Amplified Fragment Length Polymorphism Patterns Research Articles

Genetic Variation of Monilinia fructicola Population in Korea.

Brown rot disease, caused by Monilinia spp., poses a significant threat to pome and stone fruit crops globally, resulting in substantial economic losses during pre- and post-harvest stages. Monilinia fructigena, M. laxa, and M. fructicola are identified as the key agents responsible for brown rot disease. In this study, we employed the amplified fragment length polymorphism (AFLP) method to assess the genetic diversity of 86 strains of Monilinia spp. isolated from major stone fruit cultivation regions in South Korea. Specifically, strains were collected from Chungcheong, Gangwon, Gyeonggi, Gyeongsang, and Jeolla provinces (-do). A comparative analysis of strain characteristics, such as isolation locations, host plants, and responses to chemical fungicides, was conducted. AFLP phylogenetic classification using 20 primer pairs revealed the presence of three distinct groups, with strains from Jeolla province consistently forming a separate group at a high frequency. Furthermore, M. fructicola was divided into three groups by the AFLP pattern. Principal coordinate analysis and PERMANOVA were applied to compare strain information, such as origin, host, and fungicide sensitivity, revealing significant partition patterns for AFLP according to geographic origin and host plants. This study represents the utilization of AFLP methodology to investigate the genetic variability among M. fructicol isolates, highlighting the importance of continuous monitoring and management of variations in the brown rot pathogen.

No wonder, it is a hybrid. Natural hybridization between Jacobaea vulgaris and J. erucifolia revealed by molecular marker systems and its potential ecological impact.

Progressive changes in the environment are related to modifications of the habitat. Introducing exotic species, and interbreeding between species can lead to processes that in the case of rare species or small populations threatens their integrity. Given the declining trends of many populations due to increased hybridization, early recognition of hybrids becomes important in conservation management. Natural hybridization is prevalent in Jacobaea. There are many naturally occurring interspecific hybrids in this genus, including those between Jacobaea vulgaris and its relatives. Although Jacobaea erucifolia and J. vulgaris often co-occur and are considered closely related, apart from the few reports of German botanists on the existence of such hybrids, there is no information on research confirming hybridization between them. Morphologically intermediate individuals, found in the sympatric distributions of J. vulgaris and J. erucifolia, were hypothesized to be their hybrids. Two molecular marker systems (nuclear and chloroplast DNA markers) were employed to test this hypothesis and characterize putative hybrids. Nuclear and chloroplast DNA sequencing results and taxon-specific amplified fragment length polymorphism (AFLP) fragment distribution analysis confirmed the hybrid nature of all 25 putative hybrids. The AFLP patterns of most hybrids demonstrated a closer relationship to J. erucifolia, suggesting frequent backcrossing. Moreover, they showed that several individuals previously described as pure were probably also of hybrid origin, backcrosses to J. erucifolia and J. vulgaris. This study provides the first molecular confirmation that natural hybrids between J. vulgaris and J. erucifolia occur in Poland. Hybridization appeared to be bidirectional but asymmetrical with J. vulgaris as the usual maternal parent.

Candidemia among Hospitalized Pediatric Patients Caused by Several Clonal Lineages of Candida parapsilosis.

Candida parapsilosis is the second most common cause of candidemia in some geographical areas and in children in particular. Yet, the proportion among children varies, for example, from 10.4% in Denmark to 24.7% in Tehran, Iran. As this species is also known to cause hospital outbreaks, we explored if the relatively high number of C. parapsilosis pediatric cases in Tehran could in part be explained by undiscovered clonal outbreaks. Among 56 C. parapsilosis complex isolates, 50 C. parapsilosis were genotyped by Amplified Fragment Length Polymorphism (AFLP) fingerprinting and microsatellite typing and analyzed for nucleotide polymorphisms by FKS1 and ERG11 sequencing. AFLP fingerprinting grouped Iranian isolates in two main clusters. Microsatellite typing separated the isolates into five clonal lineages, of which four were shared with Danish isolates, and with no correlation to the AFLP patterns. ERG11 and FKS1 sequencing revealed few polymorphisms in ERG11 leading to amino-acid substitutions (D133Y, Q250K, I302T, and R398I), with no influence on azole-susceptibilities. Collectively, this study demonstrated that there were no clonal outbreaks at the Iranian pediatric ward. Although possible transmission of a diverse C. parapsilosis community within the hospital cannot be ruled out, the study also emphasizes the necessity of applying appropriately discriminatory methods for outbreak investigation.

Molecular characterization and antifungal susceptibility testing of Cryptococcus neoformans sensu stricto from southern Brazil.

Cryptococcosis is acquired from the environment by the inhalation of Cryptococcus cells and may establish from an asymptomatic latent infection into pneumonia or meningoencephalitis. The genetic diversity of a Cryptococcus neoformans species complex has been investigated by several molecular tools, such as multi-locus sequence typing, amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism and microsatellite analysis. This study aimed to investigate the genotype distributions and antifungal susceptibility profiles of C. neoformans sensu lato isolates from southern Brazil. We studied 219 C. neoformans sensu lato isolates with mating- and serotyping, AFLP fingerprinting, microsatellite typing and antifungal susceptibility testing.Results/Key findings. Among the isolates, 136 (69 %) were from HIV-positive patients. Only C. neoformans mating-type α and serotype A were observed. AFLP fingerprinting analysis divided the isolates into AFLP1/VNI (n=172; 78.5 %), AFLP1A/VNII (n=19; 8.7 %), AFLP1B/VNII (n=4; 1.8 %) and a new AFLP pattern AFLP1C (n=23; 10.5 %). All isolates were susceptible to tested antifungals and no correlation between antifungal susceptibility and genotypes was observed. Through microsatellite analysis, most isolates clustered in a major microsatellite complex and Simpson's diversity index of this population was D=0.9856. The majority of C. neoformans sensu stricto infections occurred in HIV-positive patients. C. neoformans AFLP1/VNI was the most frequent genotype and all antifungal drugs had high in vitro activity against this species. Microsatellite analyses showed a high genetic diversity within the regional C. neoformans sensu stricto population, and correlation between environmental and clinical isolates, as well as a temporal and geographic relationship.

Viability, purity, and genetic stability of entomopathogenic fungi species using different preservation methods

Preservation methods for entomopathogenic fungi (EPF) require effective protocols to ensure uniform processes and to avoid alterations during storage. The aim of this study was to preserve Beauveria bassiana, Metarhizium acridum, M. anisopliae, M. rileyi, Isaria javanica, Hirsutella thompsonii, H. citriformis and Lecanicillium lecanii in mineral oil (MO), sterile water (SW), silica gel (SG), lyophilisation (L), ultracold-freezing at −70 °C, and cryopreservation at −196 °C. The viability and purity of the fungi were then verified: phenotypic characteristics were evaluated qualitatively at 6, 12 and 24 m. Genetic stability was tested by amplified fragment length polymorphisms (AFLP) analysis at 24 m. Of the eight species of EPF, three remained viable in SW, five in MO and L, six at −70 °C, seven in SG, and eight at −196 °C. No significant changes were observed in AFLP patterns at 24 m of storage. The most effective preservation methods for EPF were SG, L, −70 and −196 °C. Beauveria bassiana, M. acridum, M. anisopliae, M. rileyi and I. javanica remained stable with all methods, while the remaining species were less compatible. The optimisation of preservation methods for EPF facilitates the development of reliable protocols to ensure their inherent characteristics in culture collections.

Genetic stability of ectomycorrhizal fungi is not affected by cryopreservation at -130°C or cold storage with repeated sub-cultivations over a period of 2years.

Cryopreservation is considered the most reliable method for storage of filamentous fungi including ectomycorrhizal (ECM) fungi. A number of studies, however, have reported genetic changes in fungus cultures following cryopreservation. In the present study, the genetic stability of six ECM fungus isolates was analyzed using amplified fragment length polymorphism (AFLP). The isolates were preserved for 2years either by cryopreservation (at -130°C) or by storage at 4°C with regular sub-cultivation. A third preservation treatment consisting of isolates maintained on Petri dishes at 22-23°C for 2years (i.e., without any sub-cultivation) was included and used as a control. The differences observed in AFLP patterns between the three preservation methods remained within the range of the total error generated by the AFLP procedure (6.85%). Therefore, cryopreservation at -130°C and cold storage with regular sub-cultivation did not affect the genetic stability of the ECM fungus isolates, and both methods can be used for the routine storage of ECM fungus isolates over a period of 2years.

Analysis of drought tolerance and genetic and epigenetic variations in a somatic hybrid between Ipomoea batatas (L.) Lam. and I. triloba L.

The somatic hybrid KT1 was previously obtained from protoplast fusion between sweetpotato (Ipomoea batatas (L.) Lam.) cv. Kokei No. 14 and its wild relative I. triloba L. However, its genetic and epigenetic variations have not been investigated. This study showed that KT1 exhibited significantly higher drought tolerance compared to the cultivated parent Kokei No. 14. The content of proline and activities of superoxide dismutase (SOD) and photosynthesis were significantly increased, while malonaldehyde (MDA) content was significantly decreased compared to Kokei No. 14 under drought stress. KT1 also showed higher expression level of well-known drought stress-responsive genes compared to Kokei No. 14 under drought stress. Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) analyses indicated that KT1 had AFLP and MSAP band patterns consisting of both parent specific bands and changed bands. Further analysis demonstrated that in KT1 the proportions of Kokei No. 14 specific genome components and methylation sites were much greater than those of I. triloba. KT1 had the same chloroplast and mitochondrial genomes as Kokei No. 14. These results will aid in developing the useful genes of I. triloba and understanding the evolution and phylogeny of the cultivated sweetpotato.

Heat-resistant, extended-spectrum β-lactamase-producing Klebsiella pneumoniae in endoscope-mediated outbreak

We describe an outbreak with an extended-spectrum β-lactamase-producing Klebsiella pneumoniae strain in an intensive care unit in a secondary care hospital in Norway. The outbreak source was a fibreoptic intubation endoscope in which the outbreak strain survived despite chemothermal disinfection in a decontaminator designated for such use. The genetic marker clpK, which increases microbial heat resistance, has previously been described in K.pneumoniae outbreak strains. To investigate the role of clpK in biofilm formation and heat-shock stability in the outbreak strain. The outbreak investigation was done by review of clinical records, screening of patients and culture from intubation endoscopes and bronchoscopes. Amplified fragment length polymorphism was used to identify the outbreak strain. clpK detection was performed by polymerase chain reaction, followed by mutant construction and heat-shock assays. Five patients and one intubation endoscope contained K.pneumoniae with the same amplified fragment length polymorphism pattern. The outbreak strain contained the clpK genetic marker, which rendered the strain its increased heat resistance. The survival rate of the strain grown as biofilm following heat treatment was also strongly dependent on clpK. Although clpK has been associated with clinical isolates of K.pneumoniae in earlier outbreaks, this is the first time that a ClpK-producing strain has been isolated from an environmental outbreak source. Heat resistance of certain K.pneumoniae strains may facilitate survival in biofilms on medical equipment and hence increase the potential of those strains to persist and disperse in the hospital environment.

Vicariance, long-distance dispersal, and regional extinction-recolonization dynamics explain the disjunct circumpolar distribution of the arctic-alpine plant Silene acaulis.

Many arctic-alpine species have vast geographic ranges, but these may encompass substantial gaps whose origins are poorly understood. Here we address the phylogeographic history of Silene acaulis, a perennial cushion plant with a circumpolar distribution except for a large gap in Siberia. We assessed genetic variation in a range-wide sample of 103 populations using plastid DNA (pDNA) sequences and AFLPs (amplified fragment length polymorphisms). We constructed a haplotype network and performed Bayesian phylogenetic analyses based on plastid sequences. We visualized AFLP patterns using principal coordinate analysis, identified genetic groups using the program structure, and estimated genetic diversity and rarity indices by geographic region. The history of the main pDNA lineages was estimated to span several glaciations. AFLP data revealed a distinct division between Beringia/North America and Europe/East Greenland. These two regions shared only one of 17 pDNA haplotypes. Populations on opposite sides of the Siberian range gap (Ural Mountains and Chukotka) were genetically distinct and appear to have resulted from postglacial leading-edge colonizations. We inferred two refugia in North America (Beringia and the southern Rocky Mountains) and two in Europe (central-southern Europe and northern Europe/East Greenland). Patterns in the East Atlantic region suggested transoceanic long-distance dispersal events. Silene acaulis has a highly dynamic history characterized by vicariance, regional extinction, and recolonization, with persistence in at least four refugia. Long-distance dispersal explains patterns across the Atlantic Ocean, but we found no evidence of dispersal across the Siberian range gap.

Clonal Structure of Two Wild Lotus (Nelumbo nucifera Gaertn.) Populations Inferred from Amplified Fragment Length Polymorphism Fingerprints

The lotus Nelumbo nucifera often forms dense populations at the margins of lakes and ponds. Many aquatic plant species such as N. nucifera are characterized by the ability to reproduce both sexually and asexually, which can affect the genetic diversity of populations growing in different types of environments. We compared the clonal diversities of two natural lotus populations in Japan. Diversity was quantified by DNA fingerprinting of amplified fragment length polymorphisms (AFLPs) in leaves sampled across each population. The DNA fingerprints revealed different amounts of diversity in each population. In 162 samples from Lake Kasumigaura, where lotus has grown continuously for many years, 35 AFLP patterns were detected. One particular clone was distributed widely, suggesting that growth of seed-derived plants was inhibited because of the superior competitive ability of rhizomes, resulting in low genetic diversity within the population. In 214 samples from Uchi-numa Pond in Miyagi Prefecture, where the lotus population was totally destroyed by heavy rains in 1998 and is now recovering, 213 different AFLP profiles were detected. The spatial distance between samples from Uchi-numa Pond was correlated with the genetic distance. These results suggest that, in lotus, sexual reproduction is favored when rapidly varying water level damages the population, because small seedlings are better able to survive in a low-density population. In contrast, vegetative reproduction is favored when environmental conditions are stable.

A PRELIMINARY AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) PRIMERS SELECTION FOR SPATHOGLOTTIS SPECIES

Spathoglottis is a native terrestrial orchid that attracting horticultural interest as a landscaping plant. Distinguishing orchid at species level based on morphological characteristics is unconvincing as some characteristic may sensitive to environmental. Therefore, molecular characterization could be an alternative method in characterizing Spathoglottis species. Amplified Fragment Length Polymorphism (AFLP) marker was selected to characterize the Spathoglottis species. Among 56 AFLP primer combinations tested on Spathoglottis plicata, only eight primer combinations gave clear scorable polymorphic bands. A total of 60 accessions of Spathoglottis species, hybrid and outgroup (Dendrobium) were analysed using the eight primer combinations and generated 891 polymorphic bands with 98.41% in average. The AFLP patterns showed that the Spathoglottis species sampled were clearly different.

Citrus somatic hybrid: an alternative system to study rapid structural and epigenetic reorganization in allotetraploid genomes

Citrus somatic hybrids produced in the past years provide a novel opportunity to study the immediate effects of allopolyploidization on genome structure and methylation. Here, we present a first attempt to investigate the alterations in genome structure and methylation in three sets of citrus somatic allotetraploids and their diploid parents using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. Our results indicate that all the allotetraploids mainly have the AFLP and MSAP banding patterns containing specific bands from both parents plus some alterations. The incidences of the AFLP polymorphic bands in allotetraploids show a range from 4.61 to 7.88 %, while from 12.50 to 15.67 % of the sites are methylated. In addition, the proportions of callus-parent-specific DNA structure and methylation alterations are much greater than those of leaf-parent-specific alterations in the somatic hybrids. Furthermore, we find that the somatic hybrids take on a greater divergence from the callus parent and a closer relationship to leaf parent in all groups of plants by dendrogram analysis based on AFLP or MSAP data. Taken together, our results suggest that somatic hybrids are very useful in elucidating the immediate changes that occur in newly synthesized allotetraploid.

History of Oilseed Rape Cropping and Geographic Origin Affect the Genetic Structure of Plasmodiophora brassicae Populations

The soilborne pathogen Plasmodiophora brassicae causes clubroot on Brassica crops, a common disease in many oilseed rape growing regions. Here, we investigate genetic diversity and geographic differentiation of P. brassicae populations from different regions in Germany. We compared three regions that differ in oilseed rape cropping history, oilseed rape acreage, and incidence of clubroot. These regions were either spatially separated or separated by the former inner German border. Plasmodiophora isolates were collected from 59 fields (29, 17, and 13 fields per region, respectively) and 174 amplified fragment length polymorphism (AFLP) markers were analyzed. Every field isolate showed a unique genotype pattern; that is, no genotype was shared among the regions and different fields. The mean gene diversity was 0.27, suggesting that P. brassicae is a genetically diverse species. The comparison of indexes (gene diversity, genotypic diversity, and linkage disequilibrium) between the regions does not support our hypotheses that cropping history, oilseed rape acreage, and incidence of clubroot affect these estimates. Principal component analysis (PCA), fixation index (FST), and generalized linear model (GLM) were suitable to specify regional differences. PCA revealed two clusters of isolates based on the geographic origin of the isolates and FST showed that these clusters were highly differentiated. Hypotheses about association of genotypes with different spatial scales were tested with GLM: the region, reflecting the cropping history, and the individual field had a significant effect on the AFLP pattern. We propose that individual field isolates represent a discrete population and that geographic differentiation results from low levels of gene flow due to the limited dispersal of this soilborne pathogen and from localized selection pressure as unifying force on the genotypes.

Rapid alterations of DNA sequence and cytosine methylation induced by somatic hybridization between Brassica oleracea L. var. italica and Brassica nigra (L.) Koch

Somatic hybrids were produced between hypocotyl protoplasts of Brassica oleracea L. var. italica (broccoli) and mesophyll protoplasts of B. nigra (black mustard) using polyethylene glycol—mediated protoplast fusion. A total of fifteen somatic hybrids derived from six calli (no. 1, 3, 8, 21, 38 and 44) were obtained. Cytological analysis showed that all the hybrids possessed 2n = 34, the sum of the parental chromosomes and the genomic in situ hybridization analysis revealed their BBCC genome constitutes. Moreover, all the hybrids exhibited different type of meiosis abnormalities, which were more usually observed in pollen mother cells at metaphase II/anaphase II (MII/AII, 16.1–39.6 %) than at metaphase I/anaphase I (MI/AI, 7.8–15.2 %). Simple sequence repeat analysis revealed that all the hybrids showed the same cytoplasmic genome as broccoli. Structure and methylation-variation of the nuclear were investigated by amplified fragment length polymorphism (AFLP) and DNA methylation-sensitive amplification polymorphism (MSAP). Our results indicated that all the hybrids mainly had the AFLP and MSAP banding patterns from the addition of two parents plus some alterations. The incidences of the AFLP polymorphic bands in the hybrids showed a range of 9.8–18.7 % while the DNA methylation alteration in the hybrid no. 38 was 4.07 %. This result suggested that somatic hybridization could induce more DNA sequence changes than methylation alterations in the early stage of allotetraploid hybrids.

Molecular Genetic and Hybridization Studies ofDiorhabdaspp. Released for Biological Control ofTamarix

AbstractThe genusDiorhabda(Coleoptera: Chrysomelidae) was recently revised, using morphological characters, into five tamarisk-feeding species, four of which have been used in the tamarisk (Tamarixspp.) biological control program in North America and are the subject of these studies. The taxonomic revision is here supported using molecular genetic and hybridization studies. FourDiorhabdaspecies separated into five clades using cytochrome c oxidase subunit 1 sequence data withDiorhabda elongataseparating into two clades. Amplified fragment length polymorphism (AFLP) analysis using genomic DNA revealed only four clades, which corresponded to the four morphospecies. Hybridization between the four species yielded viable eggs in F1crosses but viability was significantly lower than achieved with intraspecific crosses. Crosses involvingDiorhabda carinulataand the other three species resulted in low F2egg viability, whereas crosses betweenD. elongata, Diorhabda sublineataandDiorhabda carinataresulted in > 40% F2egg viability. Crosses betweenD. carinulataand the other three species resulted in high mortality ofD. carinulatafemales due to genital mismatch. AFLP patterns combined with principal coordinates analysis enabled effective separation betweenD. elongataandD. sublineata, providing a method to measure genetic introgression in the field.

Application of AFLP markers in fishery and seafood

Several sociological, health and conservation arguments request acorrect labelling of seafood products and fish species. Nowadays, moleculargenetics is a useful tool for food chain traceability, particularly in regardsto species identification. Among the variety of PCRbased molecular markers,AFLPs (Amplified Fragment Length Polymorphisms) have recently been used toinvestigate genomes of different complexities. This paper assesses thepotential use of the AFLP technology to determine fish and seafood species inprocessed commercial products, domestic stocks and research in the field ofmolecular ecology. In particular a species database of fish, molluscs andcrustaceans has been created with the aim to identify species of origin ofseafood products by previously defined AFLP patterns. Researchers in the fieldof molecular ecology and evolution require versatile and low-cost genetictyping methods. The AFLP method was introduced 20 years ago and shows manyfeatures that fulfil these requirements. With good quality genomic DNA at hand,it is relatively easy to generate anonymous multilocus DNA profiles in mostspecies and the start-up time before data can be generated is often less than aweek. These aspects include classical problems such as studies of populationgenetic structure and phylogenetic reconstructions, and also new challengessuch as finding markers for genes governing adaptations in wild populations andmodifications of the protocol that makes it possible to measure expressionvariation of multiple genes (cDNA-AFLP) and the distribution of DNAmethylation. We hope this review will help molecular ecologists to identifywhen AFLP is likely to be superior to other more established methods, such asmicrosatellites, SNP (single nucleotide polymorphism) analyses and multigeneDNA sequencing.

Genetic variation among natural isolates of the ectomycorrhizal hypogenous fungus, Rhizopogon roseolus from Japanese pine forests inferred using AFLP markers

Genetic variation among 45 Rhizopogon roseolus isolates from 21 different regions of Japan were inferred using amplified fragment length polymorphism (AFLP) markers. Using three primer pair combinations, AFLP analysis reproducibly produced a total of 223 DNA fragments, 74.4% of which were polymorphic. Pairwise dissimilarity of AFLP patterns between isolates ranged from 0.043 to 0.228. Cluster analysis and principal coordinate analysis of AFLP data generally showed four major clusters from geographically distinct areas. The findings suggested that the Japanese populations of R. roseolus from different geographical regions can be distinguished based on AFLP characters.

Genetic Linkage Map and Diversity Analysis of Tahitian Vanilla (Vanilla ×tahitensis, Orchidaceae)

ABSTRACTThe first vanilla genetic linkage map was constructed by analyzing the progeny of an interspecific cross between Vanilla ×tahitensis J. W. Moore (pro sp.) and Vanilla pompona Schiede. The two‐way pseudotestcross mapping strategy was used and a total of 225 amplified fragment length polymorphism (AFLP) markers were mapped on 18 linkage groups covering 1035.85 cM. Thirteen tahitian vanilla morphotypes, one Vanilla planifolia Andrews and recent hybrids were compared by measuring seven morphological traits and by genotyping, with 539 AFLP fragments. The morphotypes studied showed relatively homogenous stem traits but differed in their bean and leaf traits as in their AFLP pattern. Genetic variability and AFLP genotyping comparison suggest a diversification in French Polynesia based on sexual reproduction and are consistent with the hypothesis of a single introduction event. These results are the first step for vanilla breeding program and will be also useful for tahitian vanilla resource preservation.

AFLP Fingerprinting of Tricoderma reesei Strains

Amplified Fragment Length Polymorphism (AFLP), a novel molecular fingerprinting technique, modi- fied by using one PstI restriction enzyme, has been applied to assess the genomic diversity of Trichoderma reesei strains and mutants deriving from different world collections. The genetic relationship among all AFLP pattern based on the Jaccard's similarity coefficient were transformed to build an unweighted pair group method with arithmetic mean dendrogram. The statistical analysis of obtained results enabled the classification of T. reesei strains into two main clusters. Nine of them were classified as group I and next four strains into group II. The analysis revealed the existence of one subgroup composed of 291 and 292 strains within group II. Six subgroups were present within group I, the first formed by 289; the second com- posed by 290 and 298; the third formed by 300; the fourth including 293 and 294; the fifth formed by 296; and the sixth composed by 295 and 299. In general, the AFLP analysis has differentiated all strains tested in this study. AFLPs revealed the lower level of diversity among group II, whereas for strains in group I, the level of diversity was higher.

Biogeography of symbiotic and other endophytic bacteria isolated from medicinal Glycyrrhiza species in China

A total of 159 endophytic bacteria were isolated from surface-sterilized root nodules of wild perennial Glycyrrhiza legumes growing on 40 sites in central and northwestern China. Amplified fragment length polymorphism (AFLP) genomic fingerprinting and sequencing of partial 16S rRNA genes revealed that the collection mainly consisted of Mesorhizobium, Rhizobium, Sinorhizobium, Agrobacterium and Paenibacillus species. Based on symbiotic properties with the legume hosts Glycyrrhiza uralensis and Glycyrrhiza glabra, we divided the nodulating species into true and sporadic symbionts. Five distinct Mesorhizobium groups represented true symbionts of the host plants, the majority of strains inducing N2-fixing nodules. Sporadic symbionts consisted of either species with infrequent occurrence (Rhizobium galegae, Rhizobium leguminosarum) or species with weak (Sinorhizobium meliloti, Rhizobium gallicum) or no N2 fixation ability (Rhizobium giardinii, Rhizobium cellulosilyticum, Phyllobacterium sp.). Multivariate analyses revealed that the host plant species and geographic location explained only a small part (14.4%) of the total variation in bacterial AFLP patterns, with the host plant explaining slightly more (9.9%) than geography (6.9%). However, strains isolated from G. glabra were clearly separated from those from G. uralensis, and strains obtained from central China were well separated from those originating from Xinjiang in the northwest, indicating both host preference and regional endemism.