Dissecting the Most Complex Regions of the Mammalian Genome; the Sex Chromosomes

Abstract

Despite the unique genetic contribution of sex chromosomes, their studies generally lag behind the rest of the genome. Therefore, the aim of this thesis is to advance the assembly, annotation of sex chromosomes in domestic species. First, we present the first draft assembly of the alpaca Y. We sequenced and assembled flow-sorted Y-DNA resulting in 20,060,146 bp (652 contigs). Flow-sorted Ys were used for cDNA selection from testis and provided transcripts for annotation. Contigs with known Y genes were confirmed to be male specific (MSY) by BLAST against the female reference and PCR on male and female gDNA. Pseudoautosomal (PAR) contigs were identified by annotating known PAR genes and high homology with X. The final assembly comprised 4.5 Mb of MSY and 3.7 Mb of PAR. We annotated 15 gametologs and 20 PAR genes with HSFY and RBMY being confirmed as multicopy by qPCR. We demarcated the pseudoautosomal boundary (PAB) in contig419 between SHROOM2 and HSFY, within CLDN34. Most gametologs are shared among mammals, though WWC3Y is Y-specific only in camelids and horses. Secondly, we used trio-binning of a hinny – the F1 hybrid of a male horse and female donkey, to improve the complex genomics regions and obtained new chromosomally-assigned genomes for horse (ECAnp4) and donkey (EASnp2). Our specific goal was to refine complex regions of the X-chromosomes and define the PABs. Sequence analysis of the hinny and 4 PAB-Y and PAB-X BACs allowed demarcation of horse PAB at 1.8 Mb and identification of XKR3Y as the boundary gene. Improved horse X chromosome revealed that the PAB of EquCab3 had been misassembled. The new donkey X assembly showed significant discordance to the existing reference but not to the horse, indicating gross misassembles in EquAsi1 which were rectified in EASnp2. Comparison to the PAB of ECAnp4 and a drop in the sequencing depth of male reads placed the donkey PAB at 1.88 Mb. Two complex loci DXZ4 and ETSTY7 were collapsed in both horse and donkey references but corrected in EASnp2 and ECAnp4. This work represents a meaningful improvement in the study of camelid/equid sex chromosomes.