Assessment of genetic diversity and population structure of an endemic Moroccan tree (<Emphasis Type="Italic">Argania spinosa</Emphasis> L.) based in IRAP and ISSR markers and implications for conservation

Argan Tree is well known for its precious oil extracted from its seeds particularly used for the nutritional and cosmetic benefits. Because of the high international demand, the argan tree suffers from overexploitation and its cultivation is rare. Thus, the assessment of the genetic variation of this endemic tree is critically important for designing conservation strategies. In the present study and for the first time, genetic diversity of the global natural distribution of argan tree (Argania spinosa L.) in Morocco was assessed. Four IRAP (inter-retrotransposon amplified polymorphism) primer combinations and seven ISSR (inter-simple sequence repeat) primers amplified 164 and 248 scorable polymorphic bands respectively. Polymorphic information content (PIC = 0.27), resolving power (Rp = 15) and marker index (MI = 10.81) generated by IRAP primer combinations were almost identical to those generated by ISSR primers (PIC = 0.27, Rp = 9.16 and MI = 12). AMOVA analysis showed that 49% of the genetic variation was partitioned within populations which is supported by Nei’s genetic differentiation (Gst = 0.5391) and the overall estimate of gene flow (Nm) being 0.4274. The STRUCTURE analysis, PCoA (principal coordinate analysis) and UPGMA (unweighted pair-group method with arithmetic mean) based on the combined data matrices of IRAP and ISSR divided the 240 argan genotypes into two groups. The strong differentiation observed might be due to the geographical distribution of argan tree. Our results provide crucial insight for genetic conservation programs of this genetic resource.     

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.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Population genetics of wild Hizikia fusiformis (Sargassaceae, Phaeophyta) along China’s coast

Hizikia fusiformis is one of the important commercially cultivated seaweeds in China. Inter-simple sequence repeats (ISSR) and sequence-related amplified polymorphism (SRAP) markers were used to assess genetic structure of nine wild H. fusiformis populations collected along the coast of China. Of the 255 bands generated by 21 ISSR primers, 99.61% were polymorphic and 99.71% of 344 bands amplified by 30 SRAP primers were polymorphic. The tested high genetic diversities show that the average Nei’s genetic diversity (H) were 0.1519 and 0.1624, and average Shannon’s information index (I) were 0.2248 and 0.2400 in ISSR and SRAP analyses, respectively. Unweighted pair group method with arithmetic mean (UPGMA) dendrograms of the nine populations were divided into two main groups. The ISSR and SRAP analyses values of gene differentiation (G _ST, 0.5955, 0.5486, respectively) indicate that high variation exists among the nine populations, likely due to external interferences and limitation of gene flow (N _m?=?0.3397, 0.4114). Our study indicated that human activities and herbivore overgrazing had influenced the natural Hizikia populations and that the understanding of population genetics would be helpful in sustainable utilization and biomass conservation of Sargassaceae resources.     

Evaluation of genetic diversity in Lentinula edodes strains using RAPD, ISSR and SRAP markers

Random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers were used to evaluate the genetic diversity among 23 elite Lentinula edodes strains in China. A total of 138, 77 and 144 bands were detected by 16 RAPD primers, 5 ISSR primers and 23 SRAP primer combinations, among which 58.8%, 73.5% and 56.3% was polymorphic, respectively. By UPGMA clustering, a dendrogram was constructed based on each analysis. The three dendrograms showed that 23 L. edodes strains were clustered into three or four groups. The grouping exhibited similar structure and was generally consistent with their pedigrees. Twenty-three L. edodes strains shared great similarity indicated that the low level of genetic diversity of L. edodes strains and their relationship between each other. The important source of breeding material, such as wild and exotic types, must be introduced in order to broaden genetic base and decreases genetic vulnerability of L. edodes.     

Molecular Characterization of <Emphasis Type="Italic">Cyclamen</Emphasis> Species Collected from Different Parts of Turkey by RAPD and SRAP Markers

The genus Cyclamen (family Myrsinaceae) contains about 20 species, most of which occur in the Mediterranean region. Turkey has critically important Cyclamen genetic resources. Molecular characterization of plant materials collected from different regions of Turkey in which Cyclamen species grow naturally, namely Adana, Antalya, Ayd?n, Mu?la, ?zmir, Denizli, Kahramanmara?, Osmaniye, Eski?ehir, Trabzon, and Rize provinces, was performed using RAPD and SRAP markers. DNA was successfully amplified by 30 RAPD primers and 14 SRAP primer pairs. Among the 470 bands generated by the RAPD primers, 467 were polymorphic. The number of bands detected by a single primer set ranged from 11 to 22 (average of 15.6). The percentage polymorphism was 99.3 % based on the RAPD data. In the SRAP analysis, a total of 216 bands were generated, showing 100 % polymorphism. The number of bands detected by a single primer set ranged from 9 to 22 (average of 15.4). All data were scored and UPGMA dendrograms were constructed with similar results in both marker systems, i.e., different species from nine provinces of Turkey were separated from each other in the dendrograms with the same species being clustered together.     

Genetic Diversity of Pleurotus pulmonarius Revealed by RAPD,ISSR, and SRAP Fingerprinting

Pleurotus pulmonarius is one of the most widely cultivated and popular edible fungi in the genus Pleurotus. Three molecular markers were used to analyze the genetic diversity of 15 Chinese P. pulmonarius cultivars. In total, 21 random amplified polymorphic DNA (RAPD), 20 inter-simple sequence repeat (ISSR), and 20 sequence-related amplified polymorphism (SRAP) primers or primer pairs were selected for generating data based on their clear banding profiles produced. With the use of these RAPD, ISSR, and SRAP primers or primer pairs, a total of 361 RAPD, 283 ISSR, and 131 SRAP fragments were detected, of which 287 (79.5 %) RAPD, 211 (74.6 %) ISSR, and 98 (74.8 %) SRAP fragments were polymorphic. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of these three methods were structured similarly, grouping the 15 tested strains into four clades. Subsequently, visual DNA fingerprinting and cluster analysis were performed to evaluate the resolving power of the combined RAPD, ISSR, and SRAP markers in the differentiation among these strains. The results of this study demonstrated that each method above could efficiently differentiate P. pulmonarius cultivars and could thus be considered an efficient tool for surveying genetic diversity of P. pulmonarius.     

Genetic diversity in Vicia amoena (Fabaceae) germplasm resource in China using SRAP and ISSR markers

The genetic diversity in 17 germplasms of Vicia amoena L. from north China was analyzed using SRAP and ISSR markers. Three hundred and sixty-eight (94.11%) polymorphic bands (211 and 157 obtained from 20 pairs of SRAP and ISSR primers, respectively) were scored. Although SRAP was more effective than ISSR markers with higher PIC, RP and larger variation range of genetic distance, both the markers were useful for assessing V. amoena genetic diversity. Cluster analysis showed that the 17 germplasms were clustered into 5 groups. The results of principal coordinate analysis supported UPGMA clustering. The germplasms from source areas where the annual average temperature ranged from −1.0 to 5 °C exhibited the highest level of genetic diversity with the highest PPI, I and H. These results have important implications in genome mapping, breeding purposes, and germplasm conservation.     

A comparative analysis of genetic diversity in medicinal Chrysanthemum morifolium based on morphology, ISSR and SRAP markers

The diversity and genetic relationship among 29 populations of Chrysanthemum morifolium, one of Chrysanthemum indicum and one of Chrysanthemum nankingense from China were analyzed using morphological traits and molecular markers. Twenty morphological traits were scored as well as 182 ISSR marker-fragments, as amplified by 22 primers [the percentage of polymorphic bands (PPB): 81.87%], and 243 SRAP marker-fragments as generated by 26 primer pairs (PPB: 75.72%). Mantel’s test indicated significant correlation (r = 0.624) of morphological trait and SRAP. By contrast, the morphological trait showed low correlation with ISSR (r = 0.246). Cluster analysis showed groupings of the accessions according to all four methods correlated well with their geographic region of origin, and most populations from the south of China were classified into one cluster and most populations from the north of China were classified into another cluster. Finally, an appropriate strategy for conserving the C. morifolium germplasm was proposed.     

Identification and genetic relationships among nine Albizzia species based on morphological and molecular markers

Abstract

Identifying germplasm is an important component for efficient and effective management of plant genetic resources. This investigation was undertaken for the identification and analysis of genetic variation within 9 species of Albizzia through 33 morphological parameters, and 15 Random Amplified Polymorphic DNA (RAPD) and 17 Inter Simple Sequence Repeat (ISSR) primers. The use of selected RAPD and ISSR primers generated a total of 163 and 201 amplified DNA fragments, respectively. High frequencies of polymorphism, 95.05% for RAPD and 96.02% for ISSR, were detected. Statistical approaches were employed to construct genetic relationships by RAPD, ISSR and morphological analysis. Cluster analysis by the unweighted pair-group method (UPGMA) of Nei's similarity generated dendograms with similar topology that gave a better reflection of the diversity and affinities between species. These molecular results were comparable to main morphological characteristics. The correlation matrices generated by RAPD and ISSR markers were highly correlated (r = 0.843 at p = 1.0), thereby indicating congruence between these two marker systems. Both morphometric data and molecular markers have the potential to analyse genetic variation among the nine species of Albizzia, thus providing a major input for management strategy of plant genetic resources.     

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.     

Comprehensive genetic discrimination of Leonurus cardiaca populations by AFLP,ISSR, RAPD and IRAP molecular markers

Leonurus cardiaca is well known for its medicinal importance. In this investigation, genotypic characterization of this species from six eco-geographical regions of Iran was evaluated by four molecular techniques (AFLP, RAPD, ISSR and IRAP). A total of 899 polymorphic fragments were detected by used molecular markers (AFLP = 356, RAPD = 325, ISSR = 113 and IRAP = 105) with an overall average polymorphism of 81.24 %. Genetic variation calculated using Shannon’s Information index (I) and Nei’s gene diversity index (H) showed high genetic diversity in studied germplasm. Also, analysis of molecular variance showed high genetic variation among (55 %) and within populations (45 %). UPGMA dendrogram constructed from combined data of molecular markers distinguished studied populations in accordance with the results obtained by each marker which all individuals were clearly differentiated into two major clusters. The correlation coefficients were statistically significant for all marker systems with the highest correlation between similarity matrixes of RAPD and ISSR markers (r = 0.82). The present results have an important implication for L. cardiaca germplasm characterization, improvement, and conservation. Furthermore, the characterized individuals exhibited a great deal of molecular variation and they seem to have a rich gene pool for breeding programs.     

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.     

Evaluation of genetic diversity amongst <Emphasis Type="Italic">Descurainia sophia</Emphasis> L. genotypes by inter-simple sequence repeat (ISSR) marker

Descurainia sophia is a valuable medicinal plant in family of Brassicaceae. To determine the range of diversity amongst D. sophia in Iran, 32 naturally distributed plants belonging to six natural populations of the Iranian plateau were investigated by inter-simple sequence repeat (ISSR) markers. The average percentage of polymorphism produced by 12 ISSR primers was 86 %. The PIC values for primers ranged from 0.22 to 0.40 and Rp values ranged between 6.5 and 19.9. The relative genetic diversity of the populations was not high (Gst =0.32). However, the value of gene flow revealed by the ISSR marker was high (Nm = 1.03). UPGMA clustering method based on Jaccard similarity coefficient grouped the genotypes into two major clusters. Graph results from Neighbor-Net Network generated after a 1000 bootstrap test using Jaccard coefficient, and STRUCTURE analysis confirmed the UPGMA clustering. The first three PCAs represented 57.31 % of the total variation. The high levels of genetic diversity were observed within populations, which is useful in breeding and conservation programs. ISSR is found to be an eligible marker to study genetic diversity of D. sophia.     

Genetic diversity assessment of a germplasm collection of Salvia miltiorrhiza Bunge. based on morphology,ISSR and SRAP markers

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.     

Characterization of <Emphasis Type="Italic">Gladiolus</Emphasis> Germplasm Using Morphological,Physiological, and Molecular Markers

Estimation of variability and genetic relationships among breeding materials is one of the important strategies in crop improvement programs. Morphological (plant height, spike length, a number of florets/spike), physiological (chlorophyll content, chlorophyll fluorescence, and rapid light curve parameters) and Directed amplification of minisatellite DNA (DAMD) markers were used to investigate the relationships among 50 Gladiolus cultivars. Cluster analysis based on morphological data, physiological characteristics, molecular markers, and cumulative data discriminated all cultivars into seven, five, seven, and six clusters in the unweighted pair-group method using arithmetic mean (UPGMA) dendrogram, respectively. The results of the principal coordinate analysis (PCoA) also supported UPGMA clustering. Variations among the Gladiolus cultivars at phenotypic level could be due to the changes in physiology, environmental conditions, and genetic variability. DAMD analysis using 10 primers produced 120 polymorphic bands with 80% polymorphism showing polymorphic information content (PIC = 0.28), Marker index (MI = 3.37), Nei’s gene diversity (h = 0.267), and Shannon’s information index (I = 0.407). Plant height showed a positive significant correlation with Spike length and Number of florets/spike (r = 0.729, p < 0.001 and r = 0.448, p = 0.001 respectively). Whereas, Spike length showed positive significant correlation with Number of florets/spike (r = 0.688, p < 0.001) and Chlorophyll content showed positive significant correlation with Electron transport rate (r = 0.863, p < 0.001). Based on significant morphological variations, high physiological performance, high genetic variability, and genetic distances between cultivars, we have been able to identify diverse cultivars of Gladiolus that could be the potential source as breeding material for further genetic improvement in this ornamental crop.     

Genetic diversity and population structure of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> Bge in China revealed by ISSR and SRAP

Salvia miltiorrhiza Bge is a traditional Chinese medicinal herb used as an important drug to cure cardiovascular diseases. In this work, inter simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP) markers, were applied to assess the level and pattern of genetic diversity in five important cultivated populations of S. miltiorrhiza. Among these populations, 120 bands were amplified by 5 ISSR primers, of which all were polymorphic, and 110 polymorphic bands (90.16%) were observed in 122 bands amplified by 6 SRAP primers. A high levels of genetic diversity at the species level was detected with Hs = 0.1951, 0.1927 respectively. Analysis of molecular variance revealed that a greater proportion of total genetic variation existed within populations (86.64 and 84.83% respectively) rather than among populations (13.36 and 15.17% respectively). Cluster analysis divided the five populations into two groups. The genetic relationships among populations have low correlation with their geographical distribution (Mantel test; r = 0.4870 and 0.5740 respectively). The study indicated that both ISSR and SRAP markers were effective and reliable for assessing the degree of genetic variation of S. miltiorrhiza. Our results suggested that random collecting, preserving and planting seeds without deliberate selection might be an efficient way to conserve genetic resources of medicinal plants. Their effective use was also discussed on the further breeding.     

中国扁蓿豆遗传多样性的ISSR分析

利用ISSR标记,对来源于中国7个省(市)的44个扁蓿豆居群的亲缘关系进行了分析。从80条ISSR引物中筛选出多态性强、重复性好的16条引物,对44个扁蓿豆居群基因组DNA进行扩增,共扩增出133条谱带,平均每个引物扩增出8.31条带,其中多态性带115条,多态性位点百分率为87.08%。扁蓿豆种质间遗传相似系数变化范围在0.436～0.908之间。利用UPGMA聚类分析,44个扁蓿豆居群被划分为8个类群,与地理来源有较高的相关性。     

Genetic diversity and structure of <Emphasis Type="Italic">Capparis spinosa</Emphasis> L. in Iran as revealed by ISSR markers

Capparis spinosa L. (caper bush) is an economically and ecologically important perennial shrub that grows across different regions of Iran. In this study, the genetic diversity and population structure of Iranian genepool of C. spinosa is evaluated using Inter Simple Sequence Repeat (ISSR) markers. Using 10 ISSR primers, 387 DNA fragments (bands) were amplified from the genomic DNA of 92 individuals belonging to twenty-one populations of C. spinosa, of which 378 (97.7%) were polymorphic. High level of genetic diversity (percentage of polymorphic loci = 98.2%, h = 0.1382, I = 0.243), high genetic differentiation (G_st = 0.5234) and low gene flow (Nm = 0.4553) among populations were observed. Caper bush populations were divided into 4 groups in the dendrogram, PCoA plot and Bayesian clustering results, mostly corresponded to their geographic regions. The results showed that there are value in sampling Iranian caper bush populations to look for valuable alleles for use in plant breeding programs.     

长筒石蒜种质资源的RAPD及ISSR研究

借助RAPD及ISSR分子标记对长筒石蒜的种质资源进行了初步的研究,结果表明：RAPD扩增得到的77个位点中,53个位点具有多态性,约占总数的68．8％;ISSR扩增得到的67个位点中,其中62个位点具有多态性,约占总数的92．5％。因此,长筒石蒜遗传多样性是十分丰富的,可以作为育种材料储备种质资源。从UPGMA聚类图来看,依据花色区分的3种类型,被聚在一起,表明亲缘关系较近。因此,在未来的长筒石蒜种质资源利用中,不同花色的长筒石蒜可以作为品系进行开发。