Amplified Fragment Length Polymorphism Bands Research Articles

IDENTIFICATION OF AN AFLP MOLECULAR MARKER LINKED TO PARTHENOCARPY GENE IN MONOECIOUS CUCUMBER

In order to identify molecular markers linked to parthenocarpy in cucumber (Cucumis sativus L.), parthenocarpy and non-parthenocarpy DNA pools were created from F2 crosses between a highly parthenocarpic monoecious line '6457' and a non-parthenocarpic line '6429' according to the principle of bulk segregant analysis (BSA). The amplified fragment length polymorphism (AFLP) technique was employed with 256 primer combinations to detect the polymorphisms between the DNA pools. In total, 8544 scorable AFLP bands were generated, yielding an average of 33.38 fragments per primer combination. In the non-parthenocarpy DNA pool, a 325 bp specific fragment was amplified with the primer E41/M47, the band did not appear in the parthenocarpic pool. This marker, designated AGG/CAA325 was validated with DNA from the F2 population. Linkage analysis using MapMaker 3.0 indicated that the genetic distance from the marker to the non-parthenocarpy locus was 9.7 cM.

Widespread amplification of amplified fragment length polymorphisms (AFLPs) in marine Antarctic animals

Although recent years have witnessed a rapid growth in the number of genetic studies of Antarctic organisms, relatively few studies have so far used nuclear markers, possibly due to the perceived cost and difficulty of isolating markers such as microsatellites. However, an often overlooked alternative is to use amplified fragment length polymorphisms (AFLPs), a versatile and low-cost method capable of generating large numbers of predominantly nuclear loci in virtually any organism. We conducted a literature review of population genetic studies of Antarctic organisms, finding that fewer than 10% used AFLPs. Moreover, a strong taxonomic bias was found, with studies employing mitochondrial DNA or microsatellites focussing predominantly on animals, while those using AFLPs were mostly of plants or lower organisms. Consequently, we explored the extent to which AFLPs amplify across a range of Antarctic marine animal taxa by genotyping eight individuals each of twelve different species, ranging from echinoderms through soft corals to pelagic fish, at four selective primer combinations. AFLPs readily amplified across all of the taxa, generating between 32 and 84 loci per species, with on average 56.5% of these being polymorphic. In general, levels of polymorphism bore little relationship with expectations based on larger populations of broadcast-spawning species being more variable, though we did find a tentative positive correlation between the number of AFLP bands amplified and a measure of effective population size. Our study lends further support for the utility and ease of use of AFLPs and their suitability for studies of Antarctic species across a wide range of taxa.

Epigenetic instability in genetically stable micropropagated plants of Gardenia jasminoides Ellis

Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant.

English

  To study the genetic and phylogenetic relationship of gobioid fishes in China, the representatives of 10 gobioid fishes from 2 subfamilies in China were examined by amplified fragment length polymorphism (AFLP) analysis. We established 220 AFLP bands for 45 individuals from the 10 species, and the percentage of polymorphic bands was 100%. The percentage of polymorphic loci within species ranged from 3.61 to 58.56%. Chaeturichthys stigmatias showed the greatest percentage of polymorphic loci (58.56%), the highest Nei’s genetic diversity (0.2421 ± 0.2190) and Shannon’s information index (0.3506 ± 0.3092), while Pterogobius zacalles showed the lowest percentage polymorphic loci (3.61%), the lowest Nei’s genetic diversity (0.0150 ± 0.0778) and lowest Shannon’s information index (0.0219 ± 0.1136). The topology of UPGMA tree showed that the individuals from the same species clustered together and the 10 species formed two major clades. One clade consisted Cryptocentrus filifer, P. zacalles, Tridentiger trigonocephalus,Chaeturichthys hexanema, C. stigmatias, Acanthogobius flavimanus andSynechogobius ommaturus, and the other clade consisted Odontamblyopus rubicundus, Trypauchen vagina and Ctenotrypauchen microcephalus. The results agreed with the traditional taxonomy of the morphological characters. AFLP fingerprints were successfully used to study the phylogenetic relationship of the gobioid fishes and it identified species origins of morphologically similar taxa.   Key words: Phylogenetic, amplified fragment length polymorphism (AFLP), gobiidae, Amblyopinae, gobiinae.

The relative contribution of band number to phylogenetic accuracy in AFLP data sets

We examined the effect of increasing the number of sampled amplified fragment length polymorphism (AFLP) bands to reconstruct an accurate and well-supported AFLP-based phylogeny. In silico AFLP was performed using simulated DNA sequences evolving along balanced and unbalanced model trees with recent, uniform and ancient radiations and average branch lengths (from the most internal node to the tip) ranging from 0.02 to 0.05 substitutions per site. Trees were estimated by minimum evolution (ME) and maximum parsimony (MP) methods from both DNA sequences and virtual AFLP fingerprints. The comparison of the true tree with the estimated AFLP trees suggests that moderate numbers of AFLP bands are necessary to recover the correct topology with high bootstrap support values (i.e. >70%). Fewer numbers of bands are necessary for shorter tree lengths and for balanced than for unbalanced tree topologies. However, branch length estimation was rather unreliable and did not improve substantially after a certain number of bands were sampled. These results hold for different levels of genome coverage and number of taxa analysed. In silico AFLP using bacterial genomic DNA sequences recovered a well-supported tree topology that mirrored an empirical phylogeny based on a set of 31 orthologous gene sequences when as few as 263 AFLP bands were scored. These results suggest that AFLPs may be an efficient alternative to traditional DNA sequencing for accurate topology reconstruction of shallow trees when not very short ancestral branches exist.

Genetic Risk Assessment of a Threatened Remnant Population of Hairy Prairie-Clover (Dalea villosa var. villosa) in the Canadian Prairie

Hairy prairie-clover [Dalea villosa (Nutt.) Spreng. var. villosa] is a threatened Canadian wildflower. To facilitate the efforts of conserving this threatened plant, amplified fragment length polymorphism (AFLP) technique was applied to assess genetic diversity in a remnant hairy prairie-clover population in the Canadian Prairie. Three AFLP primer pairs were employed to genotype 610 individual plants from the population and 15 plants from a North Dakota composite population, and 100 polymorphic AFLP bands were analyzed. The assayed plants displayed 23% AFLP variation present between the remnant population and the North Dakota composite population, but maintained a high level (91%) of AFLP variation within patches of the remnant population. The individual genetic distinctiveness measured by average AFLP dissimilarity was positively associated with latitude and negatively with elevation. The among-patch AFLP variation was significantly related to inter-patch distance, indicating local genetic differentiation within the remnant population. However, the proportions of within-patch AFLP variation were not associated with any patch characteristics assessed (i.e., patch size, perimeter, nearest neighbor distance, mean inter-patch distance). No fine-scale genetic structure was found within three large patches, suggesting little genetic correlations present for plants five meters apart. Some genetically distinctive and diverse patches were also identified. These findings indicate that the genetic risk of the remnant hairy prairie-clover population in the Canadian Prairie is low.

Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.

Amplified fragment length polymorphisms (AFLPs) analysis of species of Solanum section Solanum (Solanaceae ) from Uganda

The taxonomy of species belonging to Solanum section Solanum (sometimes referred to as the Solanum nigrum complex or black nightshades) is known to be difficult and has resulted in extensive synonymy. Yet, these species play a significant role in nutrition and food security, especially in developing countries. The amplified fragment length polymorphisms (AFLPs) technique was used to assess the genetic relationships among 107 accessions representing eight species of the section Solanum and to obtain new insight into the taxonomic status of the S. nigrum complex. Four primer combinations yielded 510 AFLP bands, only 29 of which were monomorphic. Neighbor-joining and principal coordinates analyses were performed. The AFLP data only partially correlated with earlier classifications based on morphology. We have been able to confirm the presence of only 5 out of eight species of this complex previously recognized from Uganda, that is, Solanum americanum, Solanum scabrum, Solanum hirsutum, Solanum florulentum/Solanum tarderemotum and Solanum villosum . In addition, S. villosum did not separate in accordance with previous subspecific ranks. It is likely that neither Solanum grossidentatum, S. nigrum nor Solanum sarrachoides occurred in Uganda. Also, there was no correlation between geographic localities for the Ugandan material and the AFLP results, indicating that most of the studied species are introduced. Key words: Amplified fragment length polymorphisms (AFLPs), Solanum nigum complex, Africa, genetic variation, taxonomy, Uganda.

First Evidence for Hybrid Breakdown in the Backcross of Olive Flounder, Paralichthys olivaceus, and Summer Flounder, Paralichthys dentatus

Hybrid and parental backcross experiments were performed using olive flounder, Paralichthys olivaceus, summer flounder, Paralichthys dentatus, and their female F(1) hybrids to examine hybrid fitness in the backcross. Fertilization rate, hatching rate, and combined fitness measure (CFM, product of fertilization rate and hatching rate) were detected and the results showed that the CFM of hybrid and backcross generations was reduced significantly compared to pure olive flounder crosses. Genomic inheritance from parents to backcross progeny was also detected by amplified fragment length polymorphism (AFLP). Almost all AFLP bands observed in parents were presented in backcross progeny (backcross1, 218/227; backcross2, 265/282), although 9 and 17 parental bands were missing in backcross1 and backcross2, respectively. Novel bands (absent in parents) also occurred in progeny at the frequencies of 5.63% (13/231) in backcross1 and 3.28% (9/274) in backcross2. Additionally, 27.40 and 31.18% of AFLP markers deviated from expected Mendelian ratio in backcross1 and backcross2, respectively. The present study suggests that genetic incompatibilities may exist between olive flounder and summer flounder. Furthermore, possible implications of segregation distortion for reduced hybrid fitness in backcross generations are discussed.

Application of AFLP markers for population genetic study on half-smooth tongue sole Cynoglossus semilaevis

The genetic diversity of wild and hatchery populations of half-smooth tongue sole Cynoglossus semilaevis, based on observation of amplified fragment length polymorphism (AFLP) was described. Two hundred individuals from four wild populations, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ), and one hatchery population, Mingbo (MB), were screened using eight different AFLP primer combinations. A total of 384 loci were screened in the five studied populations. 48.4%, 51.3%, 50.7%, 49.3% and 45.8% of these loci were polymorphic among the individuals tested in the LZ, WH, QD, RZ and MB populations, respectively. The number of polymorphic loci detected by single primer combinations ranged from 17 to 35. The average heterozygosity of the LZ, WH, QD, RZ and MB populations was 0.072, 0.093, 0.092, 0.090 and 0.063, respectively. The WH population showed the highest genetic diversity in terms of total number of AFLP bands, total number of polymorphic bands, average heterozygosity and percentage of low frequency (0–0.2) polymorphic loci among all the populations, while the LZ population was the lowest among the wild populations. Compared with the wild populations, the hatchery population showed a low genetic viability.

Enhanced AFLP genome scans detect local adaptation in high‐altitude populations of a small rodent (Microtus arvalis)

Adaptation to adverse environmental conditions such as high altitude requires physiological and/or morphological changes. Genome scans provide a means to identify the genetic basis of such adaptations without previous knowledge about the particular genetic variants or traits under selection. In this study, we scanned 3027 amplified fragment length polymorphisms (AFLP) in four populations of the common vole Microtus arvalis for loci associated with local adaptation and high altitude. We investigated voles from two populations at high elevation (~2000 m a.s.l.) representing the upper limit of the altitudinal distribution of the species and two geographically close low-altitude populations (<600 m a.s.l.). Statistical analysis incorporated a new Bayesian F(ST) outlier approach specifically developed for AFLP markers, which considers the intensity of AFLP bands instead of mere presence/absence and allows to derive population-based estimates of allele frequencies and F(IS) values. Computer simulations showed that this approach increases the statistical power of the detection of AFLP markers under selection almost to the power of single nucleotide polymorphism (SNP) data without compromising specificity. Our enhanced genome scan resulted in 20 prime candidate markers for positive selection, which show mostly extremely high allele frequency differences between the low- and high-altitude populations. The comparison of global- and pairwise-enhanced genome scans demonstrated further that very strong selective signatures may also be associated with single populations suggesting the importance of local adaptation in alpine populations of common voles.

Morphological and physiological characterization of two new pineapple somaclones derived from in vitro culture

We previously reported a partial agricultural and amplified fragment length polymorphism (AFLP) characterization of two new pineapple somaclones (P3R5 and Dwarf) derived from in vitro culture of the donor cv. Red Spanish Pinar. Both somaclonal variants showed different AFLP banding patterns compared to the donor cultivar, although they were separated by less than 0.09 U of genetic distance. The present report shows data of various indicators of morphology and physiology of P3R5 and Dwarf D leaves. The stoma diameter, number of stomata per square millimiter, diameter of leaf vascular tissue, thickness of the leaf aquiferous parenchyma, and thickness of the leaf photosynthetic parenchyma were measured. The photosynthetic rate, the transpiration rate, the water use efficiency, the internal leaf CO2 concentration, and the chlorophyll pigment contents were recorded as well. Between the somaclonal variant P3R5 and the donor genotype, statistically significant differences were recorded in all indicators with the exception of the stoma diameter and the photosynthetic rate. Comparing the somaclonal variant Dwarf and the cv. Red Spanish Pinar (donor), statistically significant differences were also recorded in all parameters except in the stoma diameter and in the transpiration rate. This investigation was performed to demonstrate that small changes in the pineapple DNA may result in relevant phenotypic modifications.

Allogyogenetic Progeny are Produced from a Hybrid Abalone Cross of FemaleHaliotis diversicolorand MaleHaliotis Discus Discus

ABSTRACT Interspecific hybrid families of female Haliotis diversicolor × male H. discus discus were produced and analyzed using amplified fragment length polymorphism (AFLP) technology to reveal the genetic makeup of F1 progenies. The survival rates of the hybrid F1 were very low, ranging from 0–0.13%. Twenty hybrid F1 from 3 families along with 3 different female parents and their common male parent were analyzed with 3 AFLP primer combinations. In total, 266 markers were detected. Genetic relationships among the progenies and the parents were evaluated by generating a similarity and genetic distance matrix. The genetic divergence between Haliotis diversicolor and Haliotis discus was at a high level, with genetic distance ranging from 1.471–1.492. The AFLP band patterns of hybrid F1 progeny were similar to those of the female parents, but were quite different from that of the male parent. The mean genetic distance between hybrid F1 and their female parents were 0.024–0.039, slightly less than that among ...

Bayesian statistical treatment of the fluorescence of AFLP bands leads to accurate genetic structure inference

Ever since the introduction of allozymes in the 1960s, evolutionary biologists and ecologists have continued to search for more powerful molecular markers to estimate important parameters such as effective population size and migration rates and to make inferences about the demographic history of populations, the relationships between individuals and the genetic architecture of phenotypic variation (Bensch & Akesson 2005; Bonin et al. 2007). Choosing a marker requires a thorough consideration of the trade-offs associated with the different techniques and the type of data obtained from them. Some markers can be very informative but require substantial amounts of start-up time (e.g. microsatellites), while others require very little time but are much less polymorphic. Amplified fragment length polymorphism (AFLP) is a firmly established molecular marker technique that falls in this latter category. AFLPs are widely distributed throughout the genome and can be used on organisms for which there is no a priori sequence information (Meudt & Clarke 2007). These properties together with their moderate cost and short start-up time have made them the method of choice for many molecular ecology studies of wild species (Bensch & Akesson 2005). However, they have a major disadvantage, they are dominant. This represents a very important limitation because many statistical genetics methods appropriate for molecular ecology studies require the use of codominant markers. In this issue, Foll et al. (2010) present an innovative hierarchical Bayesian method that overcomes this limitation. The proposed approach represents a comprehensive statistical treatment of the fluorescence of AFLP bands and leads to accurate inferences about the genetic structure of natural populations. Besides allowing a quasi-codominant treatment of AFLPs, this new method also solves the difficult problems posed by subjectivity in the scoring of AFLP bands.

Strong Population Genetic Structure in a Broadcast-Spawning Antarctic Marine Invertebrate

Although studies of population genetic structure are commonplace, a strong bias exists toward species from low latitudes and with relatively poor dispersal capabilities. Consequently, we used 280 amplified fragment length polymorphism bands to explore patterns of genetic differentiation among 8 populations of a high latitude broadcast-spawning marine mollusc, the Antarctic limpet Nacella concinna. Over 300 individuals were sampled along a latitudinal gradient spanning the Antarctic Peninsula from Adelaide Island to King George Island (67°-62°S), then to Signy Island (60°S) and South Georgia (54°S). Populations from the Antarctic Peninsula exhibited little genetic structure but were themselves strongly differentiated from both Signy and South Georgia. This finding was analytically highly robust and implies the presence of significant oceanographic barriers to gene flow in a species long regarded as a classic example of a widely dispersing broadcast spawner.

Partial isolation of the genomic region linked with apomixis in Paspalum simplex

Apomixis is a form of asexual reproduction through seed and has the potential to be applied, to great benefit, to agriculture. Understanding the genetic control of apomixis has proven to be a challenging task because the trait is mainly present in wild species and genetic mapping is often impaired by a block of recombination. A physical mapping approach has therefore been undertaken to unlock the genetic control of apomixis in Paspalum simplex Morong, a species with a relatively small genome and which exhibits a degree of genetic synteny with rice. In this paper, we report on the construction of a bacterial artificial chromosome library for Paspalum simplex with a coverage of approximately three genome equivalents and an average insert size of 94 kb. The BAC library was screened with 19 sequence characterized amplified region markers which were 100% linked to apomixis and a recombinant SCAR marker, all developed through a bulked segregant analysis strategy. A mini-sequencing procedure reported in the literature greatly aided the direct development of SCAR markers from amplified fragment length polymorphism bands. Several BAC clones linked to apomixis were identified and assembled into seven contigs and 18 singletons. Two of the BAC clones identified contained independently isolated markers. This is the first such report in an apomictic model that lacks recombination at the locus. We believe that extension of the contigs coupled to high-throughput sequencing will help the understanding of the genomic structure of the apomixis locus in P. simplex.

Contemporary habitat loss reduces genetic diversity in an ecologically specialized butterfly

AbstractAim This study investigated the influence of contemporary habitat loss on the genetic diversity and structure of animal species using a common, but ecologically specialized, butterfly, Theclinesthes albocincta (Lepidoptera: Lycaenidae), as a model.Location South Australia.Methods We used amplified fragment length polymorphism (AFLP) and allozyme datasets to investigate the genetic structure and genetic diversity among populations of T. albocincta in a fragmented landscape and compared this diversity and structure with that of populations in two nearby landscapes that have more continuous distributions of butterflies and their habitat. Butterflies were sampled from 15 sites and genotyped, first using 363 informative AFLP bands and then using 17 polymorphic allozyme loci (n = 248 and 254, respectively). We complemented these analyses with phylogeographic information based on mitochondrial DNA (mtDNA) haplotype information derived from a previous study in the same landscapes.Results Both datasets indicated a relatively high level of genetic structuring across the sampling range (AFLP, FST = 0.34; allozyme, FST = 0.13): structure was greatest among populations in the fragmented landscape (AFLP, FST = 0.15; allozyme, FST = 0.13). Populations in the fragmented landscape also had significantly lower genetic diversity than populations in the other two landscapes: there were no detectable differences in genetic diversity between the two continuous landscapes. There was also evidence (r2 = 0.33) of an isolation by distance effect across the sampled range of the species.Main conclusions The multiple lines of evidence, presented within a phylogeographic context, support the hypothesis that contemporary habitat fragmentation has been a major driver of genetic erosion and differentiation in this species. Theclinesthes albocincta populations in the fragmented landscape are thus likely to be at greater risk of extinction because of reduced genetic diversity, their isolation from conspecific subpopulations in other landscapes, and other extrinsic forces acting on their small population sizes. Our study provides compelling evidence that habitat loss and fragmentation have significant rapid impacts on the genetic diversity and structure of butterfly populations, especially specialist species with particular habitat preferences and poor dispersal abilities.

Comparative Assessment of SSR and AFLP Markers for Evaluation of Genetic Diversity and Conservation of Fig, Ficus carica L., Genetic Resources in Tunisia

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.

Population Structure ofPhthorimaea operculella(Lepidoptera: Gelechiidae) in the United States

The potato tuberworm, Phthorimaea operculella Zeller, is one of the most damaging pests of potatoes worldwide. Although tuberworm was recorded in California as early as 1856, it was first reported in damaging numbers in Oregon and Washington in the early 2000s. The objective of this study was to provide baseline information on the population structure of potato tuberworm in the United States. Potato tuberworm adults were collected from potato fields in the major production regions in the United States. Amplified fragment length polymorphisms (AFLP) were used to determine the genetic population structure of potato tuberworm. We used 219 individuals and 335 polymorphic AFLP bands to infer the geographic population structure of potato tuberworm. Posterior probabilities calculated using the model-based clustering method implemented in STRUCTURE, and F(st) values calculated using AFLP-SURV confirmed the existence of two distinct populations in the United States (one mostly associated to the western United States and the other one mostly associated to the eastern United States). This study provides baseline data for the molecular characterization of potato tuberworm populations, which will aid in tracking the origin of future invasions within the United States.

Investigation of possible horizontal gene transfer from transgenic rice to soil microorganisms in paddy rice field

In order to monitor the possibility of horizontal gene transfer between transgenic rice and microorganisms in paddy rice field, the gene flow from bifunctional fusion (TPSP) rice containing trehalose-6-phosphate synthase and phosphatase to microorganisms in soils was investigated. The soil samples collected every month from the paddy rice field during June, 2004 to March, 2006 were investigated by multiplex PCR, Southern hybridization, and amplified fragment length polymorphism (AFLP). The TPSP gene from soil genomics DNAs was not detected by PCR. Soil genomic DNAs were not shown its homologies on the Southern blotting data, indicating that gene-transfer did not occur during the last two years in paddy rice field. In addition, the AFLP band patterns produced by both soil genomic DNAs extracted from transgenic and non-transgenic rice field appeared similar to each other when analyzed by NTSYSpc program. Thus, these data suggest that transgenic rice does not give a significant impact on the communities of soil microorganisms although long-term observation may be needed.