The complete mitochondrial genome of Gobio huanghensis (Cypriniformes: Cyprinidae) in the Yellow River, China

Abstract Gobio huanghensis, a member of the eponymous genus within the Cyprinidae, family of the Cypriniformes order, is an endemic fish species found exclusively in the upper reaches of the Yellow River, spanning from Yinchuan to Lanzhou. This study presents the first comprehensive report of the complete mitochondrial genome sequence of G. huanghensis, encompassing 16,604 base pairs (bp) with a nucleotide composition of 26.3% cytosine (C), 17.6% guanine (G), 29.4% adenine (A), and 26.7% thymine (T). In congruence with other species in the Gobio genus, its mitochondrial genome comprises 37 genes, including two ribosomal RNA genes, 13 protein-coding genes (PCGs), and 22 transfer RNA genes. Notably, COX1 initiates with the start codon GTG, distinct from the typical ATG start codon of other PCGs. The mitogenome exhibits four types of stop codons: TAA, TAG, TA-, and T--. Phylogenetic analyses, grounded in complete mitochondrial sequences, position G. huanghensis at the forefront of one of two major clusters within the genus Gobio, corroborating existing morphological classifications. These findings offer valuable theoretical insights for the taxonomic classification, conservation, and population genetics of G. huanghensis.


Introduction
Gobio huanghensis (Lo, Yao and Chen, 1977), a species within the genus Gobio, and subfamily Gobioninae (Teleostei: Cypriniformes: Cyprinidae), is native exclusively to the upper reaches of the Yellow River (Huang He) in China (Wang 1991).This species inhabits the river stretch between Yinchuan and Lanzhou, primarily feeding on benthic organisms.G. huanghensis typically spawns in May and June in areas characterized by gravelly bottoms and gentle currents (Wang 1991).However, due to the adverse impacts of human activities, the population of G. huanghensis has witnessed a significant decline (Qi and Yang 2009), leading to its inclusion in the China Species Red List (Wang and Xie 2009).Previous studies on G. huanghensis primarily centered around resource utilization, geographical distribution, and various biological and ecological aspects (Wu and Wu 1991;Chen 1998).While Qi and Yang (2009) provided CYTB sequences, comprehensive molecular and genetic data for the species remain sparse.In this study, we report the sequencing of the complete mitochondrial genome of G. huanghensis for the first time.This effort aims to furnish essential data for advanced genetic studies, biodiversity monitoring, and the conservation of this species.

Materials
A specimen of G. huanghensis (Figure 1) was collected from Jingyuan County, Baiyin City, Gansu Province, China, at coordinates 104 � 37 0 48.63 00 E, 36 � 34 0 6.14 00 N, utilizing a cage net.Morphological identification was conducted in accordance with methodologies established by Wang (1991) and Chen (1998).G. huanghensis can be differentiated from other species in the Gobio genus based on several distinct characteristics: (1) the anus is situated midway between the base of the pelvic fin and the onset of the anal fin.(2) The species pos- sess small eyes, with the head length exceeding 6.5 times the diameter of the eye.(3) The barbels are notably thick and elongated, extending rearward beyond the posterior margin of the anterior opercular bone.The collected specimen has been preserved and cataloged in the specimen room of the Gansu Fisheries Research Institute, Lanzhou, China, under the voucher number GSSCS-2021-0058 (Tai Wang, aqhongqi@qq.com).

Methods
Genomic DNA was isolated from muscle tissue using the Magnetic Animal Tissue Genomic DNA Kit (Xu et al. 2018), provided by Tiangen Biochemical Technology (Beijing) Co., Ltd.(Beijing, China).The concentration and purity of the nucleic acids were assessed using a NanoDROP 8000 ultramicro spectrophotometer.Primers were designed referencing the genomes of Rhinogobio cylindricus (KU379652) and R. ventralis (KU379653), and synthesized through OPC purification, with details available in Supplementary Material S1.The mitochondrial genome was amplified via polymerase chain reaction (PCR) using a 30 lL Taq PCR Master Mix.This reaction included genomic DNA at 0.67 ng/lL, primers at 0.33 nM, dNTPs at 0.2 mM, MgCl 2 at 1.5 mM, and Taq polymerase at 0.05 U/lL.The PCR protocol involved initial denaturation at 95 � C for 5 min, followed by 35 cycles of denaturation at 95 � C for 30 s, annealing at 60 � C for 30 s, and extension at 72 � C for 1 min, concluding with a final elongation at 72 � C for 5 min.Agarose gel electrophoresis of the 16 primer pairs is detailed in Supplementary Material S2.The amplified PCR products were sequenced using the ABI 3730xl DNA Analyzer from Applied Biosystems, Inc. (Foster City, CA).The segmented sequences obtained are listed in Supplementary Material S3.Sequence assembly was conducted using SeqMan (DNASTAR Inc., Madison, WI), with manual corrections applied for sequencing errors (Meyer 1994).Overlap sizes and positions are included in Supplementary Material S4.Annotation of the mitochondrial genome and a map of the mitogenome were performed using the MITOS WebServer (Bernt et al. 2013).Using Blast, we identified eight complete mitochondrial sequences from the Gobio genus in the NCBI database, each with an identity exceeding 91.8% (Table 1).Gnathopogon herzensteini and G. imberbis, members Table 1.Names, GenBank accession numbers, and references of 10 fish mitogenomes used in phylogeny reconstruction. of the subfamily Gobioninae, were selected as outgroups.

Species Accession number Reference
Mitogenome sequences of 10 species were aligned using ClustalW in MEGA 11 (Kumar et al. 2018).Phylogenetic analysis was carried out using MEGA11 to construct a maximumlikelihood (ML) phylogenetic tree with 1000 bootstrap replicates.

Results
The complete mitochondrial genome sequence of G. huanghensis spans 16,604 base pairs (bp) and has been submitted to the GenBank database under the accession number OM302528.This mitochondrial genome comprises 13 proteincoding genes (PCGs: ND1-ND6 and ND4L, COX1-COX3, CYTB, ATP6, and ATP8), two ribosomal RNA genes (16S rRNA and 12S rRNA), 22 transfer RNA genes (tRNAs), and a control region (Figure 2).The nucleotide composition is characterized by 29.4% adenine (A), 26.7% thymine (T), 26.3% cytosine (C), and 17.6% guanine (G).In the case of G. huanghensis, all PCGs initiate with the ATG start codon, with the exception of COX1, which starts with GTG.There are four types of stop codons employed: TAA serves as the stop codon for COX1, ATP8, ND4L, ND5, and ND6; TAG for ND1; T-for ND2, COX2, COX3, and CYTB; and TA-for ATP6, ND3, and ND4.As shown in Figure 3, the mitochondrial genome of Gobio species forms two distinct sister clades.Genes encoded on the L-strand and H-strand are shown outside and inside the circle, respectively.The arrows represent direction of transcription.The inner circle represents the GC content.

Discussion and conclusions
This study successfully presents the first complete assembly and annotation of the mitochondrial genome for G. huanghensis.The gene composition and arrangement in the mitochondrial genome of G. huanghensis are found to be consistent with those observed in other Gobio species' mitogenomes (Ge et al. 2020;Yi and Fu 2020).Phylogenetic analysis using ML, based on the complete mitochondrial genomes of G. huanghensis and seven additional Gobio species, reveals that G. huanghensis forms a sister-group relationship with G. cynocephalus, G. rivuloides, and G. coriparoides.The mitochondrial genomic data acquired from G. huanghensis in this study will be instrumental in providing a genetic foundation for DNA barcoding, population genetics studies, and the conservation efforts of this endemic species.

Figure 3 .
Figure 3. ML phylogenetic tree reconstructed using the mitochondrial genomes of eight Gobio fish species.The tree topology was evaluated with 1000 bootstrap replicates.Bootstrap values at the nodes correspond to the support values for the ML methods.A scale of 0.02 indicates genetic variation in the mitochondrial genome, equating to two differences per 100 nucleotides.