Expanding the view of Clock and cycle gene evolution in Diptera.

We expanded the view of Clock (Clk) and cycle (cyc) gene evolution in Diptera by studying the fruit fly Anastrepha fraterculus (Afra), a Brachycera. Despite the high conservation of clock genes amongst insect groups, striking structural and functional differences of some clocks have appeared throughout evolution. Clk and cyc nucleotide sequences and corresponding proteins were characterized, along with their mRNA expression data, to provide an evolutionary overview in the two major groups of Diptera: Lower Diptera and Higher Brachycera. We found that AfraCYC lacks the BMAL (Brain and muscle ARNT-like) C-terminus region (BCTR) domain and is constitutively expressed, suggesting that AfraCLK has the main transactivation function, which is corroborated by the presence of poly-Q repeats and an oscillatory pattern. Our analysis suggests that the loss of BCTR in CYC is not exclusive of drosophilids, as it also occurs in other Acalyptratae flies such as tephritids and drosophilids, however, but it is also present in some Calyptratae, such as Muscidae, Calliphoridae and Sarcophagidae. This indicates that BCTR is missing from CYC of all higher-level Brachycera and that it was lost during the evolution of Lower Brachycera. Thus, we can infer that CLK protein may play the main role in the CLK\CYC transcription complex in these flies, like in its Drosophila orthologues.

Transcriptome analysis of female reproductive tissues of Anastrepha obliqua and molecular evolution of eggshell proteins in the fraterculus group.

The investigation of cDNA libraries has been an important tool for the identification of new genes in nonmodel species such as the fruit flies from the Anastrepha fraterculus group. In the present study, we constructed a cDNA library from the female reproductive tissues of Anastrepha obliqua aiming to identify genes with high evolutionary rates. We sequenced 2304 clones obtained from the female reproductive tissues of A. obliqua flies. The expressed sequence tags generated a total of 816 unigenes which were classified into different protein classes. Among these,we identified chorionic and vitelline protein genes as being among the most highly expressed. We used unigene sequences to amplify a set of chorionic and vitelline genes, involved in the formation of the eggshell,in species of the fraterculus group. Four chorionic genes and two vitelline genes showed evidence of positive selection along the Anastrepha and/or Tephritidae lineage. The signal of selection detected for Vm26Aa was possibly generated by a gene duplication event. The rapid evolutionary rates indicate that these genes could serve as important markers in population and evolutionary studies, not only for species of this group, but possibly also for other Diptera.

Epistasis affecting litter size in mice.

Litter size is an important reproductive trait as it makes a major contribution to fitness. Generally, traits closely related to fitness show low heritability perhaps because of the corrosive effects of directional natural selection on the additive genetic variance. Nonetheless, low heritability does not imply, necessarily, a complete absence of genetic variation because genetic interactions (epistasis and dominance) contribute to variation in traits displaying strong heterosis in crosses, such as litter size. In our study, we investigated the genetic architecture of litter size in 166 females from an F2 intercross of the SM/J and LG/J inbred mouse strains. Litter size had a low heritability (h2 = 12%) and a low repeatability (r = 33%). Using interval-mapping methods, we located two quantitative trait loci (QTL) affecting litter size at locations D7Mit21 + 0 cM and D12Mit6 + 8 cM, on chromosomes 7 and 12 respectively. These QTL accounted for 12.6% of the variance in litter size. In a two-way genome-wide epistasis scan we found eight QTL interacting epistatically involving chromosomes 2, 4, 5, 11, 14, 15 and 18. Taken together, the QTL and their interactions explain nearly 49% (39.5% adjusted multiple r2) of the phenotypic variation for litter size in this cross, an increase of 36% over the direct effects of the QTL. This indicates the importance of epistasis as a component of the genetic architecture of litter size and fitness in our intercross population.

Obtaining genetic markers by using double-stringency PCR with microsatellites and arbitrary primers.

A technique to obtain genetic markers is described. PCR is performed at two different annealing temperatures in a single reaction mixture. During the first cycles, a single microsatellite oligonucleotide is used as primer to amplify DNA between two microsatellite regions. The annealing temperature and consequent stringency is then lowered, and in further cycles a random-amplified polymorphic DNA (RAPD) primer exponentially amplifies parts of the previously amplified fragments, which contain sequences complementary to the RAPD primer. The first round of specific amplification is found to be crucial for obtaining consistent, repeatable results, as compared with the results obtained by RAPD alone. This technique was proven to produce genetically informative markers, as revealed by crosses performed with Drosophila mercatorum strains, and it can be used for genetic mapping or population genetics studies.