Exploring genetic diversity and Population structure of five Aegilops species with inter-primer binding site (iPBS) markers

Molecular Biology Reports - Turkey is not only a center of origin for wheat, but also contains wild forms of various cereals. Turkey, located in the Fertile Crescent, has conserved its genetic...     

Utility of iPBS retrotransposons markers for molecular characterization of African Gnetum species

Abstract

Species of African Gnetum are lianas used as vegetables, medicines and for generating income. Despite the taxonomic confusion, identification of new species and diverse morphological characters in African Gnetum, molecular markers on these plants are lacking. However, the inter-primer binding site (iPBS) retrotransposons markers could be simple and excellent molecular markers for African Gnetum. The objective of this study was to determine the efficiency of iPBS markers in detecting genetic differentiation in African Gnetum species. A set of 21 iPBS markers were analysed on 14 accessions including G. africanum Welw., G. buchholzianum Engl. and the recently identified species G. latispicum. Six best selected primers generated 103 bands in G. africanum, 95 in G. buchholzianum and 24 in G. latispicum. Cluster analysis divided the accessions into two major groups. The first group contained all the accessions of G. africanum, whereas the second group was further divided in two subgroups representing accessions of G. buchholzianum and G. latispicum. Additionally, the Jaccard similarity coefficient indicated a close relationship between accessions of G. buchholzianum and G. latispicum. The iPBS marker system revealed genetic differentiation within African Gnetum and could be useful for evaluating genetic diversity, conservation, taxonomy and evolution studies.     

Molecular characterization of eight Indian Snakehead species (Pisces: Perciformes Channidae) using RAPD markers

Murrels (Perciformes; Channidei; Channidae) are unique group of freshwater air breathing fishes having a confined distribution to African and Asian continents. The phylogenetic relationship among eight Channid species viz. Channa aurantimaculata, Channa bleheri, Channa diplogramma, Channa gachua, Channa marulius, Channa punctatus, Channa stewartii and Channa striatus were investigated using RAPD markers. Eight random oligodecamers viz. OPAC03, OPAC05, OPAC07, OPAC09, OPAC19, OPA10, OPA11 and OPA16 were used to generate the RAPD profile. Estimates of Nei’s (Genetics, 89:583–590, 1978) unbiased genetic distance (D) demonstrated sufficient genetic divergence to discriminate the samples of different species and the values ranged from 0.3292 to 0.800 The present RAPD analyses strongly substantiate the view of earlier morphological and osteological studies of Channid species, the closer association among species in “gachua” and “marulius” groups.     

Molecular diversity analysis of grape varieties based on iPBS markers

Thirty-five grape varieties were evaluated for their molecular diversities based on inter-primer binding site (iPBS) markers. Fifteen selected iPBS primers generated a total of 99 polymorphic DNA bands with 86.3% polymorphism. The effectiveness of iPBS marker is comparable to or even more efficient than other retrotransposon-based markers in grape. The differentiation between cultivated and wild grape varieties were clearly showed by both UPGMA cluster analysis and PCoA analysis revealing that Chinese cultivated and wild grape germplasm are highly divergent and possess abundant genetic diversities. This study also confirmed that the iPBS marker is a simple, informative, reproducible and suitable method for grape genetic diversity evaluation.     

Development and characterization of polymorphic microsatellite markers in Cyclobalanopsis glauca (Fagaceae)

? Premise of the study: We isolated and characterized polymorphic microsatellite loci in Cyclobalanopsis glauca (Fagaceae), an evergreen broadleaved monoecious tree, to provide tools for analyzing parentage and mating system. ? Methods and Results: Thirteen polymorphic microsatellite markers were developed and tested in three C. glauca populations. The number of alleles per locus varied from two to 22. The observed and expected heterozygosities within populations were 0.000-0.967 and 0.033-0.949, respectively. ? Conclusions: These polymorphic primers showed high levels of polymorphism within tested populations, and can be used in parentage analysis and mating system estimation of C. glauca.     

Molecular characterization of the microsomal tamoxifen binding site

Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification of the AEBS and give new insight into a novel molecular mechanism of action for drugs of clinical value.     

Molecular characterization of India Ginseng Withania somnifera (L) using ISSR markers

Molecular Biology Reports - Ashwagandha (Withania somnifera (L.) Dunal), popularly known as Indian ginseng or winter cherry is a multipurpose plant of immense therapeutic value in the ayurvedic and...     

Molecular characterisation of Trichoderma species using SRAP markers

Trichoderma are commonly used as bio control agents in various agro ecosystems. They are known to produce a variety of compounds that induce resistance responses in plants. Among different species of Trichoderma, T. harzianum, T. viride, T. koningii and T. hamatum are commercially used as bio control agents. In the present study, four commercially important species of Trichoderma isolated from coffee ecosystem were screened with sequence related amplified polymorphism (SRAP) markers. Among 48 SRAP primer pairs tested, 29 primers were polymorphic and generated 316 distinct scorable fragments. Out of 347 amplified fragments, 177 fragments were found polymorphic with an average of 6.10 fragments per primer combination. The average polymorphism information content (PIC) and resolving power (Rp) of the 29 polymorphic SRAP primer pair were 0.42 and 14.62, respectively. The UPGMA dendrogram clearly divided Trichoderma species into two broad clusters. The highest homology (83.0%) was observed between T. viride and T. Harzianum and the lowest homology (74.0%) was observed between T. Harzianum and T. konangii. Further, among 29 polymorphic SRAP markers screened, four primer pairs (ME1-EM3, ME1-EM20, ME1-EM22 and ME2-EM4) produced unique fragments specific to each species. These markers can be useful in easy and rapid identification of the species.     

Population structure and genetic diversity of Lithocarpus litseifolius (Fagaceae) assessed using microsatellite markers

Lithocarpus litseifolius (Fagaceae), known as tree of Chinese sweet tea, is a native commercial plant distributed in south China and adjacent areas. This study used ten microsatellite markers to investigate the genetic variation and population structure of 11 populations of L. litseifolius from the main production regions in China. All of the tested loci proved to be effective for the species. The number of alleles per locus ranged between 3 and 39, and mean observed and expected heterozygosity was 0.43 and 0.52, respectively. The deficiency in heterozygosity may be the result of human interference and harvesting activities. Analysis of molecular variance revealed that most of the variation was within populations, whereas differentiation among populations was insignificant, and that there was no significant correlation between genetic distance and geographic distance (r = 0.4089, p = 0.9940). However, A Bayesian model‐based analysis sorted all 11 populations into two (when k = 2) or three (when k = 3) clusters with higher probabilities and showed a geographical pattern which is probably related to the topography of China. Our results provide important information for protecting, sustainably using, and improving the current resources of L. litseifolius.     

Chromosome number of some South American species ofNothofagus (Fagaceae)

The observed chromosome numbers for four deciduous species of South AmericanNothofagus (Sect.Nothofagus) are 2n=26. This chromosome count is the first report on the South American species of the genus, and is the same number as reported for the New Zealand counterparts of the evergreen sectionCalusparassus. Furthermore, a significant difference between the karyotypes of two subsections within the Sect.Nothofagus has been recognized.     

Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers

Buffalograss [Buchloe dactyloides (Nutt.) Englem] germplasm has a broad resource of genetic diversity that can be used for turfgrass, forage and conservation. Buffalograss is the only native grass that is presently used as a turfgrass in the Great Plains region of North America. Its low growth habit, drought tolerance and reduced requirement for fertilizer and pesticides contribute to interest in its use. The objectives of this study were to use sequence-related amplified polymorphism (SRAP) markers in the evaluation of genetic diversity and phenetic relationships in a diverse collection of 53 buffalograss germplasms, and to identify buffalograss ploidy levels using flow cytometry. Based on their DNA contents, buffalograss genotypes were grouped into four sets, corresponding to their ploidy levels. Thirty-four SRAP primer combinations were used. This is the first report of the detection of differentiating diploid, tetraploid, pentaploid and hexaploid buffalograss genotypes, representing diverse locations of origin, using SRAP markers. Cluster analysis by the unweighted pair-group method with arithmetic averages based on genetic similarity matrices indicated that there were eight clusters. The coefficients of genetic distance among the genotypes ranged from 0.33 up to 0.99 and averaged D=0.66. The genetic diversity estimate, He, averaged 0.35. These results demonstrated that genotypes with potential traits for turfgrass improvement could readily be distinguished, based on SRAP. The use of PCR-based technologies such as SRAP is an effective tool for estimating genetic diversity, identifying unique genotypes as new sources of alleles for enhancing turf characteristics, and for analyzing the evolutionary and historical development of cultivars at the genomic level in a buffalograss breeding program.Communicated by B. Friebe     

Molecular characterization of an international cacao collection using microsatellite markers

Plant germplasm collections invariably contain varying levels of genetic redundancy, which hinders the efficient conservation and utilization of plant germplasm. Reduction of genetic redundancies is an essential step to improve the accuracy and efficiency of genebank management. The present study targeted the assessment of genetic redundancy and genetic structure in an international cacao (Theobroma cacao L.) collection maintained in Costa Rica. A total of 688 cacao accessions maintained in this collection were genotyped with 15 simple sequence repeat (SSR) loci, using a capillary electrophoresis genotyping system. The SSR markers provided a high resolution among the accessions. Thirty-six synonymously labeled sets, involving 135 accessions were identified based on the matching of multilocus SSR profiles. After the elimination of synonymous sets, the level of redundancy caused by closely related accessions in the collection was assessed using a simulated sampling scheme that compared allelic diversity in different sample sizes. The result of the simulation suggested that a random sample of 113 accessions could capture 90% of the total allelic diversity in this collection. Principal Coordinate Analysis revealed that the Trinitario hybrids from Costa Rica shared a high similarity among groups as well as among individual accessions. The analysis of the genetic structure illustrated that the within-country/within-region difference accounted for 84.6% of the total molecular variation whereas the among-country/among-region difference accounted for 15.4%. The Brazilian germplasm contributed most to this collection in terms of total alleles and private alleles. The intercountry/interregion relationship by cluster analysis largely agreed with the geographical origin of each germplasm group and supported the hypothesis that the Upper Amazon region is the center of diversity for cacao. The results of the present study indicated that the CATIE International Cacao Collection contains a high level of genetic redundancy. It should be possible to rationalize this collection by reducing redundancy and ensuring optimal representation of the genetic diversity from distinct germplasm groups. The results also demonstrated that SSR markers, together with the statistical tools for individual identification and redundancy assessment, are technically practical and sufficiently informative to assist the management of a tropical plant germplasm collection.     

Molecular characterization of UV-treated sugar beet somaclones using RFLP markers

Sugar beet plants regenerated from UV-treated calluses were examined by restriction fragment length polymorphism (RFLP) analysis to determine the extent of somaclonal variation occurring at the DNA level. In total, 50 random sugar beet DNA sequences were used to screen 42 somaclones for genetic alterations. Three polymorphisms were detected among the 7 644 alleles analysed. From these data a mutation frequency of 0.03 ± 0.02% per allele was estimated. This frequency is in agreement with similar studies of somaclonal DNA variation using molecular markers and lies in the upper range of the spontaneous gene mutation frequencies found in plants. The two probegenotype combinations showing independent polymorphisms, were further analysed using the restriction enzymes Bam HI, Eco RI, Eco RV and Hind III. Both polymorphisms are likely to result from structural rearrangements rather than from point mutations. Differences in methylation among 10 of the investigated somaclones were tested for by comparing Hpa II and Msp I generated RFLP patterns. The somaclones showed extensive methylation, but no differences in their degree of methylation. Cytological analysis revealed 34 diploid, 8 tetraploid, but no aneuploid plants.     

Microsatellite markers for northern red oak (Fagaceae: Quercus rubra)

We provide primer sequences for 14 (GA) _n microsatellite loci developed from northern red oak, an important timber species. We screened loci using two sets of samples. A parent–offspring set included DNA from seven acorns collected from one mother tree along with maternal DNA, to determine that all progeny carried a maternal allele at each locus. The other set was comprised of 10 adult trees sampled from Indiana old‐growth forest, providing a measure of diversity revealed by each locus.     

Molecular characterization of Lathyrus species using chloroplast DNA trnH-psbA

Lathyrus L. is an important genus contributing in human food, animal feed and fodder. The genetic variation is studied among and within six species sampled over a large geographical area: Lathyrus cicera, Lathyrus sativus, Lathyrus sylvestris, Lathyrus tuberosus, Lathyrus ochrus and Lathyrus aphaca. The phylogenetic relationship among these species was assessed using sequences of chloroplast DNA trnH-psbA (intergenic spacer). The highly polymorphic spacer' length was 330 bp. The phylogenetic analyses using Maximum Parsimony and Genetic Distances, agreed with the universal taxonomy of Kupicha. L. sativus and L. cicera could be considered as sister species, sharing a common ancestor.     

Molecular characterization of olive (Olea europaea L.) Sicilian cultivars using SSR markers

Olive (Olea europaea L.) is one of the economically most important fruit crops for the Mediterranean area, with production being mainly destined to oil extraction. In Sicily, olive has been cultivated since ancient times and its germplasm is characterized by a wide genetic diversity that could be related to its domestication and spread in ancient times, and to some reproductive biological peculiarities as self-incompatibility. This analysis was conducted on 65 genotypes with the purpose of characterizing a large collection of Sicilian accessions (47 genotypes) and to compare them with varieties coming from Southern Italy and from the most important countries of the Mediterranean basin. With this aim we used 8 simple sequence repeat (SSR) markers, which detected a total of 74 alleles and identified an average of 19.5 genotypes in the population investigated. A larger variability than expected was found in the analyzed genotypes, some synonymies already reported in literature were confirmed, but also some cultivars considered as identical were discriminated such as in the case of Castriciana, Ogliarola messinese and Passalunara. The whole study revealed a wide intraspecific variability within the gene pool examined, independently from the geographical origin.     

Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers

Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the “Bayoud” disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S- (Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars (“Medjool”, “Ashrasi”, “Gish Rabi”, “Khineze”, and yellow- and red-“Kabkab”) have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.     

Molecular characterization of Syrian date palm cultivars using plasmid-like DNA markers

Date palm (Phoenix dactylifera L.) is one of the most important domesticated fruit trees in the Near East and North African countries. This tree has been, for several decades, in serious threat of being completely destroyed by the "Bayoud" disease caused by Fusarium oxysporum f. sp. albedinis. In this study, 18 Syrian date palm cultivars and four male trees were analyzed according to the identity of mitochondrial plasmid-like DNAs. A PCR strategy that employs plasmid-like DNAs-specific primer pair was used. These primers amplify a product of either 373-bp or 265-bp that corresponds to the S-(Bayoud-susceptible) or the R-plasmid (Bayoud-resistant), respectively. Generated data revealed that only six cultivars ('Medjool', 'Ashrasi', 'Gish Rabi', 'Khineze', and yellow- and red-'Kabkab') have the S-plasmid, suggesting their susceptibility to the fusariosis, while the remaining 12 cultivars and the four male trees contain the R-plasmid, suggesting their resistance to the fusariosis. The PCR process applied here has been proved efficient for the rapid screening for the presence of the S and R DNAs in Syrian date palm. PCR markers developed in this study could be useful for the screening of date palm lines growing in the field. The availability of such diagnostic tool for plasmid characterization in date palm would also be of great importance in establishing propagation and breeding programs of date palm in Syria.