Перевірка суб’єктів господарювання державного сектора економіки: ревізія чи аудит?

The house sparrow (Passer domesticus) is an important model species in ecology and evolution. However, until recently, genomic resources for molecular ecological projects have been lacking in this species. Here, we present transcriptome sequencing data (RNA-Seq) from three different house sparrow tissues (spleen, blood and bursa). These tissues were specifically chosen to obtain a diverse representation of expressed genes and to maximize the yield of immune-related gene functions. After de novo assembly, 15250 contigs were identified, representing sequence data from a total of 8756 known avian genes (as inferred from the closely related zebra finch). The transcriptome assembly contain sequence data from nine manually annotated MHC genes, including an almost complete MHC class I coding sequence. There were 407, 303 and 68 genes overexpressed in spleen, blood and bursa, respectively. Gene ontology terms related to ribosomal function were associated with overexpression in spleen and oxygen transport functions with overexpression in blood. In addition to the transcript sequences, we provide 327 gene-linked microsatellites (SSRs) with sufficient flanking sequences for primer design, and 3177 single-nucleotide polymorphisms (SNPs) within genes, that can be used in follow-up molecular ecology studies of this ecological well-studied species

AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning

AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N-terminal active site domain and a C-terminal peptidoglycan-binding domain. The active site domain is homologous to that of muramidase-2 of Enterococcus hirae, however, RP-HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N-acetylglucosaminidase. In the C-terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall-hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning

Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies

Next-generation sequencing of transcriptomes (RNA-Seq) is being used increasingly in studies of nonmodel organisms. Here, we evaluate the effectiveness of normalising cDNA libraries prior to sequencing in a small-scale study of the zebra finch. We find that assemblies produced from normalised libraries had a larger number of contigs but used fewer reads compared to unnormalised libraries. Considerably more genes were also detected using the contigs produced from normalised cDNA, and microsatellite discovery was up to 73% more efficient in these. There was a positive correlation between the detected expression level of genes in normalised and unnormalised cDNA, and there was no difference in the number of genes identified as being differentially expressed between blood and spleen for the normalised and unnormalised libraries. We conclude that normalised cDNA libraries are preferable for many applications of RNA-Seq and that these can also be used in quantitative gene expression studies

A method for genotype validation and primer assessment in heterozygote-deficient species, as demonstrated in the prosobranch mollusc Hydrobia ulvae

BACKGROUND: In studies where microsatellite markers are employed, it is essential that the primers designed will reliably and consistently amplify target loci. In populations conforming to Hardy-Weinberg equilibrium (HWE), screening for unreliable markers often relies on the identification of heterozygote deficiencies and subsequent departures from HWE. However, since many populations naturally deviate from HWE, such as many marine invertebrates, it can be difficult to distinguish heterozygote deficiencies resulting from unreliable markers from natural processes. Thus, studies of populations that are suspected to deviate from HWE naturally would benefit from a method to validate genotype data-sets and test the reliability of the designed primers. Levels of heterozygosity are reported for the prosobranch mollusc Hydrobia ulvae (Pennant) together with a method of genotype validation and primer assessment that utilises two primer sets for each locus. Microsatellite loci presented are the first described for the species Hydrobia ulvae; the five loci presented will be of value in further study of populations of H. ulvae. RESULTS: We have developed a novel method of testing primer reliability in naturally heterozygote deficient populations. After the design of an initial primer set, genotyping in 48 Hydrobia ulvae specimens using a single primer set (Primer set_A) revealed heterozygote deficiency in six of the seven loci examined. Redesign of six of the primer pairs (Primer set_B), re-genotyping of the successful individuals from Primer set_A using Primer set_B, and comparison of genotypes between the two primer sets, enabled the identification of two loci (Hulv-06 & Hulv-07) that showed a high degree of discrepancy between primer sets A and B (0% & only 25% alleles matching, respectively), suggesting unreliability in these primers. The discrepancies included changes from heterozygotes to homozygotes or vice versa, and some individuals who also displayed new alleles of unexpected sizes. Of the other four loci examined (Hulv-01, Hulv-03, Hulv-04, & Hulv-05), all showed more than 95% agreement between primer sets. Hulv-01, Hulv-03, & Hulv-05 displayed similar levels of heterozygosity with both primer sets suggesting that these loci are indeed heterozygote deficient, while Hulv-08 showed no deficiency in either primer set. CONCLUSION: The simple method described to identify unreliable markers will prove a useful technique for many population studies, and also emphasises the dangers in using a single primer set and assuming marker reliability in populations shown to naturally deviate from HWE

Genomic associations with bill length and disease reveal drift and selection across island bird populations

Island species provide excellent models for investigating how selection and drift operate in wild populations, and for determining how these processes act to influence local adaptation and speciation. Here, we examine the role of selection and drift in shaping genomic and phenotypic variation across recently separated populations of Berthelot's pipit (Anthus berthelotii), a passerine bird endemic to three archipelagos in the Atlantic. We first characterized genetic diversity and population structuring that supported previous inferences of a history of recent colonizations and bottlenecks. We then tested for regions of the genome associated with the ecologically important traits of bill length and malaria infection, both of which vary substantially across populations in this species. We identified a SNP associated with variation in bill length among individuals, islands, and archipelagos; patterns of variation at this SNP suggest that both phenotypic and genotypic variation in bill length is largely shaped by founder effects. Malaria was associated with SNPs near/within genes involved in the immune response, but this relationship was not consistent among archipelagos, supporting the view that disease resistance is complex and rapidly evolving. Although we found little evidence for divergent selection at candidate loci for bill length and malaria resistance, genome scan analyses pointed to several genes related to immunity and metabolism as having important roles in divergence and adaptation. Our findings highlight the utility and challenges involved with combining association mapping and population genetic analysis in nonequilibrium populations, to disentangle the effects of drift and selection on shaping genotypes and phenotypes

A new marker based on the avian spindlin gene that is able to sex most birds, including species problematic to sex with CHD markers

We have developed a new marker (Z43B) that can be successfully used to identify the sex of most birds (69%), including species difficult or impossible to sex with other markers. We utilized the zebra finch Taeniopygia guttata EST microsatellite sequence (CK309496) which displays sequence homology to the 5′ untranslated region (UTR) of the avian spindlin gene. This gene is known to be present on the Z and W chromosomes. To maximize cross-species utility, the primer set was designed from a consensus sequence created from homologs of CK309496 that were isolated from multiple distantly related species. Both the forward and reverse primer sequences were 100% identical to 14 avian species, including the Z chromosome of eight species and the chicken Gallus gallus W chromosome, as well as the saltwater crocodile Crocodylus porosus. The Z43B primer set was assessed by genotyping individuals of known sex belonging to 61 non-ratite species and a single ratite. The Z and W amplicons differed in size making it possible to distinguish between males (ZZ) and females (ZW) for the majority (69%) of non-ratite species tested, comprising 10 orders of birds. We predict that this marker will be useful for obtaining sex-typing data for ca 6,869 species of birds (69% of non-ratites but not galliforms). A wide range of species could be sex-typed including passerines, shorebirds, eagles, falcons, bee-eaters, cranes, shags, parrots, penguins, ducks, and a ratite species, the brown kiwi, Apteryx australis. Those species sexed include species impossible or problematic to sex-type with other markers (magpie, albatross, petrel, eagle, falcon, crane, and penguin species)

Conserved microsatellite markers of high cross-species utility for flying, ground and tree squirrels

Many squirrel species around the world are threatened by forest loss and fragmentation. To facilitate studies of squirrel biodiversity, particularly of flying squirrels in Southeast Asia, we identified Hylopetes, Menetes, Glaucomys and Sciurus squirrel microsatellite sequences with homologs in a second squirrel species (Spermophilus tridecemlineatus), designed 40 consensus markers and tested three squirrel species. When tested in four individuals per species, 26 markers were variable in Hylopetes phayrei, 25 markers in H. lepidus and 25 markers in Menetes berdmorei. Eleven markers were selected from 14 that were polymorphic in all three species. Cross-species utility was confirmed for these 11 markers in seven additional squirrel species, including: the flying squirrels H. phayrei, H. lepidus, H. spadiceus and Petaurista petaurista; a ground squirrel, M. berdmorei; and the tree squirrels, Callosciurus caniceps and C. finlaysoni. The other markers that were variable in one or multiple species are also useful for those specific species

Identification of 24 new microsatellite loci in the sweat bee Lasioglossum malachurum (Hymenoptera: Halictidae)

OBJECTIVE: The objective here is to identify highly polymorphic microsatellite loci for the Palaearctic sweat bee Lasioglossum malachurum. Sweat bees (Hymenoptera: Halictidae) are widespread pollinators that exhibit an unusually large range of social behaviours from non-social, where each female nests alone, to eusocial, where a single queen reproduces while the other members of the colony help to rear her offspring. They thus represent excellent models for understanding social evolution. RESULTS: 24 new microsatellite loci were successfully optimized. When amplified across 23-40 unrelated females, the number of alleles per locus ranged from 3 to 17 and the observed heterozygosities 0.45 to 0.95. Only one locus showed evidence of significant deviation from Hardy-Weinberg equilibrium. No evidence of linkage disequilibrium was found. These 24 loci will enable researchers to gain greater understanding of colony relationships within this species, an important model for the study of eusociality. Furthermore, 22 of the same loci were also successfully amplified in L. calceatum, suggesting that these loci may be useful for investigating the ecology and evolution of sweat bees in general

Autosomal and Z-linked microsatellite markers enhanced for cross-species utility and assessed in a range of birds, including species of conservation concern

Microsatellite markers were designed to be of utility for genotyping multiple species of birds, including those of conservation concern, hence saving resources and enabling species/genome comparisons. We used the proven approach of Dawson et al. (Mol Ecol Resour 10:475–494, 2010) and assessed markers in multiple species, including nine species of conservation interest. We ensured both primer sequences matched multiple species (13 loci) or designed primer sets from expressed sequence tags (2 loci). Eleven primer sets were 100 % identical to the zebra finch (Taeniopygia guttata) and a second passerine species and/or the chicken (Gallus gallus). All 15 loci were polymorphic when assessed in a non-source species (Gouldian finch, Erythrura gouldiae) suggesting utility in multiple species. Four of the five Z-linked loci were assessed in at least nine additional species each (including ratites). All were variable in multiple species, demonstrating cross-species utility and potential for identifying Z chromosome rearrangements