REVIEW ARTICLE | 1 Analyses of Wild Ungulates Mitogenome Toni TEŠIJA1 (✉) Toni SAFNER1,2 Summary In the last two decades, mitochondrial DNA (mtDNA) has become one of the most used genetic markers for studying evolution and phylogeny. Understanding mitochondrial genome (mitogenome) structure, gene arrangements, base compositions and noncoding regions can reveal valuable information on various organisms, including wild ungulates. A huge number of sequenced mammalian mitogenomes are available on GenBank, including 8074 mitogenome sequences of wild ungulates. The Next-Generation Sequencing (NGS) approach can reveal mitogenome regions that can explain the variation in populations. Such variations could show ungulates’ resistance to pathogens or processes of their adaptation. Despite the extensive number of new sequenced mitogenomes, there is still a lot of ambiguity around the mitogenome architecture. Methods for NGS, genome assembly and annotation are still in the process of development, aiming to make these tools powerful enough to reveal enormous knowledge crucial for assessing wild ungulate species’ conservation status, and their ecological status. In this review paper, we describe the methods and principles derived from mtDNA studies on wild ungulate species. We outline basic sequence preparation methods and specialized software for mitogenome assembly and annotation. Additionally, we present several different approaches of mitogenome comparison. Key words mitochondrial DNA, mitogenome, mtDNA assembly, mtDNA annotation, wild ungulates, phylogeny 1 University of Zagreb, Faculty of Agriculture, Department of Plant Breeding, Genetics and Biometrics, Svetošimunska cesta 25, 10000 Zagreb, Croatia 2 Centre of Excellence for Biodiversity and Molecular Plant Breeding (CoE CroP-BioDiv), Svetošimunska cesta 25, 10000 Zagreb, Croatia ✉ Corresponding author: [email protected] Received: May 28, 2020 | Accepted: September 23, 2020 aCS Agric. conspec. sci. Vol. 86 (2021) No. 1 (1-12) 2 | Toni TEŠIJA, Toni SAFNER INTRODUCTION can be passed to progeny. On the other hand, the number of such mutations accumulating in the coding region is small, since they The Structure of Mitochondrial DNA might be lethal, and only lesser number of them is passed down to future generations (Gupta et al., 2015). Mitochondria are organelles in eukaryotic cells responsible for producing energy, which play a pivotal role in mammals’ Since the D-loop region is the most rapidly evolving part of temperature regulation (Boore, 1999; Wallace, 2007). During the mitogenome (Upholt and Dawid, 1977; Walberg and Clayton, the oxidative phosphorylation (OXPHOS) process (that results 1981), where the largest number of mutations is accumulated, in adenosine triphosphate ATP synthesis), mitochondria oxidize it is commonly used as a molecular marker for finding genetic metabolic substrates in order to generate energy and water. differentiation between animal species, as well as between The metabolic processes differ between taxa (da Fonseca et al., individuals of the same species (Gupta et al., 2015). 2008), essentially depending on species’ metabolic requirements Another mtDNA region with high diversity is cytochrome (e.g. body size, diet, high altitudes). Mammal mtDNA is a c oxidase subunit 1 (COI), which is often used as animal DNA small (~16 kb) circular molecule with two strands (Heavy or barcoding system. Animal barcoding systems are used to provide H strand and Light or L strand), divided in two parts: coding additional information for species that are not correctly identified and non-coding. The coding part contains 13 protein-coding by typical techniques based on morphology (Naseem et al., 2020). genes (PCGs) for OXPHOS, the 22 transfer RNA genes (tRNA) Hebert et al. (2003) proposed an approach where, based on the for protein translation and 2 ribosomal RNA genes (rRNA) nucleotide sequence diversity in the COI gene, it is possible to for the mitochondrial ribosome. This part is highly conserved differentiate species from diverse taxonomic groups. for mammalian mitogenomes and has a low rate of gene rearrangements compared to the nuclear genome (Wolstenholme, 1992). The H strand encodes 12 PCGs, 2 rRNA genes and 14 tRNA D-loop Region Structure and Repeating Sequences genes and it is rich in guanin. The L strand encodes one subunit Most of the mammals have similar D-loop location and (ND6) and 8 tRNA genes (Barshad et al., 2018; Barchiesi and organization. The D-loop is located between the genes encoding Vascotto, 2019). The non-coding parts of mtDNA comprise three tRNAs Pro and Phe and it consists of three main domains. The first parts: the control region (CR, Displacement loop, D-loop), the domain is called the right domain (or conserved sequence blocks, OL region (the origin of L-strand replication), and the intergenic CBS) and is adjacent to the tRNA gene encoding Phe. This region regions. The D-loop is long about 700-1,300 bp in length while the contains crucial regulatory parts for replication, transcription of OL is long about 30 bp. The intergenic regions (spacers) are located both strands and the segment with conserved sequence blocks within some genes. MtDNA genes do not have introns, except (CBSs) involved in the processing of RNA primers for synthesis of for the intergenic spacers that are limited to a few bases, or, in H-strand. The second is the left domain (or the extended terminal some cases, completely absent from the mitogenome (Taanman, associated sequenced domain, ETAS), which is adjacent to the 1999). Dromedary camel (C. dromedaries) has a total of 116 bp tRNA gene encoding Pro. In this region synthesis of the heavy of intergenic regions (22 different locations) in its mitogenome strand is paused. The third part is the central conserved domain. (Manee et al., 2019), which is identical or very similar for species It is presumed to be the site of origin for replication and the most within the same genus. DNA and RNA synthesis start in D-loop highly conserved region of the D-loop (Saccone et al., 1991; Sbisà regions (Saccone et al., 2000), which are well conserved (Garesse et al., 1997; Shadel and Clayton, 1997; Boore, 1999). and Vallejo, 2001). However, other parts of the D-loop regions present the most polymorphic region of mtDNA (Upholt and As mentioned before, the main factors defining the variability Dawid, 1977; Shadel and Clayton, 1997). Those variable parts of of the D-loop region are processes during replication, but there the D-loop evolve four to five times faster in comparison to other are other contributing factors such as the different numbers of regions of mitogenome (Fumagalli et al., 1996). repeating sequences (RS). These regions are common in the ETAS and CSB domains (Polziehn and Strobeck, 2002; An et al., 2010; The replication of mammalian mitogenome is asymmetric Meadows et al., 2011). Furthermore, the highest rate of sequence since the two strands are synthesized from two distinct replication variation between individuals is found in hypervariable region 1 sites. The H strand replication site (OH) is located at the D-loop and hypervariable region 2 (Miller et al., 1996; Jazin et al., 1998). region while L strand is in the OL region located between the The D-loop is responsible for length variations of mitogenomes due genes encoding tRNAs asparagine and cystine (Hixson et al., to the number of RS across different taxa (Brown et al., 1996; Xu 1986). During replication, the H strand is replicated first, and et al., 2005; Meadows et al., 2011; Cui et al., 2012). In mammalian the parental H strand remains single until it is paired with D-loop regions, the RS have been found in five positions (RS 1-5). newly synthesized L strand. Before pairing, the parental H RS1 and RS2 are found in the ETAS domain of the D-loop, where strand is exposed to oxidative damage because it is only partially the H strand replication pauses; RS3, RS4, and RS5 are found in protected by proteins. This process is one of the causes of large the CBS domain, upstream of the site for the H strand origin of number of mutations in mtDNA. In addition, nuclear proteins replication (Hoelzel et al., 1994). that are transported from cytosol into mitochondria are directly involved in mitochondrial function and can cause mutations Mitogenome Sequence Variations in mitochondrial genes (Patrushev et al., 2014). The rate of the mutations caused by those reasons is the highest in the D-loop In the coding sequence, the first and second positions of the region of mtDNA (Brown and Simpson, 1982; Reyes et al., 1998). codon are often stable, but the third position mutates fast (Kimura, Mutations accumulating in D-loop region are not lethal, and they 1968). The mtDNA does not have the same repair system as the aCS Agric. conspec. sci. Vol. 86 (2021) No. 1 Analyses of Wild Ungulates Mitogenome | 3 nuclear DNA, which means it is exposed to rapid molecular and investigated the relationship between the compositional evolution (Matosiuk et al., 2014). The changes in base composition features of all three positions in amino acids and third positions might lead to modifications in amino acid composition (Foster et of fourfold degenerate codons. They concluded that transcribed H al., 1997). The changes in genome sequence are divided into four strand is richer in G base compared to L strand (L strand mRNAs different types: sequence rearrangements, additions, deletions and are very poor in G, particularly in the third position), and that A substitutions (Brown, 1985). The most common type of mutations nucleotide is more represented on L strand, while C and T differ in mitogenome are nucleotide substitutions. The substitution among species. rate has been estimated to be about 5-10 times greater than in the nuclear DNA, although rates vary between different parts of Why Study Mitochondrial DNA? the mitogenome (Brown et al., 1979). The substitutions can be detected as synonymous (i.e. silent) or nonsynonymous (Jukes MtDNA studies are a valuable tool for understanding and Bhurshan, 1986).