Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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​Re-sequencing of Whole Genome Jordanian Awassi Ewes using Hiseq Sequencing Technology: Major Step Towards Sheep Genomic Selection

Hussein Migdadi1,*, Nizar Haddad1, Ruba AlOmari1, Mohammad Brake2, Mustafa AlShdaifat1, Sadeer Amasha1, Monther sadder3
1National Agricultural Research Center, Baqa, 19381, Jordan.
2Department of Science, Jerash University, Jordan.
3Department of Horticulture and Crop Science, Faculty of Agriculture, University of Jordan Amman, Jordan.
Background: Jordanian Awassi sheep (Ovis aries) is the dominant fat tail sheep breed that appeals to customers because of its various production systems, including fiber, meat and milk. This report is the first whole ewe genome sequence (WGS) of O. aries submitted in the NCBI database from Jordan.

Methods: 64 Paired-end sequencing libraries were constructed and subjected to Illumina Hiseq 2500 sequencing system. High-quality reads were aligned against the reference sheep genome and detecting comprehensive sources (SNPs, InDels, SV, CNVs) of genetic variations. We have deposited data sequences at the NCBI under SRA (sequence reads archives) under the accession numbers SRR11128863, PRJNA574879.

Result: Genome resequencing of Jordanian Awassi ewe was carried out with approximately 93.88 Gb with a mapping rate and effective mapping depths were 99.28% and 36.32. Around 19 million SNPs, 3,6 million InDels, 35,180 Structure variation and 13,524 copy number variation among the Jordanian ewe genome were detected. This wide range of genetic variation provides a framework for further genetic studies that will help understand the molecular basis underlying phenotypic variation of economically important traits in sheep and improve intrinsic defects in domestic sheep breeds.
Domestication of sheep (Ovis aries) in the Fertile Crescent is back to 11,000 years ago (Zeder, 2008). It spread west throughout Europe, south into North Africa and east into Asia (Chessa et al., 2009) and became adapted to a wide range of environments and has been selected for varied production systems, including fiber, meat and milk. During domestication, natural and artificial selection play a role in remarkable changes occur in sheep behavior, physiological and morphological appearance and other essential traits (Wang et al., 2019) and a great variety of sheep breeds have been developed (Larson et al., 2014). Before wool and milk, man first reared the sheep for access to meat (Chessa et al., 2009), which played an essential role in human society, increased human subsistence stability and spread globally. The environmental heterogeneity and differences affect the phenotypic and genetic variation across sheep populations in climatic factors. Studying the impact of climate on selective signatures well help in the detection of the genetic basis of local adaptation and speciation in response to changing climates (Joost et al., 2007), which will provide insight into functionally important variants (Nielsen et al., 2007; Sah and Dixit, 2021) and will help in developing appropriate breeding programs under scenarios of future climate changes (Howden et al., 2007). Approximately 25% of the world’s sheep breeds have belonged to fat-tailed sheep (Farid et al., 1983). These breeds can survive in harsh environments and satisfy human intake of dietary fat.

Jordan has only Awassi sheep of various ecotypes and strains that originated and were genetically developed for many past generations (Al-Atiyat et al., 2012). However, more information and details of Jordanian Awassi sheep breeds, including morphological, phenotypical and production characteristics and genetic diversity and structure, were reported. Awassi sheep (Ovis aries) is the dominant fat tail sheep breed, which is also dominant in Mediterranean countries. They well characterized it for their carpet wool, body weight and morphological characteristics at different ages (Tabbaa, 2003). Based on mitochondrial DNA (mtDNA) and microsatellite-based evidence, (Al-Atiyat et al., 2018) and (Jawasreh et al., 2018) reported that Jordanian Awassi has common haplotype groups (B and A) and group C with Saudi, Kuwaiti, Egypt and Yemen sheep. Progress toward genetic improvements of sheep using conventional crossbreeding methods for traits associated with meat, milk, wool and reproduction was slow.

National Agricultural Research center set up objectives to decipher molecular mechanisms of the response of locally adapted genotypes and breeds to environmental stress and use the information generated to select elite lines and breeds that cope with harsh environmental conditions. 

The first whole-genome resequencing of Jordanian Awassi ram was published (Haddad et al., 2020), which reported the availability of wide genome variability (e.g., SNPs, InDel, SV, CNV) that could be helpful in the signature’s detection of selection to reveal new insight into the mechanism of advanced breeding, artificial selection and genes associated essential traits. Genetic studies on Jordanian Awassi revealed genetic variability among and within breeds (Al-Atiyat et al., 2014; Jawasreh et al., 2018), genetic parameters related to growth performance, carcass characteristics and meat quality (Jawasreh et al., 2019). To increase our knowledge of Awassi sheep evolution and breeding, national research on sheep breeding strategy will focus on selection, including an advanced genotyping test for economic traits found in Awassi. To advance genome selection techniques, speed up Awassi sheep improvement, putative selection, under standing disease inheritance and get insights into the genetic control of milk, meat and wool production, we conducted whole-genome resequencing of Jordanian Awassi ewe.
Animal Samples
All animals were handled according to the ethical approval of relevant national legislation (Approval Code No. (G/6) 2006): Instructions and conditions for the acquirer of test animals and testing them.
Sample collection and DNA extraction
Whole-blood samples (10 mL) via EDTA-coated vacutainer tubes were collected from 55 individuals of Awassi landraces from three main Awassi sheep-producing areas in Jordan to minimize potential bias because of overrepresentation of local effects (e.g., inbreeding) and kept in the freezer until DNA extraction. Genomic DNA was extracted using the DNA purification kit (Promega, Wisconsin, USA) following the manufacturer’s instructions.
Genome sequencing
For genome sequencing, 0.5 μg of genomic DNA from each sample fragmented and after electrophoresis, DNA fragments of the desired length are gel purified. 64 Paired-end sequencing libraries were constructed according to the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA) and subjected to Illumina Hiseq 2500 sequencing system (Illumina, San Diego, CA, USA). 

Raw reads contain adapters, unknown or low-quality bases discarded. The parameters of SOAPnuke (Chen et al., 2018) software for this project is “-n 0.05 -l 20-q 0.2 -G -Q 2.” The filter steps included removing reads more than half of the bases’ qualities are less than five and get clean reads. High-quality reads were aligned against the reference sheep genome assembly using Burrows-Wheeler Aligner (BWA; Li and Durbin 2009) software. BAM format files are used to store the alignment information. After fixing mate-pair information, they were adding read group information and marking duplicate reads caused by polymerase chain reaction, variant calling and detecting single-nucleotide polymorphisms (SNPs) and small Insertion/Deletions (InDels) by GATK software ( (McKenna et al., 2010). BreakDancer software ( (Chen et al., 2009) used for the identification of Structure Variants (SVs) and Copy Number Variants (CNVs). At each stage of the analysis, quality control was applied for the alignment and the called variant (Fig 1). The genome raw data sequences of Jordanian Awassi sheep have been deposited with the NCBI under SRA (sequence reads archives) under the following accession number SRR11128863, PRJNA574879. SRA records accessible with the following link: https://www.ncbi.

Fig 1: The pipeline of Standard Bioinformatics Analysis. The pipeline based on the family of SOAP software. QC; quality control.

Pools of DNA of fifty-five Jordanian Awassi ewe sheep (Fig 2), were sequenced at approximately 60× coverage and then analyzed jointly with publicly available the sheep reference genome covering with an average of 97.78% of the reference genome (Jiang et al., 2014). 99.22 Gb of genome data were generated in the present study. For quality insurance, the raw data was modified by deleting the adapter pollution in reads and then the reads which contain over 50% low-quality bases (quality value <= 12) were removed and the stringent quality filtering yielded 93.88 Gb of genome data.

Fig 2: A typical Awassi sheep (Ovis aries) is the dominant fat tail sheep breed in Jordan.

Results in Table (1) summarized the raw and cleaned reads of the whole genome sequence. The Q20 base rate of each lane is above 97%, so the data quality is very high (Fujimoto et al., 2010).

Table 1: Summary of the sequencing and alignment results from raw and clean reads in the Jordanian Awassi ewe Sheep.

The genome size is 2,584,832,510 bp and the effective size is 2,560,768,911bp. Burrows-Wheeler Aligner (BWA) software used for sequence reads alignment to the reference genome. The mapping rate and the final effective mapping depth were 99.28% and 36.32. Based on the consensus sequence, the polymorphic loci between our breed and the reference filtered and a high-fidelity SNP data set generated (Table 2). 18,994,659 SNPs among Jordanian ewe genomes. The SNP results are listed as CDS, exon and genes. In this study, 152,684 synonymous and 102,531 non-synonymous SNPs were annotated among this sheep genome. Furthermore, the distribution of predicted large-effect SNPs is presented in Fig 3. Premature stop codons were 1016 and 1071 were disrupt splicing donor or acceptor sites.

Table 2: Summary of genome-wide SNPs and their annotation distribution in the Jordanian Awassi ewe Sheep.

Fig 3: Number of SNPs generated and the distribution of predicted large-effect of SNPs.

Short InDel detection and annotation
The Genome Analysis Toolkit (GATK) was used to detect InDels. 3,626,673 InDels generated and distributed as 1,782,830 insertions and 1,843,843 were deletions (Table 3). Furthermore, the frameshift InDel based on the InDel annotation has resulted in 43,614.

Table 3: Insertion and deletion, annotation, distribution and a frameshift mutation in CDS in the genome of Jordanian Awassi ewe Sheep.

Structure and copy number variation detection and annotation
The structural variation detected includes deletion, insertion, duplication, inversion and transposition. 43.14% were deletion type of SV. Over 189326 were in gene sites. The SV results are presented in Table (4).

Table 4: Structure variation annotation, distribution in the genome of Jordanian Awassi ewe Sheep.

Copy Number Variation (CNV) is a basic form of structural variation among individuals of the same species. Genome regions deleted or duplicated on some chromosomes between the sequencing individual and the reference have corresponded to CNVs. The CNV results listed in Table (5); Over 72% of the CNV were down-regulated. Recently, the whole-genome resequencing of Jordanian Awassi Ram (Ovis aries) using Hiseq sequencing technology was reported (Haddad et al., 2020). The report considered this research as the first step towards sheep genomic selection of Awassi ram. Based on this report, the authors urged similar work to be performed on Awassi ewe’s genome. They justified that whole-genome resequencing of Jordanian Awassi Ram was considered a preliminary stage requiring similar whole sequencing of Awassi ewe’s genome to start Awassi sheep genomic research selection. Genome-wide studies used High-throughput sequencing and screening technologies on sheep are reported. They included single-nucleotide polymorphisms, induction variations, structural variation, copy number variants at the whole-genome level, transcriptome and DNA methylation sequences and comprehensive information on functional genes genetic variants associated with economically important traits. Studies to decipher the genetic basis of animal domestication are dramatically increased and considered an active research area. Genomic signatures related to domestication and improvement were found in response to demographic and selective differences between wild and domestic populations (Florian J. Alberto et al., 2018). (Li et al., 2020) stated that the availability of whole-genome sequences provides an opportunity to study domestication at the gene mutation level. They re-sequenced the whole genomes of 248 wild, landrace and improved sheep and detected genomic regions: targets of domestication, breeding and selection. Furthermore, they found non-synonymous mutations in candidate genes and significant differences in their allele frequency distributions across breeds, such as PDGFD (platelet-derived growth factor D) gene for fat deposition in the tails of sheep. (Pan et al., 2018) performed whole-genome sequencing of 99 sheep and identified candidate genes revealed local adaptations of the sheep associated with sensory perception, muscle strength, eating habits, mating process and aggressive behavior.

Table 5: Copy number variation distribution and annotation distribution in the the genome of Jordanian Awassi ewe Sheep.

Furthermore, (Florian J. Alberto et al., 2018) determined certain genomic regions carrying genes involved in the nervous system, immunity and productivity traits differentiating domestic breeds of sheep and goats from wild populations. (Chang et al., 2018), reported two novel miRNAs from the Ovis aries may play an important role in inducing embryonic development, sex regulation, production of sperm and ovulation. Moreover, (Chang et al., 2020), identified few genes serve as potential biomarkers for monitoring disease progression and abortion risk during pregnancy in sheep.

(Adam Abied et al., 2020) used high-density SNP Chip data to characterize the auto-zygosity of five local Chinese sheep breeds belonging to different geographical locations identified by the genomic regions with high runs of homozygosity frequencies within individuals of each breed. These regions included candidate genes associated with disease resistance traits, the innate and adaptive immune response, digestion and metabolism, growth, body size and developments. (Brake et al., 2021) demonstrated using the complete mitochondrial genome of the Awassi-Jo breed (Ovis aries) data to construct the phylogenetic tree. They showed that Awassi-Jo diverged earlier than related breeds (Turkey, Italy, Germany and Netherland) with a common ancestor in haplogroup HB. The results revealed the importance of mitochondrial data in studying sheep evolution and domestication.
Consequent whole-genome resequencing (WGR) has provided geneticists with the opportunity to investigate the genome in depth. These investigations have been recent and cut-edge technology applied to many world sheep breeds. WGR provided contributions to a better understanding of evolutionary forces’ effects, population dynamics, the discovery of genes, selecting signatures and starting specific maps and genomes. The present work describes the whole-genome resequencing of Jordanian Awassi ewe, which experienced specific selection pressures over several generations. The wide range of genetic variation provides a framework for further genetic studies that will help understand the molecular basis underlying phenotypic variation of economically important traits in sheep and improve intrinsic defects in domestic sheep breeds.

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