Communication Complete Mitogenome of “Pumpo” (Bos taurus), a Top Bull from a Peruvian Genetic Nucleus, and Its Phylogenetic Analysis Richard Estrada 1 , Deyanira Figueroa 1 , Yolanda Romero 1 , Wuesley Yusmein Alvarez-García 1 , Diorman Rojas 1, Wigoberto Alvarado 2, Jorge L. Maicelo 2, Carlos Quilcate 1 and Carlos I. Arbizu 3,* 1 Dirección de Desarrollo Tecnológico Agrario, Instituto Nacional de Innovación Agraria (INIA), Lima 15024, Peru; richard.estrada.bioinfo@gmail.com (R.E.); deyanirafigueroa66@gmail.com (D.F.); yolanda.bioinfo@gmail.com (Y.R.); walvarezg@unc.edu.pe (W.Y.A.-G.); diormanr@gmail.com (D.R.); promegnacional@inia.gob.pe (C.Q.) 2 Facultad de Ingeniería Zootecnista, Agronegocios y Biotecnología, Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas (UNTRM), Cl. Higos Urco 342, Chachapoyas 01001, Peru; wigoberto.alvarado@untrm.edu.pe (W.A.); jmaicelo@untrm.edu.pe (J.L.M.) 3 Facultad de Ingeniería y Ciencias Agrarias, Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas (UNTRM), Cl. Higos Urco 342, Amazonas 01001, Peru * Correspondence: carlos.arbizu@untrm.edu.pe Abstract: The mitochondrial genome of Pumpo (Bos taurus), a prominent breed contributing to livestock farming, was sequenced using the Illumina HiSeq 2500 platform. Assembly and annotation of the mitochondrial genome were achieved through a multifaceted approach employing bioinfor- matics tools such as Trim Galore, SPAdes, and Geseq, followed by meticulous manual inspection. Additionally, analyses covering tRNA secondary structure and codon usage bias were conducted for comprehensive characterization. The 16,341 base pair mitochondrial genome comprises 13 protein- coding genes, 22 tRNA genes, and 2 rRNA genes. Phylogenetic analysis places Pumpo within a clade predominantly composed of European cattle, reflecting its prevalence in Europe. This comprehen- sive study underscores the importance of mitochondrial genome analysis in understanding cattle Citation: Estrada, R.; Figueroa, D.; evolution and highlights the potential of genetic improvement programs in livestock farming, thus Romero, Y.; Alvarez-García, W.Y.; contributing to enhanced livestock practices. Rojas, D.; Alvarado, W.; Maicelo, J.L.; Quilcate, C.; Arbizu, C.I. Complete Keywords: cattle; NGS; phylogenomics; INIA Mitogenome of “Pumpo” (Bos taurus), a Top Bull from a Peruvian Genetic Nucleus, and Its Phylogenetic Analysis. Curr. Issues Mol. Biol. 2024, 1. Introduction 46, 5352–5363. https://doi.org/ 10.3390/cimb46060320 Cattle were domesticated about 12,000 years ago due to their capacity to provide food, transportation, leather, and manure as fertilizer, among others [1,2]. According to FAO [3], Academic Editor: Tomasz Popławski the human population is projected to reach 10 billion by 2050, demanding more efficient Received: 22 April 2024 cattle production. The last census reported that there are 5,156,044 heads of cattle in Peru [4], Revised: 22 May 2024 which is 14.1% higher than what was reported in the 1994 agricultural census. Most of Accepted: 26 May 2024 these cattle populations are Creole cattle (64.03%); however, due to the low productivity of Published: 28 May 2024 these animals, a breeding improvement strategy to take advantage of the heterosis effect is to crossbreed these Creole cattle with other specialized breeds such as Simmental, Brown Swiss, Gyr, and others [5]. This is why in the 1970s, animals of the Simmental breed arrived in Peru, thanks to an agreement with the German Technical Cooperation, whose objective Copyright: © 2024 by the authors. was the use of fresh semen and frozen semen from imported bulls [6]. Licensee MDPI, Basel, Switzerland. The Simmental cattle, also known as Fleckvieh, stand out in southern Germany for This article is an open access article their ability to reach live weights of up to 850 kg due to their late maturation and intensive distributed under the terms and fattening [7]. This breed not only provides high yields of milk and meat but is also conditions of the Creative Commons distinguished by a notable accumulation of proteins, contributing to its popularity in Attribution (CC BY) license (https:// livestock farming due to its excellent fertility and profitability [8,9]. According to the 2022 creativecommons.org/licenses/by/ National Agricultural Survey [10], 238,125 cattle farmers are using high-quality purebred 4.0/). Curr. Issues Mol. Biol. 2024, 46, 5352–5363. https://doi.org/10.3390/cimb46060320 https://www.mdpi.com/journal/cimb Curr. Issues Mol. Biol. 2024, 46 5353 or improved breeders, or employing semen or embryos to reproduce or improve their livestock, including the Simmental breed. Although the exact Simmental cattle population is unknown, there is a growing demand among farmers. In Peru, the first Simmental bull, sent to the National Semen Bank in 2002, initiated the widespread use of artificial insemination for this breed. Offspring produced with semen from bulls of German, Austrian, Canadian, and Swiss origin are distributed nationwide [11]. In 2020, INIA-MINAGRI developed 10 genetic nuclei in various regions, producing 4000 embryos and 710,000 high-quality semen straws to enhance milk and meat production of breeds such as Simmental [12]. In the Amazonas region of Peru, the Simmental breed is crucial for livestock farming, providing sustenance to numerous families due to its hardiness and dual-purpose aptitude, adapting to diverse agroecological zones in provinces like Utcubamba and Bagua, underscoring its importance in the local economy [13]. The study of the mitochondrial genome is pivotal in unraveling genetic diversity and evolutionary history within cattle breeds [14]. Moreover, its application extends to the conservation of indigenous and rare breeds, as demonstrated by studies such as those con- cerning Zhangmu cattle [15]. Mitochondrial genome sequencing further aids in identifying beneficial genetic variants crucial for genetic improvement programs, as evidenced in the comprehensive assembly of the Simmental cattle genome [16]. Additionally, investigations into the origins and dispersal of Bos taurus, including the Simmental breed, benefit from mitochondrial genome analysis, providing valuable insights into phylogenetic structures and adaptive strategies [17]. The objective of this study was to analyze the complete mitogenome of “Pumpo”, a distinguished bull within the Peruvian genetic nucleus of the National Institute of Agrar- ian Innovation (INIA for its acronym in Spanish), and to perform a phylogenetic analysis. This analysis aims to deepen our understanding of the genetic composition and evolution- ary history of the Simmental breed, particularly its adaptation and performance in various agroecological zones, thereby contributing to the improvement of livestock farming practices. 2. Materials and Methods 2.1. Sampling The study subject was a Simmental–Fleckvieh breed bull (Peruvian National Register Number 135, born in 2016) from the Central Genetic Nucleus of the National Institute of Agrarian Innovation, located at the Donoso Agricultural Experiment Station (EEA Donoso in Spanish), which is a government herd where a cattle genetic nucleus is established, located in Huaral, Lima (128 masl; 11◦31′18′′ S and 77◦14′06′′ W). The bull was healthy and without known genetic diseases. Data registers show that from May 2021 to date, 30,382 semen straws have been collected from Pumpo. Blood sampling was performed at the EEA Donoso and was collected from the bull’s tail using a vacutainer containing EDTA as an anticoagulant and immediately transported to the laboratory for DNA extraction. This study was conducted by following the Peruvian National Law No. 30407: “Animal Protection and Welfare”. 2.2. DNA Extraction and Sequencing Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Fitch- burg, WI, USA), adhering to the protocols provided by the manufacturer. The integrity and concentration of the isolated genomic DNA were determined using agarose gel elec- trophoresis and a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA), respectively. Subsequently, an Illumina paired-end (2 × 150 bp) genomic library was prepared according to Illumina’s established procedures (Illumina, San Diego, CA, USA) and sequenced on an Illumina HiSeq 2500 system by GENEWIZ (South Plainfield, NJ, USA). The sequencing library was clustered on a flow cell, which was then placed in the Illumina sequencing instrument as per the manufacturer’s directions. The Illumina Control Software was employed for image analysis and base calling. The raw sequencing data (.bcl files) obtained from the sequencing process were converted into fastq format using the Curr. Issues Mol. Biol. 2024, 46 5354 Illumina bcl2fastq 2.17 software, with the protocol allowing for a single mismatch in the index sequence identification. 2.3. Assembly and Annotation of the Mitogenome Adapters and reads of inferior quality were eliminated using the default parameters in the TrimGalore v0.6.7 and Trimmomatic v0.36 software [18]. Utilizing the trimmed data, we assembled the mitochondrial genome via the GetOrganelle [19] pipeline, incorporating tools such as SPAdes v3.11.1 [20], bowtie2 v.2.4.2 [21], and BLAST+ v2.11 [22] in the pro- cess. Annotations for the protein-coding genes, transfer RNAs (tRNAs), and rRNA genes within the mitochondrial genome were generated using the automated mitochondrial gene annotators available online through Geseq in the CHLOROBOX web service [23]. This was followed by manual inspection. Analysis of the tRNA secondary structure was conducted using tRNAs-can-SE 2.0 [24]. The termination codon was excluded. Subse- quently, 13 protein-coding genes (PCGs) were merged using the Concatenate Sequence Alignment feature, and codon utilization was examined using the relative synonymous codon usage (RSCU) function within MEGA v11 [25]. A graphical representation of the circular mitochondrial genome was produced using OGDRAW v1.3.1 [26]. 2.4. Phylogenetic Analysis To ascertain the genetic affiliation of Pumpo, we analyzed 49 mitochondrial genomes from other Bos species cataloged in GenBank, complemented by a species from the genus Bison (Bison bison), a member of the same subfamily Bovinae, serving as an outgroup (Ta- ble S1). Alignment of each genome was conducted using the software MAFFT v7.475 [27], followed by the construction of the most accurate maximum likelihood (ML) tree based on a GTR + GAMMA evolutionary model. This step was succeeded by 1000 nonparamet- ric bootstrap analyses using RAxML v8.2.11 [28]. The inferred phylogenetic trees were visualized using iTOL [29]. 3. Results 3.1. Genome Size and Organization The complete mitochondrial genome of Pumpo spans 16,341 base pairs (bp). This genome comprises 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes (Figure 1). The heavy (H) strand harbored the majority of genes, totaling 27, while the light (L) strand housed 9 genes. The elemental composition of this genome was distributed as follows: 24.44% Adenine (A), 25.13% Thymine (T), 25.62% Cytosine (C), and 24.81% Guanine (G). The most extensive overlap region, spanning 48 bp, is located between the tRNALeu and Nd5 genes. Additionally, the widest intergenic spacer, covering 32 bp, lies between the tRNACys and tRNATyr genes (Table 1). The complete mitochondrial genome sequence has been deposited in the GenBank database under accession number PP780079. The corre- sponding BioProject, BioSample, and SRA identifiers are PRJNA1097623, SAMN40874274, and SRR28589481, respectively. The assembly coverage was 150×. 3.2. Protein Coding Genes (PCGs) and Codon Usage In the mitogenome of Pumpo, 13 PCGs spanning a total length of 12,309 bp were identified. This accounts for approximately 75.22% of the entire genome. Additionally, this mitochondrial genome is responsible for the synthesis of 4181 amino acids. These PCGs consist of seven NADH dehydrogenase subunits, two ATPase subunits, and a gene corresponding to cytochrome b. It is noteworthy that PCGs exhibit a bias towards AT base composition, ranging from 33.5% for the Nd4 gene to 95.39% for the Cox2 gene. Furthermore, the length of PCGs showed wide variability, ranging from 200 bp for Atp8 to 1772 bp for Nad5. In terms of the length of proteins encoded by these genes, it ranged from 66 to 590 amino acids (Table 2). The most frequent start and stop codons were ATG and TAA, respectively. In contrast, the Nd1, Nd2, Cox3, Nd3, and Nd4 genes exhibited incomplete stop codons, represented as TA- or T-. PCGs contained the following five codons with the Curr. Issues Mol. Biol. 2024, 46 5355 Curr. Issues Mol. Biol. 2024, 46, FORh PiEgEhRe RsEtVRIESWC U values: CUA (2.87), CGA (2.67), UCC (2.13), ACA (1.99), and GUA (14. 84) (Figure 2). Figure 1. The mitochondrial genome map of Pumpo, a top bull from the Peruvian genetic nucleus Fiogfu IrNeI1A.. The mitochondrial genome map of Pumpo, a top bull from the Peruvian genetic nucleus of INIA. Table 1. Gene organization of the mitochondrial genome of Pumpo cattle. Table 1. Gene organization of the mitochondrial genome of Pumpo cattle. Intergenic Spacer Gene Nucleotide Positions Size (bp) Strand * (bpIn) Gene Nucleotide Positions Size (bp) Strand * tergenic tRNAPhe 365–432 68 H S p acer (bp) 12S rRNA tR4N3A3P–h1e388 365–439256 68 H H tRNAVal 121S3r8R9N–1A455 433–138687 956 H H 16S rRNA tR1N45A6V–a3l 026 1389–11455751 67 H H tRNALeu2 163S0r2R7N–3A101 1456–307256 1571 H H Nd1 3104–4059 956 H 2 tRNAIIe 4060–4128 69 H Curr. Issues Mol. Biol. 2024, 46 5356 Table 1. Cont. Gene Nucleotide Positions Size (bp) Strand * IntergenicSpacer (bp) tRNALeu2 3027–3101 75 H Nd1 3104–4059 956 H 2 tRNAIIe 4060–4128 69 H tRNAGin 4126–4197 72 L −1 tRNAMet 4200–4268 69 H 2 Nd2 4269–5310 1042 H tRNATrp 5311–5377 67 H tRNAAla 5379–5447 69 L tRNAAsn 5449–5521 73 L 1 tRNACys 5554–5620 67 L 2 tRNATyr 5621–5688 68 L Cox1 5690–7234 1545 H 1 tRNASer2 7332–7302 31 L −1 tRNAAsp 7307–7378 72 H 4 Cox2 7377–8060 684 H −1 tRNALys 8064–8130 67 H 3 Atp8 8132–8332 201 H 1 Atp6 8293–8973 681 H 60 Cox3 8973–9756 784 H −1 tRNAGly 9757–9825 69 H Nd3 9826–10,171 346 H tRNAArg 10,173–10,241 69 H −1 Nd4L 10,242–10,538 297 H Nd4 10,532–11,909 1378 H −5 tRNAHis 11,910–11,979 70 H tRNASer 11,980–12,039 60 H tRNALeu 12,041–12,111 71 H 1 Nd5 12,112–13,932 1821 H Nd6 13,916–14,443 528 L −17 tRNAGlu 14,444–14,512 69 L CytB 14,514–15,656 1143 H 1 tRNAThr 15,661–15,729 69 H 4 tRNAPro 15,729–15,794 66 L −1 D-loop 1–364, 15,795–16,341 364, 547 H * Strand: H (Heavy), L (Light). Table 2. Features of protein-coding genes detected in the mitochondrial genome of Pumpo cattle. Gene Gene Length A + T Content Start/Stop Protein Length(bp) (%) Codon (aa) Nd1 955 85.79 ATG/TA- 319 Nd2 1041 86.2 ATG/TA- 348 Cox1 1544 80.3 ATG/TAA 514 Cox2 750 95.39 ATG/TAA 227 Atp8 200 57 ATG/TAA 66 Atp6 679 50 ATG/TAA 227 Cox3 783 57.25 ATG/TA- 262 Nd3 346 57.54 ATA/TA- 116 Nd4L 296 52.56 ATG/TAA 98 Nd4 1377 33.5 ATG/T- 460 Nd5 1772 77.32 ATA/TAA 590 Nd6 527 59.02 ATG/TAA 175 CytB 1139 54.86 ATG/AGA 379 Total 12,309 4181 Curr. Issues Mol. BCioul.rr2.0 I2s4su, 4es6 Mol. Biol. 2024, 46, FOR PEER REVIEW 6 5357 Figure 2. The rFeliagtuivre 2sy. nTohney rmeloautisvec osdyonnonuysmagoeus(R cSoCdUon) oufsathge m(RiStoCcUho) nodf rtihael gmeintocmheo’nsdprrioalt eginen- omes protein- coding genes ofcPoudminpgo g,eanteosp obf uPlul mfropmo, tah teoPpe bruvlli afrnomge ntheeti cPenruucvleiauns goefnINetIiAc n. ucleus of INIA. 3.3. Ribosomal TRaNbAle, 2T.r FaenastfuerreRs NofA p,raontedinN-coond-iCnogd ginengeRs edgeitoencsted in the mitochondrial genome of Pumpo cattle. A total of 22 transfer RNA (tRNA) genes were identified, with total lengths ranging Gene from 63 bGpefnoer tLReNngAtPhh e(btop)7 4 bpAf +o rTt RCNonAtLeenut2 ((%Ta)b leS1t)a. rTt/hSetoHp sCtroadnodnh arborePdro1t4eitnR NLeAngth (aa) Nd1 genes, while the 9L5s5t rand encoded 8 tR8N5A.79g enes. All theseAtTRGN/ATAg-e nes exhibited the ch3a1r9- Nd2 acteristic cloverl1e0a4f1s econdary structur8e6, .w2 ith two exceptAioTnGs:/TthAe-t RNALys and tRNAS3e4r81 Cox1 genes (Figure 3)1. 5T4h4e total length of the80t.w3 o ribosomal RANTAG(/rTRANAA ) genes (12S and 1561S4) Cox2 amounted to 252575b0p . These genes were95d.3el9i mited by tRNAPThGe /aTnAdAtR NALeu2 (Table 1). T2h2e7 Atp8 control region (D2-0l0o op), with a total len5g7th of 911 bp, waAs TdGel/iTmAitAed by the tRNAPro an66d Atp6 tRNAPhe genes (T6a7b9l e 1). 50 ATG/TAA 227 Cox3 783 57.25 ATG/TA- 262 Nd3 3.4. Phylogenetic A34n6a lysis 57.54 ATA/TA- 116 Nd4L An exhausti2v9e6p hylogenetic analys5i2s.5o6f the mitochondArTiaGl g/TenAoAm es of Bos species ava9i8l - Nd4 able in GenBank13w7a7s performed using3t3h.5e maximum likeAlihToGo/dT-( ML) inference meth4o6d0- Nd5 ology, obtaining1h7i7g2h bootstrap suppo7r7t.v32a lues. Three mAaiTnAc/lTadAeAs were identified in 5th9e0 phylogenetic tree. The species B. taurus was located within a monophyletic clade that is Nd6 divided into sub5c2la7d e 1 and subclade 529, .w02h ile B. primigenAiuTsGa/nTdABA. indicus form separ1a7te5 CytB clades. Members11o3f9B . gaurus, B. frontal5is4,.8a6n d B. javanicusAwTeGre/AgGroAup ed in clade 2. On 3th7e9 Total other hand, B. g1r2u,n3n0i9e ns and B. mutus con stituted clade 3, which is closely related to cla4d1e8s1 1 and 2. Pumpo was placed in subclade 1, along with other cattle specimens from France, Germany, Spai3n.,3I. tRaliyb,oUsormuaglu RaNy,AM, oTnragnoslfiear, RMNaAlta, ,aanndd NConh-iCnao.diOnng Rtheegiootnhse r hand, subclade 2 was mainly compAo steodtaol focfa 2tt2l etrfarnomsfeIrt aRlNy,APo (rttRuNgaAl), Mgeenxeisc ow, eErgey ipdte,nPtaifiraegdu, awyi,tahn tdotPael rluengths ranging (Figure 4). from 63 bp for tRNAPhe to 74 bp for tRNALeu2 (Table 1). The H strand harbored 14 tRNA genes, while the L strand encoded 8 tRNA genes. All these tRNA genes exhibited the char- acteristic cloverleaf secondary structure, with two exceptions: the tRNALys and tRNASer1 genes (Figure 3). The total length of the two ribosomal RNA (rRNA) genes (12S and 16S) amounted to 2525 bp. These genes were delimited by tRNAPhe and tRNALeu2 (Table 1). The control region (D-loop), with a total length of 911 bp, was delimited by the tRNAPro and tRNAPhe genes (Table 1). Curr. Issues Mol. Biol. 2024, 46, FOR PEER REVIEW 7 Curr. Issues Mol. Biol. 2024, 46 5358 Figure 3.. TThhe epprerdedicitcetde dsesceocnodnadrayr ystrsutrcutuctruerse os fo 2f22 t2ratnrasnfesrf eRrNRAN Agengesn efsrofmro mthet hmeitmogiteongoemnoem oef Pouf mPupmo.p o. Curr. Issues Mol. Biol. 2024, 46, FOR PEER REVIEW 8 3.4. Phylogenetic Analysis An exhaustive phylogenetic analysis of the mitochondrial genomes of Bos species available in GenBank was performed using the maximum likelihood (ML) inference meth- odology, obtaining high bootstrap support values. Three main clades were identified in the phylogenetic tree. The species B. taurus was located within a monophyletic clade that is divided into subclade 1 and subclade 2, while B. primigenius and B. indicus form separate clades. Members of B. gaurus, B. frontalis, and B. javanicus were grouped in clade 2. On the other hand, B. grunniens and B. mutus constituted clade 3, which is closely related to clades 1 and 2. Pumpo was placed in subclade 1, along with other cattle specimens from France, Germany, Spain, Italy, Uruguay, Mongolia, Malta, and China. On the other hand, sub- Curr. Issues Mol. Biol. 2024, 46 clade 2 was mainly composed of cattle from Italy, Portugal, Mexico, Egypt, Paraguay,5 a35n9d Peru (Figure 4). FFigiguurere4 4. .T Thheep phhyylologgeenneetitcict rtreeee, ,c coonnsstrturuccteteddu ussininggm maaxximimuumml ilkikeelilhihoooodda annddb baaseseddo onnm mitiotocchhoonnddriraial l ggeennoommicics eseqquueenncceessf rforommB Bososs pspeceiceise,s,d disipsplalayyssb booootsttsrtarapps usuppppoortrtv vaalulueesse xexclculusisviveelylyf oforrb brarnanchcheses rerceecievivininggo ovverer7 700%%s usuppppoortr.t.B Bisiosnonb ibsiosnonw waassd deesisgignnaatetedda asst htheeo ouutgtgrorouuppi nint hthisisa nanalaylysissi.s. 4. Discussion Sequencing and characterization of the mitochondrial genome of Pumpo were per- formed, revealing a length of 16,341 bp. This dimension aligns with sizes observed in other mitogenomes in the Bovinae subfamily, such as Bos taurus (16,339 bp), B. indicus (16,339 bp), B. frontalis (16,340 bp), B. grunniens (16,324 bp), and B. gaurus (16,345 bp) [30–34]. These findings confirm the similarity in size among mitogenomes in this subfamily. Additionally, the gene order and structure were found to be consistent with those previously reported for B. taurus mitogenomes [30,35,36]. The analysis of protein-coding genes in B. taurus revealed a pattern of codon usage reflecting the typical bias observed in various organisms such as different Bos species [30,35], stemming from a combination of evolutionary and biological factors [37]. The convergence Curr. Issues Mol. Biol. 2024, 46 5360 of these codon usage patterns suggests the influence of lineage-specific factors, including translational selection, tRNA availability, and protein structure, as highlighted in previous studies [38,39]. Understanding how these patterns affect protein structure and function can shed light on the biology and evolution of domestic cattle, as well as have practical applications in genetic improvement and agricultural biotechnology [40]. To comprehend the evolutionary connection of Pumpo, a phylogenetic tree was con- structed using maximum likelihood inference methodology alongside other cattle breeds and Bos species. The mitochondrial genome phylogeny exhibited similarities with those of other Bos genera [30,31,33]. The phylogenetic tree displayed three taxonomic clusters, with the first clade experiencing a bifurcation resulting in the formation of two distinct subclades. Pumpo is grouped within subclade 1, where European cattle predominate, given that this Simmental breed is particularly prevalent in Europe [41]. This breed has been extensively researched; a comprehensive analysis of genetic variability revealed complex phylogenetic patterns, reflecting diverse genetic and selective influences over time [42]. Furthermore, the study of identifying candidate genes associated with key productive and reproductive traits underscores the significance of human selection in the genetic evolution of this breed [43]. Subdivision of the B. taurus and B. indicus species was also observed [44,45]. Pumpo was part of a genetic group that has been the subject of a population genetics study using SNP data. This analysis revealed a significant correlation with specimens of the Simmental breed [46]. Mitogenomes are crucial in evolutionary studies and forensic applications, presenting significant advantages and limitations. The precise assembly of mitogenomes is challenging due to errors and missing sequences from short-read sequencing, although long-read strategies improve accuracy [47]. Additionally, certain mitogenes are more prone to transfer between mitochondria and the nucleus, causing losses or parallel transfers in different lineages [48]. Despite limitations, mitogenomes provide valuable information about the evolutionary history and genetic diversity of Bos cattle, including evidence of genetic contributions from ancient Chinese cattle to southern Chinese taurine cattle [49]. To enhance research, it is crucial to explore emerging technologies such as long-read sequencing and integration with nuclear genomic data, which will contribute to a better understanding of evolutionary dynamics and conservation of cattle species, including Pumpo. 5. Conclusions The sequencing and analysis of the mitochondrial genome of Pumpo confirmed its similarity in size and structure to other mitogenomes within the Bovinae subfamily, partic- ularly B. taurus. The examination of protein-coding genes in B. taurus revealed codon usage patterns typical of various organisms, shedding light on evolutionary and biological influ- ences. Furthermore, phylogenetic analysis placed Pumpo within a clade predominantly occupied by European cattle breeds, indicating its evolutionary relationship with these lineages. Despite challenges in mitogenome assembly and gene transfer, mitogenomes remain invaluable for understanding the evolutionary history and genetic diversity of Bos cattle. Continued exploration of advanced sequencing technologies and integration with nuclear genomic data will further enhance our understanding of Pumpo and other cattle species’ evolutionary dynamics and conservation needs. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cimb46060320/s1, Table S1. Species, breed, origin, accession code, and clade assignment of the 45 individuals examined in this study. Author Contributions: Conceptualization, C.I.A. and W.A.; methodology, D.R., R.E., D.F., Y.R. and W.A.; software, R.E.; validation, W.Y.A.-G., Y.R. and D.R.; formal analysis, R.E., D.F. and Y.R.; investigation, R.E., D.F., Y.R., C.I.A. and W.A.; resources, C.Q. and J.L.M.; data curation, R.E., Y.R. and D.F.; writing—original draft preparation, R.E., D.F., Y.R., C.I.A. and C.Q.; writing—review and editing, R.E., D.F., Y.R., W.A. and C.I.A.; visualization, W.Y.A.-G. and C.Q.; supervision, W.Y.A.-G. Curr. Issues Mol. Biol. 2024, 46 5361 and C.I.A.; project administration, C.Q., J.L.M. and C.I.A.; funding acquisition, C.I.A., W.Y.A.-G. and J.L.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the following research project: “Mejoramiento de la disponi- bilidad de material genético de ganado bovino con alto valor a nivel nacional 7 departamentos” of the Ministry of Agrarian Development and Irrigation (MIDAGRI) of the Peruvian Government, with grant number CUI 2432072. Institutional Review Board Statement: The study was conducted according to Peruvian National Law No. 30407: “Animal Protection and Welfare”. Data Availability Statement: The associated Bioproject, Biosample, and Sequence Read Archive (SRA) numbers are PRJNA1097623, SAMN40874274, and SRR28589481, respectively. Acknowledgments: We acknowledge the ‘PROMEG NACIONAL’ team for supporting the logistic activities. C.I.A. thanks Vicecerrectorado de Investigación of UNTRM. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Pitt, D.; Sevane, N.; Nicolazzi, E.L.; MacHugh, D.E.; Park, S.D.E.; Colli, L.; Martinez, R.; Bruford, M.W.; Orozco-terWengel, P. 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