Genetic diversity of Nostoc microsymbionts from Gunnera tinctoria revealed by PCR-STRR fingerprinting

The cyanobacteria belonging to the genus Nostoc fix atmospheric nitrogen, both as free-living organisms and in symbiotic associations with a wide range of hosts, including bryophytes, gymnosperms (cycads), the small water fern Azolla (Pteridophyte), the angiosperm genus Gunnera, and fungi (lichens). The Gunnera–Nostoc symbiosis is the only one that involves a flowering plant. In Chile, 12 species of Gunnera have been described with a broad distribution in the temperate region. We examined the genetic diversity of Nostoc symbionts from three populations of Gunnera tinctoria from Abtao, Chiloé Island, southern Chile, and microsymbionts from other two species of Gunnera from southern Chile, using PCR amplification of STRR (short tandemly repeated repetitive) sequences of the Nostoc infected tissue. To our knowledge, this is the first report of PCR fingerprinting obtained directly from symbiotic tissue of Gunnera. Genetic analyses revealed that Nostoc symbionts exhibit important genetic diversity among host plants, both within and between Gunnera populations. It was also found that only one Nostoc strain, or closely related strains, established symbiosis with an individual plant host.     

Symbiosis of Astragalus cicer with its microsymbionts: partial nodC gene sequence, host plant specificity, and root nodule structure

Astragalus cicer (cicer milkvetch) nodule bacteria were investigated for host plant specificity and partial nodC gene sequences, whilst their native host was studied for the microscopic structure of root nodules. The strains under investigation formed nodules not only on the original host but also on Astragalus glycyphyllos, Astragalus sinicus, Lotus corniculatus, and Phaseolus vulgaris. The nodules induced on the cicer milkvetch were classified as indeterminate and characterized by apical, persistent meristem, a large bacteroid region with infected and uninfected cells, and elongated bacteroids singly located inside peribacteroid membranes. By comparison of the partial nodC gene sequences of a representative strain of astragali rhizobia to those contained in the GenBank database, a close symbiotic relationship of A. cicer microsymbionts to Rhizobium sp. (Oxytropis) was found.     

Numerical Analysis of Astragalus cicer Microsymbionts

Thirty-seven rhizobium strains, isolated from root nodules of Astragalus cicer (L.) (cicer milkvetch) deriving from different geographic regions, were compared with the representative strains of the known rhizobial species and genera by numerical analysis of phenotypic characteristics. Our results indicated that Astragalus cicer rhizobia were related to the bacteria of Mesorhizobium species and formed two major phena. One phenon, localized on Mesorhizobium loti branch, contained strains from Poland. Another cluster, placed in the vicinity of M. tianshanense, M. mediterraneum, M. ciceri, and M. huakuii, comprised cicer milkvetch nodule isolates from Canada, Ukraine, and one strain from Poland. The relationship of Astragalus cicer microsymbionts to bacteria of the Mesorhizobium species was also supported by phage typing. Received: 10 February 2000 / Accepted: 8 March 2000     

Genetic diversity in tetraploid switchgrass revealed by AFLP marker polymorphisms

Switchgrass (Panicum virgatum) is a perennial warm-season grass native to North America that has been identified as a dedicated cellulosic biofuel crop. We quantified genetic diversity in tetraploid switchgrass germplasm collected at Oklahoma State University and characterized genetic relatedness among the collections from distinct regions. Fifty-six tetraploid accessions, including seven upland and 49 lowland genotypes from throughout the US, were examined. The amplified fragment length polymorphism (AFLP) procedure was utilized to generate DNA profiling patterns that were scored visually. Sixteen selective AFLP primer combinations were used to amplify 452 polymorphic bands. The accessions' genetic similarity coefficients, UPGMA (unweighted pair-group method with arithmetic averaging) cluster analysis and principle coordinate analysis, were performed. The upland and lowland accessions clustered according to ecotypes, with one exception (TN104). Genetic similarity coefficients among the accessions ranged from 0.73 to 0.95. Analysis of molecular variance (AMOVA) was performed, showing significant differences between the upland and lowland genotypes. The trnL marker confirmed that TN104 was a lowland genotype, but the trnL marker identification of upland and lowland genotypes was not consistent with the AFLP analysis in two germplasms (Miami and AR4).     

Genetic diversity of Carica papaya as revealed by AFLP markers.

Genetic relationships among Carica papaya cultivars, breeding lines, unimproved germplasm, and related species were established using amplified fragment length polymorphism (AFLP) markers. Seventy-one papaya accessions and related species were analyzed with nine EcoRI-MseI primer combinations. A total of 186 informative AFLP markers was generated and analyzed. Cluster analysis suggested limited genetic variation in papaya, with an average genetic similarity among 63 papaya accessions of 0.880. Genetic diversity among cultivars derived from the same or similar gene pools was smaller, such as Hawaiian Solo hermaphrodite cultivars and Australian dioecious cultivars with genetic similarity at 0.921 and 0.912, respectively. The results indicated that self-pollinated hermaphrodite cultivars were as variable as open-pollinated dioecious cultivars. Genetic diversity between C. papaya and six other Carica species was also evaluated. Carica papaya shared the least genetic similarity with these species, with an average genetic similarity of 0.432; the average genetic similarity among the six other species was 0.729. The results from AFLP markers provided detailed estimates of the genetic variation within and among papaya cultivars, and supported the notion that C. papaya diverged from the rest of Carica species early in the evolution of this genus.     

Inhibition of ruminal cellulose fermentation by extracts of the perennial legume cicer milkvetch (Astragalus cicer).

Cicer milkvetch (Astragalus cicer L.) is a perennial legume used as a pasture or rangeland plant for ruminants. A study was undertaken to determine whether reported variations in its ruminal digestibility may be related to the presence of an antinutritive material. In vitro fermentation of neutral detergent fiber (NDF) of cicer milkvetch by mixed rumen microflora was poorer than was the fermentation of NDF in alfalfa (Medicago sativa L.). Fermentation of cicer milkvetch NDF was improved by preextraction of the ground herbage with water for 3 h at 39 degrees C. Such water extracts selectively inhibited in vitro fermentation of pure cellulose by mixed ruminal microflora and by pure cultures of the ruminal bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85. Inhibition of the cellulose fermentation by mixed ruminal microflora was dependent upon the concentration of cicer milkvetch extract and was overcome upon prolonged incubation. Pure cultures exposed to the extract did not recover from inhibition, even after long incubation times, unless the inhibitory agent was removed (viz., by dilution of inhibited cultures into fresh medium). The extract did not affect the fermentation of cellobiose by R. flavefaciens but did cause some inhibition of cellobiose fermentation by F. succinogenes. Moreover, the extracts did not inhibit hydrolysis of crystalline cellulose, carboxymethyl cellulose, or p-nitrophenylcellobioside by supernatants of these pure cultures of cellulolytic bacteria or by a commercial cellulase preparation from the fungus Trichoderma reesei. The agent caused cellulose-adherent cells to detach from cellulose fibers, suggesting that the agent may act, at least in part, by disrupting the glycocalyx necessary for adherence to, and rapid digestion of, cellulose.     

Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting

RAPD fingerprinting was used for strain identification and the assessment of genetic diversity within a field population of Bradyrhizobium japonicum . Total genomic DNAs from 13 field isolates and two inoculant strains were amplified using six different 10-mer primers. Different and informative band patterns were obtained for all strains analysed. Cluster analysis unexpectedly revealed that none of the field isolates was identical to inoculant strains which were regularly used for soybean inoculation. Among field isolates two highly divergent groups were determined. The results indicate that RAPD is a very discriminative and efficient method for differentiating and studying genetic diversity of B. japonicum strains.     

中国马铃薯晚疫病菌AFLP遗传多样性分析

The genetic diversity of the populations of Phytophthora infestans from some major potato production regions in China were detected by amplified restriction fragment polymorphism (AFLP) analysis. Among 200 combinations of primer pair screened, 12 combinations could generate consistent polymorphic bands using six tested isolates. The twelve combinations were used to amplify the genomic DNA of 50 isolates collected in China from 1997 to 2002. A total of 922 AFLP bands were obtained, and 530 of them,covering 57.5%, showed polymorphism. Cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA) separated 50 isolates into five AFLP groups which were correlated to groups defined by geographical origin, however, they were not correlated to groups defined by mating type, or response to metalaxyl and virulence. Parameters of genetic diversity calculated by POPGENE software indicated that the genetic diversity level of Phytophtora infestans population in China was not high.     

中国马铃薯晚疫病菌AFLP遗传多样性分析

应用AFLP分子标记检测了我国部分马铃薯主要产区马铃薯晚疫病菌的遗传多样性及不同地区菌株间的亲缘关系。在200对引物组合中,利用6个菌株筛选出12对多态性好、带型清晰的引物组合。利用这12对引物组合对1997-2002年间采自我国黑龙江、河北、四川和云南4省的50株菌株进行了PCR扩增,共扩增出922条谱带,其中多态性标记530条,占57.5%。利用NTSYSpc软件中UPGMA算法构建了我国马铃薯晚疫病菌的亲缘关系树状图,聚类分析结果表明我国马铃薯晚疫病菌的遗传多样性与病原菌的地理来源有一定的相关性,而与交配型、生理小种和对甲霜灵的抗性无明显的相关性。用POPGENE软件计算了各群体间的遗传多样性参数,结果表明我国马铃薯晚疫病菌的遗传多样性程度不高,不同地区种群间分化不明显。     

Genomic diversity and relationship of Bacillus thuringiensis and Bacillus cereus by multi-REP-PCR fingerprinting

The genomic diversity and relationship among 56 Bacillus thuringiensis and Bacillus cereus type strains were investigated by multi-REP-PCR fingerprinting consisting of three PCR reactions targeting the enterobacterial ERIC1 and ERIC2 and the streptococcal BOXA1R consensus sequences. A total of 113 polymorphic bands were generated in the REP-PCR profiles that allowed tracing of a single dendrogram with three major groups. Bacillus cereus strains clustered together in the A and B groups. Most of the B. thuringiensis strains clustered in group C, which included groups of serovars with a within-group similarity higher than 40% as follows: darmstadiensis, israelensis, and morrisoni; aizawai, kenyae, pakistani, and thompsoni; canadensis, entomocidus, galleriae, kurstaki, and tolworthi; alesti, dendrolimus, and kurstaki; and finitimus, sotto, and thuringiensis. Multi-REP-PCR fingerprinting clustered B. thuringiensis serovars in agreement with previously developed multilocus sequence typing schemes, indicating that it represents a rapid shortcut for addressing the genetic relationship of unknown strains with the major known serovars.     

电融合法产生骆驼刺与鹰嘴紫云英属间体细胞杂种

采用电融合法获得了骆驼刺和鹰嘴紫云英属间体细胞杂种。骆驼刺原生质体来自发根农杆菌A4转化系细胞,并经碘乙酰胺处理;鹰嘴紫云英原生质体从甲硫氨酸抗性系细胞分离。双亲及同源融合产物均不能在无激素的筛选培养基上持续分裂,融合后的杂种细胞由于双亲生理互补效应可恢复持续分裂能力而被筛选出来。本实验优化了电融合参数,如直流脉冲、交流脉冲和脉冲次数。融合产物经培养获得的杂种细胞系经形态学、染色体数目检查、生化及随机扩增多态性DNA分析,鉴定出10个杂种克隆,并从3个杂种克隆再生了小植株。     

Differentiation of European cattle by AFLP fingerprinting

The Neolithic introduction of domestic cattle into Europe was followed by differential adaptation, selection, migration and genetic isolation, leading ultimately to the emergence of specialized breeds. We have studied the differentiation of European cattle by amplified fragment length polymorphism (AFLP) fingerprinting. Combining AFLP data sets from two laboratories yielded 81 biallelic polymorphic markers scored in 19-22 individual animals from 51 breeds. Model-based clustering differentiated Podolian cattle as well as French and Alpine breeds from other European cattle. AFLP genetic distances correlated well with microsatellite-based genetic distances calculated for the same breeds. However, the AFLP data emphasized the divergence of taurine and indicine cattle relative to the variation among European breeds and indicated an Eastern influence on Italian and Hungarian Podolian breeds. This probably reflects import from the East after the original introduction of domestic cattle into Europe. Our data suggest that Italian cattle breeds are relatively diverse at the DNA sequence level.     

Analysis of genetic diversity in Aconitum kongboense L. revealed by AFLP markers

The systematic evaluation of the molecular diversity encompassed in Aconitum kongboense L. inbreds or parental lines offers an efficient means of exploiting the heterosis in A. kongboense as well as for management of biodiversity. An excellent and novel DNA-based molecular, amplified fragment length polymorphisms (AFLPs) markers, was firstly used to analysis the genetic diversity in A. kongboense genotypes. Out of 256 primers screened, a total of ten combinations successfully produced scorable, clear, reproducible and relatively high polymorphism bands, 64.12% of which were polymorphic. The values of number of polymorphic loci (NPL), percentage of polymorphic loci (PPB), observed number of alleles (Na) and effective number of alleles (Ne) were highest in population 3 (NPL = 77, PPB = 68.75%, Na = 1.688 ± 0.466, Ne = 1.412 ± 0.397) and lowest in population 2 (NPL = 57, PPB = 50.89%, Na = 1.509 ± 0.502, Ne = 1.273 ± 0.340). In addition, Jaccard's similarity coefficients among different populations varied from 0.45 to 1.00 with an average of 0.55. The data collected will contribute to identification, rational exploitation and conservation of germplasms of A. kongboense, and potentially useful to aid its breeding.     

Genetic diversity in white clover and its progenitors as revealed by DNA fingerprinting

The genetic diversity and ancestral relationships of a number of Trifolium species was revealed by using the amplified fragment length polymorphism (AFLP) and the random amplified polymorphic DNA (RAPD) markers. Both markers produced few species-specific markers. Using distance and parsimony methods, in NTSYS-pc and PAUP software programs, we clearly differentiated the accessions of white clover from other closely related progenitors. The phylogenetic trees, produced by PAUP, also reinforced the close affinity of T. nigrescens and the allopolyploid white clover in support of former views that this diploid species could have been the donor of one of two genomes of the allotetraploid T. repens. In addition, the dendrograms, produced by NTSYS-pc, also indicated close affinity of T. nigrescens and T. occidentale to the accessions of T. repens. These data is congruent with karyological and phylogenetic affinities between the white clover and T. occidentale. The relationships between the examined accessions, in the T. repens gene pool, may be regarded to indicate the presence of shared alleles between T. repens, T. occidentale and T. uniflorum. Further, T. occidentale showed close phylogenetic relations to T. pallescens.     

Genetic diversity in apricot revealed by AFLP markers: species and cultivar comparisons

The genetic diversity of apricot ( Prunus armeniaca; 2n = 16) was studied using AFLP markers. Forty seven apricot cultivars were selected from the following geographic regions: Europe, North America, North Africa, Turkey, Iran and China. Five EcoRI- MseI AFLP primer combinations revealed 416 legible bands, of which 379 were polymorphic markers. A similarity matrix was prepared using the simple matching coefficient of similarity. A UPGMA dendrogram demonstrated a gradient of decreasing genetic diversity of varieties from the former USSR to Southern Europe. This is coherent with the historical dissemination of apricot from its center of origin in Asia. The American cultivars were intermediate demonstrating a different genetic base than the European and/or Mediterranean cultivars. Euclidean distances from the first ten Factorial Component Analysis coordinate axes were used to generate a tree using the Ward algorithm. The results of these analyses were evaluated based on the known geographic origins and agronomic characteristics of the cultivars studied. Four cultivar groups were identified: Diversification, Geographically Adaptable, Continental Europe and Mediterranean Basin. To evaluate the relationship of the common apricot with some closely related species, one or two accessions of the following related species or sub-species from within the section Armeniaca were included in the analysis: Prunus armeniaca var. ansu, Prunus mume, Prunus brigantiaca, Prunus dasycarpa, and Prunus holosericea. A Neighbour Joining dendrogram was made using the similarity matrix. The P. holosericea accession fell well within the cultivar group, thus supporting its classification as a variant of P. armeniaca. The P. armeniaca var. ansu accession was sister to the common apricot cluster with a bootstrap value of 96%. P. mume was farther removed. P. brigantiaca was the most-distant from the common apricots. P. dasycarpa was intermediate between P. brigantiaca and P. mume, in accord with its plum-apricot hybrid origin. The results have a direct application for the selection of new breeding progenitors.     

Application of the AFLP method to differentiate Genista tinctoria microsymbionts

The high-resolution amplified fragment length polymorphism technique (AFLP), with single PstI restriction endonuclease and two selective primers (PstI-G and PstI-GC), was used for genomotyping and study of the genomic relationships between Genista tinctoria microsymbionts sampled in England, Poland, and Ukraine. Out of 906 amplification products obtained with both selective primers, 537 markers were polymorphic and could be used to differentiate studied nodule isolates. Cluster analysis, based on AFLP patterns from PCR reaction with PstI-G and PstI-GC primers, separated Genista tinctoria rhizobia into three subgroups according to their geographic origin. The results presented in this paper emphasize the role of AFLP analysis in taxonomic and ecological studies of rhizobia.     

Genetic diversity among summer and winter Beauveria bassiana populations as revealed by AFLP analysis

The genetic diversity of Beauveria bassiana was investigated by comparing 40 isolates collected from summer and overwintering populations of Sunn pest from different areas in Syria and Turkey, using amplified fragment length polymorphism (AFLP) markers. Considerable genetic variability among B. bassiana isolates was revealed. The examined isolates were divided into three distinct clusters (A, B, and C). Within these clusters, the summer isolates from Syria and Turkey were grouped together in three sub-clusters (A3, A4, and B2). Also, principal coordinate analyses (PCA) showed clear separation (62.5%) between summer and winter isolates. These differences in the genetic structure may be explained by the variety of eco-geography over the sampled areas of B. bassiana isolates. This information on genetic variation among summer and winter B. bassiana isolates is helpful in designing an effective integrated pest management program for Sunn pest.     

Genetic diversity and phylogenetic relationships in alder,Alnus firma, revealed by AFLP

Molecular markers for alder,Alnus firma Sieb. et Zucc, have not been studied extensively. Here, we used amplified fragment length polymorphism (AFLP) to investigate genetic relationships among 15 natural populations. EcoRI-ACG + Msel-CTG combinations revealed the highest polymorphism (62.2%). A total of 171 DNA fragments were identified. On average, 58.1% of the AFLP markers that were generated using four primer pairs were polymorphic. Diversity was insignificant among the populations. The combination of a wind-pollinated, outcrossing breeding system along with large population sizes, and the ability to regenerate by stump sprouting may explain the high level of genetic diversity within this species. The majority (98%) of the genetic variance resided within populations. The average number of individuals that were exchanged between populations per generation was very high (N _em = 12.3). Gene dispersal in alder is apparently by seed dispersalvia water and human activity as well as through pollen. Five individuals per population were claded in the same cluster.