GENETIC STRUCTURE OF CAMELUS DROMEDARIUS POPULATIONS

THROUGH GENOME-WIDE RAD SEQUENCING

Elena Ciani1, Pamela Burger2 and the International Camel Consortium for Genetic Improvement and Conservation (ICC-GIC)

1Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Italy (elena.ciani@uniba.it)

2Research Institute of Wildlife Ecology, Vetmeduni Vienna, Vienna, Austria (pamela.burger@vetmeduni.ac.at)

Introduction

• The development of cost-effective high-throughput SNP chip technologies in the last decade for major livestock species represents an outstanding breakthrough that has allowed them to enter the genomic era.

• Despite its worldwide relevance, the dromedary (Camelus dromedarius)

has tended to be excluded from the livestock ”genomic revolution” until recently. Indeed, no genome-wide screening tool is today available and no population genomics study has so far been published for this species.

• To tackle this discrepancy, we will adopt a genome-wide Restriction site Associated DNA (RAD) sequencing approach, a method that combines traditional DNA shearing via endonucleases with the Illumina Next Generation Sequencing technology to simultaneously genotype tens to hundreds of thousands of single nucleotide polymorphism (SNP) markers in hundreds of individuals. This technology has the major advantage of not requiring substantial prior knowledge of both genome sequence and variability, while achieving per-site and per-individual costs below that of current SNP chip technology.

• A total of 120 animals collected from a wide geographic sampling area have been considered (Figure 1). Genomic DNA has been isolated either from whole blood using standard commercial kits, or from FTA cards® using the FTA purification reagent®, and following manufacturer’s instructions.

• We adopted a double-digest approach (ddRADseq) which has been

proved to better allow optimization of the number of loci to be sequenced and maximization of the number of sequence reads incorporatedin the analysis.

• An in silico analysis of the Camelus dromedarius (GCF_000767585) V1 whole-genome reference sequence has been carried out to identify the optimal combination of enzymes able to identify the desired number of loci to be sequenced (about 40.000)

• For large-scale multiplexing of samples (120 samples on a single Illumina HiSeq2500 lane), adapters with combinatorial sequence barcodes (in-line on R1, in-line on R2 and index TruSeq) were ligated to samples. Coverage redundancy has been optimized to allow sequence disambiguation. A coverage >20X is expected for most of the loci, which will allow enough accuracy in detecting heterozygous genotypes.

• Libraries will be paired-end sequenced . After sequence trimming, 2x110 bp sequences will be available for each locus.

Results

• The in silico analysis of the Camelus dromedarius (GCF_000767585) V1 whole-genome reference sequence highlighted the pair SphI-BstYI as the best combination of restriction enzymes able to produce about 40.000 fragments 400-530bp in size.

AKNOWLEDGEMENTS: We are grateful to the owners and collectors of the camel samples included in this study, i.e., Gräfliche Kamelheimat and Erste Kamelreitsschule (AT), A.R. Kakar (PK), O. Abdelhadi (SD), G. Konuspayeva (KZ), A. and J. Perret (KE), R. Saleh (SY), J. Juhasz and P. Nagy (UE), Youceg Cherifi and Souheil Gaouar (DZ), Davide Monaco (MA), Habib Yahyaouhi (TN), Salem Khalifa (LY). This work was mainly supported by the ENPI-CBC MED PROCAMED project (EU). Support from the Austrian Science Foundation (FWF) project grant P1084-B17 and the Austrian Academy of Sciences (APART fellowship 11506) is also aknowledged.

Results

• A first batch of 24 samples has been sequenced at the moment. Setting strict filtering parameters, a minimum coverage of 3 and a minimum genotyping threshold of 0.75, 60k polymorphic SNPs distributed over 44k RAD loci RAD were identified.

Figure 1. Countries where samples of Camelus dromedarius considered in this study were collected.

• The obtained SNP genotypes will help carry out the first population genomic study in the Camelus dromedarius species and will hopefully open the way to a larger collaborative effort for a more comprehensive characterization of the genetic diversity and reconstruction of the evolutionary history of this fascinating desert animal.

Figure 2. Picture of one dromedary camel sampled in Algeria.

Materials and Methods

Conclusions

(Photo: courtesy of Dr. Davide Monaco)