oa De Novo Sequencing Of The Highly Endangered Spix's Macaw: A Case Study In Ultra-small Population Genetics

Objectives: The purpose of this study is to sequence and annotate the genomes of the entire spix's macaw population "the world's most endangered parrot" at AWWP (of Al Wabara Wildlife Preservation). This data would be used to identify Single Nucleotide Polymorphism (SNPs) that would later be used in population studies of this species. The results would be used to guide future breeding programs to increase the genetic diversity of the spix's macaw, which will lead to the ultimate objective of the reestablishment of a self-sustaining population of Spix's Macaws in its native Caatinga habitat. Methods: DNA samples, thus far, were provided from 40 males and females at the AWWP. The spix's macaw whole genome was sequenced using Next-Generation sequencing approach. For the genome assembly, we utilized a 63bp kmer and ~1.1 billion paired 100bp reads (~66x coverage) from Illumina HiSeq 2500 instruments. These reads were distributed across libraries ranging in size from 300-1000bp for paired-end and 2000-15000bp for matepair. SNPs were detected on scaffolds >1000bp. Results: The predicted genome size is ~1.5Gb, which is similar to the parrot genome, the closest sequenced species. Scaffold N50 of 3.1Mb (longest scaffold of ~16.5Mb) and an assembly spanning >92% of the genome. The sequence is distributed across ~6500 scaffolds >500bp. Heterozygous SNPs with coverage of >20x were considered to analyze the survival rates of offspring. We noticed that the average percentage of unique heterozygous SNPs in a bird drastically dropped to below 50% when compared to a second bird. This percentage further dipped exponentially each time another bird was added to the comparison. The average percentage of unique heterozygous SNPs fell to below 10% when 8 birds were compared for unique heterozygous SNPs. This shows that random mating combinations increase the chance of generating offspring with decreased survival rates due to loss of heterozygosity. Conclusion: The initial analysis of polymorphisms confirms the existence of a very high level of inbreeding, in which a random recombination of birds could result in offspring with very high levels of homozygous SNPs by the 4th generation. Further study will be needed to identify detrimental SNP combinations. This information will be used to better understand the genetic pool and to promote better breeding results with higher offspring survival rates.