Indian J. Genet., 77(2): 321-323 (2017) DOI: 10.5958/0975-6906.2017.00044.X *Corresponding author’s e-mail: f.rahmani@urmia.ac.ir Published by the Indian Society of Genetics & Plant Breeding, F2, First Floor, NASC Complex, PB#11312, IARI, New Delhi 110 012 Online management by indianjournals.com; http://epubs.icar.org.in/journal/index.php/IJGPB Short Communication The analysis of genetic diversity in willow (Salix spp.) by ISSR markers Maryam Ghaidaminiharouni, Fatemeh Rahmani* and Ali Khodakarimi 1 Department of Biology and Institute of Biotechnology, Urmia University, Urmia, Iran; 1 Research Center for Agriculture and Natural Resources, Urmia, Iran (Received: March 2016; Revised: March 2017; Accepted: March 2017) Abstract In the present study, ISSR (Inter Simple Sequence Repeat) markers were employed to determine genetic diversity among 30 clones of Salix (S. acmophylla, S. excelsa, S. alba and S. triandra). The average percentage of polymorphic loci was 59.86. The pair-wise genetic similarity varied from 0.18 to 0.65. Nei’s gene diversity (H E ) ranged from 0.12 to 0.18 and the average expected heterozygosity (H E ) estimated 0.15. The values obtained for genetic diversity (H T = 0.20) and genetic differentiation (G ST = 0.20) indicated a low level of genetic differentiation among examined populations of Salix in North West of Iran. Key words: Genetic diversity, ISSR, polymorphism, Salix The genus Salix belongs to the Salicaceae family and comprised of 350 to 500 species worldwide in form of trees or shrubs (Argus 1997). Thirty-one species of the Salix genus were reported in Iran (Maassoumi 2009). Willows are categorized as advanced biomass crops (Keoleian and Volk 2005) with many desirable traits that make them suitable for production. ISSR markers reveal higher level of polymorphism and show a higher reproducibility than RAPD and RFLP approaches (Tsumura et al.1996). This study is the first attempt to assess genetic diversity among willow clones in Iran. Plant material consisted of S. acmophylla (6 clones), S. excels (12 clones), S. alba (9 clones) and S. triandra (3 clones). All of the studied clones were collected from different regions of the North West of Iran. Genomic DNA was extracted from fresh leaves tissue based on modified CTAB procedure (Doyle and Doyle 1987). The PCR – ISSR reaction was performed in a total volume of 25 μl using the following touchdown program: 3 min at 95 o C for 1 cycle; 30 s at 95 o C, 30 s at 65 o C and 60 s at 72 o C for 10 cycles; annealing temperature at 65 o C being subsequently reduced by 1 o C for the next 10 cycles and remained at 55 o C for the remaining 30 cycles; 10 min at 72 o C for 1 cycle. Shannon diversity index ( I ) was calculated in POPGENE 1.31 (Yeh et al. 1997). POPGENE 32 was employed to calculate number of observed alleles (Na), mean number of effective alleles (Ne), number of polymorphic loci, percentage of polymorphic loci, total heterozygosity (H T ), mean heterozygosity within the clones (H s ) and the coefficient of clone differentiation (G ST = H T -H S /H T ). Figure 1 is the representative of the extent of polymorphism observed in 20 willow clones by primer 825. The average number of scorable fragments per primer was 15.2, ranging from 9 to 23 (Table 1). Percentage of polymorphic loci was from 36.84% (S. triandra) to 80.26% (S. excelsa), with an average polymorphism of 59.86% across all four species (Table 1). The number of observed alleles per locus reached to 1.58 with the effective number of alleles equal to 1.24 (Table 2). Therefore, effective alleles accounted for 78.48% of total number of alleles. Number of polymorphic loci and Nei’s gene diversity (H E ) ranged from 56 to 122 and 0.12 to 0.18, respectively. The