Genetic characterization of Iranian native Bombyx mori strains using amplified fragment length polymorphism markers

Genetic relationships within and among seven Iranian native silkworm strains was determined by DNA fingerprinting by using amplified fragment length polymorphism (AFLP) markers. In total, 189 informative AFLP markers were generated and analyzed. Estimates of Nei's gene diversity for all loci in individual strains showed a higher degree of genetic similarity within each studied strain. The highest and the least degrees of gene diversity were related to Khorasan Pink (h = 0.1804) and Baghdadi (h = 0.1412) strains, respectively. The unweighted pair-group method with arithmetic average dendrogram revealed seven strains of silkworm, Bombyx mori (L.), resolving into two major clusters. The highest degree of genetic similarity was related to Baghdadi and Harati White, and the least degree was related to Guilan Orange and Harati Yellow. The genetic similarity estimated within and among silkworms could be explained by the pedigrees, historical and geographical distribution of the strains, effective population size, inbreeding rate, selection intensity, and gene flow. This study revealed that the variability of DNA fingerprints within and among silkworm strains could provide an essential basis for breeders in planning crossbreeding strategies to produce potentially hetrotic hybrids in addition to contributing in conservation programs.     

Trypanosoma brucei gambiense: study of population genetic structure of Central African stocks using amplified fragment length polymorphism (AFLP)

To understand the maintenance and resurgence of historical Human African Trypanosomiasis (HAT) foci, AFLP was used to genotype 100 Central African Trypanosoma brucei s.l. stocks. This technique confirmed the high genetic stability of T. b. gambiense group 1 stocks and the micro genetic variability within Central African T. b. gambiense stocks. It revealed several T. b. gambiense genotypes and allowed the identification of minor and major genotypes in HAT foci. The coexistence of these genotypes in the same focus suggests that clustering of stocks according to HAT focus does not provide the true genetic picture of trypanosome circulating within the disease focus because the minor genotypes are generally underestimated. The presence of minor and major genotypes in HAT foci may explain the persistence and the resurgence of Central African sleeping sickness foci.     

Inheritance of amplified fragment length polymorphism markers and their utility in population genetic analysis of Plecoglossus altivelis

The reproducibility, mode of inheritance and polymorphism of amplified fragment length polymorphism (AFLP) markers were examined in ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae). The AFLP markers were highly reproducible, their inheritance following Mendelian expectations. The number of fragments amplified (34–134), polymorphic ratio (0·15–0·78) and average heterozygosity (0·02–0·25) of the AFLP markers showed significant variation among six primer pairs and among ayu populations, including a landlocked Lake‐Biwa population, two amphidromous populations ( P. a. altivelis ) and two Ryukyu‐ayu populations ( P. a. ryukyuensis ). Although AFLP analysis provided similar results in intra‐population diversity and relationships among populations to those found by analyses of allozymes, microsatellites and mitochondrial DNA sequences, AFLPs showed higher polymorphisms and hence greater distinction between genetically close populations.     

Inferring population history with DIY ABC: a user-friendly approach to approximate Bayesian computation

Genetic data obtained on population samples convey information about their evolutionary history. Inference methods can extract part of this information but they require sophisticated statistical techniques that have been made available to the biologist community (through computer programs) only for simple and standard situations typically involving a small number of samples. We propose here a computer program (DIY ABC) for inference based on approximate Bayesian computation (ABC), in which scenarios can be customized by the user to fit many complex situations involving any number of populations and samples. Such scenarios involve any combination of population divergences, admixtures and population size changes. DIY ABC can be used to compare competing scenarios, estimate parameters for one or more scenarios and compute bias and precision measures for a given scenario and known values of parameters (the current version applies to unlinked microsatellite data). This article describes key methods used in the program and provides its main features. The analysis of one simulated and one real dataset, both with complex evolutionary scenarios, illustrates the main possibilities of DIY ABC. AVAILABILITY: The software DIY ABC is freely available at http://www.montpellier.inra.fr/CBGP/diyabc.     

Use of amplified fragment length polymorphism markers to assess genetic diversity of Lolium species from Portugal

We evaluated the use of amplified fragment length polymorphism (AFLP) markers to distinguish genotypes, populations and species of Lolium. Accessions of two species Lolium perenne and Lolium multiflorum and their hybrid Lolium x hybridum, collected by the Institute of Grass and Environmental Research in 1995 from locations across Portugal, were used. The genetic variation within and between populations from the extremes of latitude and altitude was determined and assessed. Three primer pair combinations generated 765 polymorphic bands. Principal coordinate analysis of similarities between 127 plants showed high dimensionality in the data. Axes 1-3 were associated primarily with species differences, axes 4-14 with population differences within species and axis 15 onwards with within population differences. UPGMA analysis confirmed the groupings. The three populations of L. perenne formed a discrete cluster widely separated from all other populations. There were two distinct groups of L. x hybridum, of which one was similar to and overlapped with L. multiflorum and the second formed a distinct cluster. Analyses of individual bands showed that every inter- and intraspecific contrast involved a different sets of bands, again confirming the high dimensionality of the data. No single band was strictly diagnostic of any population or species. Nevertheless, the UPGMA analysis showed little or no overlap between populations. Thus, despite the high ratio of within-to-between population genetic variance, the full AFLP banding pattern of each genotype is a relatively reliable fingerprint diagnostic of its parent population. The high dimensionality implies that many different factors contribute to the differences observed. This adds to the potential value of the methodology, since it implies that there is a reasonably high likelihood of finding bands relevant to a given environmental gradient or other factor influencing the distribution of genetic diversity.     

Genetic diversity and structure of urban populations of Pieris butterflies assessed using amplified fragment length polymorphism

Conservation programs in urban ecosystems need to determine the genetic background in populations of urban dwellers. We examined the genetic diversity and structure of Pieris rapae and P. melete using AFLP markers, and compared them between species and between urban and rural environments. As a result: (i). in both species, there was no reduction in genetic diversity within urban populations by direct comparison of diversity measurements, although the analysis of molecular variance suggested significant reductions in the variance within seasonal subpopulations in urban populations; (ii). P. rapae retained greater genetic diversity within species and populations; (iii). populations of both species showed significant genetic differentiation, and P. melete was more strongly subdivided; (iv). in both species, geographically close populations did not cluster with one another in the upgma analysis; (v). there was no genetic isolation due to geographical distance in either species; (vi). the genetic composition of seasonal subpopulations differed in urban populations of both species, and the genetic distances among subpopulations were correlated with seasonal differences in P. rapae and with temporal differences in P. melete. These results indicate that the genetic diversity in urban populations of both species was reduced at times, but was maintained by dispersal from genetically differentiated populations. Differences in the ability and mode of dispersal in the two species may be reflected in the degree of population subdivision and patterns of seasonal change in the genetic composition.     

A Bayesian approach to inferring population structure from dominant markers

Molecular markers derived from polymerase chain reaction (PCR) amplification of genomic DNA are an important part of the toolkit of evolutionary geneticists. Random amplified polymorphic DNA markers (RAPDs), amplified fragment length polymorphisms (AFLPs) and intersimple sequence repeat (ISSR) polymorphisms allow analysis of species for which previous DNA sequence information is lacking, but dominance makes it impossible to apply standard techniques to calculate F-statistics. We describe a Bayesian method that allows direct estimates of FST from dominant markers. In contrast to existing alternatives, we do not assume previous knowledge of the degree of within-population inbreeding. In particular, we do not assume that genotypes within populations are in Hardy-Weinberg proportions. Our estimate of FST incorporates uncertainty about the magnitude of within-population inbreeding. Simulations show that samples from even a relatively small number of loci and populations produce reliable estimates of FST. Moreover, some information about the degree of within-population inbreeding (FIS) is available from data sets with a large number of loci and populations. We illustrate the method with a reanalysis of RAPD data from 14 populations of a North American orchid, Platanthera leucophaea.     

Determination of phylogenetic relationships between some wild wheat species using amplified fragment length polymorphism (AFLP) markers

This study reports the molecular characterization, polymorphism, and phylogenetic relationships of Triticum aestivum , T. dicoccoides , T. urartu , and T. monococcum ssp. boeoticum , obtained from different locations in Anatolia, using 33 primer combinations to generate amplified fragment length polymorphism (AFLP) patterns in 31 individual plant samples. The objectives of this work were to estimate the phylogenetic relationships between these species and to investigate the genetic distance as a result of ecological and climatic factors. The origin of the A genome of polyploid wheats is also discussed. Eight hundred and seventy-five AFLP fragments had polymorphic loci, 133 of which were unique to T. monococcum ssp. boeoticum , 66 were unique to T. urartu , and 141 were unique to T. dicoccoides . Analysis using the program POPGENE showed polymorphism levels of T. monococcum ssp. boeoticum , T. urartu , and T. dicoccoides of 42.63, 32.34, and 27.71%, respectively. No correlation between genetic distance and ecological or climatic factors was recorded in this study. Our results support the hypothesis that T. urartu is a diploid ancestor of T. dicoccoides and T. aestivum .  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 67–72.     

Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation

This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) molecular markers linked to genes controlling semen freezability can be identified using amplified restriction fragment length polymorphism (AFLP) technology. Five ejaculates were collected from each of 129 boars. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and five straws (0.5 ml) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells, motility characteristics (computer-aided semen analysis; CASA), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (positive for fluorescein-labeled peanut agglutinin; PNA). Consistent between-boar variability was detected for postthaw sperm motility (P < 0.01), membrane integrity (P < 0.01), acrosome integrity (P < 0.01), and all CASA characteristics (P < 0.05). There was no significant difference between ejaculates (P > 0.05) or straws (P > 0.05) for any viability assessment. Multivariate pattern analysis of the viability data set highlighted three groups of boars producing spermatozoa with poor, average, and good postthaw recovery (42, 63, and 24 boars, respectively). DNA from Large White boars (n = 22) previously classified as good and poor freezers was screened for AFLP markers. Twenty-eight polymerase chain reaction primer combinations generated 2182 restriction fragment bands, of which 421 were polymorphic. Sixteen candidate genetic markers (P < 0.005) were identified by comparing the AFLP profile with semen freezability using logistic regression analysis. These findings support the hypothesis that there is a genetic basis for variation in postthaw semen quality between individuals, and that AFLP technology may be able to identify molecular markers linked to genes influencing this variation.     

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

COMPUTER PROGRAMS: onesamp: a program to estimate effective population size using approximate Bayesian computation

The estimation of effective population size from one sample of genotypes has been problematic because most estimators have been proven imprecise or biased. We developed a web-based program, onesamp that uses approximate Bayesian computation to estimate effective population size from a sample of microsatellite genotypes. onesamp requires an input file of sampled individuals' microsatellite genotypes along with information about several sampling and biological parameters. onesamp provides an estimate of effective population size, along with 95% credible limits. We illustrate the use of onesamp with an example data set from a re-introduced population of ibex Capra ibex.     

A comparative study of the population genetics of wild and cultivated populations of Paris polyphylla var. yunnanensis based on amplified fragment length polymorphism markers

Paris polyphylla var. yunnanensis is one of the original plants used to make the traditional medicine Paridis Rhizoma. Wild individuals have been excessively collected in recent decades, and thus, it has become increasingly endangered. Cultivation is a major method for the conservation and sustainable utilization of its wild resources. In this study, amplified fragment length polymorphism markers were used in the genetic analysis of 15 wild and 17 cultivated populations of P. polyphylla var. yunnanensis. This study revealed that cultivated populations possessed higher genetic diversity than wild ones at the species level (H = 0.2636 vs. 0.2616, respectively). However, most of the genetic variation was found within populations for both of these groups (Φ_ST = 18.83% vs. 19.39%). In the dendrogram produced using UPGMA, the 32 populations were divided into three groups (I, II, and III). In group II, both wild and cultivated populations were included, but remained in distinct clusters within this group, which showed the significant separation between the cultivated and wild populations. Furthermore, wild populations were also clustered into three subgroups (W‐I, W‐II, and W‐III), with an obvious geographic structure. In contrast, although cultivated populations were similarly placed in three subgroups (C‐I, C‐II, and C‐III), the latter two of these were not separated based on geography. Finally, the wild populations in Guizhou, China (W‐I), possessed higher genetic diversity than those in Yunnan (W‐II and W‐III). As P. polyphylla var. yunnanensis is an endangered medicinal plant, the fact that it showed richer genetic diversity in its wild populations in Guizhou means that these should be regarded as priority areas for protection and used for provenance selection. Moreover, cultivated populations also showed high genetic variation, which might be attributed to them having originated from mixed provenances, indicating that screening for superior provenances should be carried out as soon as possible.     

Construction of a high-density linkage map of Italian ryegrass (Lolium multiflorum Lam) using restriction fragment length polymorphism, amplified fragment length polymorphism, and telomeric repeat associated sequence markers.

To construct a high-density molecular linkage map of Italian ryegrass (Lolium multiflorum Lam), we used a two-way pseudo-testcross F1 population consisting of 82 individuals to analyze three types of markers: restriction fragment length polymorphism markers, which we detected by using genomic probes from Italian ryegrass as well as heterologous anchor probes from other species belonging to the Poaceae family, amplified fragment length polymorphism markers, which we detected by using PstI/MseI primer combinations, and telomeric repeat associated sequence markers. Of the restriction fragment length polymorphism probes that we generated from a PstI genomic library, 74% (239 of 323) of randomly selected probes detected hybridization patterns consistent with single-copy or low-copy genetic locus status in the screening. The 385 (mostly restriction fragment length polymorphism) markers that we selected from the 1226 original markers were grouped into seven linkage groups. The maps cover 1244.4 cM, with an average of 3.7 cM between markers. This information will prove useful for gene targeting, quantitative trait loci mapping, and marker-assisted selection in Italian ryegrass.     

Amplified fragment length polymorphism (AFLP) markers reveal that population structure of triploid dandelions (Taraxacum officinale) exhibits both clonality and recombination

Highly variable amplified fragment length polymorphism (AFLP) fingerprints of triploid apomictic dandelions obtained from three localities in an area where diploids are lacking were analysed to infer the predominant modes of reproduction. The distribution of markers was analysed using character compatibility to infer whether many genotypes agree with a tree-like structure in the data set. The presence of incompatible character state combinations (matrix incompatibility; MI) was used as a measure of genetic exchange. The detection of overrepresented genotypes, of which some were widespread, confirmed asexual reproduction. Not all genotypes were overrepresented; approximately half of the genotypes in the three localities were found only once. Because, in terms of genotype frequencies, only a part of the genetic variation is described, more important aspects of the molecular data such as relationships between markers or genotypes have been studied. The analysis of character compatibility indicated a disagreement of the data with a clonal structure. Nearly all genotypes contributed to MI and this contribution varied considerably among genotypes in each sampled locality. A gradual decrease of matrix incompatibility upon successive deletion of genotypes showing the highest contribution to MI indicated that marker distribution of virtually all genotypes disagreed with a tree-like structure in the data. This result suggested that many genotypes were separated by one or more sexual generations. Consistent with this conclusion was the fact that markers that show a low probability of contributing to MI are different in every sampled locality, which is most easily explained as the result of recombination. Apparently, asexual reproduction has resulted in overrepresented, widespread genotypes but sexual recombination has also substantially contributed to genetic variation in the sites studied.     

Clonal structure in a dwarf bamboo (Sasa senanensis) population inferred from amplified fragment length polymorphism (AFLP) fingerprints

Amplified fragment length polymorphism (AFLP) fingerprints were used to reveal clonal structure of a dense population of dwarf bamboo, Sasa senanensis, in a 10-ha study plot at Sugadaira Montane Research Center, University of Tsukuba, Nagano, Japan. We generated AFLP fingerprints for 51 leaf samples, collected at 50 m intervals, using three selective primer pairs. A total of 135-166 fragments were detected per sample, and 22 different fingerprints were identified based on 24-83 differing fragments. Our results demonstrate that the S. senanensis population in our plot consists of at least 22 clones and that the largest single clone occurs over a distance of about 300 m. Furthermore, the clone distribution pattern implies a relationship between site quality and clonal structure.     

Cleaved amplified polymorphic sequence and amplified fragment length polymorphism markers linked to the fertility restorer gene in chili pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS) in plants, which is due to failure to produce functional pollen, is a maternally inherited trait. Specific nuclear genes that sup-press CMS, termed fertility restorer (Rf) genes, have been identified in several plants. In this study, Rf-linked molecular markers in pepper (Capsicum annuum L.) were detected by bulked segregant analysis of eight amplified fragment length polymorphisms (AFLPs). Only AFRF8 was successfully converted to a cleaved amplified polymorphic sequence (CAPS) marker. This was named AFRF8CAPS and genotype determination using it agreed with that obtained with the original AFRF8. A linkage map with a total size of 54.1 cM was constructed with AFRF8CAPS and the seven AFLP markers using the Kosambi function. The AFRF8CAPS marker was shown to be closest to Rf with a genetic distance of 1.8 cM. These markers will be useful for fast and reliable detection of restorer lines during F(1) hybrid seed production and breeding programs in pepper.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

Identification of restriction fragment length polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce, using near-isogenic lines.

Near-isogenic lines were used to identify restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers linked to genes for resistance to downy mildew (Dm) in lettuce. Two pairs of near-isogenic lines that differed for Dm1 plus Dm3 and one pair of near-isogenic lines that differed for Dm11 were used as sources of DNA. Over 500 cDNAs and 212 arbitrary 10-mer oligonucleotide primers were screened for their ability to detect polymorphism between the near-isogenic lines. Four RFLP markers and four RAPD markers were identified as linked to the Dm1 and Dm3 region. Dm1 and Dm3 are members of a cluster of seven Dm genes. Marker CL922 was absolutely linked to Dm15 and Dm16, which are part of this cluster. Six RAPD markers were identified as linked to the Dm11 region. The use of RAPD markers allowed us to increase the density of markers in the two Dm regions in a short time. These regions were previously only sparsely populated with RFLP markers. The rapid screening and identification of tightly linked markers to the target genes demonstrated the potential of RAPD markers for saturating genetic maps.