Analysis of mosquito bloodmeals by DNA profiling

Abstract. Human specific genetic markers have been used to profile the human DNA found within a mosquito bloodmeal. In this technique, variable numbers of tandem repeat (VNTR) sequences are employed to prime amplification of human DNA in the polymerase chain reaction (PCR) and the radiolabeled products are analysed by high resolution denaturing gel electrophoresis. Matching of DNA profiles allows identification of the individual human host. Bloodmeals of 125 female Anopheles gambiae Giles mosquitoes, caught dead or alive in verandah-trap huts wherein two people had slept overnight protected by intact insecticide-impregnated bednets, were analysed: thirty-five out of thirty-nine profiles generated were identical to those of the sleepers under the nets. Thus the blood-fed mosquitoes found after impregnated nets have been used cannot, in most cases, be explained away by entry of already fed mosquitoes into the huts.     

Analysis of mosquito bloodmeals using RFLP markers

An important variable in the amplification of arthropod vector-borne diseases is the degree of contact between human hosts and mosquito vectors. To analyze this interaction, a DNA based method was developed to differentiate human bloodmeals from other sources in the mosquito Anopheles stephensi (Diptera: Culicidae) Liston. A portion of the host mitochondrial DNA cytochrome B genes were PCR amplified and classified to the species level based on their restriction fragment length polymorphism (RFLP). The cytochrome B sequences showed sufficient interspecific polymorphism to distinguish between human, cow, sheep, chicken, and guinea pig hosts. XhoI could distinguish human from other vertebrates whereas TaqI alone could separate the others. The importance of these results in epidemiological studies of malaria and other vector borne diseases is discussed.     

Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean

DNA amplification fingerprinting (DAF) using a high primer-to-template ratio and single, very short arbitrary primers, was used to generate amplified fragment length polymorphic markers (AFLP) in soybean (Glycine max (L.) Merr.). The inheritance of AFLPs was studied using a cross between the ancestral Glycine soja PI468.397 and Glycine max (L.) Merr. line nts382, F1 and F2 progeny. The amplification reaction was carried out with soybean genomic DNA and 8 base long oligounucleotide primers. Silver-stained 5% polyacrylamide gels containing 7 M urea detected from 11 to 28 DAF products with primers of varying GC content (ranging from 50 to 100% GC). Depending on their intensity, AFLPs were classified into three classes. DAF profiles were reproducible for different DNA extractions and gels. Forty AFLPs were detected by 26 primers when comparing G. soja and G. max. Most AFLPs were inherited as dominant Mendelian markers in F1 and F2 populations. However, abnormal inheritance occured with about 25% of polymorphisms. One marker was inherited as a maternal marker, presumably originating from organelle DNA while another showed apparent paternal inheritance. To confirm the nuclear origin and utility of dominant Mendelian markers, three DAF polymorphisms were mapped using a F11 mapping population of recombinant inbred lines from soybean cultivars Minsoy × Noir 1. The study showed that DAF-generated polymorphic markers occur frequently and reliably, that they are inherited as Mendelian dominant loci and that they can be used in genome mapping.     

Amplification and analysis of human DNA present in mosquito bloodmeals

Abstract. DNA fingerprinting should permit the identification of individual human hosts of haematophagous arthropods, providing epidemi-ologically useful information, for example, the biting rates on different people and the impact of insecticide-impregnated bednets.
Investigations reported here demonstrate that it is possible to extract, amplify and fingerprint human DNA from the bloodmeals of individual female Anopheles gambiae mosquitoes kept at 24^oC for up to 10–15 h post-ingestion.     

Molecular methods for arthropod bloodmeal identification and applications to ecological and vector-borne disease studies

DNA-based methods have greatly enhanced the sensitivity and specificity of hematophagous arthropod bloodmeal identification. A variety of methods have been applied to study the blood-feeding behaviour of mosquitoes, ticks, black flies and other blood-feeding arthropods as it relates to host-parasite interactions and pathogen transmission. Overviews of the molecular techniques used for bloodmeal identification, their advantages, disadvantages and applications are presented for DNA sequencing, group-specific polymerase chain reaction primers, restriction fragment length polymorphism, real-time polymerase chain reaction, heteroduplex analysis, reverse line-blot hybridization and DNA profiling. Technical challenges to bloodmeal identification including digestion and analysis of mixed bloodmeals are discussed. Analysis of bloodmeal identification results remains a challenge to the field, particularly with regard to incorporation of vertebrate census and ecology data. Future research directions for molecular analysis of arthropod bloodmeals are proposed.     

Genetic relationship of Porteresia coarctata Tateoka using molecular markers

ABSTRACT

Twenty-one species belonging to Oryza, including wild rices, were compared with a tetraploid (2n=48) halophytic wild rice relative, Porteresia coarctata Tateoka (=Oryza coarctata) for the genetic relatedness using AFLP and RAPD markers. Diploid and tetraploid groups were clearly separated except in the case of a few species where the clustering was unique and different. The molecular analysis has helped in positioning Porteresia in the vicinity of other wild rice species, and to better understand the pattern of species differentiation in Oryza. From our study, O. australiensis seems to be related to P. coarctata; thus, O. australiensis may be an effective “bridge” species in transferring genetic traits from P. coarctata to O. sativa. The usefulness of molecular marker systems for studying polymorphism and classification, and in clarifying genetic relationships between wild species has been confirmed.     

Development of a high-throughput microsphere-based molecular assay to identify 15 common bloodmeal hosts of Culex mosquitoes

For vectorborne infections, host selection by bloodfeeding arthropods dictates the interaction between host and pathogen. Because Culex mosquitoes that transmit West Nile virus (WNV) feed both on mammalian and avian hosts with varying competence, understanding the bloodfeeding patterns of these mosquitoes is important for understanding the transmission dynamics of WNV. Herein, we describe a new microsphere‐based assay using Luminex xMAP^® technology to rapidly identify 15 common hosts of Culex mosquitoes at our California study sites. The assay was verified with over 100 known vertebrate species samples and was used in conjunction with DNA sequencing to identify over 125 avian and mammalian host species from unknown Culex bloodmeals, more quickly and with less expense than sequencing alone. In addition, with multiplexed labelled probes, this microsphere array identified mixed bloodmeals that were difficult to discern with traditional sequencing. The microsphere set was easily expanded or reduced according to host range in a specific area, and this assay has made it possible to rapidly screen thousands of Culex spp. bloodmeals to extend our understanding of WNV transmission patterns.     

EST–SSR markers for asparagus genetic diversity evaluation and cultivar identification

Simple sequence repeat (SSR) markers generated from expressed sequence tag (EST) sequences represent useful tools for genotyping and their development is relatively easy because of the public availability of EST databases. We report design and application of EST–SSRs to assess the level of genetic diversity among thirty-five asparagus cultivars and to fingerprint DePaoli, a new variety released by University of California, Riverside. DNA was isolated from bulks of pooled cladophylls coming from five plants of each variety to reduce the number of DNA extractions and PCR reactions. Allele frequencies were estimated from the intensity of the bands in two bulks and two individual plant samples for each variety. Although asparagus varieties derive from a limited germplasm pool, eight EST–SSR loci differentiated all of the analyzed cultivars. Moreover, UPGMA (unweighted pair group method with arithmetic mean) and neighbor-joining trees, as well as principal components analysis separated the cultivars into clusters corresponding to the geographical areas where they originated.     

Molecular screening of Leishmania spp. infection and bloodmeals in sandflies from a leishmaniasis focus in southwestern Turkey

Leishmaniasis is an arthropod‐borne disease that affects approximately 2 million people worldwide annually. The aims of this study were to detect the presence of Leishmania (Kinetoplastida: Trypanosomatidae) DNA and the feeding preferences of probable vector species in an endemic focus of Leishmania infantum in Turkey. Entomological sampling was performed in August and October 2015 in Ayd?n province, where cases of human and canine leishmaniasis have been reported previously. A total of 1059 sandfly specimens comprising nine species belonging to two genera, Phlebotomus and Sergentomyia (both: Diptera: Psychodidae), and five subgenera of the Phlebotomus genus (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius and Transphlebotomus) were collected in five villages. Among all Phlebotomus specimens, Phlebotomus neglectus (39%) was noted as the most abundant species, followed by Phlebotomus tobbi (18%). Leishmania DNA was detected in pools from P. neglectus, P. tobbi and Sergentomyia dentata by kDNA polymerase chain reaction (PCR). Leishmania DNA from Phlebotomus specimens was identified as L. infantum, but Leishmania DNA from Sergentomyia spp. could not be identified to species level by ITS‐1 real‐time PCR. The detection of Leishmania DNA in wild‐caught P. neglectus and the high percentage (24.2%) of human DNA in engorged specimens suggests that P. neglectus is probably an important vector species for L. infantum in Ayd?n province.     

Identification of genetic factors for alachlor tolerance in maize by molecular markers analysis

Genetic factors controlling tolerance to the herbicide Alachlor in maize were localised by means of two different strategies. In the first approach, backcross (BC) plants, derived from pollen which had been subjected to selective pressure for resistance to the herbicide, were analysed for segregation distortion at 47 RFLP loci and compared to BC plants obtained from non-selected pollen. Preferential transmission of five chromosomal regions where putative QTLs (Quantitative Trait Loci) are localised was revealed in the BC plants from selected pollen. A second approach was based on a classical linkage analysis for segregation of the same set of RFLPs and factors controlling the trait, in a BC population of 210 individuals, by means of regression analysis. This study detected seven significant loci in four genomic regions. Overall, two loci revealed both segregation distortion and association with the expression of the trait, indicating linkage to genes expressed in both gametophytic and sporophytic phase. Three chromosomal regions appeared to carry factors involved in plant tolerance to Alachlor which are not expressed in pollen. Conversely, three loci were linked to factors selectable in pollen, but did not reveal significant association with tolerance in the plant in the segregating populations.     

分子标记技术在蚕学研究中的应用

介绍了近年来DNA分子标记技术在绢丝昆虫的进化及亲缘关系分析、家蚕品种真实性鉴定、家蚕分子连锁图的构建、基因标记和定位等方面应用的重要进展 ,并展望了家蚕分子标记辅助育种的前景     

Number of individuals and molecular markers to use in genetic differentiation studies

Molecular markers are frequently used to study genetic variation among individuals within or between populations. Differences in marker banding patterns can be used to verify if individuals do, or do not, represent distinct groups or populations. Only in 2005, more than 500 studies used molecular markers to group individuals in clusters. Such studies make use of an arbitrary number of molecular markers from each of an arbitrary number of individuals presumed to represent distinct genotypes. However, the greater the genetic variation, the more likely a larger number of individuals and markers will be needed to capture a population's genetic signature. The numbers of both, markers and individuals included thus affect the way in which individuals are organized through cluster analyses, thereby affecting the conclusions drawn. Here we present a method that provides statistical criteria to verify that individual and marker sample sizes are sufficient to accurately depict genetic differentiation among different populations. Our method uses a resampling technique to assess the reproducibility of obtaining a particular grouping pattern for specific data sets. It thus, allows to estimate the robustness of the results obtained without including additional individuals, or markers.     

Generating molecular markers from zooxanthellate cnidarians

Genetic techniques are providing tools that are necessary to answer questions concerning the ecology and evolution of cnidarians that, until recently, could not be easily addressed. In developing molecular markers for cnidarians with algal symbionts (zooxanthellae), however, caution must be used to ensure the markers in question are derived from the cnidarian host and not zooxanthellae. Unless the DNA template is from asymbiotic tissue, both host and symbiont genomes are present in the DNA template and zooxanthella-specific markers are often inadvertently generated. Steps should be taken to minimize the contamination by zooxanthella DNA in the template, and the origin of the molecular marker (host or symbiont) must be verified. Including zooxanthella-specific markers in analyses for cnidarians will confound interpretations of the results as biogeographic and phylogeographic patterns of zooxanthellae do not necessarily reflect those of the host.     

Analysis of genetic recombination in maize populations using molecular markers

Summary Variation in recombination rate is important to plant breeders since a major objective is to obtain favorable recombinants of linked genes. The ability to increase recombination (R) in circumstances in which favorable and unvavorable genes are linked (Corn Belt x exotic populations) and to decrease recombination when many favorable genes are linked (narrow-based, elite populations) would be of immense value. However, the concept of variation in recombination frequencies between linked genes has received limited attention despite its implications in breeding and genetic linkage studies. Molecular techniques have allowed better estimations of this variation. In this study, attempts were made to characterize: (1) the R values in the Pgm1-Adh1 and Adh1-Phi1 adjacent regions of chromosome 1 and the Idh2-Mdh2 region of chromosome 6 in F₂ families of three maize (Zea mays L.) populations; (2) the environmental effect on R values of F₂s from two populations. One population, NSO, was a Corn Belt synthetic, and the other two populations, CBMEX3 and CBCAR5, were composites from crosses between Corn Belt and exotic germ-plams.Wide ranges of estimated recombination ( ) values were observed among families in each population for all three chromsomal regions. The distribution of values for the Pgm1-Adh1 region showed that the F₂ families of each population fell into two broad categories: 0.30–0.50 and 0.02–0.20. No intermediates (0.21–0.29) were found. The distributions were almost normal for the Adh1-Phi1 and the Idh2-Mdh2 regions. It would appear that the major dispersion in the Pgm1-Adh1 region was controlled by the effects of a single gene, while the Adh1-Phi1 and Idh2-Mdh2 regions were only affected by polygenes. No correlation was found between recombination values of the two adjacent regions, indicating that the genes affecting recombination for the Pgm1-Adh1 region may be specific for that region.For the Pgm1-Adh1 region, no differences in values were found among the three populations. For the Adh1-Phi1 region, frequencies of CBMEX3 and NSO were not significantly different, but both had significantly greater values than CBCAR5. For the Idh2-Mdh2 region, CBMEX3 was significantly different from NSO. There were significant differences between some paired F₂ families within each population for each chromosome region.No significant differences in response to the two environments were detected in CBMEX3 and NSO for either region in chromosome 1.Published as Journal Paper No. 9498 of the Nebraska Agric Res Div, University of Nebraska, Lincoln, Neb. Research supported in part by USDA Competitive Grant 87-CRCR-2359     

DNA分子标记在雌雄异株植物性别鉴定中的应用

雌雄异株植物中不同性别的植株所产生的经济效应和生态效应存在一定的差异,在生产实践中,选取适宜性别的植株进行栽培有助于提高效率,避免不必要的浪费。然而,性别鉴定的常用方法大多是根据表型、代谢产物含量及活性等方面的差异,在成株阶段进行的,鉴定结果的可靠性和准确性都有一定的局限。近几年,DNA分子标记技术应用于雌雄异株植物的性别鉴定研究中,获得了快速准确的鉴定结果。在比较常用性别鉴定方法的基础上,主要就常用DNA分子标记在雌雄异株植物性别鉴定中的研究进展做一概述并对该领域的研究提出展望。     

Analysis of genetic stability of in vitro propagated potato microtubers using DNA markers

The genetic stability of in vitro propagated potato microtubers was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Microtubers were developed through in vitro from potato microplants using standardized protocols. The microtubers were conserved for 1 year under three different culture media and consequently microplants were regenerated for the DNA analyses. During the study, a total of 38 (10 RAPD, 11 ISSR, 12 SSR and 5 AFLP) primers produced a total of 407 (58 RAPD, 56 ISSR, 96 SSR and 197 AFLP) clear, distinct and reproducible amplicons. Cluster analysis revealed 100 % genetic similarity among the mother plant and its derivatives within the clusters by SSR, ISSR and RAPD analyses, whereas AFLP analysis revealed from 85 to 100 % genetic similarity. Dendrogram analysis based on the Jaccard’s coefficient classified the genotypes into five clusters (I–V), each cluster consisting of mother plant and its derivatives. Principal component analysis (PCA) also plotted mother plant and its genotypes of each cluster together. Based on our results, it is concluded that AFLP is the best method followed by SSR, ISSR and RAPD to detect genetic stability of in vitro conserved potato microtubers. The in vitro conservation medium (T2) is a safe method for conservation of potato microtubers to produce true-to-type plans.     

Unravelling first-generation pedigrees in wild endangered salmon populations using molecular genetic markers

There is increasing interest in the use of molecular genetic data to infer genealogical relationships among individuals in the absence of parental information. Such analyses can provide insight into mating systems and estimations of heritability in the wild. In addition, accurate pedigree reconstruction among the founders of endangered populations being reared in captivity would be invaluable. Many breeding programs for endangered species attempt to minimize loss of genetic variation and inbreeding through strategies designed to minimize global co-ancestry, but they assume a lack of relatedness among the founders. Yet populations that are the target of such programs are generally in serious demographic decline, and many of the available founders may be closely related. Here we demonstrate determination of full and half-sib relationships among the wild founders of a captive breeding program involving two endangered Atlantic salmon populations using two different approaches and associated software, pedigree and colony. A large portion of the juveniles collected in these two rivers appear to be derived from surprisingly few females mating with a large number of males, probably small precocious parr. Another group of potential founders, obtained from a local hatchery, clearly originated from a small number of full-sib crosses. These results allowed us to prioritize individuals on the basis of conservation value, and are expected to help minimize loss of genetic variation through time. In addition, insight is provided into the number of contributing parents and the mating systems that produced this last generation of endangered wild Atlantic salmon.     

Deterministic paternity exclusion using RAPD markers

The Random Amplified Polymorphic DNA (RAPD) technique can potentially provide hundreds of polymorphic markers for use by ecologists studying mating systems in natural populations. We consider here the implications of the dominance displayed by RAPD markers for deterministic paternity assignment. Our goal was to provide a means for assessing the costs associated with such a study for ecologists who might be considering the use of RAPD markers for paternity analysis. The theoretical expected proportion of offspring for which all males except the true father can be exlucded (P(ET)) is calculated for both dominant and codominant marker systems. The ability to assign paternity unambiguously generally increases with the number of loci and the frequency of the recessive allele (but only up to a point), and decreases with increasing sample size (number of individuals surveyed). The gain in P(ET) with decreasing sample size is unexpectedly slight. Not surprisingly, the performance of dominant markers at paternity exclusion is, in general, greatly exceeded by codominant markers, with the exception of the case in which the frequency of the recessive allele is high at all loci. In this case, codominant markers perform only slightly better than do dominant markers. Thus, a researcher should expect to score more than 50 RAPD loci for each offspring for most applications of paternity exclusion analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of genetic variation in tomato （Solanum lycopersicum L.） inbred lines using SSR molecular markers

A study was conducted to determine the genetic diversity of 39 determinate and indeterminate tomato inbred lines collected from China, Japan, S. Korea, and USA. Using 35 SSR polymorphic markers, a total of 150 alleles were found with moderate levels of diversity, and a high number of unique alleles existing in these tomato lines. The mean number of alleles per locus was 4.3 and the average polymorphism information content (PIC) was 0.31. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering at genetic similarity value of 0.85 grouped the inbred lines into four groups, where one USA cultivar formed a separate and more distant cluster. The most similar inbred lines are from USA, both with determinate type, whereas the most different lines are from USA (Us-16) and Japan (Ja-2) with determinate and indeterminate growth habit, respectively. Clustering was consistent with the known information regarding geographical location and growth habit. The genetic distance information reported in this study might be used by breeders when planning future crosses among these inbred lines.