Amplified Fragment Length Polymorphism Markers Research Articles

Analysis of the genetic diversity and population structure of Perinereis aibuhitensis in China using TRAP and AFLP markers

Perinereis aibuhitensis is found in the coastal waters of northwestern Pacific. Different ecotypes are distributed throughout the coastal sea beach based on their ecology and behavior, but relatively little is known about the population structure of this species. In this study, we estimated the genetic relationships within P. aibuhitensis using Target Region Amplified Polymorphisms (TRAP) and Amplified Fragment Length Polymorphisms (AFLP) that were derived from related populations on the coasts of China. All populations were uniquely fingerprinted using the TRAP and AFLP marker method. The mean genetic distance was estimated to be 0.1859 based on the TRAPs and AFLPs. Using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) by Molecular Evolutionary Genetics Analysis (MEGA) 6.06 software, Principal Coordinate Analysis (PCA) and STRUCTURE analysis, the phylogenetic tree of the ten P. aibuhitensis populations was separated into four major clusters; however, the mantel test indicated that there was no significant correlation between the genetic and geographical distances due to gene differentiation and gene flow (P > 0.005). The genetic diversity was low for P. aibuhitensis at the population level compared with the species level. Finally, an appropriate strategy for conserving the P. aibuhitensis germplasm is proposed.

Assessment of genetic diversity among faba bean genotypes using agro-morphological and molecular markers

Forty faba bean (Vicia faba L.) genotypes were evaluated for their agro-morphological performance and molecular diversity under Central Region of Saudi Arabia conditions during 2010–11 and 2011–12 seasons. Field performance results showed that faba genotypes exhibited a significant amount of variation for their agro-morphological studied parameters. Giza40 recorded the tallest genotype (139.5cm), highest number of seeds per plants (100.8), and the highest seed yield per plant (70.8g). The best performing genotypes were Giza40, FLIP03-014FB, Gazira1 and Goff1. Genetic variability among genotypes was determined using Sequence Related Amplified Polymorphism (SRAP) and Amplified Fragment Length Polymorphism (AFLP) markers. A total of 183 amplified fragments (alleles) and 1758 polymorphic fragments (bands) in SRAP and 202 alleles and 716 bands in AFLP were obtained using six SRAP and four AFLP primer combinations respectively. Polymorphism information content (PIC) values for AFLP and SRAP markers were higher than 0.8, indicating the existence of a considerable amount of genetic diversity among faba tested genotypes. The UPGMA based clustering of faba genotypes was largely based on origin and/or genetic background. Result of cluster analysis based on SRAP showed weak and not significant correlation while, it was highly significant based on AFLP analysis with agro-morphological characters (r=0.01, p>0.54 and r=0.26, p<0.004 respectively). Combined SRAP and AFLP markers proved to be significantly useful for genetic diversity assessment at molecular level. They exhibited high discrimination power, and were able to distinguish the faba bean genotypes with high efficiency and accuracy levels.

The possible physical barrier and coastal dispersal strategy for Japanese grenadier anchovy, Coilia nasus in the East China Sea and Yellow Sea: evidence from AFLP markers.

In order to ascertain the taxonomic status of the Ariake Sea population of Japanese grenadier anchovy, Coilia nasus, and assess the contemporary possible genetic barrier between the west and east coastal waters of the East China Sea, we used amplified fragment length polymorphism (AFLP) markers to detect the genetic structure of C. nasus, in the East China Sea and Yellow Sea. Eighty-one individuals of C. nasus were collected from five locations and 12 individuals of Coilia mystus were sampled from the Yangtze River Estuary. A total of 371 loci were detected by five primer combinations, 310 of which were polymorphic (83.56%). Analysis of molecular variation (AMOVA) and pairwise fixation index (FST) revealed significant genetic differentiation among five samples, indicating limited gene flow among populations. The dendrogram for populations by neighbor-joining (NJ) cluster analysis provided evidence of a clear relationship between genetic and geographic patterns, supporting significant genetic differentiation between China coastal populations and Ariake Sea populations. Compared to the genetic divergence between C. nasus and C. mystus, the level of genetic differentiation between China and the Ariake Sea populations of C. nasus is obvious below the species level, indicating isolated populations of C. nasus in the Ariake Sea. Isolation by distance analysis revealed that direct ocean distance with deep-water at the continental slope and high salinity between west and east coastal waters of the East China Sea served as major physical barrier to C. nasus, supporting the coastal dispersal pattern in this estuarine species, and rejecting offshore dispersal strategy.

Continuous within-plant variation as a source of intraspecific functional diversity: Patterns, magnitude, and genetic correlates of leaf variability in Helleborus foetidus (Ranunculaceae).

Continuous within-plant variation in quantitative traits of reiterated, homologous structures is a component of intraspecific variation, but its contribution to functional diversity remains largely unexplored. For the perennial Helleborus foetidus, we measured functional leaf traits to quantify the contribution of within-plant variation to intraspecific functional variance and evaluate whether within-plant variability itself deserves separate consideration. Within-individual variation in eight leaf traits was quantified for 138 plants sampled from 10 widely spaced locations in the Sierra de Cazorla, southeastern Spain. An amplified fragment length polymorphism (AFLP) technique was used to look for associations between within-plant variability and specific AFLP markers. Leaflets from basal positions in ramets were longer, heavier, had greater surface area and larger stomata, and lower specific area, stomatal index, and stomatal density than those from distal positions. Continuous variation between leaves from the same ramet was the main source of population-wide variance for most traits. Within-plant variability differed among populations. Individuals differed in within-plant variability, which was largely independent of trait means and associated with genetic characteristics. Up to four AFLP markers were associated with the within-plant variability level of a given leaf trait. Subindividual variability in continuous leaf traits was independent of plant means and related to genetic features. The within-individual component generally exceeded the between-individual component of intraspecific variance. Within-plant variation may broaden the ecological breadth and enhance stability and persistence of plant populations and communities and may provide novel insights when incorporated in trait-based community ecology models.

Effects of domestication on genetic diversity in Chimonanthus praecox: Evidence from chloroplast DNA and amplified fragment length polymorphism data

AbstractChimonanthus praecox (L.) Link is a widely cultivated endemic winter‐flowering plant in China that has a long cultivation history. Genetic diversity and genetic structure were compared between wild and cultivated groups to reveal the geographic origin of the cultivated genotypes using chloroplast DNA (cpDNA) sequences and amplified fragment length polymorphism (AFLP) markers. Nine haplotypes were identified using three combined chloroplast fragments. Based on chloroplast data, the wild group showed greater genetic variation and genetic differentiation and a lower measure of gene flow compared to the cultivated group. The AFLP markers also supported this trend. More than 40% of the cpDNA haplotypes were shared between wild and cultivated groups, with shared haplotypes originating from multiple wild populations, suggesting multiple origins of cultivated plants. Moreover, principal coordinate analysis, UPGMA, and structure analysis of AFLP markers revealed that two wild populations clustered with most of the cultivated populations of Ch. praecox, suggesting that most of the cultivated populations mainly originated from these two populations. The combined cpDNA and AFLP results indicated that modern cultivated Ch. praecox experienced multiple events of origin involving two geographic origins, eastern China (Tianmu Mountain) and southwestern China (the border of Hunan–Guangxi–Sichuan–Guizhou).

Intraspecific variability in frost hardiness of Fagus sylvatica L.

This study investigated the intraspecific variability of frost hardiness of Fagus sylvatica. We tested for local adaptation by relating the frost hardiness of different provenances to the climatic conditions at the populations’ origin and searched for genetic markers that coincided with frost hardiness. Twenty provenances of F. sylvatica were selected covering the major part of the climatic gradient within the species’ range. Frost hardiness was assessed in winter and tested in a climate test chamber by exposing buds to different freezing temperatures and estimating LT 50-values by the electrolyte leakage method. Additionally, the genotypes of all investigated provenances were analyzed using amplified fragment length polymorphism (AFLP) fingerprinting. The frost hardiness differed up to 10.3 K between provenances. In contrast to our expectation, we did not find any relationship between LT 50 and climate variables. Although the populations were not well differentiated by AFLP markers, the first PCoA axis of all loci of seven different primers was strongly related to LT 50-values. Linear regressions showed that frost hardiness could be predicted from the presence/absence of 12 loci. The high intraspecific variation in frost hardiness revealed a high potential of this species to different climates. The ability to withstand low temperatures was neither related to the species’ phylogeography, nor to the current climatic conditions of provenances. This points to a more recent evolution of frost hardiness and points to a link of frost hardiness to other characteristics (e.g., drought tolerance), which might have been subjected to other selection pressures than low temperatures.

Diversity in the breadfruit complex (Artocarpus, Moraceae): genetic characterization of critical germplasm

Breadfruit (Artocarpus altilis, Moraceae) is a traditional staple crop in Oceania and has been introduced throughout the tropics. This study examines important germplasm collections of breadfruit and its closest wild relatives and aims to (1) characterize genetic diversity, including identification of unknown and duplicate accessions, (2) evaluate genetic structure and hybridization within the breadfruit complex, and (3) compare utility of microsatellite markers to previously reported amplified fragment length polymorphism (AFLP) and isozyme markers in differentiating among cultivars. Data for 19 microsatellite loci were collected for 349 individuals (representing 255 accessions) including breadfruit (A. altilis), two wild relatives (Artocarpus camansi and Artocarpus mariannensis), and putative hybrids (A. altilis × A. mariannensis). Accessions were of mixed ploidy and regional origin, but predominantly from Oceania. Microsatellite loci collectively had a polymorphic information content (PIC) of 0.627 and distinguished 197 unique genotypes sorted into 129 different lineages, but a single genotype accounts for 49 % of all triploid breadfruit examined. Triploid hybrids and diploid A. altilis exhibited the highest levels of diversity as measured by allele number and gene diversity. Most accessions (75 %) of unknown origin matched either a known genotype or lineage group in the collection. Putative hybrids all had genetic contributions from A. mariannensis but ranged in the level of genetic contribution from A. altilis. Microsatellite markers were found to be more informative than isozyme markers and slightly less informative, with regard to accession discrimination, than AFLP markers. This set of microsatellite markers and the dataset presented here will be valuable for breadfruit germplasm management and conservation.

Identification of molecular markers associated with fruit traits in olive and assessment of olive core collection with AFLP markers and fruit traits.

The purpose of this study was to characterize olive core collection with amplified fragment length polymorphism (AFLP) markers and fruit traits and to determine AFLP markers significantly associated with these fruit characters in olive. A total of 168 polymorphic AFLP markers generated by five primer combinations and nine fruit traits were used to characterize relationships between 18 olive cultivars. Although all olive cultivars were discriminated from each other by either AFLP markers (<0.75 similarity level) or fruit traits, clustering based on the AFLP markers and fruit traits was not significantly correlated (r = 0.13). Partial clustering of olive cultivars by AFLP markers according to their geographical origin was observed. Associations of AFLP markers with fruits were determined using a multiple-regression analysis with stepwise addition of AFLP markers. Significant associations between eight AFLP markers and fruit traits were identified. While five AFLP markers demonstrated significant negative correlation with fruit and stone weight, width and length and total polyphenols (P < 0.05), three AFLP markers displayed significant positive correlation with α-tocopherol and γ-tocopherol (P < 0.01). This is the first report on the association of molecular markers with fruit traits in olive. Molecular markers associated with morphological and agronomic traits could be utilized for the breeding of olive cultivars. However, the association power of these markers needs to be confirmed in larger populations, and highly correlated markers should then be converted to PCR-based DNA markers such as sequence-characterized amplified region markers for better utilization.

Genetic structure of Pilosocereus gounellei (Cactaceae) as revealed by AFLP marker to guide proposals for improvement and restoration of degraded areas in Caatinga biome.

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate DNA polymorphism in Pilosocereus gounellei with the aim of differentiating samples grown in different Brazilian semiarid regions. Seven primer pairs were used to amplify 703 AFLP markers, of which 700 (99.21%) markers were polymorphic. The percentage of polymorphic markers ranged from 95.3% for the primer combination E-AAG/M-CTT to 100% for E-ACC/M-CAT, E-ACC/M-CAA, E-AGC/M-CAG, E-ACT/M-CTA, and E-AGG/M-CTG. The largest number of informative markers (126) was detected using the primer combination E-AAC/M-CTA. Polymorphism of the amplified DNA fragments ranged from 72.55% (in sample from Piauí State) to 82.79% (in samples from Rio Grande Norte State), with an average of 75.39%. Despite the high genetic diversity of AFLP markers in xiquexique, analysis using the STRUCTURE software identified relatively homogeneous clusters of xiquexique from the same location, indicating a differentiation at the molecular level, among the plant samples from different regions of the Caatinga biome. The AFLP methodology identified genetically homogeneous and contrasting plants, as well as plants from different regions with common DNA markers. Seeds from such plants can be used for further propagation of plants for establishment of biodiversity conservation units and restoration of degraded areas of the Caatinga biome.

Analysis of genetic diversity identified by amplified fragment length polymorphism marker in hybrid wheat.

Amplified fragment length polymorphism markers were used to assess genetic diversity in 10 male sterile wheat crop lines (hetero-cytoplasm with the same nucleus) in relation to a restorer wheat line. These male sterile lines were evaluated using 64 amplified fragment length polymorphism primer combinations, and 13 primers produced polymorphic bands, generating a total 682 fragments. Of the 682 fragments, 113 were polymorphic. The polymorphic information content and marker index values demonstrated the utility of the primer combinations used in the present study. Unweighted pair group method with arithmetic mean and principal coordinate analysis of the genotypic data revealed clustering of accessions based on genetic relationships, and accessions were separated into 2 groups with their restorer line. Jaccard's similarity coefficient values suggested good variability among the male sterile lines, indicating their utility in breeding programs. The fallouts of analysis of molecular variance showed large within-group population variation, accounting for 77% of variation, while among-group comparison accounted for 23% of the total molecular variation, which was statistically significant. The molecular diversity observed in this study will be useful for selecting appropriate accessions for plant improvement and hybridization through molecular-breeding approaches and for developing suitable conservation strategies.

Development of AFLP markers associated with zucchini yellow mosaicvirus resistance in cucumber (Cucumis sativus L.)

Zucchini yellow mosaic virus (ZYMV) is one of the most important pathogens that cause significant yield losses in many cucurbit crops including cucumber (Cucumis sativus L). ZYMV resistance in cucumber is inherited by a single recessive gene. The purpose of this study was to identify molecular markers linked to the gene conferring ZYMV resistance in cucumber. We developed a population of 188 F2 plants derived from inbred cucumber lines. Individual F2 plants were self-pollinated to generate F3 populations. Ten randomly selected plants from each F3 population were tested for ZYMV resistance. We used a bulk segregant analysis method to identify putative molecular markers linked to ZYMV resistance. Using bulked DNA samples with parental lines and F1, a total of 170 sequence-related amplified polymorphism (SRAP), 586 simple sequence repeat (SSR), and 308 amplified fragment length polymorphism (AFLP) primer combinations were screened. Neither polymorphic SRAP nor SSR markers were linked with ZYMV resistance. Among the 308 AFLP primer combinations tested, an AFLP marker in the E-ACA/MCA primer combination showed significant association among parental lines, F1, and resistant and susceptible plants. The combination of E-ACA/M-CA was achieved on parental lines, F1, and 188 F2 individuals for confirmation of the marker segregation on the F2 population. We found that the combination of E-ACA/M-CA was linked to the zym locus with 6.91 cM.

Characterization of the Solanum Nigrum Complex of Kenya by AFLP Markers

Species of the Solanum nigrum complex are known to have a long tradition as alternative food- or fodder plants and a significant medical value in parts of Africa, India, Indonesia and China. Mature berries are often edible and cooked leaves are used as herbs and vegetables. The taxonomy of the S. nigrum complex is difficult, especially because of extensive synonymy. Amplified fragment length polymorphism (AFLP) technique was used to assess the genetic relationship of twelve accessions from Kenya and Germany. Distances on the basis of molecular markers were only partially correlated to morphological descriptions. Intermixing of S. nigrum and S. scabrum accessions suggests close relationship of both species.

Genetic diversity of Kenyan native oyster mushroom (Pleurotus)

Members of the genus Pleurotus, also commonly known as oyster mushroom, are well known for their socioeconomic and biotechnological potentials. Despite being one of the most important edible fungi, the scarce information about the genetic diversity of the species in natural populations has limited their sustainable utilization. A total of 71 isolates of Pleurotus species were collected from three natural populations: 25 isolates were obtained from Kakamega forest, 34 isolates from Arabuko Sokoke forest and 12 isolates from Mount Kenya forest. Amplified fragment length polymorphism (AFLP) was applied to thirteen isolates of locally grown Pleurotus species obtained from laboratory samples using five primer pair combinations. AFLP markers and internal transcribed spacer (ITS) sequences of the ribosomal DNA were used to estimate the genetic diversity and evaluate phylogenetic relationships, respectively, among and within populations. The five primer pair combinations generated 293 polymorphic loci across the 84 isolates. The mean genetic diversity among the populations was 0.25 with the population from Arabuko Sokoke having higher (0.27) diversity estimates compared to Mount Kenya population (0.24). Diversity between the isolates from the natural population (0.25) and commercial cultivars (0.24) did not differ significantly. However, diversity was greater within (89%; P > 0.001) populations than among populations. Homology search analysis against the GenBank database using 16 rDNA ITS sequences randomly selected from the two clades of AFLP dendrogram revealed three mushroom species: P. djamor, P. floridanus and P. sapidus; the three mushrooms form part of the diversity of Pleurotus species in Kenya. The broad diversity within the Kenyan Pleurotus species suggests the possibility of obtaining native strains suitable for commercial cultivation.

Genetic variation in wild populations of the tuber crop Amorphophallus konjac (Araceae) in central China as revealed by AFLP markers.

Amorphophallus konjac is an economically important crop. In order to provide baseline information for sustainable development and conservation of the wild plant resources of A. konjac, we studied the genetic diversity and population structure of this species using amplified fragment length polymorphism (AFLP) molecular markers. We sampled 139 individuals from 10 wild populations of A. konjac in central China. Using five AFLP primer combinations, we scored a total of 270 DNA fragments, most of which were polymorphic (98.2%). Percentage of polymorphic loci, Nei's genetic diversity index, and Shannon's information index showed high levels of genetic variation within A. konjac populations. Analysis of molecular variance indicated that most of the variance (68%) resided within populations. The coefficient of genetic differentiation between populations was 0.348 and the estimated gene flow was 0.469, indicating that there was limited gene flow among the populations. Unweighted pair group method with arithmetic mean cluster analysis and principal coordinates analysis indicated that geographically close populations were more likely to cluster together. The Mantel test revealed a significant correlation between geographic and genetic distances (R2 = 0.2521, P < 0.05). The special insect-pollination system of A. konjac and the complex geography of central China are likely to have contributed to the current pattern of genetic variation of this species. In the present study, we provide several suggestions on the future protection of the wild plant genetic resources of A. konjac.

Genetic Diversity Analysis of Jatropha Species from Costa Rica Using AFLP Markers

The genetic diversity from species of the genus Jatropha collected from Costa Rica was analyzed by AFLP (amplified fragments length polymorphism). The study consisted of 114 accessions from 15 populations of 4 different species: J. curcas, J. costaricensis, J. gossypifolia and J. stevensii. These were collected from different locations in Costa Rica. Three different primers were used, resulting in 428 loci, and they were classified in three categories: unique and double bands (UBD), rare bands (RB) and shared bands (SB). The UBD were excluded for a total of 339 polymorphic loci used for the UPGMA dendrogram and principal component analysis (PCA). The species that obtained the highest average of polymorphic loci was J. curcas, which obtained the highest percentage of polymorphic loci (80.24%), followed by J. gossypifolia (79.35%), J. costaricensis (78.76%), and finally J. stevensii (40.71%). The average for the polymorphic loci was of 69.76%. Thus, the phylogeny of the Jatropha species in Costa Rica was elucidated, showing J. curcas more related to J. stevensii and this one with J. costaricensis, and J. gossypifolia as the most distant member of the genus. For the best of our knowledge this is the first report of a genetic analysis of J. costaricensis and J. stevensii. The obtained molecular evidence showed high levels of polymorphisms in the present study compared with reports from Africa, India, and China. The molecular diversity estimated in our analysis, together with agronomical or morphological data, can be very useful for plant breeding programs, given the importance of Jatropha species in oil production. Interestingly, Central American Jatropha material can be used to increase the genetic base of J. curcas populations localized in Asia and Africa, where reduced genetic diversity has been reported.

Molecular and chemical characterization of vetiver, Chrysopogon zizanioides (L.) Roberty, germplasm

Due to the economic interests in vetiver, Chrysopogon zizanioides (L.) Roberty, molecular and chemical studies are essential to generate information for its sustainable exploitation. The aim of this study was to undertake a molecular and chemical characterization of vetiver accessions of the active germplasm bank of the Universidade Federal de Sergipe. The molecular characteristics of the accessions were studied using amplified fragment length polymorphism markers, with a total of 14 primer combinations that generated 442 loci, allowing us to observe that these accessions have similar genomes. The vetiver accessions were divided into three distinct groups, where accession UFS-VET005 was the most differentiated and accession UFS-VET004 had the lowest essential oil content (0.70%). The content of the chemical constituents of the essential oils was observed to vary, with a predominance of khusimol, which ranged from 18.97 to 25.02%. It was possible to divide the vetiver accessions into two groups based on chemical composition, and these groups do not correlate with the molecular grouping. Therefore, it is necessary to perform molecular and chemical analyses to characterize vetiver accessions.

Screening of molecular markers linked to dwarf trait in crape myrtle by bulked segregant analysis.

Plant height is one of the most important traits of plant architecture as it modulates both economic and ornamental values. Crape myrtle (Lagerstroemia indica L.) is a popular ornamental woody plant because of its long-lasting mid-summer bloom, rich colors, and diversified plant architecture. These traits also make it an ideal model of woody species for genetic analysis of many ornamental traits. To understand the inheritance of plant height and screen for genes modulating plant height in Lagerstroemia, segregation of the plant height trait was analyzed using the F1 population of L. fauriei (standard) x L. indica 'Pocomoke' (dwarf) with 96 seedlings, while dwarf genes were screened using the bulked segregant analysis method, combined with 28 amplified fragment length polymorphism primers and 41 simple sequence repeat primers. The results showed that the dwarf trait of crape myrtle was controlled by a major gene and modified by minor genes. An amplified fragment length polymorphism marker, M53E39-92, which was 23.33 cM from the loci controlling the dwarf trait, was screened. These results provide basic information for marker-assisted selection in Lagerstromia and cloning of dwarf genes in future studies.

Genetic variation and genetic structure of the endangered species Sinowilsonia henryi Hemsi. (Hamamelidaceae) revealed by amplified fragment length polymorphism (AFLP) markers.

Comprehensive research of genetic variation is crucial in designing conservation strategies for endangered and threatened species. Sinowilsonia henryi Hemsi. is a tertiary relic with a limited geographical distribution in the central and western areas of China. It is endangered because of climate change and habitat fragmentation over the last thousands of years. In this study, amplified fragment length polymorphism markers were utilized to estimate genetic diversity and genetic structure in and among S. henryi. In this study, Nei's genetic diversity and Shannon's information index were found to be 0.192 and 0.325 respectively, indicating a moderate-to-high genetic diversity in species. According to analysis of molecular variation results, 32% of the genetic variation was shown to be partitioned among populations, demonstrating a relatively high genetic divergence; this was supported by principal coordinate analysis and unweighted pair-group method with arithmetic average analysis. Moreover, the Mantel test showed that there was no significant correlation between genetic and geographical distances. The above results can be explained by the effects of habitat fragmentation, history traits, and gene drift. Based on the results, several implications were indicated and suggestions proposed for preservation strategies for this species.

AFLP Fingerprinting for Identification of Infra-Species Groups of Rhizoctonia solani and Waitea circinata

Patch diseases caused by Thanatephorus cucumeris (Frank) Donk and Waitea circinata Warcup and Talbot varieties (anamorphs: Rhizoctonia species) pose a serious threat to successful maintenance of several important turfgrass species. Reliance on field symptoms to identify Rhizoctonia causal agents can be difficult and misleading. Different Rhizoctonia species and Anastomosis Groups (AGs) vary in sensitivity to commonly applied fungicides and they also have different temperature ranges conducive for causing disease. Thus correct identification of the causal pathogen is important to predict disease progression and make future disease management decisions. Grouping Rhizoctonia species by anastomosis reactions is difficult and time consuming. Identification of Rhizoctonia isolates by sequencing Internal Transcribed Spacer (ITS) region can be cost prohibitive. Some Rhizoctonia isolates are difficult to sequence due to polymorphism of the ITS region. Amplified Fragment Length Polymorphism (AFLP) is a reliable and cost effective fingerprinting method for investigating genetic diversity of many organisms. No detailed analyses have been done to determine the suitability of AFLP for inferring infra-species level of Rhizoctonia isolates. The objective of the present study was to develop AFLP fingerprinting to identify infra-species level of unknown R. solani Kuhn and W. circinata isolates. Seventy-nine previously characterized R. solani (n=55) and W. circinata (n=24) isolates were analyzed with AFLP markers generated by four primer pairs. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) correctly grouped R. solani and W.circinata isolates according to their AG, AG subgroup or W.circinata variety. Principle component analysis (PCA) corroborated UPGMA clusters. To our knowledge this is the first time AFLP analysis has been tested as a method to decipher the AG, AG subgroup or W.circinata variety across a wide range of Rhizoctonia isolates.

Phylogeography ofAgkistrodon piscivoruswith Emphasis on the Western Limit of Its Range

The Cottonmouth, Agkistrodon piscivorus, is a semi-aquatic pitviper that occupies the southeastern U.S. west into Texas. Several previous studies have investigated the biogeographic history of A. piscivorus. It has been hypothesized that A. piscivorus was split into two separate populations during the last glacial maximum and climate change has impacted its distribution. Additionally, a geographically isolated population of A. piscivorus occurs at the western limit of the species' range in the Concho Valley of Texas. To investigate biogeography and population structure within A. piscivorus in Texas and throughout its range, we utilized amplified fragment length polymorphism (AFLP) and sequence data from cytochrome b (cyt-b). The AFLP data indicate a lack of gene flow between the population of A. piscivorus in the Concho Valley and other nearby populations. However, based on cyt-b, there is no genetic differentiation. The AFLP data for the entire species show a signature of two historic populations that have recently come into secondary contact. Finding two historic populations is consistent with previously published data based on mitochondrial DNA analyses; however, due to the rapid evolution rate of AFLP data, we were able to detect a high level of gene flow between these populations. We conclude that it is possible Texas and Florida served as refugia for A. piscivorus during the last glacial maximum, and, as the glaciers receded, the two populations expanded, coming into secondary contact. The subsequent gene flow has resulted in shared loci across the two populations. The difference between the conclusions drawn between our two markers and previous research is due to the different time scales that AFLP and cyt-b markers measure. The AFLP data provided a contemporary marker and cyt-b indicated historic separation.