Copy number variation of individual cattle genomes using next-generation sequencing

Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one Holstein, and one Hereford) and one indicine (Nelore) cattle. Within mapped chromosomal sequence, we identified 1265 CNV regions comprising similar to 55.6-Mbp sequence-476 of which (similar to 38%) have not previously been reported. We validated this sequence-based CNV call set with array comparative genomic hybridization (aCGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH), achieving a validation rate of 82% and a false positive rate of 8%. We further estimated absolute copy numbers for genomic segments and annotated genes in each individual. Surveys of the top 25 most variable genes revealed that the Nelore individual had the lowest copy numbers in 13 cases (similar to 52%, chi(2) test; P-value <0.05). In contrast, genes related to pathogen- and parasite-resistance, such as CATHL4 and ULBP17, were highly duplicated in the Nelore individual relative to the taurine cattle, while genes involved in lipid transport and metabolism, including APOL3 and FABP2, were highly duplicated in the beef breeds. These CNV regions also harbor genes like BPIFA2A (BSP30A) and WC1, suggesting that some CNVs may be associated with breed-specific differences in adaptation, health, and production traits. By providing the first individualized cattle CNV and segmental duplication maps and genome-wide gene copy number estimates, we enable future CNV studies into highly duplicated regions in the cattle genome.

Transposon Tn1721 distribution among strains of Xylella fastidiosa

Transposons are mobile genetic elements found within the genomes of various organisms including bacteria, fungi, plants and animals. Fragments of the transposon Tn1721 were found included in the genome of Xylella fastidiosa strain 9a5c. Regions from such fragments were PCR-amplified using specially designed primers (TNP1 and TNP2). In order to detect insertions of the Tn1721 element, both primers were used and one of them included a region of the transposon (TNP1) and the other one had the right repeat and part of the bacterial chromosome (TNP2). The PCR products obtained from strain 9a5c were used as a pattern for fragment size comparisons when DNA samples from other X. fastidiosa strains were used as template for the PCR assays. Differences were observed concerning the PCR products of such amplifications when some X. fastidiosa strains isolated from grapevine and plum were used. For the citrus-derived strains only the strains U187d and GP920b produced fragments with different sizes or weak band intensity. Such variations in the X. fastidiosa genome related to disrupted Tn1721 copies are probably due to the possibility of such a transposon element being still able to duplicate even after deletion events might have taken place and also because the bacterial strains in which the main differences were detected are derived from different host plants cultivated under different climate conditions from the one used as reference. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Mitochondrial genome sequence of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae): Mitochondrial genes utility to investigate the evolutionary history of Coleoptera and its bioluminescence

The Coleoptera order is the richest group among Metazoa, but its phylogenetics remains incompletely understood. Among Coleoptera, bioluminescence is found within the Elateroidea, but the evolution of this character remains a mystery. Mitochondrial DNA has been used extensively to reconstruct phylogenetic relationships, however, the evolution of a single gene does not always correspond to the species evolutionary history and the molecular marker choice is a key step in this type of analysis. To create a solid basis to better understand the evolutionary history of Coleoptera and its bioluminescence, we sequenced and comparatively analyzed the mitochondrial genome of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae). © 2007 Elsevier B.V. All rights reserved.

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Background. From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings. The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions. Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas. © 2010 Moreira et al; licensee BioMed Central Ltd.

Characterization and genome organization of a repetitive element associated with the nucleolus organizer region in leporinuselongatus (anostomidae: Characiformes)

Chromosome mapping and studies of the genomic organization of repetitive DNA sequences provide valuable insights that enhance our evolutionary and structural understanding of these sequences, as well as identifying chromosomal rearrangements and sex determination. This study investigated the occurrence and organization of repetitive DNA sequences in Leporinus elongatus using restriction enzyme digestion and the mapping of sequences by chromosomal fluorescence in situ hybridization (FISH). A 378-bp fragment with a 54.2% GC content was isolated after digestion with the SmaI restriction enzyme. BLASTN search found no similarity with previously described sequences, so this repetitive sequence was named LeSmaI. FISH experiments were conducted using L. elongatus and other Anostomidae species, i.e. L. macrocephalus,L. obtusidens, L. striatus, L. lacustris, L. friderici, Schizodon borellii, S. isognathus, and Abramites hypselonotus which detected signals that were unique to male and female L. elongatus individuals. Double-FISH using LeSmaI and 18S rDNA showed that LeSmaI was located in a nucleolus organizer region (NOR) in the male and female metaphases of L. elongatus. This report also discusses the role of repetitive DNA associated with NORs in the diversification of Anostomidae species karyotypes. Copyright © 2012 S. Karger AG, Basel.

Genome-Wide Analysis Reveals Selection for Important Traits in Domestic Horse Breeds

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an FST-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse. © 2013 Petersen et al.

Genetic Diversity in the Modern Horse Illustrated from Genome-Wide SNP Data

Horses were domesticated from the Eurasian steppes 5,000-6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized into closed, breeding populations (breeds). This report describes the use of a genome-wide set of autosomal SNPs and 814 horses from 36 breeds to provide the first detailed description of equine breed diversity. FST calculations, parsimony, and distance analysis demonstrated relationships among the breeds that largely reflect geographic origins and known breed histories. Low levels of population divergence were observed between breeds that are relatively early on in the process of breed development, and between those with high levels of within-breed diversity, whether due to large population size, ongoing outcrossing, or large within-breed phenotypic diversity. Populations with low within-breed diversity included those which have experienced population bottlenecks, have been under intense selective pressure, or are closed populations with long breed histories. These results provide new insights into the relationships among and the diversity within breeds of horses. In addition these results will facilitate future genome-wide association studies and investigations into genomic targets of selection. © 2013 Petersen et al.

Study of whole genome linkage disequilibrium in Nellore cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Detecting Loci under Recent Positive Selection in Dairy and Beef Cattle by Combining Different Genome-Wide Scan Methods

As the methodologies available for the detection of positive selection from genomic data vary in terms of assumptions and execution, weak correlations are expected among them. However, if there is any given signal that is consistently supported across different methodologies, it is strong evidence that the locus has been under past selection. In this paper, a straightforward frequentist approach based on the Stouffer Method to combine P-values across different tests for evidence of recent positive selection in common variations, as well as strategies for extracting biological information from the detected signals, were described and applied to high density single nucleotide polymorphism (SNP) data generated from dairy and beef cattle (taurine and indicine). The ancestral Bovinae allele state of over 440,000 SNP is also reported. Using this combination of methods, highly significant (P<3.17×10-7) population-specific sweeps pointing out to candidate genes and pathways that may be involved in beef and dairy production were identified. The most significant signal was found in the Cornichon homolog 3 gene (CNIH3) in Brown Swiss (P = 3.82×10-12), and may be involved in the regulation of pre-ovulatory luteinizing hormone surge. Other putative pathways under selection are the glucolysis/gluconeogenesis, transcription machinery and chemokine/cytokine activity in Angus; calpain-calpastatin system and ribosome biogenesis in Brown Swiss; and gangliosides deposition in milk fat globules in Gyr. The composite method, combined with the strategies applied to retrieve functional information, may be a useful tool for surveying genome-wide selective sweeps and providing insights in to the source of selection.

Genome-wide association study for backfat thickness in Canchim beef cattle using Random Forest approach

Background: Meat quality involves many traits, such as marbling, tenderness, juiciness, and backfat thickness, all of which require attention from livestock producers. Backfat thickness improvement by means of traditional selection techniques in Canchim beef cattle has been challenging due to its low heritability, and it is measured late in an animal's life. Therefore, the implementation of new methodologies for identification of single nucleotide polymorphisms (SNPs) linked to backfat thickness are an important strategy for genetic improvement of carcass and meat quality.Results: The set of SNPs identified by the random forest approach explained as much as 50% of the deregressed estimated breeding value (dEBV) variance associated with backfat thickness, and a small set of 5 SNPs were able to explain 34% of the dEBV for backfat thickness. Several quantitative trait loci (QTL) for fat-related traits were found in the surrounding areas of the SNPs, as well as many genes with roles in lipid metabolism.Conclusions: These results provided a better understanding of the backfat deposition and regulation pathways, and can be considered a starting point for future implementation of a genomic selection program for backfat thickness in Canchim beef cattle. © 2013 Mokry et al.; licensee BioMed Central Ltd.

Genome-wide association study for birth weight in Nellore cattle points to previously described orthologous genes affecting human and bovine height

Background: Birth weight (BW) is an economically important trait in beef cattle, and is associated with growth- and stature-related traits and calving difficulty. One region of the cattle genome, located on Bos primigenius taurus chromosome 14 (BTA14), has been previously shown to be associated with stature by multiple independent studies, and contains orthologous genes affecting human height. A genome-wide association study (GWAS) for BW in Brazilian Nellore cattle (Bos primigenius indicus) was performed using estimated breeding values (EBVs) of 654 progeny-tested bulls genotyped for over 777,000 single nucleotide polymorphisms (SNPs).Results: The most significant SNP (rs133012258, PGC = 1.34 × 10-9), located at BTA14:25376827, explained 4.62% of the variance in BW EBVs. The surrounding 1 Mb region presented high identity with human, pig and mouse autosomes 8, 4 and 4, respectively, and contains the orthologous height genes PLAG1, CHCHD7, MOS, RPS20, LYN, RDHE2 (SDR16C5) and PENK. The region also overlapped 28 quantitative trait loci (QTLs) previously reported in literature by linkage mapping studies in cattle, including QTLs for birth weight, mature height, carcass weight, stature, pre-weaning average daily gain, calving ease, and gestation length.Conclusions: This study presents the first GWAS applying a high-density SNP panel to identify putative chromosome regions affecting birth weight in Nellore cattle. These results suggest that the QTLs on BTA14 associated with body size in taurine cattle (Bos primigenius taurus) also affect birth weight and size in zebu cattle (Bos primigenius indicus). © 2013 Utsunomiya et al.; licensee BioMed Central Ltd.

Identification of duplicated and stress-inducible Aox2b gene co-expressed with Aox1 in species of the Medicago genus reveals a regulation linked to gene rearrangement in leguminous genomes

In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization. © 2013 Elsevier GmbH. All rights reserved.

The Genome of Anopheles darlingi, the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vectorhuman and vectorparasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles- darlingi. © 2013 The Author(s).