George E. Liu

Analysis of copy number variations among diverse cattle breeds

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or approximately 1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

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 ~55.6-Mbp sequence--476 of which (~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 (~52%, χ(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.

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

Activation of SIRT1 by Resveratrol Represses Transcription of the Gene for the Cytosolic Form of Phosphoenolpyruvate Carboxykinase (GTP) by Deacetylating Hepatic Nuclear Factor 4α

The SIRT1 activators isonicotinamide (IsoNAM), resveratrol, fisetin, and butein repressed transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C). An evolutionarily conserved binding site for hepatic nuclear factor (HNF) 4α (−272/−252) was identified, which was required for transcriptional repression of the PEPCK-C gene promoter caused by these compounds. This site contains an overlapping AP-1 binding site and is adjacent to the C/EBP binding element (−248/−234); the latter is necessary for hepatic transcription of PEPCK-C. AP-1 competed with HNF4α for binding to this site and also decreased HNF4α stimulation of transcription from the PEPCK-C gene promoter. Chromatin immunoprecipitation experiments demonstrated that HNF4α and AP-1, but not C/EBPβ, reciprocally bound to this site prior to and after treating HepG2 cells with IsoNAM. IsoNAM treatment resulted in deacetylation of HNF4α, which decreased its binding affinity to the PEPCK-C gene promoter. In HNF4α-null Chinese hamster ovary cells, IsoNAM and resveratrol failed to repress transcription from the PEPCK-C gene promoter; overexpression of HNF4α in Chinese hamster ovary cells re-established transcriptional inhibition. Exogenous SIRT1 expression repressed transcription, whereas knockdown of SIRT1 by RNA interference reversed this effect. IsoNAM decreased the level of mRNA for PEPCK-C but had no effect on mRNA for glucose-6-phosphatase in AML12 mouse hepatocytes. We conclude that SIRT1 activation inhibited transcription of the gene for PEPCK-C in part by deacetylation of HNF4α. However, SIRT1 deacetylation of other key regulatory proteins that control PEPCK-C gene transcription also likely contributed to the inhibitory effect.

Analysis of recent segmental duplications in the bovine genome.

BackgroundDuplicated sequences are an important source of gene innovation and structural variation within mammalian genomes. We performed the first systematic and genome-wide analysis of segmental duplications in the modern domesticated cattle (Bos taurus). Using two distinct computational analyses, we estimated that 3.1% (94.4 Mb) of the bovine genome consists of recently duplicated sequences (≥ 1 kb in length, ≥ 90% sequence identity). Similar to other mammalian draft assemblies, almost half (47% of 94.4 Mb) of these sequences have not been assigned to cattle chromosomes.ResultsIn this study, we provide the first experimental validation large duplications and briefly compared their distribution on two independent bovine genome assemblies using fluorescent in situ hybridization (FISH). Our analyses suggest that the (75-90%) of segmental duplications are organized into local tandem duplication clusters. Along with rodents and carnivores, these results now confidently establish tandem duplications as the most likely mammalian archetypical organization, in contrast to humans and great ape species which show a preponderance of interspersed duplications. A cross-species survey of duplicated genes and gene families indicated that duplication, positive selection and gene conversion have shaped primates, rodents, carnivores and ruminants to different degrees for their speciation and adaptation. We identified that bovine segmental duplications corresponding to genes are significantly enriched for specific biological functions such as immunity, digestion, lactation and reproduction.ConclusionOur results suggest that in most mammalian lineages segmental duplications are organized in a tandem configuration. Segmental duplications remain problematic for genome and assembly and we highlight genic regions that require higher quality sequence characterization. This study provides insights into mammalian genome evolution and generates a valuable resource for cattle genomics research.

Effect of Artificial Selection on Runs of Homozygosity in U.S. Holstein Cattle

The intensive selection programs for milk made possible by mass artificial insemination increased the similarity among the genomes of North American (NA) Holsteins tremendously since the 1960s. This migration of elite alleles has caused certain regions of the genome to have runs of homozygosity (ROH) occasionally spanning millions of continuous base pairs at a specific locus. In this study, genome signatures of artificial selection in NA Holsteins born between 1953 and 2008 were identified by comparing changes in ROH between three distinct groups under different selective pressure for milk production. The ROH regions were also used to estimate the inbreeding coefficients. The comparisons of genomic autozygosity between groups selected or unselected since 1964 for milk production revealed significant differences with respect to overall ROH frequency and distribution. These results indicate selection has increased overall autozygosity across the genome, whereas the autozygosity in an unselected line has not changed significantly across most of the chromosomes. In addition, ROH distribution was more variable across the genomes of selected animals in comparison to a more even ROH distribution for unselected animals. Further analysis of genome-wide autozygosity changes and the association between traits and haplotypes identified more than 40 genomic regions under selection on several chromosomes (Chr) including Chr 2, 7, 16 and 20. Many of these selection signatures corresponded to quantitative trait loci for milk, fat, and protein yield previously found in contemporary Holsteins.

Fine mapping of copy number variations on two cattle genome assemblies using high density SNP array

BackgroundBtau_4.0 and UMD3.1 are two distinct cattle reference genome assemblies. In our previous study using the low density BovineSNP50 array, we reported a copy number variation (CNV) analysis on Btau_4.0 with 521 animals of 21 cattle breeds, yielding 682 CNV regions with a total length of 139.8 megabases.ResultsIn this study using the high density BovineHD SNP array, we performed high resolution CNV analyses on both Btau_4.0 and UMD3.1 with 674 animals of 27 cattle breeds. We first compared CNV results derived from these two different SNP array platforms on Btau_4.0. With two thirds of the animals shared between studies, on Btau_4.0 we identified 3,346 candidate CNV regions representing 142.7 megabases (~4.70%) of the genome. With a similar total length but 5 times more event counts, the average CNVR length of current Btau_4.0 dataset is significantly shorter than the previous one (42.7 kb vs. 205 kb). Although subsets of these two results overlapped, 64% (91.6 megabases) of current dataset was not present in the previous study. We also performed similar analyses on UMD3.1 using these BovineHD SNP array results. Approximately 50% more and 20% longer CNVs were called on UMD3.1 as compared to those on Btau_4.0. However, a comparable result of CNVRs (3,438 regions with a total length 146.9 megabases) was obtained. We suspect that these results are due to the UMD3.1 assemblys efforts of placing unplaced contigs and removing unmerged alleles. Selected CNVs were further experimentally validated, achieving a 73% PCR validation rate, which is considerably higher than the previous validation rate. About 20-45% of CNV regions overlapped with cattle RefSeq genes and Ensembl genes. Panther and IPA analyses indicated that these genes provide a wide spectrum of biological processes involving immune system, lipid metabolism, cell, organism and system development.ConclusionWe present a comprehensive result of cattle CNVs at a higher resolution and sensitivity. We identified over 3,000 candidate CNV regions on both Btau_4.0 and UMD3.1, further compared current datasets with previous results, and examined the impacts of genome assemblies on CNV calling.

Cattle Sex-Specific Recombination and Genetic Control from a Large Pedigree Analysis

Meiotic recombination is an essential biological process that generates genetic diversity and ensures proper segregation of chromosomes during meiosis. From a large USDA dairy cattle pedigree with over half a million genotyped animals, we extracted 186,927 three-generation families, identified over 8.5 million maternal and paternal recombination events, and constructed sex-specific recombination maps for 59,309 autosomal SNPs. The recombination map spans for 25.5 Morgans in males and 23.2 Morgans in females, for a total studied region of 2,516 Mb (986 kb/cM in males and 1,085 kb/cM in females). The male map is 10% longer than the female map and the sex difference is most pronounced in the subtelomeric regions. We identified 1,792 male and 1,885 female putative recombination hotspots, with 720 hotspots shared between sexes. These hotspots encompass 3% of the genome but account for 25% of the genome-wide recombination events in both sexes. During the past forty years, males showed a decreasing trend in recombination rate that coincided with the artificial selection for milk production. Sex-specific GWAS analyses identified PRDM9 and CPLX1 to have significant effects on genome-wide recombination rate in both sexes. Two novel loci, NEK9 and REC114, were associated with recombination rate in both sexes, whereas three loci, MSH4, SMC3 and CEP55, affected recombination rate in females only. Among the multiple PRDM9 paralogues on the bovine genome, our GWAS of recombination hotspot usage together with linkage analysis identified the PRDM9 paralogue on chromosome 1 to be associated in the U.S. Holstein data. Given the largest sample size ever reported for such studies, our results reveal new insights into the understanding of cattle and mammalian recombination.

Comparative analysis of Alu repeats in primate genomes

Using bacteria artificial chromosome (BAC) end sequences (16.9 Mb) and high-quality alignments of genomic sequences (17.4 Mb), we performed a global assessment of the divergence distributions, phylogenies, and consensus sequences for Alu elements in primates including lemur, marmoset, macaque, baboon, and chimpanzee as compared to human. We found that in lemurs, Alu elements show a broader and more symmetric sequence divergence distribution, suggesting a steady rate of Alu retrotransposition activity among prosimians. In contrast, Alu elements in anthropoids show a skewed distribution shifted toward more ancient elements with continual declining rates in recent Alu activity along the hominoid lineage of evolution. Using an integrated approach combining mutation profile and insertion/deletion analyses, we identified nine novel lineage-specific Alu subfamilies in lemur (seven), marmoset (one), and baboon/macaque (one) containing multiple diagnostic mutations distinct from their human counterparts-Alu J, S, and Y subfamilies, respectively. Among these primates, we show that that the lemur has the lowest density of Alu repeats (55 repeats/Mb), while marmoset has the greatest abundance (188 repeats/Mb). We estimate that approximately 70% of lemur and 16% of marmoset Alu elements belong to lineage-specific subfamilies. Our analysis has provided an evolutionary framework for further classification and refinement of the Alu repeat phylogeny. The differences in the distribution and rates of Alu activity have played an important role in subtly reshaping the structure of primate genomes. The functional consequences of these changes among the diverse primate lineages over such short periods of evolutionary time are an important area of future investigation.

Genomic divergence of zebu and taurine cattle identified through high-density SNP genotyping

BackgroundNatural selection has molded evolution across all taxa. At an arguable date of around 330,000 years ago there were already at least two different types of cattle that became ancestors of nearly all modern cattle, the Bos taurus taurus more adapted to temperate climates and the tropically adapted Bos taurus indicus. After domestication, human selection exponentially intensified these differences. To better understand the genetic differences between these subspecies and detect genomic regions potentially under divergent selection, animals from the International Bovine HapMap Experiment were genotyped for over 770,000 SNP across the genome and compared using smoothed FST. The taurine sample was represented by ten breeds and the contrasting zebu cohort by three breeds.ResultsEach cattle group evidenced similar numbers of polymorphic markers well distributed across the genome. Principal components analyses and unsupervised clustering confirmed the well-characterized main division of domestic cattle. The top 1% smoothed FST, potentially associated to positive selection, contained 48 genomic regions across 17 chromosomes. Nearly half of the top FST signals (n = 22) were previously detected using a lower density SNP assay. Amongst the strongest signals were the BTA7:~50 Mb and BTA14:~25 Mb; both regions harboring candidate genes and different patterns of linkage disequilibrium that potentially represent intrinsic differences between cattle types. The bottom 1% of the smoothed FST values, potentially associated to balancing selection, included 24 regions across 13 chromosomes. These regions often overlap with copy number variants, including the highly variable region at BTA23:~24 Mb that harbors a large number of MHC genes. Under these regions, 318 unique Ensembl genes are annotated with a significant overrepresentation of immune related pathways.ConclusionsGenomic regions that are potentially linked to purifying or balancing selection processes in domestic cattle were identified. These regions are of particular interest to understand the natural and human selective pressures to which these subspecies were exposed to and how the genetic background of these populations evolved in response to environmental challenges and human manipulation.