An improved genome reference for the African cichlid, Metriaclima zebra

Funding for Open Access provided by the UMD Libraries Open Access Publishing Fund.Background: Problems associated with using draft genome assemblies are well documented and have become more pronounced with the use of short read data for de novo genome assembly. We set out to improve the draft genome assembly of the African cichlid fish, Metriaclima zebra, using a set of Pacific Biosciences SMRT sequencing reads corresponding to 16.5x coverage of the genome. Here we characterize the improvements that these long reads allowed us to make to the state-of-the-art draft genome previously assembled from short read data. Results: Our new assembly closed 68 % of the existing gaps and added 90.6Mbp of new non-gap sequence to the existing draft assembly of M. zebra. Comparison of the new assembly to the sequence of several bacterial artificial chromosome clones confirmed the accuracy of the new assembly. The closure of sequence gaps revealed thousands of new exons, allowing significant improvement in gene models. We corrected one known misassembly, and identified and fixed other likely misassemblies. 63.5 Mbp (70 %) of the new sequence was classified as repetitive and the new sequence allowed for the assembly of many more transposable elements. Conclusions: Our improvements to the M. zebra draft genome suggest that a reasonable investment in long reads could greatly improve many comparable vertebrate draft genome assemblies.https://doi.org/10.1186/s12864-015-1930-

Comparative analysis of a sex chromosome from the blackchin tilapia, Sarotherodon melanotheron

Background Inversions and other structural polymorphisms often reduce the rate of recombination between sex chromosomes, making it impossible to fine map sex-determination loci using traditional genetic mapping techniques. Here we compare distantly related species of tilapia that each segregate an XY system of sex-determination on linkage group 1. We use whole genome sequencing to identify shared sex-patterned polymorphisms, which are candidates for the ancestral sex-determination mutation. Results We found that Sarotherodon melanotheron segregates an XY system on LG1 in the same region identified in Oreochromis niloticus. Both species have higher densities of sex-patterned SNPs, as well as elevated number of ancestral copy number variants in this region when compared to the rest of the genome, but the pattern of differentiation along LG1 differs between species. The number of sex-patterned SNPs shared by the two species is small, but larger than expected by chance, suggesting that a novel Y-chromosome arose just before the divergence of the two species. We identified a shared sex-patterned SNP that alters a Gata4 binding site near Wilms tumor protein that might be responsible for sex-determination. Conclusions Shared sex-patterned SNPs, insertions and deletions suggest an ancestral sex-determination system that is common to both S. melanotheron and O. niloticus. Functional analyses are needed to evaluate shared SNPs near candidate genes that might play a role in sex-determination of these species. Interspecific variation in the sex chromosomes of tilapia species provides an excellent model system for understanding the evolution of vertebrate sex chromosomes. (Résumé d'auteur

Calibration Procedure for Fuel Flow Meters at the Nebraska Tractor Test Lab

Reports in the literature indicated several factors that can influence the accuracy of Coriolis Effect mass flow meters. A Coriolis Effect mass flow meter is used to verify tractor manufacturer’s fuel consumption claims at the Nebraska Tractor Test Laboratory (NTTL). The accuracy requirement placed on the flow meter by the Organization for Economic Co-operation and Development (OECD) in the Code 2 tractor performance test procedure is not clear, but in the most conservative interpretation is ±0.5% of each flow rate measured. Results showed a dynamic weighing calibration method was not accurate enough to obtain a calibration of the flow meter to the desired accuracy level. A static weighing calibration method developed showed no significant difference between the calibration determined by the flow meter’s manufacturer with water and the calibration determined by NTTL with No. 2 diesel fuel. Static weighing calibration tests showed that for flow rates at or above 32 kg/h, the flow meter met the ±0.5% error most conservative interpretation of tolerance on flow rate from OECD Code 2

Structure and decay of a proto-Y region in Tilapia, Oreochromis niloticus

Funding for Open Access provided by the UMD Libraries Open Access Publishing Fund.Sex-determination genes drive the evolution of adjacent chromosomal regions. Sexually antagonistic selection favors the accumulation of inversions that reduce recombination in regions adjacent to the sex-determination gene. Once established, the clonal inheritance of sex-linked inversions leads to the accumulation of deleterious alleles, repetitive elements and a gradual decay of sex-linked genes. This in turn creates selective pressures for the evolution of mechanisms that compensate for the unequal dosage of gene expression. Here we use whole genome sequencing to characterize the structure of a young sex chromosome and quantify sex-specific gene expression in the developing gonad. We found an 8.8 Mb block of strong differentiation between males and females that corresponds to the location of a previously mapped sex-determiner on linkage group 1 of Oreochromis niloticus. Putatively disruptive mutations are found in many of the genes within this region. We also found a significant female-bias in the expression of genes within the block of differentiation compared to those outside the block of differentiation. Eight candidate sex-determination genes were identified within this region. This study demonstrates a block of differentiation on linkage group 1, suggestive of an 8.8 Mb inversion encompassing the sex-determining locus. The enrichment of female-biased gene expression inside the proposed inversion suggests incomplete dosage compensation. This study helps establish a model for studying the early-to-intermediate stages of sex chromosome evolution.https://doi.org/10.1186/1471-2164-15-97

Mapping of pigmentation QTL on an anchored genome assembly of the cichlid fish, Metriaclima zebra

Pigmentation patterns are one of the most recognizable phenotypes across the animal kingdom. They play an important role in camouflage, communication, mate recognition and mate choice. Most progress on understanding the genetics of pigmentation has been achieved via mutational analysis, with relatively little work done to understand variation in natural populations. Pigment patterns vary dramatically among species of cichlid fish from Lake Malawi, and are thought to be important in speciation. In this study, we crossed two species, Metriaclima zebra and M. mbenjii, that differ in several aspects of their body and fin color. We genotyped 798 SNPs in 160 F2 male individuals to construct a linkage map that was used to identify quantitative trait loci (QTL) associated with the pigmentation traits of interest. We also used the linkage map to anchor portions of the M. zebra genome assembly. We constructed a linkage map consisting of 834 markers in 22 linkage groups that spanned over 1,933 cM. QTL analysis detected one QTL each for dorsal fin xanthophores, caudal fin xanthophores, and pelvic fin melanophores. Dorsal fin and caudal fin xanthophores share a QTL on LG12, while pelvic fin melanophores have a QTL on LG11. We used the mapped markers to anchor 66.5% of the M. zebra genome assembly. Within each QTL interval we identified several candidate genes that might play a role in pigment cell development. This is one of a few studies to identify QTL for natural variation in fish pigmentation. The QTL intervals we identified did not contain any pigmentation genes previously identified by mutagenesis studies in other species. We expect that further work on these intervals will identify new genes involved in pigment cell development in natural populations

An EST resource for tilapia based on 17 normalized libraries and assembly of 116,899 sequence tags

<p>Abstract</p> <p>Background</p> <p>Large collections of expressed sequence tags (ESTs) are a fundamental resource for analysis of gene expression and annotation of genome sequences. We generated 116,899 ESTs from 17 normalized and two non-normalized cDNA libraries representing 16 tissues from tilapia, a cichlid fish widely used in aquaculture and biological research.</p> <p>Results</p> <p>The ESTs were assembled into 20,190 contigs and 36,028 singletons for a total of 56,218 unique sequences and a total assembled length of 35,168,415 bp. Over the whole project, a unique sequence was discovered for every 2.079 sequence reads. 17,722 (31.5%) of these unique sequences had significant BLAST hits (e-value < 10^-10) to the UniProt database.</p> <p>Conclusion</p> <p>Normalization of the cDNA pools with double-stranded nuclease allowed us to efficiently sequence a large collection of ESTs. These sequences are an important resource for studies of gene expression, comparative mapping and annotation of the forthcoming tilapia genome sequence.</p

Circular DNA Intermediate in the Duplication of Nile Tilapia vasa Genes

vasa is a highly conserved RNA helicase involved in animal germ cell development. Among vertebrate species, it is typically present as a single copy per genome. Here we report the isolation and sequencing of BAC clones for Nile tilapia vasa genes. Contrary to a previous report that Nile tilapia have a single copy of the vasa gene, we find evidence for at least three vasa gene loci. The vasa gene locus was duplicated from the original site and integrated into two distant novel sites. For one of these insertions we find evidence that the duplication was mediated by a circular DNA intermediate. This mechanism of gene duplication may explain the origin of isolated gene duplicates during the evolution of fish genomes. These data provide a foundation for studying the role of multiple vasa genes in the development of tilapia gonads, and will contribute to investigations of the molecular mechanisms of sex determination and evolution in cichlid fishes

Structure and Sequence of the Sex Determining Locus in Two Wild Populations of Nile Tilapia

In domesticated strains of the Nile tilapia, phenotypic sex has been linked to genetic variants on linkage groups 1, 20 and 23. This diversity of sex-loci might reflect a naturally polymorphic sex determination system in Nile tilapia, or it might be an artefact arising from the process of domestication. Here, we searched for sex-determiners in wild populations from Kpandu, Lake Volta (Ghana-West Africa), and from Lake Koka (Ethiopia-East Africa) that have not been subjected to any genetic manipulation. We analysed lab-reared families using double-digest Restriction Associated DNA sequencing (ddRAD) and analysed wild-caught males and females with pooled whole-genome sequencing (WGS). Strong sex-linked signals were found on LG23 in both populations, and sex-linked signals with LG3 were observed in Kpandu samples. WGS uncovered blocks of high sequence coverage, suggesting the presence of B chromosomes. We confirmed the existence of a tandem amh duplication in LG23 in both populations and determined its breakpoints between the oaz1 and dot1l genes. We found two common deletions of ~5 kb in males and confirmed the presence of both amhY and amh∆Y genes. Males from Lake Koka lack both the previously reported 234 bp deletion and the 5 bp frameshift-insertion that creates a premature stop codon in amh∆Y