The genome of the cattle tick Rhipicephalus microplus, an ectoparasite with global distribution, is estimated to be 7.1Gbp in length and consists of approximately 70% repetitive DNA. We report the draft assembly of a tick genome that utilized a hybrid sequencing and assembly approach to capture the repetitive fractions of the genome. Our hybrid approach produced an assembly consisting of 2.0Gbp represented in 195,170 scaffolds with a N50 of 60,284bp. The Rmi v2.0 assembly is 51.46% repetitive with a large fraction of unclassified repeats, short interspersed elements, long interspersed elements and long terminal repeats. We identified 38,827 putative R. microplus gene loci, of which 24,758 were protein coding genes (≥100 amino acids). OrthoMCL comparative analysis against 11 selected species including insects and vertebrates identified 10,835 and 3,423 protein coding gene loci that are unique to R. microplus or common to both R. microplus and Ixodes scapularis ticks, respectively. We identified 191 microRNA loci, of which 168 have similarity to known miRNAs and 23 represent novel miRNA families. We identified the genomic loci of several highly divergent R. microplus esterases with sequence similarity to acetylcholinesterase. Additionally we report the finding of a novel cytochrome P450 CYP41 homolog that shows similar protein folding structures to known CYP41 proteins known to be involved in acaricide resistance.

Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

Two of the major challenges in the field of genomics are first to obtain a correct assembly of DNA sequence data and then mapping known markers onto a genome sequence template to obtain an accurate physical map. Traditionally, the efforts of genome sequence assembly and physical mapping were split into two totally different datasets. This was primarily due to the incompatibility of data generated: physical mapping technologies were based on DNA fragment lengths (i.e., SNaPshot, optical mapping, agarose gel, polyacrylamide gel or capillary electrophoresis, restriction digest-based fingerprinting technology), whereas DNA sequence data is comprised of an A, G, C, and T letter code. Herein, we describe whole-genome profiling (WGPTM) – a technology that solves this dilemma. This new application of bacterial artificial chromosome (BAC) cloning in combination with short-read next-generation sequencing (NGS) technology eliminates the incompatibilities between physical mapping datasets and genome sequencing datasets. WGP is a physical mapping technology that uses Illumina NGS reads on systematically pooled BAC clones to create an ultra-high-density physical map of the genome using DNA sequencing data. In this chapter, we will explore what WGP is, how to perform WGP, and compare the advantages and disadvantages of WGP. We will show that WGP is a superior physical mapping technology that can improve the accuracy of any de novo whole-genome sequencing assembly as well as for finding genetic variations in resequencing projects.

We present whole genome profiling (WGP), a novel next-generation sequencing-based physical mapping technology for construction of bacterial artificial chromosome (BAC) contigs of complex genomes, using Arabidopsis thaliana as an example. WGP leverages short read sequences derived from restriction fragments of two-dimensionally pooled BAC clones to generate sequence tags. These sequence tags are assigned to individual BAC clones, followed by assembly of BAC contigs based on shared regions containing identical sequence tags. Following in silico analysis of WGP sequence tags and simulation of a map of Arabidopsis chromosome 4 and maize, a WGP map of Arabidopsis thaliana ecotype Columbia was constructed de novo using a six-genome equivalent BAC library. Validation of the WGP map using the Columbia reference sequence confirmed that 350 BAC contigs (98%) were assembled correctly, spanning 97% of the 102-Mb calculated genome coverage. We demonstrate that WGP maps can also be generated for more complex plant genomes and will serve as excellent scaffolds to anchor genetic linkage maps and integrate whole genome sequence data.

The Consortium for Snake Genomics is in the process of sequencing the genome and creating transcriptomic resources for the Burmese python. Here, we describe how this will be done, what analyses this work will include, and provide a timeline.

In the U.S., 30 to 100 million cats are estimated to survive in a feral environment, representing a major health, environmental and welfare issue. A single-dose contraceptive vaccine has several potential applications in felids, most notably for control of feral cat populations. In this study, we evaluated the safety and effectiveness of a single-dose luteinizing hormone-releasing hormone (LHRH) vaccine for contraception in cats. The vaccine was comprised of a recombinant ovalbumin-LHRH (ova-LHRH) fusion protein as the antigen mixed with adjuvant containing cytosine guanine oligodeoxynucleotide (CpG ODN). The single-dose vaccine contained both non-encapsulated and encapsulated forms of ova-LHRH; the latter presented the antigen in agarose microbeads for slow release. Fifteen young adult, intact female cats were randomly assigned to one of the following treatment groups (n=5 cats/group): 1) two injections of non-encapsulated ova-LHRH (350 μg each), 8 weeks apart (positive control); 2) one injection of a mixture of encapsulated (440 μg) and non-encapsulated (350 μg) ova-LHRH; and 3) one injection of saline (negative control). Injection sites were monitored for inflammatory responses. Blood samples were collected bi-weekly for one year, and LHRH antibody production assessed using a validated radioimmunoassay. Progestin metabolite profiles were monitored via a validated enzyme immunoassay in fecal samples collected every 2 to 3 days for one year. Fertility was assessed by housing a proven breeder male with females for four months beginning six months after initial injection. In the saline group, percent binding of 125I-LHRH in serum was zero throughout the study. At the end of the study (weeks 38-44 post-injection), antibody binding activity was higher (P < 0.05) in the two-dose group (63.4% ± 9.8; mean ± SEM) compared to the single-dose group (27.0% ± 7.9). Prior to immunization, all 15 females exhibited estrual behavior indicative of ovarian cyclicity, and most females (13/15) appeared to be spontaneous ovulators based on elevated fecal progestin levels lasting 5 weeks or longer. In the saline group, all five females continued to ovulate spontaneously throughout the study until pregnancy. In the single-dose group, four of five females were no longer ovulating by the beginning of the fertility trial; however, one of these four resumed ovulatory activity towards the end of the trial and became pregnant. The remaining female in this group ovulated regularly until pregnancy. In the two-dose group, three of five cats were spontaneous ovulators initially; all three ceased ovulating by the time of the fertility trial. One of these cats showed elevated progestin levels towards the end of this time period, but did not become pregnant. All females (5/5) in the saline group became pregnant (kittens/litter, 3.6; litter size, 1-5). No pregnancy occurred in the two-dose group. In the single-dose group, two of five females became pregnant (litter size, 3-4). Subcutaneous granulomas were observed at the injection sites in all immunized females, with the most severe granulomas occurring in the two-dose group including two with ulceration. Granulomas reduced in size spontaneously but were still palpable one year after injection. In conclusion, the single-dose LHRH vaccine provided some fertility control but adjustment of dose and/or delivery system may be needed for more consistent, long-lasting contraception. (Morris Animal Foundation grant # D08FE-029). (platform)

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

J. Anim. Sci. Vol. 86, E-Suppl. 2/J. Dairy Sci. Vol. 91, E-Suppl. 1 W150 Immunosterilization of bitches with an anti-LHRH vaccine using CpG ODN as an adjuvant. R. Zanella1, M. Ragagnin de Lima*4, J. J. Reeves1, V. Conforti2, D. DeAvila1, A Ferreira Marques4, S. A. Messina1, R Bogden3, and E. L. Zanella4, 1Washington State University, Pullman, 2Cincinnati Zoo & Botanical Garden, Cincinnati, OH, 3Amplicon Express, Pullman, WA, 4Universidade de Passo Fundo, Passo Fundo, RS, Brazil.

Background Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented. Principal Findings We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before). Conclusions Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.

Two field trials were conducted in Brazil to evaluate LHRH immunocastration of Bos indicus bulls (d 0 = 2 yr of age). In Study I, 72 bulls were assigned randomly to one of three treatment groups: LHRH0-immunized, castrated, and intact. Immunized animals (n = 25) received a primary and two booster injections of ovalbumin-LHRH-7 and thioredoxin-LHRH-7 fusion proteins on d 0, 141, and 287. Twenty-three bulls were surgically castrated on d 141, and 24 served as intact controls. All animals were slaughtered on d 385, at approximately 3 yr of age. In Study II, 216 bulls were assigned randomly to the same three treatments as in Study I; however, because of a drought in the area, bulls were kept on pasture an additional year, and a fourth treatment was added, in which one-half the LHRH-immunized bulls received an additional booster on d 639 (fourth immunization). All animals in Study II were slaughtered on d 741 (4 yr of age). Luteinizing hormone-releasing hormone antibodies increased following each immunization for immunized bulls, but they were not detectable in castrate or intact animals in either study. Consequently, scrotal circumference was suppressed in immunized bulls compared with intact controls in both studies. By d 287, serum concentrations of testosterone in LHRH-immunized bulls were decreased compared with intact controls (P < 0.01). In both studies, testes and epididymal weights at slaughter were greater (P < 0.01) for intact (500 +/- 17 and 60 +/- 2 g, respectively) than for immunized bulls (173 +/- 22 and 26 +/- 2 g, respectively) and fourth immunization bulls (78 +/- 23 and 20 +/- 2 g, respectively; Study II). At the end of each study, BW was greater (P < 0.01) for intact bulls than for castrated and LHRH-immunized animals. In these two studies, the efficacy of the LHRH fusion proteins to induce an effect similar to that of surgical castration was considered 92 and 93%, respectively. These data support the concept that immunocastration of bulls at 2 yr of age was successful and that it has practical application as a tool for producing grass-fattened bulls in Brazil.

The effectiveness of a luteinizing hormone-releasing hormone (LHRH) fusion protein vaccine or surgical castration, at two years of age, on growth and carcass characteristics of Bos indicus bulls was evaluated. Seventy Nelore-cross bulls were divided into three groups: (1) immunized, (2) castrated and (3) intact control. At slaughter (three years of age), intact bulls had higher body weights, ADG, carcass weights, and muscle percentage compared to immunized and surgically castrated animals. Both castrated and immunized animals had greater marbling and percent carcass fat than the intact bulls. Average tenderness scores were inferior for intact bulls compared to immunized and castrated animals, but these differences were not significant (P>0.05). Juiciness, flavor, thawing, nor cooking losses differed significantly among the three groups. Immunocastration was effective in producing carcass traits similar to that of surgical castration. Therefore, immunization with LHRH fusion proteins appears to have practical utility in the management and castration of grazing bulls.

Six novel microsatellite loci are identified from genomic DNA of the threatened New Mexico Ridge-nosed Rattlesnake (Crotalus willardi obscurus). Data from the Animas Mountains (New Mexico) population demonstrate these loci: W are highly variable with 5-24 alleles per locus, expected heterozygosities between 0.35 and 0.92, and observed heterozygosities between 0.32 and 0.91; (ii) are sufficiently variable for assigning parentage with total exclusionary power for the first parent of 0.96, and 0.99 for the second parent; and (iii) amplify similar size fragments in other rattlesnakes (C. atrox, C. lutosus, C. scutulatus, and C. tigris).