Xiangzhe Zhang

Genotyping by Genome Reducing and Sequencing for Outbred Animals

Next-generation sequencing (NGS) approaches are widely used in genome-wide genetic marker discovery and genotyping. However, current NGS approaches are not easy to apply to general outbred populations (human and some major farm animals) for SNP identification because of the high level of heterogeneity and phase ambiguity in the haplotype. Here, we reported a new method for SNP genotyping, called genotyping by genome reducing and sequencing (GGRS) to genotype outbred species. Through an improved procedure for library preparation and a marker discovery and genotyping pipeline, the GGRS approach can genotype outbred species cost-effectively and high-reproducibly. We also evaluated the efficiency and accuracy of our approach for high-density SNP discovery and genotyping in a large genome pig species (2.8 Gb), for which more than 70,000 single nucleotide polymorphisms (SNPs) can be identified for an expenditure of only

WRKY gene family evolution in Arabidopsis thaliana

80 (USD)/sample.

FoxO gene family evolution in vertebrates

The Arabidopsis thaliana WRKY proteins are characterized by a sequence of 60 amino acids including WRKY domain. It is well established that these proteins are involved in the regulation of various physiological programs unique to plants including pathogen defense, senescence and response to environmental stresses, which attracts attention of the scientific community as to how this family might have evolved. We tried to satisfy this curiosity and analyze reasons for duplications of these gene sequences leading to their diversified gene actions. The WRKY sequences available in Arabidopsis thaliana were used to evaluate selection pressure following duplication events. A phylogenetic tree was constructed and the WRKY family was divided into five sub-families. After that, tests were conducted to decide whether positive or purified selection played key role in these events. Our results suggest that purifying selection played major role during the evolution of this family. Some amino acid changes were also detected in specific branches of phylogeny suggesting that relaxed constraints might also have contributed to functional divergence among sub-families. Sites relaxed from purifying selection were identified and mapped onto the structural and functional regions of the WRKY1 protein. These analyses will enhance our understanding of the precise role played by natural selection to create functional diversity in WRKY family.

A New Genotype Imputation Method with Tolerance to High Missing Rate and Rare Variants

BackgroundForkhead box, class O (FoxO) belongs to the large family of forkhead transcription factors that are characterized by a conserved forkhead box DNA-binding domain. To date, the FoxO group has four mammalian members: FoxO1, FoxO3a, FoxO4 and FoxO6, which are orthologs of DAF16, an insulin-responsive transcription factor involved in regulating longevity of worms and flies. The degree of homology between these four members is high, especially in the forkhead domain, which contains the DNA-binding interface. Yet, mouse FoxO knockouts have revealed that each FoxO gene has its unique role in the physiological process. Whether the functional divergences are primarily due to adaptive selection pressure or relaxed selective constraint remains an open question. As such, this study aims to address the evolutionary mode of FoxO, which may lead to the functional divergence.ResultsSequence similarity searches have performed in genome and scaffold data to identify homologues of FoxO in vertebrates. Phylogenetic analysis was used to characterize the family evolutionary history by identifying two duplications early in vertebrate evolution. To determine the mode of evolution in vertebrates, we performed a rigorous statistical analysis with FoxO gene sequences, including relative rate ratio tests, branch-specific dN/dSratio tests, site-specific dN/dSratio tests, branch-site dN/dSratio tests and clade level amino acid conservation/variation patterns analysis. Our results suggest that FoxO is constrained by strong purifying selection except four sites in FoxO6, which have undergone positive Darwinian selection. The functional divergence in this family is best explained by either relaxed purifying selection or positive selection.ConclusionWe present a phylogeny describing the evolutionary history of the FoxO gene family and show that the genes have evolved through duplications followed by purifying selection except for four sites in FoxO6 fixed by positive selection lie mostly within the non-conserved optimal PKB motif in the C-terminal part. Relaxed selection may play important roles in the process of functional differentiation evolved through gene duplications as well.

Genetic diversity and population structure of six Chinese indigenous pig breeds in the Taihu Lake region revealed by sequencing data.

We report a novel algorithm, iBLUP, to impute missing genotypes by simultaneously and comprehensively using identity by descent and linkage disequilibrium information. The simulation studies showed that the algorithm exhibited drastically tolerance to high missing rate, especially for rare variants than other common imputation methods, e.g. BEAGLE and fastPHASE. At a missing rate of 70%, the accuracy of BEAGLE and fastPHASE dropped to 0.82 and 0.74 respectively while iBLUP retained an accuracy of 0.95. For minor allele, the accuracy of BEAGLE and fastPHASE decreased to −0.1 and 0.03, while iBLUP still had an accuracy of 0.61.We implemented the algorithm in a publicly available software package also named iBLUP. The application of iBLUP for processing real sequencing data in an outbred pig population was demonstrated.

Comparative analysis of MTF-1 binding sites between human and mouse.

Summary The Chinese indigenous pig breeds in the Taihu Lake region are the most prolific pig breeds in the world. In this study, we investigated the genetic diversity and population structure of six breeds, including Meishan, Erhualian, Mi, Fengjing, Shawutou and Jiaxing Black, in this region using whole‐genome SNP data. A high SNP with proportions of polymorphic markers ranging from 0.925 to 0.995 was exhibited by the Chinese indigenous pigs in the Taihu Lake region. The allelic richness and expected heterozygosity also were calculated and indicated that the genetic diversity of the Meishan breed was the greatest, whereas that of the Fengjing breed was the lowest. The genetic differentiation, as indicated by the fixation index, exhibited an overall mean of 0.149. Both neighbor‐joining tree and principal components analysis were able to distinguish the breeds from each other, but structure analysis indicated that the Mi and Erhualian breeds exhibited similar major signals of admixture. With this genome‐wide comprehensive survey of the genetic diversity and population structure of the indigenous Chinese pigs in the Taihu Lake region, we confirmed the rationality of the current breed classification of the pigs in this region.

Comparative analysis of vertebrate PEPT1 and PEPT2 genes

MTF-1 is a crucial transcription factor involved in the cellular response to heavy-metal load and other stresses by specifically binding to metal response elements (MREs). Thus far only a handful of direct target genes are known for this transcription factor, limiting our understanding of the biological network it governs. In this article we try to employ a computational strategy based on the generation of literature-based positional weight matrices (PWM) and log-likelihood scoring of the candidate binding sites (BSs) for identification of direct targets of the transcription factor MTF-1 in human and mouse. Through comparisons, we explore the conservation and unique characteristics between two species. Our results show that the numbers of MREs differ dramatically between species and their positions relative to their cognate promoter is also flexible. Importantly, we identify a set of target genes generally well conserved between human and mouse. Finally, by combining expression analysis we provide two putative targets (HMGCR and CYP51A), which regulate lipid metabolism conserved in human and mouse. Overall, interspecies comparison from our study may provide some valuable information for further studying human Wilson disease (WD) using mouse model systems.

The Molecular Evolutionary Patterns of the Insulin/FOXO Signaling Pathway.

The plasma membrane transport proteins belong to SoLute Carrier 15 (SLC15) family and two members of this family have been characterized extensively in higher vertebrates, namely PEPT1 and PEPT2. Despite many efforts have made to define a pharmacophore model for efficient binding and transporting of substrates, there is not a comprehensive study performed to elucidate the evolutionary mechanisms among the SLC15 family members and to statistically evaluate sequence conservation and functional divergence between members. In this study, we compared and contrasted the rates and patterns of molecular evolution of 2 PEPT genes. Phylogenetic tree assembly with all available vertebrate PEPTs suggests that the PEPTs originated by duplications and diverged from a common protein at the base of the eukaryotic tree. Topological structure demonstrates both members share the similar hydrophobic domains (TMDs), which have been constrained by purifying selection. Although both genes show qualitatively similar patterns, their rates of evolution differ significantly due to an increased rate of synonymous substitutions in the structural domains in one copy, suggesting substantial differences in functional constraint on each gene. Site-specific profiles were established by posterior probability analysis revealing significantly divergent regions mainly locate at the hydrophobic region between predicted transmembrane domains 9 and 10 of the proteins. Thus, these results provide the evidence that several amino acid residues with reduced selective constraints are largely responsible for functional divergence between the paralogous PEPTs. These findings may provide a starting point for further experimental verifications.

A genome-wide scan for selection signatures in Yorkshire and Landrace pigs based on sequencing data.

The insulin/insulin growth factor-1 (IGF1)/FOXO (IIF) signal transduction pathway plays a core role in the endocrine system. Although the components of this pathway have been well characterized, the evolutionary pattern remains poorly understood. Here, we perform a comprehensive analysis to study whether the differences of signaling transduction elements exist as well as to determine whether the genes are subject to equivalent evolutionary forces and how natural selection shapes the evolution pattern of proteins in an interacting system. Our results demonstrate that most IIF pathway components are present throughout all animal phyla investigated here, and they are under strong selective constraint. Remarkably, we detect that the components in the middle of the pathway undergo stronger purifying selection, which is different from previous similar reports. We also find that the dN/dS may be influenced by quite complicated factors including codon bias, protein length among others.

Identification of pleiotropic genes and gene sets underlying growth and immunity traits: a case study on Meishan pigs

Pigs have experienced dramatic selection due to domestication, which has led to many different phenotypes when compared to their wild counterparts, especially in the last several decades. Currently, genome-wide scans in both cattle and humans showing positive selection footprints have been investigated. However, few studies have focused on porcine selection footprints, particularly on a genome-wide scale. Surveying for selection footprints across porcine genomes can be quite valuable for revealing the genetic mechanisms of phenotypic diversity. Here, we employed a medium sequencing depth (5–20x/site per individual, on average) approach called genotyping by genome reducing and sequencing (GGRS) to detect genome-wide selection signatures of two domestic pig breeds (Yorkshire and Landrace) that have been under intensive selection for traits of muscle development, growth and behavior. The relative extended haplotype homozygosity test, which identifies selection signatures by measuring the characteristics of haplotypes’ frequency distribution within a single population, was also applied to identify potential positively selected regions. As a result, signatures of positive selection were found in each breed. However, most selection signatures were population specific and related to genomic regions containing genes for biological categories including brain development, metabolism, growth and olfaction. Furthermore, the result of the gene set enrichment analysis indicated that selected regions of the two breeds presented a different over-representation of genes in the Gene Ontology annotations and Kyoto Encyclopedia of Genes and Genomes pathways. Our results revealed a genome-wide map of selection footprints in pigs and may help us better understand the mechanisms of selection in pig breeding.