Diversity of benzimidazole-resistance alleles in populations of small ruminant parasites

The resistance of gastro-intestinal nematodes of small ruminants (sheep and goat) to benzimidazole anthelmintic drugs seems to be linked primarily to a single mutation in the isotype 1 beta-tubulin gene. This study was carried out to investigate the origin and diversity of benzimidazole-resistance alleles in trichostrongylid nematodes. We sequenced a 550 bp fragment of the isotype 1 beta-tubulin gene from several benzimidazole-resistant Teladorsagia circumcincta populations isolated from dairy goat farms in the central and south-western France. We also sequenced the same beta-tubulin fragment from Trichostrongylus colubriformis and Haemonchus contortus populations in south-western France. We found eight benzimidazole-resistance alleles in all T. circumcincta populations studied, six in H. contortus populations, and only one in T. colubriformis populations. In most cases, only one benzimidazole-resistance allele was present in T. circumcincta and H. contortus populations, but two alleles were found in a fewer number of them. Some T. circumcincta populations shared the same benzimidazole-resistance allele whereas some others had a specific benzimidazole-resistance allele. Similar findings were obtained for H. contortus. As no parasites are introduced once the flock of dairy goat farms has been constituted, these data indicate for the three studied species that rare pre-existing benzimidazole-resistance alleles already present before the isolation of populations had been selected. On the other hand, the fact that some benzimidazole-resistance alleles were specific to one population of T. circumcincta or H. contortus, seems to be in agreement with the hypothesis of the selection of spontaneous mutations. Thus, the origin of benzimidazole-resistance alleles in trichostrongylid nematodes seems to involve primarily the selection of rare alleles and possibly of spontaneous mutations.     

Amplified fragment length polymorphism analysis of genetic diversity of Haemonchus contortus during selection for drug resistance.

For the first time we used amplified fragment length polymorphism on individual nematode parasites to analyse the genetic diversity between and within isolates during consecutive stages of increased benzimidazole resistance and of increased levamisole resistance of Haemonchus contortus. The genetic diversity of the H. contortus genome turned out to be unusually high, within and between the isolates. The difference between individuals of an isolate could be as high as between individuals of two different mammalian species that do not interbreed. During benzimidazole selection the genetic constitution of the population was changed, but did not lead to a decrease in the genetic diversity. The selection for levamisole resistance resulted in a limited reduction of the genetic diversity only after the first selection step. The extensive genetic diversity apparently has allowed a fast and flexible response of H. contortus to drug selection as shown by the appearance of drug resistant isolates. This selection however has little or no effect on the extent of the genetic diversity of these resistant isolates. Implications for more sustainable control methods are discussed.     

Estimating levels of inbreeding using AFLP markers

In the absence of detailed pedigree records, researchers have attempted to estimate individuals' levels of inbreeding using molecular markers, generally making use of heterozygosity measures based on microsatellite markers. Here we report and validate a method for estimating an individual's inbreeding coefficient, f, using amplified fragment length polymorphism (AFLP) markers. We use simulations to confirm that our measure scales appropriately with f when allele frequencies can be estimated from a subset of outbred individuals. We also present an approach for obtaining satisfactory estimates even in the absence of an independent set of known outbred individuals from which to estimate allele frequencies. We then test our method against empirical data from 179 wild and captive-bred old-field mice, Peromyscus polionotus subgriseus, comprising pedigree-based estimates of f, along with genetic data from 94 AFLP markers and 12 microsatellites. Inbreeding estimates based on both AFLP and microsatellite markers were found to correlate strongly with pedigree-based inbreeding coefficients. Owing to their ease of amplification in any species, AFLP markers may prove to be a valuable new tool for estimating f in natural populations and for examining correlations between heterozygosity and fitness.     

Global patterns reveal strong population structure in Haemonchus contortus, a nematode parasite of domesticated ruminants

We have examined the global population genetic structure of Haemonchus contortus. The genetic variability was studied using both amplified fragment length polymorphism (AFLP) and nad4 sequences of the mitochondrial genome. To examine the performance and information content of the two different marker systems, comparative assessment of population genetic diversity was undertaken in 19 isolates of H. contortus, a parasitic nematode of small ruminants. A total of 150 individual adult worms representing 14 countries from all inhabited continents were analysed. Altogether 1,429 informative AFLP markers were generated using four different primer combinations. Also, the genetic variation was high, which agrees with results from previous AFLP studies of nematode parasites of livestock. The genetic structure was high, indicating limited gene flow between the different isolates and populations from each continent mostly formed monophyletic groups in the phylogenetic analysis. However, for isolates representing Australia, Greece and one laboratory strain that originated from South Africa (WRS), there was no clear genetic relationship between the isolates and the distance between their geographical origins. Basically the same pattern was observed for the mitochondrial marker, although the phylogenetic analysis was less resolved than for AFLP. In contrast with previous findings on the population genetic structure of H. contortus, the calculation of population structure gave high values (Nst=0.59). The strong structure was present also for the four Swedish isolates (Nst=0.16) representing a small geographical area.     

人类短串联重复序列HUMTH01基因座的遗传多态性

作者用扩增片段长度多态技术分析了人类短串联重复序列ＨＵＭＴＨ０１基因型及等位基因频率在中国成都地区汉族群体和德国科隆地区白人中的分布。用同步电泳技术比较不同引物ＰＣＲ产物分型结果,评估不同实验室ＨＵＭＴＨ０１群体数据的可比性。计算了２５个群体间ＨＵＭＴＨ０１ＳＴＲ的遗传距离,并构建了系统树。ＨＵＭＴＨ０１ＳＴＲ系统树分析不仅与传统遗传标记结果一致,并且获得了群体遗传的新线索。     

Construction of a genetic linkage map based on amplified fragment length polymorphism markers and development of sequence-tagged site markers for marker-assisted selection of the sporeless trait in the oyster mushroom (Pleurotus eryngii)

A large number of spores from fruiting bodies can lead to allergic reactions and other problems during the cultivation of edible mushrooms, including Pleurotus eryngii (DC.) Quél. A cultivar harboring a sporulation-deficient (sporeless) mutation would be useful for preventing these problems, but traditional breeding requires extensive time and labor. In this study, using a sporeless P. eryngii strain, we constructed a genetic linkage map to introduce a molecular breeding program like marker-assisted selection. Based on the segregation of 294 amplified fragment length polymorphism markers, two mating type factors, and the sporeless trait, the linkage map consisted of 11 linkage groups with a total length of 837.2 centimorgans (cM). The gene region responsible for the sporeless trait was located in linkage group IX with 32 amplified fragment length polymorphism markers and the B mating type factor. We also identified eight markers closely linked (within 1.2 cM) to the sporeless locus using bulked-segregant analysis-based amplified fragment length polymorphism. One such amplified fragment length polymorphism marker was converted into two sequence-tagged site markers, SD488-I and SD488-II. Using 14 wild isolates, sequence-tagged site analysis indicated the potential usefulness of the combination of two sequence-tagged site markers in cross-breeding of the sporeless strain. It also suggested that a map constructed for P. eryngii has adequate accuracy for marker-assisted selection.     

Microsatellite analysis reveals marked genetic differentiation between Haemonchus contortus laboratory isolates and provides a rapid system of genetic fingerprinting

Many of the Haemonchus contortus isolates currently used for experimental work were originally derived from different regions of the world and are commonly exchanged between laboratories. In most cases, these are largely genetically uncharacterised other than the analyses conducted on specific genes of interest. We have used a panel of eight microsatellite markers to genetically characterise five different commonly used H. contortus isolates including MHco3 (ISE), the isolate chosen for full genome sequencing as part of the H. contortus genome project. There is an extremely high level of genetic differentiation between each of the isolates except the two which have a common origin, MHco1 (MOSI) and MHco3 (ISE). We have investigated the amplification of microsatellite markers from pooled DNA as a potential method for fingerprinting different isolates. Good estimates of the true allele frequencies can be made by amplification from either pooled adult DNA or bulk L3 DNA for seven out of the eight markers tested. Both single worm genotyping and bulk DNA fingerprinting revealed no genetic differentiation between adult worms in the host and larvae derived from faecal culture. Furthermore, none of the eight markers showed genetic changes when isolates were passaged through different individual hosts. Hence the microsatellite genotyping of bulk larval DNA samples provides a simple and rapid method to genetically define and monitor laboratory isolates, and to determine their relationship with particular field populations.     

Increased inbreeding and strong kinship structure in Taxus baccata estimated from both AFLP and SSR data

Habitat fragmentation can have severe genetic consequences for trees, such as increased inbreeding and decreased effective population size. In effect, local populations suffer from reduction of genetic variation, and thus loss of adaptive capacity, which consequently increases their risk of extinction. In Europe, Taxus baccata is among a number of tree species experiencing strong habitat fragmentation. However, there is little empirical data on the population genetic consequences of fragmentation for this species. This study aimed to characterize local genetic structure in two natural remnants of English yew in Poland based on both amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers. We introduced a Bayesian approach that estimates the average inbreeding coefficient using AFLP (dominant) markers. Results showed that, in spite of high dispersal potential (bird-mediated seed dispersal and wind-mediated pollen dispersal), English yew populations show strong kinship structure, with a spatial extent of 50–100 m, depending on the population. The estimated inbreeding levels ranged from 0.016 to 0.063, depending on the population and marker used. Several patterns were evident: (1) AFLP markers showed stronger kinship structure than SSRs; (2) AFLP markers provided higher inbreeding estimates than SSRs; and (3) kinship structure and inbreeding were more pronounced in denser populations regardless of the marker used. Our results suggest that, because both kinship structure and (bi-parental) inbreeding exist in populations of English yew, gene dispersal can be fairly limited in this species. Furthermore, at a local scale, gene dispersal intensity can be more limited in a dense population.     

Pedigree and marker information requirements to monitor genetic variability

There are several measures available to describe the genetic variability of populations. The average inbreeding coefficient of a population based on pedigree information is a frequently chosen option. Due to the developments in molecular genetics it is also possible to calculate inbreeding coefficients based on genetic marker information. A simulation study was carried out involving ten sires and 50 dams. The animals were mated over a period of 20 discrete generations. The population size was kept constant. Different situations with regard to the level of polymorphism and initial allele frequencies and mating scheme (random mating, avoidance of full sib mating, avoidance of full sib and half sib mating) were considered. Pedigree inbreeding coefficients of the last generation using full pedigree or 10, 5 and 2 generations of the pedigree were calculated. Marker inbreeding coefficients based on different sets of microsatellite loci were also investigated. Under random mating, pedigree-inbreeding coefficients are clearly more closely related to true autozygosity (i.e., the actual proportion of loci with alleles identical by descent) than marker-inbreeding coefficients. If mating is not random, the demands on the quality and quantity of pedigree records increase. Greater attention must be paid to the correct parentage of the animals.     

A single linkage group confers dominant resistance to Bacillus thuringiensis δ‐endotoxin Cry1Ac in Helicoverpa armigera

The BKBT strain of Helicoverpa armigera was derived from a susceptible BK77 strain (collected from Bouake, Cote D’Ivoire in 1977) through 30 generations of selection with activated Bacillus thuringiensis δ‐endotoxin Cry1Ac. Unlike recessive inheritance of Cry1Ac resistance in H. armigera from previous reports, resistance to activated Cry1Ac in the BKBT strain is dominant. A backcross approach was used to map dominant resistance to Cry1Ac in the BKBT strain. One hundred and forty‐seven informative amplified fragment length polymorphism (AFLP) DNA markers covered all 31 linkage groups of H. armigera. Five AFLP markers linked to Cry1Ac resistance in the BKBT strain were on the same autosomal linkage group, which is the only linkage group contributing dominant Cry1Ac resistance in the BKBT strain of H. armigera.     

DNA polymorphisms in the chicken growth hormone gene: response to selection for disease resistance and association with egg production

Analysis of the growth hormone (GH) gene in 12 strains of White Leghorn chickens revealed restriction fragment length polymorphisms (RFLPs) at three Msp I sites and at a Sac I site. Based on linkage disequilibrium analysis, they gave rise to eight different alleles (i.e. combinations of RFLPs), with five occurring at frequencies above 5% in at least one strain. Pairs of GH–RFLPs were at near maximal linkage disequilibrium, suggesting either a lack of recombination or the presence of selection pressure during evolution of the GH gene. Allele frequencies were determined in 12 non-inbred strains derived from three different genetic bases. These strains had been selected either for an array of egg production traits, resistance to Marek's disease or resistance to avian leukosis. Selection for disease resistance was consistently correlated with an increase in the frequency of one of the alleles. One strain segregated for only two alleles, which differed by three RFLPs. Analysis of variance in this strain indicated that the GH allele co-selected with resistance was associated with a delayed onset of ovulation but a higher persistency of ovulation as age progressed, resulting in an overall increase of egg production by 15% (age at first egg to 497 days). The resistance-associated GH allele was dominant for the onset of ovulation and recessive for the persistency of egg production. There was no significant effect of the GH genotype on juvenile body weight, egg weight or egg specific gravity.     

新疆维吾尔族和汉族亚甲基四氢叶酸还原酶基因多态性研究

目的:研究新疆维吾尔族、汉族两民族亚甲基四氢叶酸还原酶(Methyleneterahyofolate reductase MTHFR)基因多态性的分布情况,获取新疆维吾尔族与汉族MTHFR 1298位群体遗传学数据。方法:应用PCR-RFLP技术检测新疆维吾尔族及汉族MTHFR1298位多态性位点基因频率及基因型频率。结果:新疆维吾尔族、汉族MTHFR 1298位C等位基因分布频率分别为12%、23%,P<0.05有统计学差异性,且新疆维吾尔族MTHFR 1298位C等位基因分布频率与现有报道的少数民族贵州苗族、布依族具有统计学差异。结论:MTHFR 1298位多态性在不同民族具有差异性:MTHFR 1298位多态性在新疆维吾尔族和汉族有民族差异;新疆新疆维吾尔族MTHFR 1298位C等位基因频率与贵州苗族、布依族少数民族之间具有民族差异性。     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

生化标记和分子标记在蕈菌研究中的应用现状

本文介绍了目前在蕈菌研究中的酯酶同工酶标记,RAPD标记,RFLP标记,AFLP标记,简单重复序列标记和电泳核型等分子标记和生化标记在蕈菌遗传育种、菌株鉴定、遗传多样性研究、亲缘关系和基因定位等方面的研究、应用现状,包括原理、应用领域及最新研究进展。     

Microsatellite markers of genetic diversity and population structure of Carica papaya

We assessed the genetic diversity of 96 papaya accessions by molecular characterisation using microsatellite markers. Fifteen polymorphic primers were selected. Accessions, which were classified as Common, Formosa and Solo according to fruit types, were evaluated for allele frequency, heterozygosity, polymorphism information content (PIC), inbreeding coefficient (f) and the genetic diversity structure. Fifteen primers amplified 68 alleles with an average of 4.53 per locus. PIC values ranged from 0.19 to 0.69. The observed heterozygosity (H_O) was low for all selected microsatellites. High f estimates (0.58) and excess of homozygotes indicated inbreeding, mainly caused by the tendency to select hermaphrodite plants for succeeding generations. Analysis of molecular variance showed that most of the variation (98%) resides within subpopulation. The genetic analysis based on Bayesian statistics proved to be sensitive enough to detect relationships among the papaya accessions, grouping them into six clusters, irrespective of their classification types.     

Allozyme variation in Anopheles stephensi Liston from Pakistan (Diptera: Culicidae)

Allozyme variability was analyzed at 16 loci in 11 lines of Anopheles stephensi Liston from Pakistan. Six lines were considered as samples from natural populations. For these lines the mean number of alleles was 1.31-1.63, the degree of polymorphism was 0.188-0.375, the observed heterozygosity was 0.065-0.086, and the genetic distance ranged from 0.001 to 0.016. No population-specific alleles were found. Interbreeding was considerable (mean Fit = 0.183). Differences in allele frequencies were due considerable (mean Fit = 0.183). Differences in allele frequencies were due primarily to local inbreeding (Fis greater than Fst at most loci). The Lahore line, reared for more than 20 generations, had more homozygotes than the other lines. A line refractory to Plasmodium falciparum and a genetic sexing line exhibited decreased allozyme variability. The latter line showed reduced staining intensity at 10 loci. Linkage studies are recommended for the following loci with rare alleles: Acp, Gapdh, Icd-1, Icd-2, Mpi, and Pgd.     

Genetic Variability of the β-Tubulin Genes in Benzimidazole-Susceptible and -Resistant Strains of Haemonchus Contortus

Benzimidazole anthelmintics are the most common chemotherapeutic agents used to remove intestinal helminths from farm animals. The development of drug resistance within helminth populations is wide-spread and can render these drugs essentially useless. The mechanism of benzimidazole resistance appears to be common to many species ranging from fungi to nematodes and involves alterations in the genes encoding β-tubulin. During the selection process resulting in resistance, there must be quantitative changes in the population gene pool. Knowledge of these changes would indicate the mechanisms underlying the spread of resistance in the population, which in turn could be used to design more effective drug administration strategies. To this end we have identified allelic variation at two β-tubulin genes in Haemonchus contortus using restriction map analysis of individual adults. Extremely high levels of variation were identified at both loci within a susceptible strain. In two independently derived benzimidazole resistant strains, allele frequencies at both loci were significantly different from the susceptible strain but not from each other. The same alleles at both loci, in both resistant strains, were favored by selection with benzimidazoles, suggesting that both loci are involved in determining benzimidazole resistance. These data confirm that changes in allele frequency, rather than novel genetic rearrangements induced by exposure to the drug, explain the changes associated with benzimidazole resistance. These results also show that any DNA based test for the development of benzimidazole resistance must take into account the frequency of alleles present in the population and not simply test for the presence or absence of specific allelic types.     

Impact of multispores in vitro subcultivation of Glomus sp. MUCL 43194 (DAOM 197198) on vegetative compatibility and genetic diversity detected by AFLP

Vegetative compatibility and amplified fragment length polymorphism (AFLP) genotyping of in vitro multispores clonal lineages, issued from the same ancestor culture of the arbuscular mycorrhizal fungal strain MUCL 43194 and subcultured several generations in different locations, was assessed. Vegetative compatibility was studied by confronting the germ tubes of two spores from the same or different clonal lineages and stained with nitrotetrazolium blue–Trypan blue and diamidinophenylindole to detect hyphal fusions and nuclei, respectively. Further AFLP analysis of single spores was performed to assess the genetic profile and Dice similarity between clonal lineages. Germ tubes of spores distant by as many as 69 generations were capable of fusing. The anastomosis frequencies averaged 69% between spores from the same clonal lineage, 57% between spores from different clonal lineages, and 0% between spores belonging to different strains. The AFLP patterns showed similarities averaging 92% within clonal lineages and 86% between clonal lineages. Each spore presented unique genotype and some of them shared more markers with spores from different lineages than within the same lineage. We showed that MUCL 43194 maintained self-recognition for long periods of subcultures in vitro and that spores involved in compatibility tests had different genotypes. Our findings suggest that MUCL 43194 maintains genetic diversity by means of anastomoses.     

Genetic analysis of Helicobacter pylori strain populations colonizing the stomach at different times postinfection

Genetic diversity of the human gastric pathogen Helicobacter pylori in an individual host has been observed; whether this diversity represents diversification of a founding strain or a mixed infection with distinct strain populations is not clear. To examine this issue, we analyzed multiple single-colony isolates from two to four separate stomach biopsies of eight adult and four pediatric patients from a high-incidence Mexican population. Eleven of the 12 patients contained isolates with identical random amplified polymorphic DNA, amplified fragment length polymorphism, and vacA allele molecular footprints, whereas a single adult patient had two distinct profiles. Comparative genomic hybridization using whole-genome microarrays (array CGH) revealed variation in 24 to 67 genes in isolates from patients with similar molecular footprints. The one patient with distinct profiles contained two strain populations differing at 113 gene loci, including the cag pathogenicity island virulence genes. The two strain populations in this single host had different spatial distributions in the stomach and exhibited very limited genetic exchange. The total genetic divergence and pairwise genetic divergence between isolates from adults and isolates from children were not statistically different. We also analyzed isolates obtained 15 and 90 days after experimental infection of humans and found no evidence of genetic divergence, indicating that transmission to a new host does not induce rapid genetic changes in the bacterial population in the human stomach. Our data suggest that humans are infected with a population of closely related strains that vary at a small number of gene loci, that this population of strains may already be present when an infection is acquired, and that even during superinfection genetic exchange among distinct strains is rare.     

载脂蛋白E基因多态性与动脉粥样硬化性脑梗塞关系的研究

应用聚合酶链反应(PCR)扩增ApoE基因外显子4中编码112位和158位氨基酸的 DNA片段,将该长为292bp的PCR产物以HhaI酶切,根据其限制性片段长度多态性图谱确定ApoE基因型。对广东汉族人群的50例动脉粥样硬化脑梗塞(ACI)患者和50例健康对照者的ApoE基因多态性频率的研究结果表明:所研究的人群中,ApoE基因多态性频率与ACI没有关联。ACI患者中各基因型亚组之间血清TG和TC浓度无显著性差异。 Abstract:The polymerase chain reaction(PCR)and restriction fragment length polymorphism techniques(RFLPs)were used to study the relation between apolipoprotein E gene polymorphism and atherosclerotic cerebral infarction(ACI).A 292 bp DNA fragment containing codes for the 112 and 158 amino acid residues in the fourth exon of ApoE gene was amplified by PCR.The PCR products were digested with HhaI.The polymorphism patterns of Apo E gene,allele frequencies in 50 Chinese healthy and 50 patients with ACI,and the serum levels of TG and TC in the different subgenotypes of 50 patients with ACI were detected.,no statistical significant differences of alleles frequencies were found between the cases with ACI and the control,and no statistical significant differences of the serum levels of TG and TC were found among the different subgenotypes of 50 patients with ACI.These results suggested that ApoE gene polymorphism was not associated with the development of ACI.