Lianming Du

Worldwide patterns of genomic variation and admixture in gray wolves

The gray wolf (Canis lupus) is a widely distributed top predator and ancestor of the domestic dog. To address questions about wolf relationships to each other and dogs, we assembled and analyzed a data set of 34 canine genomes. The divergence between New and Old World wolves is the earliest branching event and is followed by the divergence of Old World wolves and dogs, confirming that the dog was domesticated in the Old World. However, no single wolf population is more closely related to dogs, supporting the hypothesis that dogs were derived from an extinct wolf population. All extant wolves have a surprisingly recent common ancestry and experienced a dramatic population decline beginning at least ∼30 thousand years ago (kya). We suggest this crisis was related to the colonization of Eurasia by modern human hunter-gatherers, who competed with wolves for limited prey but also domesticated them, leading to a compensatory population expansion of dogs. We found extensive admixture between dogs and wolves, with up to 25% of Eurasian wolf genomes showing signs of dog ancestry. Dogs have influenced the recent history of wolves through admixture and vice versa, potentially enhancing adaptation. Simple scenarios of dog domestication are confounded by admixture, and studies that do not take admixture into account with specific demographic models are problematic.

Hypoxia adaptations in the grey wolf (Canis lupus chanco) from Qinghai-Tibet Plateau.

The Tibetan grey wolf (Canis lupus chanco) occupies habitats on the Qinghai-Tibet Plateau, a high altitude (>3000 m) environment where low oxygen tension exerts unique selection pressure on individuals to adapt to hypoxic conditions. To identify genes involved in hypoxia adaptation, we generated complete genome sequences of nine Chinese wolves from high and low altitude populations at an average coverage of 25× coverage. We found that, beginning about 55,000 years ago, the highland Tibetan grey wolf suffered a more substantial population decline than lowland wolves. Positively selected hypoxia-related genes in highland wolves are enriched in the HIF signaling pathway (P = 1.57E-6), ATP binding (P = 5.62E-5), and response to an oxygen-containing compound (P≤5.30E-4). Of these positively selected hypoxia-related genes, three genes (EPAS1, ANGPT1, and RYR2) had at least one specific fixed non-synonymous SNP in highland wolves based on the nine genome data. Our re-sequencing studies on a large panel of individuals showed a frequency difference greater than 58% between highland and lowland wolves for these specific fixed non-synonymous SNPs and a high degree of LD surrounding the three genes, which imply strong selection. Past studies have shown that EPAS1 and ANGPT1 are important in the response to hypoxic stress, and RYR2 is involved in heart function. These three genes also exhibited significant signals of natural selection in high altitude human populations, which suggest similar evolutionary constraints on natural selection in wolves and humans of the Qinghai-Tibet Plateau.

MSDB: A User-Friendly Program for Reporting Distribution and Building Databases of Microsatellites from Genome Sequences

Microsatellite Search and Building Database (MSDB) is a new Perl program providing a user-friendly interface for identification and building databases of microsatellites from complete genome sequences. The general aims of MSDB are to use the database to store the information of microsatellites and to facilitate the management, classification, and statistics of microsatellites. A user-friendly interface facilitates the treatment of large datasets. The program is powerful in finding various types of pure, compound, and complex microsatellites from sequences as well as generating a detailed statistical report in worksheet format. MSDB also contains other two subprograms: SWR, which is used to export microsatellites from the database to meet users requirements, and SWP, which is used to automatically invoke R to draw a sliding window plot for displaying the distribution of density or frequency of identified microsatellites. MSDB is freely available under the GNU General Public license for Windows and Linux from the following website: http://msdb.biosv.com/.

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system

BackgroundThe giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda.ResultsBy screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What’s more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system.ConclusionThe microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda.

Whole-Genome Sequencing of Tibetan Macaque (Macaca thibetana) Provides New Insight into the Macaque Evolutionary History

Macaques are the most widely distributed nonhuman primates and used as animal models in biomedical research. The availability of full-genome sequences from them would be essential to both biomedical and primate evolutionary studies. Previous studies have reported whole-genome sequences from rhesus macaque (Macaca mulatta) and cynomolgus macaque (M. fascicularis, CE), both of which belong to the fascicularis group. Here, we present a 37-fold coverage genome sequence of the Tibetan macaque (M. thibetana; TM). TM is an endemic species to China belonging to the sinica group. On the basis of mapping to the rhesus macaque genome, we identified approximately 11.9 million single-nucleotide variants), of which 3.9 million were TM specific, as assessed by comparison two Chinese rhesus macaques (CR) and two CE genomes. Some genes carried TM-specific homozygous nonsynonymous variants (TSHNVs), which were scored as deleterious in human by both PolyPhen-2 and SIFT (Sorting Tolerant From Intolerant) and were enriched in the eye disease genes. In total, 273 immune response and disease-related genes carried at least one TSHNV. The heterozygosity rates of two CRs (0.002617 and 0.002612) and two CEs (0.003004 and 0.003179) were approximately three times higher than that of TM (0.000898). Polymerase chain reaction resequencing of 18 TM individuals showed that 29 TSHNVs exhibited high allele frequencies, thus confirming their low heterozygosity. Genome-wide genetic divergence analysis demonstrated that TM was more closely related to CR than to CE. We further detected unusual low divergence regions between TM and CR. In addition, after applying statistical criteria to detect putative introgression regions (PIRs) in the TM genome, up to 239,620 kb PIRs (8.84% of the genome) were identified. Given that TM and CR have overlapping geographical distributions, had the same refuge during the Middle Pleistocene, and show similar mating behaviors, it is highly likely that there was an ancient introgression event between them. Moreover, demographic inferences revealed that TM exhibited a similar demographic history as other macaques until 0.5 Ma, but then it maintained a lower effective population size until present time. Our study has provided new insight into the macaque evolutionary history, confirming hybridization events between macaque species groups based on genome-wide data.

First insights into the giant panda (Ailuropoda melanoleuca) blood transcriptome: a resource for novel gene loci and immunogenetics

The giant panda (Ailuropoda melanoleuca) is one of the most famous flagship species for conservation, and its draft genome has recently been assembled. However, the transcriptome is not yet available. In this study, the blood transcriptomes of three pandas were characterized and about 160 million sequencing reads were generated using Illumina HiSeq 2000 paired‐end sequencing technology. The assembly yielded 92 598 transcripts with an average length of 1626 bp and N50 length of 2842 bp. Based on a sequence similarity search against nonredundant (nr) protein database, a total of 38 522 (41.6%) transcripts were annotated. Of these annotated transcripts, 25 142 and 8272 transcripts were assigned to gene ontology terms and clusters of orthologous group, respectively. A search against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 9098 (9.83%) transcripts mapped to 324 KEGG pathways, and the best represented functional categories of pathways were signal transduction and immune system. We have also identified 23 460 microsatellites, 43 560 SNPs as well as 21 456 alternative splicing events in the assembly. Additionally, a total of 24 341 complete open reading frames (ORFs) were detected from the assembly where 1492 ORFs were found to be novel gene loci as these have not been annotated so far in any public database.

Genome-Wide Survey and Analysis of Microsatellite Sequences in Bovid Species.

Microsatellites or simple sequence repeats (SSRs) have become the most popular source of genetic markers, which are ubiquitously distributed in many eukaryotic and prokaryotic genomes. This is the first study examining and comparing SSRs in completely sequenced genomes of the Bovidae. We analyzed and compared the number of SSRs, relative abundance, relative density, guanine-cytosine (GC) content and proportion of SSRs in six taxonomically different bovid species: Bos taurus, Bubalus bubalis, Bos mutus, Ovis aries, Capra hircus, and Pantholops hodgsonii. Our analysis revealed that, based on our search criteria, the total number of perfect SSRs found ranged from 663,079 to 806,907 and covered from 0.44% to 0.48% of the bovid genomes. Relative abundance and density of SSRs in these Bovinae genomes were non-significantly correlated with genome size (Pearson, r < 0.420, p > 0.05). Perfect mononucleotide SSRs were the most abundant, followed by the pattern: perfect di- > tri- > penta- > tetra- > hexanucleotide SSRs. Generally, the number of SSRs, relative abundance, and relative density of SSRs decreased as the motif repeat length increased in each species of Bovidae. The most GC-content was in trinucleotide SSRs and the least was in the mononucleotide SSRs in the six bovid genomes. The GC-contents of tri- and pentanucleotide SSRs showed a great deal of similarity among different chromosomes of B. taurus, O. aries, and C. hircus. SSR number of all chromosomes in the B. taurus, O.aries, and C. hircus is closely positively correlated with chromosome sequence size (Pearson, r > 0.980, p < 0.01) and significantly negatively correlated with GC-content (Pearson, r < -0.638, p < 0.01). Relative abundance and density of SSRs in all chromosomes of the three species were significantly negatively correlated with GC-content (Pearson, r < -0.333, P < 0.05) but not significantly correlated with chromosome sequence size (Pearson, r < -0.185, P > 0.05). Relative abundances of the same nucleotide SSR type showed great similarity among different chromosomes of B. taurus, O. aries, and C. hircus.

Phylogenomics and Evolutionary Dynamics of the Family Actinomycetaceae

The family Actinomycetaceae comprises several important pathogens that impose serious threat to human health and cause substantial infections of economically important animals. However, the phylogeny and evolutionary dynamic of this family are poorly characterized. Here, we provide detailed description of the genome characteristics of Trueperella pyogenes, a prevalent opportunistic bacterium that belongs to the family Actinomycetaceae, and the results of comparative genomics analyses suggested that T. pyogenes was a more versatile pathogen than Arcanobacterium haemolyticum in adapting various environments. We then performed phylogenetic analyses at the genomic level and showed that, on the whole, the established members of the family Actinomycetaceae were clearly separated with high bootstrap values but confused with the dominant genus Actinomyces, because the species of genus Actinomyces were divided into three main groups with different G+C content. Although T. pyogenes and A. haemolyticum were found to share the same branch as previously determined, our results of single nucleotide polymorphism tree and genome clustering as well as predicted intercellular metabolic analyses provide evidence that they are phylogenetic neighbors. Finally, we found that the gene gain/loss events occurring in each species may play an important role during the evolution of Actinomycetaceae from free-living to a specific lifestyle.

GenScalpel: An Application for Sequence Retrieval and Extraction from the GenBank flatfile

GenScalpel is a program designed for the retrieval and extraction of specified sequences from large-scale sequence sets in NCBI GenBank flatfile format. This routine task in bioinformatics analysis is a pressing need for laboratory biologists. Another objective of application development is to respond to the new form of the NCBI Nucleotide Sequence Database, which was updated in November 2011. In addition to a powerful sequence refinement application, GenScalpel provides convenient functions for web-based sequence downloading or multiple files batch processing. This note discusses major applications of the program and includes example data sets to demonstrate its performance. The program is written in PERL. GenScalpel, including installation packages for Windows and Linux systems as well as the accompanying documentation, are available free of charge at http://genscalpel.biosv.com/.

Krait: an ultrafast tool for genome-wide survey of microsatellites and primer design

Summary Microsatellites are found to be related with various diseases and widely used in population genetics as genetic markers. However, it remains a challenge to identify microsatellite from large genome and screen microsatellites for primer design from a huge result dataset. Here, we present Krait, a robust and flexible tool for fast investigation of microsatellites in DNA sequences. Krait is designed to identify all types of perfect or imperfect microsatellites on a whole genomic sequence, and is also applicable to identification of compound microsatellites. Primer3 was seamlessly integrated into Krait so that users can design primer for microsatellite amplification in an efficient way. Additionally, Krait can export microsatellite results in FASTA or GFF3 format for further analysis and generate statistical report as well as plotting. Availability and implementation Krait is freely available at https://github.com/lmdu/krait under GPL2 License, implemented in C and Python, and supported on Windows, Linux and Mac operating systems. Contact chizhang_swmu@126.com. Supplementary information Supplementary data are available at Bioinformatics online.