Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo): genome assembly and analysis.

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo) and chicken (Gallus gallus) genomes.

BACKGROUND: A robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. RESULTS: The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. CONCLUSION: The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

A BAC/BIBAC-based physical map of chickpea, Cicer arietinum L.

BACKGROUND: Chickpea (Cicer arietinum L.) is the third most important pulse crop worldwide. Despite its importance, relatively little is known about its genome. The availability of a genome-wide physical map allows rapid fine mapping of QTL, development of high-density genome maps, and sequencing of the entire genome. However, no such a physical map has been developed in chickpea. RESULTS: We present a genome-wide, BAC/BIBAC-based physical map of chickpea developed by fingerprint analysis. Four chickpea BAC and BIBAC libraries, two of which were constructed in this study, were used. A total of 67,584 clones were fingerprinted, and 64,211 (~11.7 x) of the fingerprints validated and used in the physical map assembly. The physical map consists of 1,945 BAC/BIBAC contigs, with each containing an average of 28.3 clones and having an average physical length of 559 kb. The contigs collectively span approximately 1,088 Mb. By using the physical map, we identified the BAC/BIBAC contigs containing or closely linked to QTL4.1 for resistance to Didymella rabiei (RDR) and QTL8 for days to first flower (DTF), thus further verifying the physical map and confirming its utility in fine mapping and cloning of QTL. CONCLUSION: The physical map represents the first genome-wide, BAC/BIBAC-based physical map of chickpea. This map, along with other genomic resources previously developed in the species and the genome sequences of related species (soybean, Medicago and Lotus), will provide a foundation necessary for many areas of advanced genomics research in chickpea and other legume species. The inclusion of transformation-ready BIBACs in the map greatly facilitates its utility in functional analysis of the legume genomes.

Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research.

Megabase-sized DNA is crucial to modern genomics research of all organisms. Among the preparation methods developed, the nuclei method is the simplest and most widely used for preparing high-quality megabase-sized DNA from divergent organisms. In this method, nuclei are first isolated by physically grinding the source tissues. The nontarget cytoplast organellar genomes and metabolites are removed by centrifugation and washing, thus maximizing the utility of the method and substantially improving the digestibility and clonability of the resultant DNA. The nuclei are then embedded in an agarose matrix containing numerous pores, allowing the access of restriction enzymes while preventing the DNA from physical shearing. DNA is extracted from the nuclei, purified and subsequently manipulated in the agarose matrix. Here we describe the nuclei method that we have successfully used to prepare high-quality megabase-sized DNA from hundreds of plant, animal, fish, insect, algal and microbial species. The entire protocol takes ∼3 d.

Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research.

Large-insert BAC (bacterial artificial chromosome) and BIBAC (binary BAC) libraries are essential for modern genomics research for all organisms. We helped pioneer the BAC and BIBAC technologies, and by using them we have constructed hundreds of BAC and BIBAC libraries for different species of plants, animals, marine animals, insects, algae and microbes. These libraries have been used globally for different aspects of genomics research. Here we describe the procedure with the latest improvements that we have made and used for construction of BIBAC libraries. The procedure includes the preparation of BIBAC vectors, the preparation of clonable fragments of the desired size from the source DNA, the construction and transformation of BIBACs and, finally, the characterization and assembly of BIBAC libraries. We also specify the modifications necessary for construction of BAC libraries using the protocol. The entire protocol takes ∼7 d.

Alteration of lipid metabolism in cells infected with human cytomegalovirus.

The human cytomegalovirus (HCMV) envelope contains 12 virus-encoded glycoproteins and glycoprotein complexes but the lipid composition of the envelope has not been clearly defined. Given the specificity of the interactions between integral membrane proteins and lipids, it is likely that the lipid content of the virion envelope is regulated during infection. In an effort to determine the effects of HCMV infection on lipid metabolism, we have used PCR array technology to investigate how infection affects the expression of genes involved in lipoprotein signaling and cholesterol homeostasis pathways. Our results indicate that HCMV infection leads to down-regulation of the ABCA1 transporter. Decreased levels of ABCA1 appear to be the result of enhanced calpain-mediated cleavage in virus-infected cells. In addition, our data also show that HCMV infection inhibits the development of the foam cell phenotype in conditionally permissive THP-1 derived macrophages