Identifying Genetic Factors Influencing Sperm Mobility Phenotype in Chicken using Genome Wide Association Studies, Primordial Germ Cell Transplantation, and RNAseq.

Sperm mobility is a major determinant of male fertility in chicken. In spite of low heritability of reproductive traits, sperm mobility has high heritability index which suggests presence of quantitative trait loci (QTLs) governing the trait. Our research focused on three objectives: i) to identify the QTLs affecting low mobility phenotype in chicken, ii) to understand the impact of Sertoli-cells and germ cells interactions in influencing the mobility phenotype and iii) to identify the genes and gene networks differentially expressed in male and female PGCs. To detect the QTLs, genome wide association studies (GWAS) was conducted which revealed the presence of multiple minor alleles influencing the trait and indicated the role of epistasis. The second section of research involved isolation, culture and transfer of primordial germ cells (PGCs) to create high line germ line chimera chicken carrying low line PGCs. We established the culture of chicken PGCs isolated from the embryonic blood in a feeder free culture conditions but could not detect the presence of low line genotype in the semen of transgenic males. Our final study involved RNA-sequencing (RNAseq) of male and female PGCs to identify differentially expressed genes from their transcriptomes. We identified five candidate genes: 3-hydroxy-3-methylglutaryl CoA reductase (HMGCA), germ cell-less (GCL), SWIM (zinc finger SWIM domain containing transcription factor), SLC1A1 (solute carrier family 1 member 1), UBE2R2L (ubiquitin conjugating enzyme) and validated their expression level in male and female PGCs by RT-qPCR. GCL was exclusively expressed in males while SLC1A1 & UBE2R2L were expressed only in female cPGCs. This present study provides novel gender specific germ cell markers in the broiler chicken. These results will help in elucidating the genetic programming of gender specific germ line development in broilers

Genome Analysis of Staphylococcus agnetis, an Agent of Lameness in Broiler Chickens.

Lameness in broiler chickens is a significant animal welfare and financial issue. Lameness can be enhanced by rearing young broilers on wire flooring. We have identified Staphylococcus agnetis as significantly involved in bacterial chondronecrosis with osteomyelitis (BCO) in proximal tibia and femorae, leading to lameness in broiler chickens in the wire floor system. Administration of S. agnetis in water induces lameness. Previously reported in some cases of cattle mastitis, this is the first report of this poorly described pathogen in chickens. We used long and short read next generation sequencing to assemble single finished contigs for the genome and a large plasmid from the chicken pathogen. Comparison of the S. agnetis genome to those of other pathogenic Staphylococci shows that S.agnetis contains a distinct repertoire of virulence determinants. Additionally, the S. agnetis genome has several regions that differ substantially from the genomes of other pathogenic Staphylococci. Comparison of our finished genome to a recent draft genome for a cattle mastitis isolate suggests that future investigations focus on the evolutionary epidemiology of this emerging pathogen of domestic animals

Summary data of assembly and annotation of the genome of <i>S</i>. <i>agnetis</i> 908.

<p>Summary data of assembly and annotation of the genome of <i>S</i>. <i>agnetis</i> 908.</p

Representative BCO lesions for proximal femora.

<p>Severity of the lesions are scored by macroscopic examination: Femoral Head Separation, FHS (Panel A), Femoral Head Transitional degeneration, FHT (Panel B), Femoral Head Necrosis, FHN (Panels C and D) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143336#pone.0143336.ref001" target="_blank">1</a>].</p

Phylogenetic trees of toxin virulence factors.

<p>Trees were constructed using the closest homologs to <i>S</i>. <i>agnetis</i> toxin proteins from BLASTp searches of refseq proteins from <i>S</i>. <i>hyicus</i>, S.chromgenes, <i>S</i>. <i>pseudintermedius</i> and <i>S</i>. <i>aureus</i>. Tress are for Superantigen-like protein homologs (Panel A), beta hemolysin (Panel B), and exfoliative toxin A (Panel C). The <i>S</i>. <i>agnetis</i> ortholog are prefixed with the ORF number from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143336#pone.0143336.s001" target="_blank">S1 Table</a>. Details of the alignment strategy are in Materials and Methods.</p

Bacterial species from lame birds based on bone lesion.

<p>Lesion designations were <b>Normal-</b> no macroscopic abnormalities of the proximal femur or tibia; <b>FHS</b>- proximal femoral head separation; <b>FHT-</b>proximal femoral head transitional degeneration; <b>FHN-</b>proximal femoral head necrosis; <b>THN-</b> mild proximal tibial head necrosis; <b>THNs</b>-"severe" THN in which the growth plate was imminently threatened or damaged; and, <b>THNc</b>- "caseous" THN in which caseous exudates or bacterial sequestrae were macroscopically evident [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143336#pone.0143336.ref001" target="_blank">1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143336#pone.0143336.ref029" target="_blank">29</a>]. Species identification and Total number of infections diagnosed was as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143336#pone.0143336.t001" target="_blank">Table 1</a>.</p><p>Bacterial species from lame birds based on bone lesion.</p

Bacterial species from lame birds based on site sampled.

<p>A total of 24 lame birds were sampled from all five locations and bacterial colonies diagnosed by PCR-sequencing of a portion of the 16S rDNA. The number of infection sites diagnosed excludes sampled that either did not show bacterial growth, failed in the PCR, or yielded poor sequence data.</p><p>Bacterial species from lame birds based on site sampled.</p

Phylogenetic tree of Staphylococcus species based on 16S rDNA sequences.

<p>Full length 16S rDNA sequences for the indicated species and isolates were obtained from NCBI and aligned using ClustalW and used to generate a Neighbor Joining tree (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143336#sec002" target="_blank">Materials and Methods</a>).</p

BLAST Ring Image comparing Staphylococcus genomes.

<p>BLAST Ring Image Generator version 0.95 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143336#pone.0143336.ref035" target="_blank">35</a>] was used to analyse the <i>S</i>. <i>agnetis</i> 908 2.4 Mb contig by position (inner ring 1), GC skew (ring 2), and for GC content (ring 3). BLASTn conservation (minimum threshold 50%) in other Staphylococcus genomes proceed outward <i>S</i>. <i>agnetis</i> CBRMN20813338, <i>S</i>. <i>hyicus</i> ATCC11249, <i>S</i>. <i>pseudintermedius</i> HKU10-03, <i>S saprophyticus</i> ATCC15305, and then five <i>S</i>. <i>aureus</i> genomes for strains FPR3757, TW20, ST228, N315, JH1, and 04–02981. The outer ring indicates features described in the text for the <i>S</i>. <i>agnetis</i> 908 genome.</p

Phylogenetic tree of fibronectin binding proteins from <i>S</i>. <i>agnetis</i> with homologs from <i>S</i>. <i>hyicus</i>.

<p>The protein sequences predicted for the seven ORFs from <i>S</i>. <i>agnetis</i> (see text) were aligned using Clustal W to the five predicted fibronectin proteins for the <i>S</i>. <i>hyicus</i> ATCC11294 genome (AJC entries are accession numbers) representing the closest orthologs in NCBI for the proteins from <i>S</i>. <i>agnetis</i>. The phylogenetic tree includes bootstrap significance for 2500 iterations.</p