Genomic variability among high pristinamycin-producing recombinants of Streptomyces pristinaespiralis revealed by amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP) was used to analyze genomic variability between high pristinamycin-producing recombinants of Streptomyces pristinaespiralis produced by genome shuffling and their ancestral strain. The AFLP fingerprints obtained with two restriction enzyme combinations of ApaI/TaqI and PstI/SacII showed together that there was no major polymorphism (less than 10%) between these high yield recombinants and their ancestor. However, the unique polymorphic bands, which might be related to the yield increasing of pristinamycin, could be distinguished from all the recombinants. Clustering analysis further indicated that the recombinants with similar ability of pristinamycin production had similar genomic variability.     

Phylogenetic relationships of turfgrasses as revealed by restriction fragment analysis of chloroplast DNA

Chloroplast DNAs (cpDNAs) were analyzed in order to clarify the phylogenetic relationships among turfgrasses. Physical maps of cpDNAs from Agrostis stolonifera and Zoysia japonica, which are representative species of cool (C3 type) and warm (C4 type) season turfgrasses, respectively, were constructed with four restriction enzymes, i.e., PstI, SalI, SacI, and XhoI. The genome structures of these cpDNAs were found to be similar to each other in terms of genome size and gene orders, showing thereby a similarity to other grass cpDNAs. CpDNAs of 5 species of cool season turfgrasses and 6 species of warm season turfgrasses as well as four species of cereals, distributed among 14 genera of Gramineae, were digested with PstI, XhoI, and BamHI, and their restriction fragment patterns were compared. Their genome sizes were estimated to be 135–140 kbp. Each species showed characteristic RFLP patterns. On the basis of the frequency of commonly shared fragments, a dendrogram showing the phylogenetic relationships among their cpDNAs was constructed. This dendrogram shows that turfgrasses can be divided into three major groups; these correspond to the subfamilies. Cool and warm season turfgrasses are clearly distinguishable from each other, and the latter can be further classified into two subgroups that correspond to Eragrostoideae and Panicoideae. Our classification of turfgrasses and cereals by RFLP analysis of cpDNA agreed in principal with their conventional taxonomy, except for the location of Festuca and Lolium.Contribution no. 101 from the Kihara Institute for Biological Research, Yokohama City University, Yokohama 232, Japan     

Phenetic relationships and levels of variability detected by restriction fragment length polymorphism and random amplified polymorphic DNA analysis of cultivated and wild accessions of Lycopersicon esculentum.

Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to assess the variability in tomato germplasm (Lycopersicon esculentum Mill.), which included 46 accessions from the following groups: vintage cultivars, modern cultivars, South American regional cultivars, and wild L. esculentum van cerasiforme. Two L. cheesmanii accessions served as an outgroup. The 48 accessions were monomorphic at 135 of the 215 RAPDs loci and 31 of the 48 RFLP loci that were assayed. Alleles were identified that distinguished the five groups and many of the cultivars. The frequency of polymorphic loci was low in vintage cultivars, with less than 2.8% of the loci being variable within the group. In contrast, introgression of wild germplasm into modern cultivars has increased the polymorphic loci to 11.6%, whereas within the group of regional cultivars linkage drag and outcrossing may be responsible for the further increase to 20.3%. These levels of genetic variability were lower in comparison to the 24.5% polymorphic loci of cerasiforme, a group that may contain unutilized desirable traits. The small genetic distance from the vintage cultivars to several of the widely distributed regionals and cerasiformes indicated that proximity of vintage cultivars in Latin America may have resulted in intercrossing of these materials with the wilder germplasm. RAPDs appear promising for both germplasm fingerprinting and as a predictor of genetic diversity for plant breeding applications.     

Genetic relationship in ecotypes of Leymus chinensis revealed by polymorphism of amplified DNA fragment lengths

Leymus chinensis (Trin.) Tzvel is a perennial grass in the tribe Gramineae and important forage in Northern China. Knowledge of its genetic diversity is a prerequisite for using modern breeding techniques. Amplified fragment length polymorphism (AFLP) was first used to evaluate the genetic relationship among and within three ecotypes. Distinct clusters were produced based on AFLP markers. All accessions from the same ecotype were grouped in a cluster except accessions 6. According to AFLP profile ecotype-specific bands differ from each other. The genetic differentiation within the ecotype of the species was much smaller than that among ecotypes. Self-incompatibility in this species contributes to evident genetic differentiation together with environment. These results indicate that ecotypes were distinguished visually similarly to genetic variation. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 5, pp. 764–770. The text was submitted by the authors in English.     

Genetic diversity among elite Sorghum lines revealed by restriction fragment length polymorphisms and random amplified polymorphic DNAs

The genetic diversity of sorghum, as compared to corn, is less well characterized at the genetic and molecular levels despite its worldwide economic importance. The objectives of this study were to: (1) investigate genetic diversity for restriction fragment length polymorphism (RFLPs) and random amplified polymorphic DNAs (RAPDs) in elite sorghum lines, (2) compare similarities based on molecular markers with pedigree relationships, and (3) examine the potential of RFLPs and RAPDs for assigning sorghum lines to the A/B (sterile) and R (restorer) groups. Using four restriction enzymes, polymorphism was detected with 61% of the RFLP probes used, compared to 77% of the random primers. One hundred and sixteen (64%) probe-enzyme combinations yielded multiple-band profiles compared to 98% of the random primers. RFLP profiles generated 290 polymorphic bands compared to 177 polymorphic RAPDs. Pair-wise comparisons of polymorphic RFLPs and RAPDs were used to calculate Nei and Jaccard coefficients. These were employed to generate phenograms using UPGMA and neighborjoining clustering methods. Analysis of RFLP data with Jaccard's coefficient and neighbor-joining clustering produced the phenogram with the closest topology to the known pedigree.Contribution of the College of Agricultural Sciences, Texas Tech University, Journal No. T-4-365     

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Summary Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.Contributions no. 45 and no. 490 from the Kihara Institute for Biological Research, Yokohama City University and the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, respectively.     

Phylogenetic relationships among Secale species revealed by amplified fragment length polymorphisms.

Amplified fragment length polymorphism (AFLP) data were utilized to analyze the phylogenetic relationships among 29 accessions representing 14 of the most commonly recognized ranked species or subspecies in the genus Secale. We observed 789 AFLP markers of 1130 fragments utilizing 18 P-/M- and E-/M- primer combinations. All polymorphic fragments were used to construct phenetic and phylogenetic trees. The resulting phenogram and cladogram had similar tree topologies. Cluster analysis showed that Secale sylvestre was the most distantly related to all other ryes. Annual forms were grouped together, and the perennial forms appeared more closely related to each other. This suggested that life cycle could have played an important role in determining the relationships among Secale species. Secale sylvestre was considered to be the most ancient species, whereas Secale cereale was the most recently evolved species. Amplified fragment length polymorphism analysis clearly separated all Secale species into only 3 major species groups, within the genus Secale: S. sylvestre, Secale montanum (syn. Secale strictum) for perennial forms, and S. cereale for annual forms. This study demonstrated that the AFLP approach is a useful tool for discriminating species differences, and also gave a much better resolution in discerning genetic relationships among Secale species as compared with previous studies using other approaches.     

DNA variation in the wild plant Arabidopsis thaliana revealed by amplified fragment length polymorphism analysis.

To investigate the level and pattern of DNA variation of Arabidopsis thaliana at the entire genome level, AFLP analysis was conducted for 38 ecotypes distributed throughout the world. Ten pairs of selective primers were used to detect a total of 472 bands, of which 374 (79. 2%) were polymorphic. The frequency distribution of polymorphic bands was skewed toward an excess of singleton variation. On the basis of AFLP variation, nucleotide diversity for the entire genome was estimated to be 0.0106, which was within the range reported previously for specific nuclear genes. The frequency distribution of pairwise distance was bimodal because of an ecotype (Fl-3) with a large number of unique bands. Linkage disequilibrium between polymorphic AFLPs was tested. The proportion of significant linkage disequilibria was close to random expectation after neglecting the ecotype Fl-3. This result indicates that the effect of recombination could not be ignored in this selfing species. A neighbor-joining tree was constructed on the basis of the AFLP variation. This tree has a star-like topology and shows no clear association between ecotype and geographic origin, suggesting a recent spread of this plant species and limited migration between its habitats.     

Genetic variability in the species Rhizopus stolonifer, assessed by random amplified polymorphic DNA analysis

Rhizopus stolonifer is an important post-harvest pathogenic fungus. Recent taxonomic findings based on morphological and growth characteristics led to a dramatic reduction in the number of accepted species within the genus. The aim of this study was to examine this situation with molecular markers. Twenty-nine R. stolonifer strains isolated from various locations and substrates were characterized by random amplified polymorphic DNA (RAPD) analysis. The numerical analysis of the RAPD data revealed four main clusters with extremely high dissimilarity values, but only low or moderate variability was observed within these groups. These results suggest a high genetic heterogeneity in the case of R. stolonifer: isolates of R. stolonifer var. stolonifer, R. stolonifer var. reflexus and R. niveus displayed species-level genetic distances, which gives rise to considerations that they might be separate species.     

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.     

Genetic variability in species of bats revealed by RAPD analysis

Random amplified polymorphic DNA molecular marker was utilized as a means of analyzing genetic variability in seven bat species: Molossus molossus, M. rufus, Eumops glaucinus, E. perotis, Myotis nigricans, Eptesicus furinalis, and Artibeus planirostris. The determination of genetic diversity was based on 741 bands produced by a 20-random primer set. Only eight bands were considered monomorphic to one species. The greatest number of bands and the most polymorphic condition were exhibited by M. molossus, followed by M. nigricans, A. planirostris, E. furinalis, E. glaucinus, M. rufus, and E. perotis. Nei's genetic diversity index in the seven species considering the 20 primers was not greater than 0.22, but some primers were capable of detecting values between 0.39 and 0.49. Nei's unbiased genetic distance values and the UPGMA clustering pattern show that M. molossus and M. rufus have a close genetic relationship, unlike that observed between E. perotis and E. glaucinus. The latter was clustered with A. planirostris and E. furinalis. The low values for genetic diversity and distance observed indicate a genetic conservatism in the seven species. The fluorescent in situ hybridization experiments did not confirm a monomorphic condition for the eight bands identified, demonstrating that the monomorphic bands obtained by random amplified polymorphic DNA are insufficient for the identification of bat species.     

Phylogenetic relationships in the stone fruit group of Prunus as revealed by restriction fragment analysis of chloroplast DNA.

In order to clarify the genetic relationships among stone fruits, a restriction fragment analysis of chloroplast DNAs (cpDNAs) was applied to cultivated Prunus species, whose genetic diagnoses are difficult because of their heterogeneity and long life span. Chloroplast DNAs (cpDNAs) were extracted from leaves of nine stone fruit accessions covering six species of Prunus. A restriction fragment analysis was conducted by gel electrophoresis after digestion with these endonucleases. The genome sizes of the cpDNAs were about 135-139 kbp, and the fruits were classified into seven chloroplast genome types according to their restriction fragment patterns. Two peach cultivars and nectarine were found to harbor identical plastomes, differing from those of two wild peaches and the European plum. This suggests that two cultivated peaches (P. persica) did not receive the cytoplasm from the wild peaches, P. mira and P. davidiana. Phylogenetic relationships among these types were then estimated based on the shared common fragments among the species.     

Genetic diversity of Echinacea species based upon amplified fragment length polymorphism markers.

The taxonomy of Echinacea is based on morphological characters and has varied depending on the monographer. The genus consists of either nine species and four varieties or four species and eight varieties. We have used amplified fragment length polymorphisms (AFLP) to assess genetic diversity and phenetic relationships among nine species and three varieties of Echinacea (sensu McGregor). A total of 1086 fragments, of which approximately 90% were polymorphic among Echinacea taxa, were generated from six primer combinations. Nei and Li's genetic distance coefficient and the neighbor-joining algorithm were employed to construct a phenetic tree. Genetic distance results indicate that all Echinacea species are closely related, and the average pairwise distance between populations was approximately three times the intrapopulation distances. The topology of the neighbor-joining tree strongly supports two major clades, one containing Echinacea purpurea, Echinacea sanguinea, and Echinacea simulata and the other containing the remainder of the Echinacea taxa (sensu McGregor). The species composition within the clades differs between our AFLP data and the morphometric treatment offered by Binns and colleagues. We also discuss the suitability of AFLP in determining phylogenetic relationships.     

Genetic analysis of nuclear DNA restriction fragment patterns.

Restriction fragment length polymorphism (RFLP) analysis in the broad sense is the analysis of differences in restriction fragment pattern produced by defined target segments within or between cell compartments, cell types, etc., in a single individual or in different individuals. Thus both molecular hybridization and DNA amplification by two-primer extension using the polymerase chain reaction can define target segments for RFLP analysis. The two techniques are outlined with special consideration of characteristics important for genetic analysis. The mode of inheritance of restriction fragment patterns as a prerequisite for their use as genetic markers in inheritance studies is explained, leading to criticism of common usage. The importance of internal restriction sites for the determination of allelic variation is stressed. It is shown that, if target segments are under the control of a single nuclear diploid restriction fragment locus, then complete reconstruction of all parental target segments requires controlled crosses between individuals of like restriction fragment pattern.     

Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L.

CpDNA variation among 52 tree samples belonging to 25 different taxa of Coffea and two species of Psilanthus was assessed by RFLP analysis on both the total chloroplast genome and the atpB-rbcL intergenic region. Twelve variable characters were distinguished allowing the identification of 12 different plastomes. The low sequence divergence observed might suggest that Coffea is a young genus. The results were in contradiction with the present classification into two genera. Additionally, cpDNA inheritance was studied in interspecific hybrids between C. arabica and C. canephora, and in an intraspecific progeny of C. canephora, using PCR-based markers. Both studies showed exclusively maternal inheritance of cpDNA.     

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.     

Genetic variation of Calycophyllum spruceanum in the Peruvian Amazon Basin, revealed by amplified fragment length polymorphism (AFLP) analysis

An understanding of the level, structure and origin of genetic variation within and among populations of tropical trees is essential for devising optimum management strategies for their sustainable utilization and conservation. Here, amplified fragment length polymorphism (AFLP) analysis was used to partition genetic variation within and among nine populations of the predominantly riverine tree, Calycophyllum spruceanum , sampled across a wide geographical range along river tributaries of the Peruvian Amazon Basin. Analysis of molecular variance ( AMOVA ) employed 65 AFLP markers and revealed most variation among individuals within populations (91%), although variation among populations was highly significant ( P < 0.001). Calculation of genetic distances and nested AMOVA indicated a degree of structuring among populations based on geographical proximity, although clustering did not depend on geographical distance alone. No firm evidence was obtained for unidirectional seed dispersal by water playing an important role in determining genetic structure over the geographical range sampled. Implications of data for optimising genetic management of the species are discussed and areas for further study identified.     

Genetic characterization of Erwinia amylovora strains by amplified fragment length polymorphism

AIMS: Erwinia amylovora is one of the most important pathogens of pear and apple and is subject to strict quarantine regulations worldwide, although its patterns of dispersal are largely unknown. Previous attempts to fingerprint E. amylovora strains by molecular techniques have detected very little polymorphism because of the high genetic homogeneity of this bacterium. Our aim was to establish and test a typing method to quantify genetic diversity among strains of this plant pathogen. METHODS AND RESULTS: Twenty-two strains from different hosts and geographical locations were examined by PCR fingerprinting with four primers and by amplified fragment length polymorphism (AFLP) with four selected combinations of primers with a single base extension. PCR fingerprinting revealed little polymorphism producing the same amplification patterns for 17 strains, while the combined AFLP patterns yielded 78 polymorphic bands (34% of total bands) and allowed the differentiation of all but two strains. Clustering of strains in the resulting dendrogram was not correlated with host, year or country of isolation, and questions previous genealogies based on PFGE patterns. CONCLUSIONS: The AFLP technique allowed the detection of an unprecedented number of genetic markers in E. amylovora and proved to be the most useful tool so far for discriminating among strains of this pathogen. The results obtained in this study strongly suggest the occurrence of multiple introductions of the pathogen in Spain and other European countries. SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in understanding the ecology of fire blight is the lack of typing techniques with a high power of discrimination. This study demonstrates the high resolution and the usefulness of the AFLP technique to differentiate among E. amylovora strains.     

Inter-strain relationships among wine leuconostocs and their divergence from other Leuconostoc species, as revealed by low frequency restriction fragment analysis of genomic DNA

Thirty Leuconostoc oenos strains, representing 28 different isolates, were distributed into 20 genomic groups according to PFGE patterns of restriction digests. The 8 bp-specific enzymes Sfi I, Not I and Asc I cleaved the Leuc. oenos DNA in a mean of 17, 11 and four fragments respectively and Sma I produced more than 50 fragments per genome. The strain differentiating capacity of the four enzymes was similar; only two related genomic groups failed to be distinguished by Asc I or Not I. Genomic relationships between Leuc. oenos strains were quantified by numerical analysis of Not I and Sfi I banding patterns. More than half of the strains, including the starters ML34 and PSU-1, formed a major cluster. The average size of the Leuc. oenos genome was estimated as 1.86 Mb. Although similar values were obtained for the genomes of Leuc. mesenteroides, Leuc. pseudomesenteroides, Leuc. gelidum and Leuc. citreum, a significant divergence between wine and non-wine species was inferred from comparisons of genome cleavage frequencies, determined with five different enzymes.     

Genetic structure of the genus Lemna L. (Lemnaceae) as revealed by amplified fragment length polymorphism

Duckweeds (Lemnaceae) are extremely reduced in morphology, which made their taxonomy a challenge for a long time. The amplified fragment length polymorphism (AFLP) marker technique was applied to solve this problem. 84 clones of the genus Lemna were investigated representing all 13 accepted Lemna species. By neighbour-joining (NJ) analysis, 10 out of these 13 species were clearly recognized: L. minor, L. obscura, L. turionifera, L. japonica, L. disperma, L. aequinoctialis, L. perpusilla, L. trisulca, L. tenera, and L. minuta. However, L. valdiviana and L. yungensis could be distinguished neither by NJ cluster analysis nor by structure analysis. Moreover, the 16 analysed clones of L. gibba were assembled into four genetically differentiated groups. Only one of these groups, which includes the standard clones 7107 (G1) and 7741 (G3), represents obviously the “true” L. gibba. At least four of the clones investigated, so far considered as L. gibba (clones 8655a, 9481, 9436b, and Tra05-L), represent evidently close relatives to L. turionifera but do not form turions under any of the conditions tested. Another group of clones (6745, 6751, and 7922) corresponds to putative hybrids and may be identical with L. parodiana, a species not accepted until now because of the difficulties of delineation on morphology alone. In conclusion, AFLP analysis offers a solid base for the identification of Lemna clones, which is particularly important in view of Lemnaceae application in biomonitoring.