Evaluation of Diversity Based on Morphological Variabilities and ISSR Molecular Markers in Iranian <Emphasis Type="Italic">Cynodon dactylon</Emphasis> (L.) Pers. Accessions to Select and Introduce Cold-Tolerant Genotypes

The main goals of the present study were to screen Iranian common bermudagrasses to find cold-tolerant accessions and evaluate their genetic and morphological variabilities. In this study, 49 accessions were collected from 18 provinces of Iran. One foreign cultivar of common bermudagrass was used as control. Morphological variation was evaluated based on 14 morphological traits to give information about taxonomic position of Iranian common bermudagrass. Data from morphological traits were evaluated to categorize all accessions as either cold sensitive or tolerant using hierarchical clustering with Ward’s method in SPSS software. Inter-Simple Sequence Repeat (ISSR) primers were employed to evaluate genetic variability of accessions. The results of our taxonomic investigation support the existence of two varieties of Cynodon dactylon in Iran: var. dactylon (hairless plant) and var. villosous (plant with hairs at leaf underside and/or upper side surfaces or exterior surfaces of sheath). All 15 primers amplified and gave clear and highly reproducible DNA fragments. In total, 152 fragments were produced, of which 144 (94.73%) being polymorphic. The polymorphic information content (PIC) values ranged from 0.700 to 0.928. The average PIC value obtained with 15 ISSR primers was 0.800, which shows that all primers were informative. Probability identity (PI) and discriminating power between all primers ranged from 0.029 to 0.185 and 0.815 to 0.971, respectively. Genetic data were converted into a binary data matrix. NTSYS software was used for data analysis. Clustering was done by the unweighted pair-group method with arithmetic averages and principle coordinate analysis, separated the accessions into six main clusters. According to both morphological and genetic diversity investigations of accessions, they can be clustered into three groups: cold sensitive, cold semi-tolerant, and cold tolerant. The most cold-tolerant accessions were: Taft, Malayear, Gorgan, Safashahr, Naein, Aligoudarz, and the foreign cultivar. This study may provide useful information for further breeding programs on common bermudagrass. Selected genotypes can be evaluated for other abiotic stresses such as drought and salinity.     

Assessment of Genetic Diversity in Ziziphus mauritiana Using Inter-Simple Sequence Repeat Markers

Genetic diversity among 47 ber accessions belonging to cultivated species (Ziziphus mauritiana Lam) and one wild accession of Ziziphus nummularia (Burm F) Willed was investigated using Inter-Simple Sequence Repeat (ISSR) markers. A total of 167 amplification products were detected with 18 ISSR primers of which 152 (89.96%) were polymorphic. Most of the primers that produced distinct bands (14 primers out of 18) contained dinucleotide repeats. Primers based on (AC)n and (AG)_n repeats produced more polymorphic bands. Genetic similarity ranging from 43.07% to 90.30% suggested that the 48 Ziziphus genotypes used in the study were divergent. Cluster analysis based on UPGMA method and Bootstrap analysis separated all the 48 genotypes in four distinct clusters. The present study has successfully distinguished morphologically similar genotypes that emphasize the use of molecular markers to the taxonomists. Morphologically similar but genetically distinct genotypes, identified using ISSR markers could be potential sources for genotype identification and to resolve controversies over misnomination of ber genotypes. Present study is the first report on the exploitation of ISSR markers in ber for genetic diversity analysis.     

Genetic homogeneity of in vitro raised plants of grapevine cv. Crimson Seedless revealed by ISSR and microsatellite markers

The present study was conducted to test the clonal homogeneity of six month old tissue culture raised plants of grapevine cv. Crimson Seedless using Inter Simple Sequence Repeat (ISSR) and Simple Sequence Repeat (SSR) markers. Visible assessment of these in vitro raised plants maintained in polyhouse did not show any morphological differences among themselves. However, to test the genetic homogeneity of these plants, we screened 50 ISSR primers out of which, 22 primers produced scorable and repeatable bands. These 22 primers were used further for assessing genetic homogeneity of in vitro raised plants of Crimson Seedless. These 22 ISSR primers generated 134 distinct band classes with a total of 3216 scorable bands. All the primers showed uniform banding pattern for all the in vitro raised plants and the mother plant. In case of 5 SSR primers (VS1, VVMD5, VVS2, VMCNG4c8 and VVMD31) used, a total of 288 scorable bands were obtained. The allele sizes ranged from 98 to 254 bp. Allelic composition of 23 in vitro raised plants and the mother plant at 5 SSR loci did not show any polymorphism. The results of the two marker systems in the present study revealed the genetic uniformity among the in vitro raised plants demonstrating the reliability of in vitro propagation system used for the cultivar.     

RAPD和ISSR标记对水稻化感种质资源遗传多态性的分析

运用RAPD和ISSR技术分析水稻化感种质资源的遗传多态性。从供试材料中筛选到具有多态性的RAPD引物12条,ISSR引物7条。RAPD引物共扩增到85条清晰的多态性条带,多态性条带比率为69．4％。ISSR引物共扩增到34条清晰的多态性条带,多态性条带比率为53．0％。对两种标记结果进行UPGMA聚类分析,结果极其类似,呈极显著的正相关(r=0．74)。聚类结果表明,地理位置相近的品种聚为一类。部分具有较强化感作用潜力的水稻品种亲缘关系很近,表明控制其化感作用性状的基因可能是等位的相同基因。而部分化感作用潜力差异显著的水稻品种聚为一类,这是由于人类在长期高产品种的定向选择过程中,水稻化感作用性状不被注意而丢失,遗传基础日益狭窄的原因。     

Genetic characterization of Iranian safflower (Carthamus tinctorius) using inter simple sequence repeats (ISSR) markers

Safflower (Carthamus tinctorious L.) is valued as a source of high quality vegetable oil. 20 ISSR primers were used to assess the genetic diversity of 18 accessions of safflower collected from different geographical regions of Iran. The ISSR primers combinations revealed 57.6 % polymorphism, among 338 genetic loci amplified from the accessions. The sum of effective number of alleles and observed number of alleles were 29.76 and 36.77, respectively. To understand genetic relationships among these cultivars, Jacquards’ similarity coefficient and UPGMA clustering algorithm were applied to the ISSR marker data set. ISSR markers grouped accessions into two main clusters and four sub clusters. Also, the principal coordinate analysis (PCoA) supported the cluster analysis results. The results showed these genotypes have high genetic diversity, and can be used for alternative safflower breeding program.     

Detecting DNA polymorphism and genetic diversity in Lentil (Lens culinaris Medik.) germplasm: comparison of ISSR and DAMD marker

Genetic diversity and interrelationships among 31 lentil genotypes were evaluated using 10 Inter-Simple Sequence Repeat (ISSR) and 10 directed amplification of minisatellite DNA region (DAMD) primers. A total of 43 and 48 polymorphic bands were amplified by ISSR and DAMD markers, respectively. Average polymorphism information content (PIC) for ISSR and DAMD markers were 0.37 and 0.41, respectively. All 31 lentil genotypes could be distinguished by ISSR markers into three groups and by DAMD markers into two groups. Various molecular markers show a different efficiency for evaluating DNA polymorphism in lentil and indicate that the patterns of variation are clearly influenced by the genetic marker used. Comparatively, the genetic diversity of examined lentil genotypes by two different marker techniques (ISSR and DAMD) was high and indicated that ISSR and DAMD are effective and promising marker systems for fingerprinting in lentil and give useful information on its genetic relationships.     

Genetic diversity analysis of Cynodon dactylon (bermudagrass) accessions and cultivars from different countries based on ISSR and SSR markers

ISSR and SSR markers were used to evaluate genetic diversity among 33 Cynodon dactylon accessions and 22 cultivars from four different countries in order to provide information on how to improve the utilization of bermudagrass germplasms. Eighty eight bands were amplified by nine SSR primer combinations and 236 bands were observed from 23 ISSR primers. The results showed that 97.7% of the SSR primers and 86.9% of the ISSR primers were polymorphic. The genetic similarity coefficients (GSC), gene diversity (He) and Shannon index (I) were 0.58–0.97, 0.27 and 0.41, respectively, for ISSR and 0.52–0.97, 0.29, and 0.43 for SSR. The UPGMA analysis clustered the 55 accessions (cultivars) into three groups. The cluster results produced by the ISSR data were close to the SSR data results. Analysis based on the combined ISSR and SSR data was more closely related to the geographical distribution of the tested germplasm.     

Genetic Diversity in Various Accessions of Pineapple [<Emphasis Type="Italic">Ananas comosus</Emphasis> (L.) Merr.] Using ISSR and SSR Markers

Inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers were used to assess the genetic diversity of 36 pineapple accessions that were introduced from 10 countries/regions. Thirteen ISSR primers amplified 96 bands, of which 91 (93.65%) were polymorphic, whereas 20 SSR primers amplified 73 bands, of which 70 (96.50%) were polymorphic. Nei’s gene diversity (h = 0.28), Shannon’s information index (I = 0.43), and polymorphism information content (PIC = 0.29) generated using the SSR primers were higher than that with ISSR primers (h =  0.23, I = 0.37, PIC = 0.24), thereby suggesting that the SSR system is more efficient than the ISSR system in assessing genetic diversity in various pineapple accessions. Mean genetic similarities were 0.74, 0.61, and 0.69, as determined using ISSR, SSR, and combined ISSR/SSR, respectively. These results suggest that the genetic diversity among pineapple accessions is very high. We clustered the 36 pineapple accessions into three or five groups on the basis of the phylogenetic trees constructed based on the results of ISSR, SSR, and combined ISSR/SSR analyses using the unweighted pair-group with arithmetic averaging (UPGMA) method. The results of principal components analysis (PCA) also supported the UPGMA clustering. These results will be useful not only for the scientific conservation and management of pineapple germplasm but also for the improvement of the current pineapple breeding strategies.     

Molecular characterization of Greek pepper (Capsicum annuum L) landraces with neutral (ISSR) and gene-based (SCoT and EST-SSR) molecular markers

Thirty landraces were selected from a Gene Bank collection comprising of pepper (Capsicum annuum L.) landraces from across Greece. The genetic variability among the 30 landraces and one commercial greek cultivar, intended for industrial use, was assessed by Inter-Simple Sequence Repeat (ISSR), Start Codon Targeted (SCoT) and Expressed Sequence Tag (EST) - Simple Sequence Repeat (SSR) molecular markers. All genotypes were clearly distinguished in the dendrograms produced, with the ISSR and SCoT dendrograms being the most consistent. Three landraces were grouped together in all dendrogams revealing a possible phylogenetic relationship. Furthermore, EST-SSR markers were combined with High Resolution Melting (HRM) analysis by which the more adequate and confident separation of the landraces was accomplished due to the unique HRM profiles produced. Genotyping the rich Greek pepper germplasm by molecular markers will aid the breeding process towards well adopted Greek pepper cultivars of higher quality and productivity.     

Morphological and molecular genetic variations of oat genotypes grown in Kermanshah,Iran

Morphological traits and molecular markers are two common methods for genetic variation studies. Molecular markers, morphological traits methods and relationship between the two were used to study genetic variation among 43 oat genotypes and varieties. For this purpose, an augmented design was conducted in three replicates at 2008–2009 cropping season in the experimental field of Campus of Agriculture and Natural Resources of Razi University, Kermanshah, Iran. Four wild oat accessions (Avena sterilis) were added to evaluated genotypes in molecular experiment. Results showed a significant variation among genotypes for all morphological traits and they were classified based on this variation in four groups by WARD cluster analysis. In molecular experiment, 28 inter simple sequence repeat (ISSR) primers amplified 206 polymorph bands. Based on Jaccard similarity matrix, similarity among genotypes was varied from 0.23 to 0.66 and cluster analysis classified genotypes in seven groups by complete linkage method. The correlation between ISSR marker and morphological traits classifications was not significant. ISSR showed to be a helpful marker for genotype identity and separation as it put wild accessions in a group.     

RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species.

Abstract: Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and phylogenetic relationships among cultivated peanut and wild species of the genus Arachis. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. Moreover, the dendrograms based on RAPD, ISSR, and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. One SSR primer was identified that could distinguish all the accessions analysed within a variety. Although the polymorphic index content varied from 0.1 to 0.5 for both ISSR and RAPD markers, primer index values were substantially higher for RAPD primers (0.35-4.65) than for SSR primers (0.35-1.73). It was possible to identify accessions, particularly those of divergent origins, by RAPD and (or) ISSR fingerprints. Based on these results, marker-based genetic improvement in A. hypogaea appears possible. None of the 486 RAPD and 330 ISSR amplification products were found to be commonly shared among 13 species of section Arachis and one species each of sections Heteranthae, Rhizomatosae, and Procumbentes. Dendrograms constructed from RAPD, ISSR, and RAPD + ISSR data showed overall similar topologies. They could be resolved into four groups corresponding to the species grouped in four taxonomic sections. The present results strongly support the view that Arachis monticola (2n = 4x = 40) and A. hypogaea are very closely related, and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species.     

Analysis of Genetic Diversity in Napier Grass (Pennisetum purpureum Schum) as Detected by RAPD and ISSR Markers

Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) markers were used to detect the DNA polymorphism among thirty Napier grass collections of wide geographical distribution. A total of 20 RAPD and 10 ISSR primers were used in this study. RAPD analysis produced 222 fragments of which, 195 were polymorphic with an average of 9.75 polymorphic fragments per primer. The ten ISSR primers produced a total of 98 fragments out of which 88 were polymorphic accounting for 89.8%. The Mantel test between two similarity matrices of the markers revealed a low correlation (r = 0.33) indicating low correspondence between polymorphism brought out by the two marker systems. The UPGMA clustering of genotypes eventhough was not similar when RAPD and ISSR derived dendrograms were compared, but showed a greater correspondence with geographical identity in both the marker systems employed. This correspondence was also evident when data from both the RAPD and ISSR markers were combined. The implications on collection and breeding of this important forage grass had been discussed.     

Comparative Evaluation of RAPD, ISSR and Anchored-SSR Markers in the Assessment of Genetic Diversity and Fingerprinting of Oilseed Brassica Genotypes

Forty-two genotypes representing oilseed Brassica species were analyzed for the level of genetic diversity and molecular identity using Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat (ISSR) and 5'-Anchored Simple Sequence Repeat (ASSR) markers. DNA profiles revealed high degree of interspecific polymorphism, while the level was considerably low within a species, particularly in B. juncea. The UPGMA clusters clearly delineated genotypes of the respective Brassica species. Comparison of cophenetic matrices indicated a high degree of correspondence between dendrograms generated by different marker systems. A minimum of 10 random primers (approximately 105 bands) were required for the RAPD profiles to generate the expected cluster. Comparatively less number of primers was required to do the same in case of ISSR (4 primers) and ASSR (3 primers). The principal component analysis revealed similar genetic relationship among the genotypes as in cluster analysis. Although none of the DNA profiles could individually identify all the B. juncea genotypes, a combined DNA profile consisting 125 markers from the informative primers of all the three DNA marker systems could do the same. A positive correlation was found among the marker utility parameters (calculated for individual primers of different marker systems) such as marker index (MI), resolving power (Rp) and discrimination coefficient (D) with the number of genotypes identified by each primer with a few exceptions. Single plant analysis for a set of five B. juncea varieties revealed absence of intra-varietal heterogeneity in case of ASSR profiles, thereby suggesting its utility in varietal identification and differentiation.     

A comparison of genetic variation among wild and cultivated Morus Species (Moraceae: Morus) as revealed by ISSR and SSR markers

In this study, inter-simple sequence repeats (ISSR) ans simple sequence repeat (SSR) markers were used to investigate genetic diversity of 27 mulberry accessions including 19 cultivated accessions (six M. multicaulis, three M. alba, two M. atropurpurea, two M. bombycis, one M. australis, two M. rotundiloba, one M. alba var. pendula, one M. alba var. macrophylla, and one M. alba var. venose) and 8 wild accessions (two M. cathayana, two M. laevigata, two M. wittiorum, one M. nigra and one M. mongolica). ISSRs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 27 mulberry genotypes. SSRs presented a higher level of polymorphism and greater information content. All index values of genetic diversity both markers analyzed using Popgene 32 software indicated that within wild species had higher genetic diversity than within cultivated species. Cultivation may caused the lose of genetic diversity of mulberry compared with wild species revealed by ISSR and SSR markers. The mean genetic similarity coefficients among all mulberry genotypes ascribed by ISSR and SSR matrices were 0.7677 and 0.6131, respectively. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. Cluster analysis of ISSR and SSR using UPGMA method revealed that the wild species are genetically distant from the domesticated species studied here. The correlation coefficients of similarity were statistically significant for both marker systems used. Principal coordinates analysis (PCA) for ISSR and SSR data also supports their UPGMA clustering. These results have an important implication for mulberry germplasm characterization, improvement, molecular systematics and conservation.     

Comparative Evaluation of SSR and ISSR Markers for Polymorphism in Landraces and Elite Varieties of Rice (Oryza sativa L)

The study aims at comparing SSR and ISSR markers for their ability to identify polymorphism in parents comprising three landraces and two elite varieties of rice. Both the SSR (0.51) and ISSR (0.46) primers showed almost similar values for Poly morphic Information Content (PIC). Maximum PIC values were observed with trinucleotide ISSR primers (0.67) followed by dinucleotide ISSR primers. In addition to the mapped SSR markers, ISSR markers used in the present study produced more polymorphic markers suggesting their utility in the survey of polymorphism between the parental lines belonging to the same sub-species of rice.     

Use of Simple Sequence Repeat Markers for Estimation of Genetic Diversity in Coconut (Cocos nucifera L) Germplasm Accessions

Simple Sequence Repeat (SSR) markers were used to determine the genetic relationship among 33 coconut germplasm accessions collected from different geographical regions. Wide range of Jaccard’s similarity coefficient (0.00–0.86) reflects the informativeness of the SSR markers. Dwarf and intermediate accessions showed highest similarity among them. The tall accessions belonging to South East Asia, South Asia and South Pacific were clustered based on their geographical regions, but dwarf and intermediate accessions were clustered separately. Clustering of accessions belonging to Atlantic and America revealed the spread of coconut from Far East to South Pacific. Principal coordinate plot and UPGMA analyses formed similar groups of the accessions.     

Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits

The genetic diversity of 39 garlic accessions was investigated using eight simple sequence repeat (SSR) primer combinations and 17 inter-simple sequence repeat (ISSR) primer combinations. A total of 109 polymorphic loci were detected among these accessions, with an average of 4.63 polymorphic loci per SSR primer combination and 4.29 polymorphic loci per ISSR primer combination. The mean effective number of alleles, the mean Nei's genetic diversity, and the mean Shannon's information index for SSR were 1.4799, 0.2870, and 0.4378, respectively; and those for ISSR were 1.4847, 0.2898 and 0.4415, respectively. Cluster analysis, using the unweighted pair-group method with arithmetic averages (UPGMA) based on the allele frequency data, classified the accessions into three groups. The results of principal component analysis (PCA) were consistent with those of the cluster analysis. PCA showed that each of these three groups exhibited significant variation in agro-morphological traits. These findings suggest that the eight SSR and 17 ISSR primers identified could define valuable markers for genetic diversity for use by plant breeders.     

Estimation of genetic diversity in some Iranian wild Prunus subgenus Cerasus accessions using inter-simple sequence repeat (ISSR) markers

As Iran is one of the main origins of Prunus germplasm. In this study, ISSR markers were used for genetic diversity evaluation of 39 accessions of subgenus Cerasus belonging to six species i.e. Prunus avium L., Prunus cerasus L., Prunus mahaleb L., Prunus incana Pall., Prunus microcarpa Boiss., and Prunus brachypetala Boiss.. With 12 ISSR primers, 151 polymorphic bands were detected with polymorphism ratio range of 81.8%–100%. The lowest similarity (0.04) was found between P. avium and P. microcarpa genotypes and the mean of similarity between all genotypes was 0.28. Cluster analysis separated improved cultivars from wild accessions. Improved cherry cultivars and rootstocks were placed closer to the P. avium than the other species. The principal coordinate analysis (PCoA) supported the cluster analysis results. The wild accessions were separated according to their species and collection sites. ISSR markers are useful techniques for genetic diversity evaluation in Prunus subgenus Cerasus.     

Genetic diversity in european and Argentinian cultivated potatoes (Solanum tuberosum subsp. tuberosum) detected by inter-simple sequence repeats (ISSRs).

In this study, the use of inter-simple sequence repeats (ISSR) to assess genetic diversity between cultivated potatoes (Solanum tuberosum subsp. tuberosum) is reported. ISSR technology rapidly reveals high polymorphic fingerprints and thus determines the genetic diversity among potato cultivars. Nine primers were selected according to the number of amplified markers and the level of polymorphism detected. Three primers (GAG(CAA)5, CTG(AG)8, and (AG)8) were used to cluster the 28 potato accessions and 77 polymorphic markers were sufficient to identify all of the accessions. Among the 15 simple sequence repeat (SSR) motifs tested, the most abundant were CAA and AG. Argentinian- and European-grown potatoes were easily distinguished, with a higher level of genetic diversity among potatoes from Argentina. An ISSR study using a limited number of cultivars and very few primers clearly differentiated between all cultivars, thus ISSR was revealed to be a good tool for the genetic identification of potato and for future germplasm-management programs.     

甘薯种质资源遗传稳定性及遗传多样性SSR分析

应用SSR标记检测国家种质徐州甘薯试管苗库中离体保存5年和8年的24份种质资源及其对应的田间圃材料的遗传稳定性,同时对24份甘薯种质的遗传多样性进行分析.20对SSR引物分析表明,24份甘薯材料扩增得到了清晰的DNA条带30条,其中多态性条带2l条,多态性百分率为70%,全部品种在2种保存方式下谱带一致,说明2种保存方式的效果相同.应用NTSYS软件对材料进行遗传相似性和UPGMA聚类分析,24份甘薯种质资源遗传相似系数在0.57～0.93之间,平均为0.74.在0.72的相似系数上24份材料可以聚成三大类,表明我国的甘薯品种种质资源遗传多样性还是比较丰富的.该研究为甘薯种质资源长期离体保存及甘薯杂交育种提供了理论依据.