Studying the genetics of Mendelian and complex traits with high-throughput genotyping and sequencing in cattle

Background For decades, identifying genes and mutations underlying inherited diseases and economically import traits has been at the core of genetic studies in domestic animals. The initial arduous task of gene mapping with microsatellites or low-density SNP panels was then followed by a often fruitless and tedious attempt to isolate the causative mutations for both monogenic and polygenic traits. The availability of high-density SNP arrays considerably accelerated the gene mapping process. More recently, advances in second-generation high-throughput sequencing (HTS) including whole genome, exome, and RNA sequencing have brought tremendous efficiency into dissecting the genetic underpinnings of phenotypes of interest. In this thesis, we describe the use of these new technologies to decipher the genetic basis of several monogenic and polygenic traits, in both forward and reverse genetic settings. Results We first describe the use of medium-density SNP arrays to dissect genetic basis of a monogenic disease (Crooked Tail Syndrome, CTS), as well as of complex phenotypes related to meiotic recombination in cattle. With regards to CTS, we first demonstrate that a common 2bp-deletion in the MRC2 gene causes the condition in Belgian Blue cattle (BBC), and that the frequency of the condition is due to a pleiotropic effect of the mutation on muscle mass. We discover a second loss-of-function mutation in the same gene MRC2, supporting the hypothesis of pleiotropy and balancing selection. With regards to recombination, we report that genetic variants in REC8 and RNF212 affect the genome-wide recombination rate in cattle. The RNF212 gene has also been shown to be associated with genome-wide recombination rate in humans, however REC8 is not supported as a candidate gene by newer data. We also demonstrated that several variants in the zinc-finger domain of a gonosomal PRDM9 paralogue influence hotspot usage in cattle, reminiscent of previous findings in human and mice. The subsequent application of HTS technology in forward genetic analyses, allowed us to identify: i) an intronic mutation in the PIGH gene that causes the skipping of exon 2, and is the cause of arthrogryposis in BBC, ii) a 3.3 kb deletion removing exons 25-27 of the FANCI gene, that causes brachyspina in Holstein-Friesian cattle. We finally used a reverse genetic approach to identify embryonic lethal (EL) mutations compromising fertility in beef (BBC) and dairy cattle (Holstein-Friesian and Jersey). We mined whole genome and exome sequence data from more than 500 animals for candidate embryonic lethal variants corresponding to loss-of-function (stop gain, frameshift and splice site variants) and disruptive missense variants. By doing so we demonstrate that against expectations domestic Bos taurus cattle are genetically more variable than humans including Africans, however, that their burden of disruptive variants is similar, amounting to ∼100 such variants per individual. We interpret this as resulting from more effective purifying selection in recent times as a result of increased inbreeding. We have genotyped thousands of animals for thousands of EL candidates, and estimate – from the observed depletion in homozygotes - that no more ∼15% of those are developmentally essential. We do not find evidence for synthetic epistasis between candidate EL mutations. By evaluating the departure from Mendelian expectations in matings between carrier sires and dams, we unambiguously demonstrate the embryonic lethal nature of nine relatively common candidate EL variants. All of them affect genes that are involved in basic cellular processes. We demonstrate that only one of these would have been detected using the haplotype-based approach that were applied thus far. The established list of loss-of-function variants is being mined for putative non-lethal, yet major phenotypic effects of medical relevance. Among these, we uncovered a 10-bp deletion in the MLPH gene, which results a premature stop codon, that causes diluted color – ‘cool gray’ – in BBC. Conclusions and Perspectives Due to the intensive use of some elite sires by means of artificial insemination in cattle, several outbursts of genetic defects have been observed in recent years. We herein demonstrate that uncovering the genetic basis of such Mendelian disorders can be very effective when integrating high-throughput genotyping and sequencing technology. Fertility in cattle and other domestic animals has declined over the last decades. Our discovery of a number of embryonic lethal variants segregating in cattle provide a complementary explanation for the decline in fertility, in addition to the theory of negative energy balance of high producing cows. The reverse genetic approach we developed for screening EL variants has at least two advantages. First, our approach also works in small sized populations. Second, our approach has higher sensitivity than the haplotype-based approach. It has been suspected for some time that most of the variants underlying complex traits map to noncoding regions. Deciphering the functional consequence of these regulatory variants has proven challenging. The ENCODE projects in human and mice provide good examples of how high-throughput functional assays based on NGS technology may enhance our knowledge of the non-coding regions of the genome. It is reasonable to anticipate that the release of ENCODE-like data in the animal research field will assist in the discovery of the genetic basis of complex traits at an unprecedented pace