Genome-wide QTL mapping of saltwater tolerance in sibling species of Anopheles (malaria vector) mosquitoes.

Although freshwater (FW) is the ancestral habitat for larval mosquitoes, multiple species independently evolved the ability to survive in saltwater (SW). Here, we use quantitative trait locus (QTL) mapping to investigate the genetic architecture of osmoregulation in Anopheles mosquitoes, vectors of human malaria. We analyzed 1134 backcross progeny from a cross between the obligate FW species An. coluzzii, and its closely related euryhaline sibling species An. merus. Tests of 2387 markers with Bayesian interval mapping and machine learning (random forests) yielded six genomic regions associated with SW tolerance. Overlap in QTL regions from both approaches enhances confidence in QTL identification. Evidence exists for synergistic as well as disruptive epistasis among loci. Intriguingly, one QTL region containing ion transporters spans the 2Rop chromosomal inversion that distinguishes these species. Rather than a simple trait controlled by one or a few loci, our data are most consistent with a complex, polygenic mode of inheritance.

RNAi knock-downs support roles for the mucin-like (AeIMUC1) gene and short-chain dehydrogenase/reductase (SDR) gene in Aedes aegypti susceptibility to Plasmodium gallinaceum.

The mosquito midgut represents the first barrier encountered by the Plasmodium parasite (Haemosporida: Plasmodiidae) when it is ingested in blood from an infected vertebrate. Previous studies identified the Aedes aegypti (L.) (Diptera: Culicidae) mucin-like (AeIMUC1) and short-chain dehydrogenase/reductase (SDR) genes as midgut-expressed candidate genes influencing susceptibility to infection by Plasmodium gallinaceum (Brumpt). We used RNA inference (RNAi) by double-stranded RNA (dsRNA) injections to examine ookinete survival to the oocyst stage following individual gene knock-downs. Double-stranded RNA gene knock-downs were performed 3 days prior to P. gallinaceum infection and oocyst development was evaluated at 7 days post-infection. Mean numbers of parasites developing to the oocyst stage were significantly reduced by 52.3% in dsAeIMUC1-injected females and by 36.5% in dsSDR-injected females compared with females injected with a dsß-gal control. The prevalence of infection was significantly reduced in dsAeIMUC1- and dsSDR-injected females compared with females injected with dsß-gal; these reductions resulted in a two- and three-fold increase in the number of uninfected individuals, respectively. Overall, these results suggest that both AeIMUC1 and SDR play a role in Ae. aegypti vector competence to P. gallinaceum.

Development of bacterial artificial chromosome library resources for parasitoid Hymenoptera (Nasonia vitripennis and Nasonia giraulti: Pteromalidae).

The species of the genus Nasonia possess qualities that make them excellent candidates for genetic and genomic studies. To increase the wealth of genomic resources for the genus we constructed publicly available bacterial artificial chromosome (BAC) libraries for Nasonia vitripennis and Nasonia giraulti. Libraries have 36 864 clones each, empty-vector contents of approximately 2% and average insert sizes of 113.1 and 97.7 Kb, respectively, representing 12 and 11 genome equivalents. The N. vitripennis library was used for genome sequence assembly and in efforts at positional cloning of a developmental gene. The genome assembly of N. vitripennis is currently composed on 6181 un-joined scaffolds. These BAC libraries can be used to identify and close regions between scaffolds of the genome assemblies of both species.

Quantitative trait loci determining autogeny and body size in the Asian tiger mosquito (Aedes albopictus).

The majority of mosquito species require a blood meal to stimulate vitellogenesis and subsequent oviposition (anautogeny), but some autogenous individuals complete their first ovarian cycle without a blood meal. Autogeny may be facultative or obligatory. In this study, we selected for an autogenous strain in the Asian tiger mosquito Aedes albopictus and examined an F(1) intercross population for quantitative trait loci (QTL) determining the autogeny trait as well as wing length as a proxy for body size. Using composite interval mapping, we identified four QTL for each trait and observed considerable overlap in genome positions between each QTL for autogeny (follicle size) and wing length. Most QTL were minor in magnitude, individually explaining <10% of the phenotypic variation. Alleles from the autogenous parent generally showed a dominance or overdominance effect on both phenotypes. Strong genetic and phenotypic correlations indicate that autogeny and wing length are determined by up to four clusters of tightly linked genes or the potential pleiotropic effects of single genes. Although females from the autogenous strain produced approximately fivefold more eggs following a blood meal than through autogeny, we suggest that the maintenance of alleles for autogeny in natural populations is likely due to balancing selection. Autogeny should be favored under conditions of limited host availability for blood feeding or increased defensive behavior by the host and adequate larval nutrition. Correlation between autogeny and body size may reflect an increased ability for larger females to accumulate sufficient nutrient reserves to support oogenesis without the requirement for a blood meal.

Investigations of dengue-2 susceptibility and body size among Aedes aegypti populations.

The mosquito Aedes aegypti (L.) (Diptera: Culicidae) is the primary global vector for dengue virus (DENV), yet considerable genetic variation exists among populations in terms of its competence to vector DENV. Variability in adult body size has also been observed among various mosquito populations and several studies have reported a relationship between body size and arbovirus dissemination, although most of these relied on artificially derived variation in body size. Here we examine the relationship between body size and disseminated DENV infection among 10 Ae. aegypti populations reared under optimum laboratory conditions. Body size variability was inferred from wing length measurements and DENV competence was evaluated as the proportion of individuals with disseminated infections following exposure to the dengue-2 JAM1409 strain. There were significant differences in mean wing lengths among populations (anova, F(9,22)= 7.10, P < 0.0001), ranging from 2.16 mm (Bangkok population) to 2.79 mm (MOYO-S [susceptible] population). We also observed significant differences among some populations in mean DENV infection rates (Waller-Duncan K-ratio t-test), ranging from 19.54% (MOYO-R [refractory] population) to 56.60% (MOYO-S population). However, we did not observe evidence for significant interactions between body size and DENV dissemination. We suggest that either the two traits are genetically independent or that our ability to detect interactions between them was limited by their respective inheritances as quantitative traits.

Genetic basis for reproductive diapause is correlated with life history traits within the Culex pipiens complex.

The evolution of late season reproductive arrest (diapause) among female Culex pipiens mosquitoes allows them to overwinter in temperate climates, while females of the sibling species Culex quinquefasciatus do not exhibit the diapause phenotype. We present results for quantitative trait loci (QTL) analyses of two independent segregating populations derived from crosses between C. pipiens and C. quinquefasciatus. QTL for diapause and three life history traits were identified and compared for genome positions and gene effects. Using a combination of composite interval mapping, single-marker analysis and all possible subsets regression, we identified multiple QTL for each trait, totalling 14 and 17 QTL for each population, respectively. Individual QTL across traits often mapped to similar genome locations, suggesting these traits may be controlled in part by genes with pleiotropic effects or multiple tightly linked genes. The majority of QTL were intermediate in magnitude in that they explained 10-25% of the phenotypic variation. The majority of QTL showed overdominance effects. We suggest that this could impact natural populations, as increased heterosis across hybrid zones may allow populations to adapt to environmental conditions via stabilizing selection, and yet maintain species identity outside these regions because of strong morphological integration, wherein related traits evolve as an integrated unit.

Sequencing of a new target genome: the Pediculus humanus humanus (Phthiraptera: Pediculidae) genome project.

The human body louse, Pediculus humanus humanus (L.), and the human head louse, Pediculus humanus capitis, belong to the hemimetabolous order Phthiraptera. The body louse is the primary vector that transmits the bacterial agents of louse-borne relapsing fever, trench fever, and epidemic typhus. The genomes of the bacterial causative agents of several of these aforementioned diseases have been sequenced. Thus, determining the body louse genome will enhance studies of host-vector-pathogen interactions. Although not important as a major disease vector, head lice are of major social concern. Resistance to traditional pesticides used to control head and body lice have developed. It is imperative that new molecular targets be discovered for the development of novel compounds to control these insects. No complete genome sequence exists for a hemimetabolous insect species primarily because hemimetabolous insects often have large (2000 Mb) to very large (up to 16,300 Mb) genomes. Fortuitously, we determined that the human body louse has one of the smallest genome sizes known in insects, suggesting it may be a suitable choice as a minimal hemimetabolous genome in which many genes have been eliminated during its adaptation to human parasitism. Because many louse species infest birds and mammals, the body louse genome-sequencing project will facilitate studies of their comparative genomics. A 6-8X coverage of the body louse genome, plus sequenced expressed sequence tags, should provide the entomological, evolutionary biology, medical, and public health communities with useful genetic information.

Application of One Sided t-tests and a Generalized Experiment Wise Error Rate to High-Density Oligonucleotide Microarray Experiments: An Example Using Arabidopsis.

MOTIVATION: A formidable challenge in the analysis of microarray data is the identification of those genes that exhibit differential expression. The objectives of this research were to examine the utility of simple ANOVA, one sided t tests, natural log transformation, and a generalized experiment wise error rate methodology for analysis of such experiments. As a test case, we analyzed a Affymetrix GeneChip microarray experiment designed to test for the effect of a CHD3 chromatin remodeling factor, PICKLE, and an inhibitor of the plant hormone gibberellin (GA), on the expression of 8256 Arabidopsis thaliana genes. RESULTS: The GFWER(k) is defined as the probability of rejecting k or more true null hypothesis at a given p level. Computing probabilities by GFWER(k) was shown to be simple to apply and, depending on the value of k, can greatly increase power. A k value as small as 2 or 3 was concluded to be adequate for large or small experiments respectively. A one sided t-test along with GFWER(2)=.05 identified 43 genes as exhibiting PICKLE-dependent expression. Expression of all 43 genes was re-examined by qRT-PCR, of which 36 (83.7%) were confirmed to exhibit PICKLE-dependent expression.

Transcriptome analysis of the cowpea weevil bruchid: identification of putative proteinases and alpha-amylases associated with food breakdown.

We describe here the first systematic work to discover insect genes involved in food breakdown using a cDNA library enriched for gut-expressed transcripts from Callosobruchus maculatus. A total of 1056 clones were screened for cDNA insert-containing plasmids, and 503 nonredundant open reading frames were discovered. Twenty-three inferred genes potentially involved in digestive processes in cowpea weevil were identified, including proteinases and amylases. The predicted catalytic sites were identified in the inferred cysteine and aspartic acid proteinases, and in alpha-amylases. Transcriptome analysis of the cowpea bruchid will potentially permit gene discovery in other beetles, an insect order of major economic and ecological importance that is poorly represented in genomic databases.

Genetic basis of heterosis explored by simple sequence repeat markers in a random-mated maize population.

The genetic basis of heterosis in crop plants has not been completely resolved. Our objective in this study was to determine the level of dominance for quantitative trait loci (QTLs) that underlie heterosis in maize (Zea mays L.). An F2 population of an elite maize single cross, LH200 x LH216, was random mated for three generations in an attempt to break up repulsion linkages that might lead to pseudo-overdominance. The population was analyzed with 160 simple-sequence repeat markers. Phenotypic data analyses indicated overdominance for grain yield and partial dominance for plant height, grain moisture and stalk lodging. A total of 28 QTLs were identified for grain yield, 16 for grain moisture, 8 for stalk lodging, and 11 for plant height. For grain yield, 24 QTLs (86%) showed overdominance. In contrast, most of the QTLs for plant height, grain moisture and stalk lodging showed partial to complete dominance. Little epistasis was detected among the QTLs for any of the traits. Our results can be interpreted in one of two ways, or a combination of both: (1) QTLs for grain yield in maize exhibit true overdominance, or (2) QTLs for grain yield show partial to complete dominance, but they are so tightly linked such that three generations of random mating failed to separate their individual effects.

Chromosomal regions associated with segregation distortion in maize.

Segregation distortion skews the genotypic frequencies from their Mendelian expectations. Our objectives in this study were to assess the frequency of occurrence of segregation distortion in maize, identify chromosomal regions consistently associated with segregation distortion, and examine the effects of gametophytic factors on linkage mapping. We constructed a simple sequence repeat (SSR) linkage map for a LH200/LH216 F(2)Syn3 (i.e., random-mated three times) population, and compared the segregation distortion in this map with the segregation distortion in three published linkage maps. Among 1,820 codominant markers across the four mapping populations, 301 (17%) showed segregation distortion ( P < 0.05). The frequency of markers showing segregation distortion ranged from 19% in the Tx303/CO159 mapping population to 36% in the B73/Mo17 mapping population. A positive relationship was found between the number of meioses and the frequency of segregation distortion detected in a population. On a given chromosome, nearly all of the markers showing segregation distortion favored the allele from the same parent. A total of 18 chromosomal regions on the ten maize chromosomes were associated with segregation distortion. The consistent location of these chromosomal regions in four populations suggested the presence of segregation distortion regions (SDRs). Three known gametophytic factors are possible genetic causes of these SDRs. As shown in previous research, segregation distortion does not affect the estimate of map distance when only one gametophytic factor is present in an SDR.

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers.

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.

Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats.

The abundance and scattered distribution of simple-sequence repeats (SSR) in eukaryotic genomes prompted us to explore the use of SSR-based oligonucleotide primers in single primer amplification reactions. In a pilot experiment, 23 primers were used across a panel of evolutionarily diverse eukaryotic genomes, including grapes, lettuce, tomato, pine, maize, salmon, chicken, Holstein cows and humans. The primers were 16-20 bases in length and represented SSRs of di-, tri-, tetra-, and pentanucleotide repeats. The results showed that tetranucleotide repeat primers were most effective in amplifying polymorphic patterns. Of 11 such primers tested, 70% produced polymorphic patterns from the DNA of one or more species. Primers representing a combination of two tetranucleotide repeats, or compound microsatellites, were equally effective. The polymorphisms contained in such fingerprints were able to identify individuals of vertebrate species as well as lines or varieties of plants. Inheritance of the polymorphic bands was studied in a maize recombinant inbred population, DE811 x B73. Thirty-two polymorphic bands, derived from two amplification patterns, were mapped as dominant markers on an existing RFLP map of the same population. The bands were distributed across nine of the ten chromosomes.

Pinning down loose ends: mapping telomeres and factors affecting their length.

A degenerately repeated sequence, proximal to the telomere heptanucleotide repeat in maize, contains restriction enzyme sites that permit the separation of telomeres from the rest of the chromosomes. Probing with a telomere-specific oligonucleotide revealed genotype-dependent telomere lengths that vary more than 25-fold in maize among the 22 inbreds that have been surveyed. These lengths were found to segregate reproducibly in a recombinant inbred family where 50% of the variation can be accounted for by three loci. The dynamic control over telomere length in maize appears to act rapidly to achieve new genotypically determined telomere lengths in the F1. Clones of telomere proximal sequences were used to map restriction fragment length loci at the distal ends of eight of 20 chromosome arms.

Improved tissue culture response of an elite maize inbred through backcross breeding, and identification of chromosomal regions important for regeneration by RFLP analysis.

The frequency of initiation of friable, embryogenic callus from immature embryos of the elite maize inbred line B73 was increased dramatically by introgression of chromosomal segments from the inbred line A188 through classical backcross breeding. Less than 0.2% of the immature B73 embryos tested (5 of 3,710) formed embryogenic callus. The breeding scheme consisted of six generations of backcrossing to B73 with selection at each generation for high frequency initiation of embryogenic cultures. BC6 individuals were selfed for four generations to select homozygous lines. The average embryogenic culture initiation frequency increased to 46% (256/561). Nearly all (91%) of the embryos from one BC6 S4 plant formed embryogenic cultures. RFLP analysis was used to determine the locations and effects of the introgressed A188 chromosomal segments. Five segments were retained through at least the fifth backcross generation. The hypothesis that one or more of these five regions contains genes controlling somatic embryogenesis in maize was tested using an F2 population of the cross A188 X Mo17. A set of five DNA markers (three of them linked) explained 82% of the observed phenotypic variance for percentage of immature embryos forming embryognic callus. Four of the five markers were located in or near introgressed A188 chromosome segments.The region marked by probe c595 on the long arm of chromosome 9 was highly associated with several measures of in vitro culture response (percent embryogenic embryos, plants per embryo, and plants per embryogenic embryo). We propose that there is a major gene (or genes) in this region in A188 that promotes embryogenic callus initiation and plant regeneration in B73, Mo17, and probably many other recalcitrant inbred lines of maize.