Sequencing, de novo Assembly and Functional Analysis of the Male Specific Region of the Horse Y Chromosome

The Y chromosome plays a central role in sex determination, male fertility, and evolution of reproductive barriers. Here we sequenced on Illumina, Roche 454 and Pacific Biosciences NGS platforms 102 BAC clones of a tiling path of the horse male specific region of Y (MSY). We generated a nearly complete 9.12 Mb assembly of the euchromatic MSY. Scaffold N50 was 299,624 bp and the maximum length of scaffolds and super-scaffolds were is 554,834 bp and 2.2 Mb, respectively. Ampliconic BACs were sequenced and assembled individually into ~3.56 Mb contigs/scaffolds. For MSY annotation, we generated 45 Gb testis RNA-Seq data from 5 horses, 2 donkeys and 2 mules. The horse testis transcriptome yielded 68,361 transcripts with over 10X coverage. Mapping RNA-seq data to the MSY draft assembly revealed 939 MSY transcripts. We are currently exploring the number of novel transcripts and variation in expression levels to annotate the MSY and test differential expression among fertile and subfertile stallions. This includes expression analysis of all MSY genes/transcripts in adult and embryonic tissues (n=26). Expression data from mules and donkeys will allow comparative analysis of the Y in equids. The equine MSY assembly fills a major gap in the equine genome sequence assembly and enables examining the contribution of Y genes to stallion reproductive biology and fertility. Being among the few MSYs sequenced and assembled to date, the findings provide new insights into Y evolution in mammals.