Oral delivery of double-stranded RNA induces prolonged and systemic gene knockdown in Metaseiulus occidentalis only after feeding on Tetranychus urticae

Metaseiulus (=Typhlodromus or Galendromus) occidentalis is an important biological control agent. Functional genomic studies on this predator have been hampered by the lack of reverse genetic tools such as RNA interference (RNAi). In the current study, we evaluated possible RNAi responses in M. occidentalis females by feeding double-stranded RNA (dsRNA) of RpL11, RpS2, RpL8, or Pros26.4 genes in 20 % sucrose solution. Females needed to subsequently feed on two-spotted spider mites (Tetranychus urticae) to elicit a nearly complete loss of egg production. The corresponding gene knockdown was robust, long-term, and was observed in the very few eggs produced (systemic or parental RNAi). Interestingly, dsRNA-mediated gene knockdown could not be induced if these predators were provided only the sucrose diet after ingesting dsRNAs; T. urticae had to be provided to elicit the RNAi response. However, the spider mite diet was not needed for sustaining the dsRNA-mediated gene knockdown once it commenced. Oral delivery of dsRNA will be a valuable tool for efficient genome-wide functional screens in this important predatory mite.     

An RNAi-Based Approach to Down-Regulate a Gene Family In Vivo

Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo.     

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma

BackgroundFew driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes.MethodsWe searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort.ResultsWe found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC.ConclusionOur integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.     

A species in decline: genetic diversity and conservation of the Victorian eastern barred bandicoot, Perameles gunnii

The eastern barred bandicoot, Perameles gunnii, has undergone a dramatic decline in distribution and abundance on the mainland of Australia during the twentieth century. In 1988 a captive breeding program was initiated to reduce the chance of extinction. With the extinction of the last wild mainland population in the early 1990s, reintroductions from captive-bred P. gunnii have met limited success, and currently only two extant populations persist in predator proof enclosures in the State of Victoria. With ~20 years of breeding, there are concerns that the genetic diversity within the breeding program has declined and may inhibit current and future success of the program. We have used ten nuclear microsatellite loci and sequencing of two partial mitochondrial genes (cytochrome oxidase I and ATPase 6) to determine genetic diversity within current Victorian P. gunnii. These diversity estimates are compared with historic samples from the captive breeding program dating back to 1995, historic samples from the last wild mainland population found at Hamilton in 1992 and contemporary Tasmanian wild populations. Results indicate that the captive P. gunnii population in the State of Victoria has lost significant genetic diversity through time. Genetic diversity is also reduced in populations at Hamilton Community Parklands and Mount Rothwell. Samples from the last wild population at Hamilton collected in 1992, along with samples from Tasmanian P. gunnii, had significantly greater genetic diversity than contemporary mainland populations. The results are discussed with reference to management options for maintaining genetic diversity within Victorian P. gunnii, including crossing Victorian and Tasmanian P. gunnii to increase genetic diversity, adaptability and evolutionary potential.     

Are captive wild boar more introgressed than free-ranging wild boar? Two case studies in Italy

Hybridization between wild boar (Sus scrofa) and domestic pig occurred in the past and still occurs today, having great evolutionary and management implications. In fact, genetic introgression from the domestic form may alter traits like behavior, reproduction rate, and immunology in wild populations, with likely demographic impacts. Thus, it is crucial to understand under what conditions hybridization occurs in S. scrofa. Captive crosses with domestic pigs (released or escaped) have been suggested to constitute the major source of the spread of domestic genes into wild boar populations. However, to date, few studies have assessed the degree of admixture in farmed animals in comparison to the surrounding wild populations. With this purpose, we analyzed microsatellite loci in wild boar sampled in breeding stations and in the local wild population in two Italian regions (Sardinia and Piedmont). Both captive populations had lower allelic richness than the corresponding wild population, but a similar expected heterozygosity. In Piedmont, introgression from the domestic form into the wild population seems to be extremely low, while there are significant signs of admixture in the sampled breeding stations. In Sardinia, instead, the captive sample did not differ significantly from the wild population, which showed moderate signs of introgression. We conclude that hybridization in nature seems to play the key role in Sardinia, while intentional hybridization in captivity is the major source of introgression in Piedmont. Our findings emphasize the need for a routine genetic monitoring of wild boar captive populations, coupled with reference data on the neighboring wild populations.     

The genetic diversity and population structure of wild soybean evaluated by chloroplast and nuclear gene sequences

Glycine soja, also called wild soybean, is the wild ancestor of domesticated soybean (Glycine max), and one of the world's major cultivated crops. Wild soybean is a valuable resource for the breeding of cultivated soybean and harbors useful genes or agronomic traits. To use and conserve this valuable resource, we conducted a study to evaluate the genetic diversity and population structure of wild soybean using the sequencing data of two nuclear loci (AF105221 and PhyB) and one chloroplast locus (trnQ-rps16) of more than 600 individuals representing 53 populations throughout the natural distribution range. The results showed that most of the variation was found within the populations and groups, but significant genetic differentiation was also detected among different eco-geographical groups. Correlations between genetic and geographical distance at all the loci were consistent with the isolation by distance gene flow model. G. soja exhibited the highest genetic diversity in middle and downstream of Yangzi River (MDYR) region, followed by North East China (NEC), and was the lowest in North West China (NWC). We concluded that both in situ and ex situ conservation strategies required for wild soybean populations, especially which are native to MDYR and NEC regions.     

No Association between Personality and Candidate Gene Polymorphisms in a Wild Bird Population

Consistency of between-individual differences in behaviour or personality is a phenomenon in populations that can have ecological consequences and evolutionary potential. One way that behaviour can evolve is to have a genetic basis. Identifying the molecular genetic basis of personality could therefore provide insight into how and why such variation is maintained, particularly in natural populations. Previously identified candidate genes for personality in birds include the dopamine receptor D4 (DRD4), and serotonin transporter (SERT). Studies of wild bird populations have shown that exploratory and bold behaviours are associated with polymorphisms in both DRD4 and SERT. Here we tested for polymorphisms in DRD4 and SERT in the Seychelles warbler (Acrocephalus sechellensis) population on Cousin Island, Seychelles, and then investigated correlations between personality and polymorphisms in these genes. We found no genetic variation in DRD4, but identified four polymorphisms in SERT that clustered into five haplotypes. There was no correlation between bold or exploratory behaviours and SERT polymorphisms/haplotypes. The null result was not due to lack of power, and indicates that there was no association between these behaviours and variation in the candidate genes tested in this population. These null findings provide important data to facilitate representative future meta-analyses on candidate personality genes.     

Comparative genetic diversity of wild and hachery populations of Korean threadsail filefish Stephanolepis cirrhifer using cross-species microsatellite markers

The threadsail filefish Stephanolepis cirrhifer is a highly commercial fisheries resource in Korea that suffers intensive anthropogenic pressure across much of its range. For basic information about its current genetic status in relation to stock enhancement, the level and distribution of genetic variation between a wild and a hatchery-bred population were investigated using 10 microsatellite markers developed for Thamnaconus modestus. High levels of polymorphism were observed between the two populations. A total of 95 different alleles were found at all loci, with some alleles being unique. The allelic variability ranged from six to 13 in the wild population and from five to 13 in the hatchery one. The average observed and expected heterozygosities were estimated to be 0.72 and 0.80 in the wild sample and 0.70 and 0.79 in the hatchery one, respectively. These results showed similar genetic variability in the hatchery population, as compared with the wild population and significant genetic differentiation between the wild population and the hatchery samples (F _ST = 0.016, P < 0.05). Genetic drift in the intensive breeding practices for stock enhancement has probably promoted differentiation between populations. Significant deviations from Hardy-Weinberg equilibrium were detected in both populations. Our results indicate that further studies using species-specific microsatellite markers will be necessary for a more reliable assessment of genetic diversity of the species.     

COSTS AND BENEFITS OF MOSQUITO REFRACTORINESS TO MALARIA PARASITES: IMPLICATIONS FOR GENETIC VARIABILITY OF MOSQUITOES AND GENETIC CONTROL OF MALARIA

The problem of fitness costs associated with host resistance to parasitism is related to the evolution of parasite virulence, population genetic diversity and the dynamics of host-parasite relationships, and proposed strategies for disease control through the genetic manipulation of mosquito vectors. Two Aedes aegypti populations, refractory and susceptible to Plasmodium gallinaceum, were previously selected from the Moyo-In-Dry strain (MOYO) through inbreeding (F = 0.5). Reproductive success and survivorship of the two populations were compared, and the influence of the parasite on mosquito fitness also was evaluated. Fitness components studied include fecundity, adult survivorship and egg-to-adult developmental time, blood-meal size, and adult body size. The refractory population has a significantly shorter egg-to-adult developmental time and a smaller body size, takes a smaller blood meal, and subsequently lays fewer eggs than the susceptible population. The mean longevity of the refractory population is significantly shorter than the susceptible population. Exposure to the parasite exhibited little effect on the survivorship and fecundity of either population. Several factors may contribute to the lower fitness of the refractory population, including founder effect, inbreeding depression, the effect of other uncharacterized genes linked to genes conferring refractoriness, and pleiotropic effects associated with these genes. The results are discussed in relation to the genetic diversity of natural mosquito populations and their implications for the genetic control of malaria.     

Comparative Genetic Diversity of Wild and Captive Populations of the Bare-Faced Curassow (Crax fasciolata) Based on Cross-Species Microsatellite Markers: Implications for Conservation and Management

The bare-faced curassow (Crax fasciolata) is a large Neotropical bird that suffers anthropogenic pressure across much of its range. A captive population is maintained for conservation management, although there has been no genetic screening of stocks. Based on the six microsatellite markers developed for Crax globulosa, the genetic variability of C. fasciolata and possible differences between a wild and a captive population were investigated. Only three loci were polymorphic, with a total of 27 alleles. More than half of these alleles were private to the wild (n = 8) or captive (n = 7) populations. Significant deviations from Hardy–Weinberg equilibrium were restricted to the captive population. Despite the number of private alleles, genetic drift has probably promoted differentiation between populations. Our results indicate that wild C. fasciolata populations are genetically impoverished and structured, but species-specific microsatellite markers will be necessary for a more reliable assessment of the species’ genetic diversity.     

Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis

The Korean starry flounder, Platichthys stellatus, is economically valuable coastal resident fish species. However, the annual catch of this fish has fluctuated and suffered major declines in Korea. We examined the genetic diversity and population structure for four wild populations and three hatchery stocks of Korean starry flounder to protect its genetic integrity using nine microsatellites. A group of 339 genotypes belonging to seven populations were screened. High degrees of polymorphism at the microsatellite loci were observed within both the wild and hatchery populations. Compared to the wild populations, genetic changes, including reduced genetic diversity and highly significant differentiation, have occurred in cultured stocks. Significant population differentiation was also observed in wild starry flounder populations. Similar degrees of inbreeding and significant Hardy–Weinberg equilibrium deviations were detected in both the wild and the hatchery populations. The genetic connectivity pattern identified four distinct metapopulations of starry flounder in Korea by clustering in the phylogenetic tree, Bayesian analyses, molecular variance analysis, PCA and multidimensional scaling analysis. A pattern of isolation-by-distance was not significant. This genetic differentiation may be the result of the co-effects of various factors, such as historic dispersal, local environment or anthropogenic activities. These results provide useful information for the genetic monitoring of P. stellatus hatchery stocks, for the genetic improvement of this species by selective breeding and for designing suitable management guidelines for the conservation of this species.     

Genome and population dynamics under selection and neutrality: an example of S-allele diversity in wild cherry (Prunus avium L.)

Self-incompatibility, a common attribute of plant development, forms a classical paradigm of balancing selection in natural populations, in particular negative frequency-dependent selection. Under negative frequency-dependent selection population genetics theory predicts that the S-locus, being in command of self-incompatibility, keeps numerous alleles in equal frequencies demonstrating a wide allelic range. Moreover, while natural populations exhibit a higher within population genetic diversity, a reduction of population differentiation and increase of effective migration rate is expected in comparison to neutral loci. Allelic frequencies were investigated in terms of distribution and genetic structure at the gametophytic self-incompatibility locus in five wild cherry (Prunus avium L.) populations. Comparisons were also made between the differentiation at the S-locus and at the SSR loci. Theoretical expectations under balancing selection were congruent to the results observed. The S-locus showed broad multiplicity (16 S-alleles), high genetic diversity, and allelic isoplethy. Genetic structure at the self-incompatibility locus was almost four times lower than at 11 nSSR loci. Analysis of molecular variance revealed that only 5?% of the total genetic variation concerns differentiation among populations. In conclusion, the wealth of S-allelic diversity found in natural wild cherry populations in Greece is useful not only in advancing basic population genetics research of self-incompatibility systems in wild cherry but also in the development of breeding programs.     

Decreasing genetic diversity in wild and captive populations of endangered Itasenpara bittering (Acheilognathus longipinnis) in the Himi region,central Japan,and recommendations for conservation

Biodiversity is increasingly declining as a result of direct human impact and structural alteration of ecosystems resulting from changes in human life styles. Itasenpara bittering (Acheilognathus longipinnis), which has been maintained in floodplain and paddy fields, is a threatened cyprinid fish endemic to central Japan. To aid in the preservation of this species, information on genetic diversity and demographic variables in wild and captive populations was obtained using microsatellite DNA analysis. Temporal changes in genetic diversity and effective population size (N _e) tended to be relatively stable in the wild Moo River population, although lower values were detected in the wild Busshouji River population, suggesting an extremely high risk of extinction in the latter. Captive populations derived from the Busshouji River population demonstrated significant genetic divergence even among intrapopulational cohorts, suggesting the influence of genetic drift caused by geographic isolation and small population size. Active maintenance of genetic diversity in captive population is a necessary part of conservation programs, as are continuous addition of wild individuals and replacement of individuals among captive populations. In addition, increasing or maintaining suitable floodplain areas and artificial habitats such as paddy fields might contribute to the conservation of genetic diversity in the Itasenpara bittering.     

Sequence-related amplified polymorphism (SRAP) of wild emmer wheat (Triticum dicoccoides) in Israel and its ecological association

Genetic diversity and population structure of 15 wild emmer wheat (Triticum dicoccoides) populations from Israel were detected by 30 sequence-related amplified polymorphism (SRAP) primer pairs. Two hundred and forty four fragments out of 438 were polymorphic. The proportion of polymorphic loci (P), the genetic diversity (He), and Shannon's information index were 0.557, 0.198, and 0.295, respectively. The population Amirim had the highest genetic variation, whereas the population of Tabigha had the lowest genetic variation. The hierarchical analysis of molecular variance (AMOVA) revealed that most of the variation was presented within populations. The value of genetic distance (D) between the populations varied from 0.027 to 0.165 with an average of 0.079, and the estimates of genetic distance were geographically independent based on the Mantel test (r = 0.105, P = 0.168). A total of 30 significant (P < 0.05) correlations were detected between 14 SRAP loci and 12 ecogeographic factors.     

Engineered underdominance allows efficient and economical introgression of traits into pest populations

A novel form of underdominance is suggested as a mechanism that is able to drive desired genes into pest populations through the release of transgenic individuals over one or more generations. Such a mechanism is urgently needed by those working to reduce the impact of malaria by releasing strains of Anopheles, the vector of the disease, that are not susceptible to malaria parasites. We use simple population genetics models to quantify the benefits conferred when heterozygous genotypes, arising from matings between introduced and wild individuals, are not viable. In a randomly mating population, underdominant systems accelerate introgression of desired alleles and allow the release of individuals to be discontinued once the frequency of transgenic alleles attains a threshold. A set of two constructs, which together are selectively neutral but lethal when one is carried without the other, are found to produce dynamics that are characteristic of underdominant systems. When these constructs are carried on non-homologous chromosomes, then the ratio of released to natural born individuals need only be greater than 3:100 for introgression to occur. Furthermore, the threshold for the gene frequencies over which the introduced genes are expected to become fixed upon discontinuing the release of transgenic individuals is surprisingly low. The location of the threshold suggests that the introduced genes are expected to spread in space, at least locally. For the first time, the prospect of a practical drive mechanism for the genetic manipulation of pest populations is raised.     

The population biology of Gehyra (Gekkonidae). III. Patterns of microgeographic variation

The distribution of allozymic and chromosomal polymorphisms was examined among central Australian populations of the chromosomally variable genus Gehyra to assess whether they typically have the small deme size and low gene flow levels required by some models of chromosomal speciation. Particular attention was given to comparisons between rock-specialists (Gehyra nana) and habitat-generalists (Gehyra variegata) to investigate whether the former have more restricted gene flow. Both allozyme and chromosome data sets showed greater among population differentiation in the rock-specialists than the habitat-generalists, consistent with predictions from a previous ecological study (Moritz, 1987), although this pattern could also be due to historical effects. This was evident from the relationships between genetic and geographic distance, the conditional frequency of alleles, and F-statistics. However, both taxa appear to have substantial levels of gene flow. This indicates that Gehyra populations typically do not meet the stringent conditions for the fixation of strongly underdominant chromosome rearrangements through strong genetic drift. A consistent deficiency of heterozygotes does, however, suggest the possibility of inbreeding which would increase the likelihood of the establishment of underdominant rearrangements.     

Genetic diversity of cultured populations of giant freshwater prawn (Macrobrachium rosenbergii) in China using mtDNA COI and 16S rDNA markers

Partial sequences of mitochondrial DNA 16S rDNA and COI genes (395 bp and 498 bp respectively) were sequenced from samples of ten cultured populations of Macrobrachium rosenbergii (Giant Freshwater Prawn – GFP) in Zhejiang, Guangdong and Guangxi Provinces in China and two wild populations of GFP from Mekong River and Dongnai River in Viet Nam. Five haplotypes of 16S rDNA were identified in the 360 samples. The wild populations displayed nucleotide diversity (π) of 0.0008 and 0.0003, and genetic diversity (h) of 0.3030 and 0.1310 in the Mekong River and Dongnai River respectively. The cultured populations displayed no significant genetic diversity. COI sequences identified 17 haplotypes based on 21 polymorphic sites. At this marker, the 12 populations showed a range of h from 0.1290 to 0.6940 and π from 0.0003 to 0.0073. The largest genetic distance (Da) among the 12 populations was 0.0065 (between ZJB and BT/DN populations) and the lowest Da was 0.0003 (between GDD and GDA populations). The wild populations had higher genetic diversity than the cultured populations, but three of cultured populations from Zhejiang (ZJA, ZJB and ZJC) had π higher than wild populations, because they originated from Thailand, Bangladesh and the Mekong River in Viet Nam.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.