Lea V, Law J, Wolters P, Powell W, Donini P. 2003. Assessment of EST- and genomic microsatellite markers for variety discrimination and genetic diversity studies in wheat. Euphytica 133, 359-366.

Lewontin RC. 1972. Testing the theory of natural selection. Nature 236, 181-182.

Liu KJ, Muse SV. 2005. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 21, 2128-2129.

Luan QF, Ouyang T, Jiang YC, Wang CX. 2008. Tulip RAPD analysis of cultivars and wild species in Xinjiang. Acta Agriculturae Universitatis Jiangxiensis 30, 656-660.

Mantovani P, van der Linden G, Maccaferri M, Sanguineti MC, Tuberosa R. 2006. Nucleotide-binding site (NBS) profiling of genetic diversity in durum wheat. Genome 49, 1473-1480.

McDowell JM, Dhandaydham M, Long TA, Aarts MG, Goff S, Holub EB, Dangl JL. 1998. Intragenic recombination and diversifying selection contribute to the evolution of downy mildew resistance at the RPP8 locus of Arabidopsis. The Plant Cell 10, 1861-1874.

Meyers BC, Shen KA, Rohani P, Gaut BS, Michelmore RW. 1998. Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection. The Plant Cell 10, 1833-1846.

Nair RA, Thomas G. 2012. Functional genetic diversity at nucleotide binding site (NBS) loci: comparisons among soft rot resistant and susceptible Zingiber taxa. Biochemical systematics and ecology 44, 196-201.

Nam BE, Nam JM, Kim JG. 2016. Effects of habitat differences on the genetic diversity of Persicaria thunbergii. Journal of Ecology and Environment 40, 11.

Parniske M, Hammond-Kosack KE, Golstein C, Thomas CM, Jones DA, Harrison K, Wulff BB, Jones JD. 1997. Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato. Cell 91, 821-832.

Pashley CH, Ellis JR, McCauley DE, Burke JM. 2006. EST databases as a source for molecular markers: lessons from Helianthus. Journal of Heredity 97, 381-388.

Peakall R, Smouse PE. 2012. GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research – an update. Bioinformatics 28, 2537-2539.

Pineda‐Martos R, Velasco L, Pérez‐Vich B. 2014. Identification, characterization and discriminatory power of microsatellite markers in the parasitic weed Orobanche cumana. Weed Research 54, 120-132.

Prevost A, Wilkinson MJ. 1999. A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theoretical and Applied Genetics 98, 107-112.

Pritchard JK, Stephens M, Donnelly P. 2000. Inference of population structure using multilocus genotype data. Genetics 155, 945-959. 

Qi-fu L, Tong O, Yan-cheng J, Cai-xia W. 2008. Tulip RAPD analysis of cultivars and wild species in Xinjiang. Acta Agriculturae Universitatis Jiangxiensis 30, 656-660.

Rosenberg NA. 2004. Distruct: a program for the graphical display of population structure. Molecular Ecology Notes 4, 137-138. 

Rungis D, Berube Y, Zhang J, Ralph S, Ritland CE, Ellis BE, Douglas C, Bohlmann J, Ritland K. 2004. Robust simple sequence repeat markers for spruce (Picea spp.) from expressed sequence tags. Theoretical and Applied Genetics 109, 1283-1294.

Schuelke M. 2000. An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology 18, 233-234.

Shahin A, van Gurp T, Peters S, Visser R, van Tuyl J, Arens P. 2012. Generation and analysis of expressed sequence tags in the extreme large genomes Lilium and Tulipa. BMC Genomics 13,640

Sharma R, Chowdhury VK, Jain S, Jain RK. 2009. A comparative study of genetic relationships among and within male and female genotypes of dioecious Jojoba (Simmondsia chinensis L. Schneider) using RAPD and ISSR markers. Asian Journal of Horticulture 4, 184-193.

Sun Q, Collins NC, Ayliffe M, Smith SM, Drake J, Pryor T, Hulbert SH. 2001. Recombination between paralogues at the rp1 rust resistance locus in maize. Genetics 158, 423-438.

Tabin Sh, Kamili AN, Ganie SA, Zargar O, Sharma V, Gupta RG. 2016. Genetic diversity and population structure of Rheum species in Kashmir Himalaya based on ISSR markers. Flora 223, 121-128.

Tang N, Mo G, van Tuyl J, Arens P, Liu J, Tang D. 2013a. Genetic diversity and structure of Lilium pumilum DC. in southeast of Qinghai–Tibet plateau. Plant Systematic and Evolution 300, 1453-1464.

Tang N, Shahin A, Bijman P, Liu J, van Tuyl J, Arens P. 2013b. Genetic diversity and structure in a collection of tulip cultivars assessed by SNP markers. Scientia Horticulturae 161, 286-292.

Turktas M, Metin ÖK, Baştuğ B, Ertuğrul F, Saraç YI, Kaya E. 2013. Molecular phylogenetic analysis of Tulipa (Liliaceae) based on noncoding plastid and nuclear DNA sequences with an emphasis on Turkey. Botanical Journal of the Linnean Society 172, 270-279.

van der Linden CG, Wouters DC, Mihalka V, Kochieva EZ, Smulders MJM, Vosman B. 2004. Efficient targeting of plant disease resistance loci using NBS profiling. Theoretical and Applied Genetics 109,384-393

van Scheepen J. 1996. Classified list and international register of tulip names. KAVB, Hillegom, The Netherlands.

Veldkamp JF, Zonneveld BJM. 2012. The infrageneric nomenclature of Tulipa (Liliaceae). Plant Systematics and Evolution 298, 87-92.

Wang F, Yang T, Burlyaeva M, Li L, Jiang J, Fang L, Redden R, Zong X. 2015. Genetic diversity of grasspea and its relative species revealed by SSR markers. PLoS One 10(:e0118542. Doi: 10.1371/journal. pone.0118542

Wang M, van den Berg R, van der Linden G, Vosman B. 2008. The utility of NBS profiling for plant systematics: a first study in tuber-bearing Solanum species. Plant systematics and evolution 276, 137.

Woodhead M, Russell J, Squirrell J, Hollingsworth M, Cardle L, Ramsay L, Gibby M, Powell W. 2003. Development of EST-SSRs from the alpine lady-fern, Athyrium distentifolium. Molecular Ecology Notes 3, 287-290.

Yanagisawa R, Kuhara T, Nishikawa T, Sochacki D, Marasek-Ciolakowska A, Okazaki K. 2012. Phylogenetic analysis of wild and garden tulips using sequences of chloroplast DNA. Acta Horticulturae 953, 103-110.

Yeh FC, Yang RC, Boyle TBJ. 1999. Popgene version 1.31: Microsoft Windows-based freeware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, and Centre for International Forestry Research, Canada. 

Zonneveld B. 2009. The systematic value of nuclear genome size for all species of Tulipa L. (Liliaceae). Plant Systematic and Evolution 281, 217-245.