Int. J. Biosci. 2014 International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print) 2222-5234 (Online) http://www.innspub.net Vol. 4, No. 1, p. 75-81, 2014 RESEARCH PAPER OPEN ACCESS The study of genetic variation for Lolium perenne using ISSR molecular markers Hooshmand Safari1*, Hooman Shirvani2, Ali Ashraf Jafari3, Sara Mahdavi4 1 Agriculture and Natural Resources Research Center, Kermanshah, Iran 2 Department of Agriculture, Payame Noor University, Iran 3 Research Institute of Forests and Rangelands, Tehran, Iran 4 Department of Agriculture, Borujerd Branch, Islamic Azad University, Borujerd, Iran Key words: Genetic diversity, Lolium perenne, accessions, ISSR molecular markers. doi: http://dx.doi.org/10.12692/ijb/4.1.75-81 Article published on January 01, 2014 Abstract Genetic variation for 12 accessions of Lolium perenne were surveyed using the number of 12 ISSR primers, that the number of 10 primers can be scored. The ISSR primers can be produced the number of 62 bands, which the polymorphism was showed for the number of 47 bands. The average of bands was 6.2 for each primer. The primer of IS9 showed the highest number of band (11 bands) and IS 15 showed the lowest number of band (3 bands). A desirable polymorphism between genotype was observed based on ISSR markers, which the primers of IS9, IS10, IS13 and IS16 were determined for genetic variation study in Lolium perenne as desirable primers. Average PIC in the used primers was 0.33 that the highest amount of PIC related to primer IS 12 that amount of PIC was 0.45 in this primers and primers IS3 and IS14 with the lowest value of PIC don’t have ability in the separation of accessions. Yatsyn (G11) had the most genetic distance with Grenisle (G7). Cluster analysis and Scatter plot based on first and second axis from principal coordinate analysis for genotypes, showed that genetic variation did not agreement with the geographical distribution. Therefore the genotypes which had the most genetic distance based on used ISSR marker can be considered as suitable material in breeding programs for using hetrosis. * Corresponding Author: Hooshmand Safari  hooshmand.safari@gmail.com 75 Safari et al. Int. J. Biosci. 2014 Introduction of DNA profiling of populations is to determine Perennial ryegrass (Lolium perenne L.) is the most diversity among populations in order to develop important grass species in temperate climates of the genetically distinct subsets of populations in a world. Perennial ryegrass is a diploid species breeding program or to check for duplicates in a gene (2n=2x=14) with a two-locus self incompatibility bank. In these cases, it may be possible to determine system, which ensures a high degree of genetic diversity among populations by profiling bulked DNA variation in populations (Bolaric et al., 2005a). of the individuals (Rouf et al., 2002). Inter Simple Lolium perenne (Poaceae) was chosen as the model Sequence Repeat (ISSR) is a dominant molecular species and small populations of 15 different cultivars marker revealed in mass. ISSR has recently been of this species were established in each experimental developed as an anonymous, RAPD–like approach community. Lolium perenne is one of most important that accesses variation in the numerous microsatellite grass species in central Europe and worldwide used regions dispersed throughout the various genomes for the sowing and regeneration of temperate and circumvents the challenge of characterizing agricultural grassland. Its productivity is stimulated individual loci that other molecular approaches by nitrogen fertilization and it has a high fodder require. They are characterized by mono-, di- or multi quality and grazing tolerance. Beside its importance - nucleotide repeats that have 4-10 repeat units’ side- as a fodder crop, perennial ryegrass is frequently used by-side. Extremely high variability combined with as a turf species in lawns (Beddows, 1967). greater robustness in repeatability experiments and less prone to changing band patterns with changes in Knowledge of genetic variation is a useful tool in constituent or DNA concentration template make genebank management, helping in the establishment them superior to other readily available marker of core collections, facilitating efficient sampling and systems in investigations of genetic variation (Fang utilization and/or and Roose, 1997). Genetic diversity based on eliminating duplicates in the gene stock), and Molecular markers for within different species of selecting of desirable genotypes to be used in Lolium was reported by many researchers (Elazreg et breeding programs (Elham et al., 2010; Vishwanath al., 2011; Vieira et al., 2004; Majidi and Mirlohi, et al., 2011). On the other hands, understanding 2010). of germplasm (identifying genetic diversity of certain species is not only useful in addressing questions about evolutionary process Thus, the present study aimed to determine and and the development of conservation strategies, but assess the genetic variability of the Lolium collection also a prerequisite for efficient use of genetic and better understand the genetic diversity structure resources in breeding programs. Interest in the of the available accessions using ISSR molecular genetic structure of natural populations of grass markers. species has been increased in the last few years due to the necessity of broadening the knowledge of genetic Materials and methods variations in cultivated species (Che and Li, 2007). Plant materials Assessment of genetic diversity with molecular In order to evaluate the genetic variation, 12 markers is a alternative. Molecular accessions of Lolium perenne were prepared from characterization can also be re-applied after years of gene bank of Research Institute of Forests and maintenance and new accessions can be related to Rangelands, Tehran, Iran (Table 1). existing promising collections. Moreover, molecular characterization of genetic diversity provides base DNA extraction and ISSR method information, which can be used to select a promising Total genomic DNA was extracted for young leaves of range of accessions for different breeding programs greenhouse-grown plants using a modified CTAB (Roldan-Ruiz et al., 2001). Often, the initial objective (Murry and Tompson, 1980) with modification 76 Safari et al. Int. J. Biosci. 2014 described by De la Rosa et al., (2002). Quality and information content (PIC) calculated for each primer quantity of extracted DNA were examined using 0.8% (Anderson et al., 1993). Similarity matrix computed agarose gel. The compounds of polymerase chain based on Dice's coefficient and cluster analysis reaction were carried out according to Table 2. performed for grouping accessions based on Dice's coefficient by UPGMA methods. Principal coordinate Template DNA was initially denatured at 92oC for 5 analysis performed to better interpret the genetic min, followed by 35 cycles of PCR amplification under variation the following parameters: denaturation for 30 molecular variance analysis performed for the three seconds at 95oC, primer annealing for 30 seconds at groups from cluster analysis. Statistical analyses were the temperature based on primer temperature done by Darwin 5 and Gen ALEX 6.2. between accessions and finally the (Temperatures of annealing in this study was 50, 55 and 60 oC) and primer extension for 1 min at 72oC. A final incubation was performed for 5 min at 72oC to Results ISSR polymorphism ensure that the primer extension reaction proceeded Genetic variation for 12 accessions of Lolium perenne to completion. The PCR amplified products were were surveyed using the number of 12 ISSR primers, separated by electrophoresis on a 1.5% agarose gels that the number of 10 primers can be scored. For all using TBE buffer. The gels were put in the Ethidium primers, the number of 62 bands was scored that bromide for 30-45 min and visualized by gel polymorphism was observed for 47 of them. The document. average of bands was 6.2 for each primer. IS9 primer with 11 bands had the highest and primer IS15 with 3 Statistical analysis bands had the lowest number of bands. The average ISSR bands were treated as binary characters and of bands for each primer was 5.1 for 12 genotypes that coded accordingly (presence =1, absence = 0). The genotype Spelga (G12) had the most and genotype Number of Fontoon (G10) had the lowest band. Band pattern of of accessions for IS9 showed in Fig. 1. of polymorphic scored bands bands (NSB), (NPB), number percentage polymorphism bands (PPB) and polymorphism Table 1. Gen bank cod and Origin of accessions of Lolium perenne. Gene bank Name code genotype 1303 Grenisle 1312 Tyrone Origin Number Gen bank cod Southern Ireland Northern Ireland G7 G8 1309 1307 Name genotype Aubisque Green Gold 1311 Napoleon Denmark G9 1308 Magician 1330 1302 1306 Fontoon Yatsyn Spelga Southern Ireland New Zealand Northern Ireland G10 G11 G12 1305 1313 1301 Carat Gilford Moy Origin Number Netherlands Southern Ireland Southern Ireland Netherlands Ireland Northern Ireland G1 G2 Table 2. Compounds of optimized ISSR reaction. To provide 20 µl 12.6 µl 2 µl 1.5 µl 0.4 µl 1.2 µl 0.3 µl 2 µl 20 µl 77 Safari et al. Compounds of a sample Water distillated twice Buffer PCR (X10) Colored manyazium (50 mmol) Nucleotides mixture (10 mmol) Primer (10 µmol) Tag polymerase DNA (10 ng) Total G3 G4 G5 G6 Int. J. Biosci. 2014 Average of polymorphism percent was 80.24%. The Primers sequences, code, number of bands scored, lowest percent of polymorphism belonged to IS15 number (50%) and the highest percent of polymorphism was polymorphic 100% for primers IS3, IS9, IS10, IS13 and IS16. Average information content (PIC) were showed for ISSR of PIC for all primers was 0.33 that the highest value primers in Table 3. of polymorphic bands bands, (PPB) and percent of polymorphism of PIC related to IS12 and the lowest belonged to IS3 and IS14 (Table 3). Table 3. ISSR primers used in this study and some summary results. ISSR code IS3 Primer sequence 5' GAGAGAGAGAGAGAGAYC 3' NSB 4 NPB 4 PPB 100% PIC 0.257 IS5 IS9 5' AG AG AG AG AG AG AG AGC 3' 5' CTCTCTCTCTCTCTCTG 3' 11 4 8 4 72.73% 100% 0.304 0.368 IS10 5' GAGAGAGAGAGAGAGARC 3' 8 8 100% 0.339 IS11 5' ACACACACACACACACC 3' 7 4 57.14% 0.344 IS12 5' TGTGTGTGTGTGTGTGG 3' 5 3 60% 0.458 IS13 5' AGAGAGAGAGAGAGAGYT 3' 3 3 100% 0.324 IS14 5' GACAGACAGACAGACA 3' 8 5 62.50% 0.280 IS15 5' GGATGGATGGATGGAT 3' 8 4 50% 0.336 IS16 5'DBDACACACACACACACA3' 4 4 100% 0.340 Average 6.2 4.7 80.24 0.335 Similarity Matrix of similarity was 0.70 [Between the accessions of Similarity matrix based on Dice’s coefficient for Yatsyn (G11) and Grenisle (G7)] to 0.87 [Between the accessions accessions of Aubisque (G1) with Grenisle (G7)]. showed that (Table 4) the average of Similarity between accessions was 0.79 and the range Table 4. Similarity matrix for studying accessions based on Dice’s coefficient. accessions G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 0.787 0.825 0.805 0.732 0.8 0.831 0.814 0.789 0.736 0.8 0.782 0.818 0.809 0.771 0.738 0.839 0.747 0.766 0.804 0.785 0.775 0.767 0.8 0.786 0.764 0.800 0.75 0.84 0.8 0.831 0.86 0.829 0.767 0.778 0.747 0.753 0.814 0.743 0.782 0.778 0.817 0.795 0.857 0.709 0.786 0.815 0.862 0.756 0.8 0.865 0.791 0.809 0.784 0.736 0.812 0.822 0.795 0.714 0.873 0.778 0.836 0.776 0.827 0.787 0.747 0.744 included accessions of Carat (G4), Green Gold (G2) Cluster Analysis grouping and Tyrone (G8), which the average similarity accessions based on Dice's coefficient (Fig. 2) were coefficient was 0.81 for this group. The third group identified the three distinctive groups. The first group consisted of Aubisque (G1), Gilford (G5), Grenisle consisted of accessions Yatsyn (G11), Spelga (G12), (G7), Napoleon (G9) and Fontoon (G10). The average Magician (G3) and Moy (6G), which the average of Dice's coefficient was 0.83 for this group. UPGMA hierarchical clustering for similarity was 0.79 for this group. The second group 78 Safari et al. Int. J. Biosci. 2014 Cluster analysis of the similarity matrix for grouping between groupings of accessions based on cluster showed, the most similar among Third group and analysis (Table 6). The results showed a significant Second group, and the most distance for accession in (P<0.01) difference between groups and the portion groups Second and first. (Table5). of variance percent for between group and within Table 5. Similar groups from the cluster analysis. group were 13% and 87%, respectively. Groups C1 C2 C3 C1 1 0.767 0.778 C2 C3 Principal Coordinate Analysis 1 0.795 Scatter plot for accessions based on first (33.25) and 1 second (17.26) axis from principal coordinate analysis Molecular Variance Analysis (Fig. 3) showed that Genetic variation did not Analysis of molecular variance was performed for matching with the geographical distribution of ISSR bands to determine of significant difference accessions. These results confirmed by cluster analysis and similarity matrix. Table 6. Molecular variance analysis. S.O.V Df SS MS Est. Var. Var% PhiPT Between group 2 27.63 13.82 1.323 13% 0.133* Within group 9 77.70 8.633 8.633 87% Total 11 105.33 9.957 100% Est. Var; Calculated variance for Within and between group %Var; Percent of variance of each source to total variance. Discussion determine of genetic diversity between and within The results revealed that ISSR markers are suitable different Lolium species (Pivoriene et al., 2008; Hu et tools for detecting the genetic variation in Lolium al., 2011; Posselt et al., 2006; Bolaric et al., 2005b). accessions. According to this study a Significant The similarity between accessions based on Dice's variation was observed among accessions. We believe coefficient was high, therefore can be stated that there that there needs a molecular markers studies as a was a low genetic variation among accessions. It was complementary studies for the morphological traits in noticed that the majority of the similarity coefficients the field. It will reduce the amount of materials for between accessions was close to 0.78. This indicated study as well as the costs of experiments. the This close relationships between the evaluated technique has been used to study a variety of plants. accessions, though they are collected from different The results of this study Ghariani et al (2003) were origin. Grouping of accessions based on cluster consistent. The Average percentage of polymorphism analysis and principal coordinate analysis indicated was 80.24% that showed a good polymorphism that genetic variations do not in agreement with the between genotypes. Between 10 used primers, the geographical distribution of accessions. primers IS3, IS9, IS10, IS13 and IS16 showed 100% polymorphism that indicate high ability for survey molecular variation and high variation among genotypes. PIC values estimate the discriminatory power of a marker. The mean PIC values for markers used in present study were 0.33. Marker with high PIC values such as IS12 could be effectively used in genetic diversity studies in Lolium. 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