Complete mitochondrial genome of golden variant of freshwater fish Labeo rajasthanicus (Cypriniformes: Cyprinidae): endemic to India

Abstract The complete mitochondrial genome of the freshwater fish species Labeo rajasthanicus was obtained, using Illumina NovaSeq 6000 with 2 × 150 bp paired-end sequencing. The mitogenome of L. rajasthanicus is 16,738 bp in length (GenBank accession no.: OQ834146), comprised of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region, i.e. D-loop. The arrangement of genes was found to be identical to other Cypriniformes fish mitogenome, available in the NCBI database. The taxonomic status of L. rajasthanicus as a valid species was debated by some researchers and it was considered a synonym of L. boggut. However, phylogenetic analysis in the present study supports the species validity of L. rajasthanicus, as it showed a distinct node well separated from L. boggut and supported by a high bootstrap value. Furtherly, the pairwise genetic divergence among studied species showed the divergence between L. rajasthanicus and L. boggut as 1.6% whereas the minimum divergence was found to be 0.13% with L. dussumieri followed by L. fimbriatus (0.58%) and L. gonius (0.63%). The complete mitogenome of L. rajasthanicus will also be useful as a baseline reference genome for the reconstruction and annotation of the mitogenome of other Labeo species.


Introduction
Labeo rajasthanicus (Datta and Majumdar 1970) belongs to the order Cypriniformes, family Cyprinidae, and subfamily Labeoninae, and is a medium-sized carp endemic to the Indian state of Rajasthan.It was first described from Jaisamand Lake of Rajasthan based on a single specimen in 1970.The species validity of L. rajasthanicus was debated as Jayaram and Dhas (2000) considered this species a synonym of Labeo boggut due to its phenotypic similarity.After more than four decades of confusion about its validity as a species, L. rajasthanicus was confirmed as a valid species in the year 2015 with the designation of a neotype (Lal et al. 2015).This species was evaluated as an important minor carp with great potential for inclusion in composite fish culture (Bansal et al. 2014).Due to its restricted distribution, this species has conservation value, and its golden variant holds great ornamental value.A farm-type golden variant of L. rajasthanicus (Pratap Sunahari) was also developed through captive breeding, which was recommended to be promoted as an ornamental fish, especially for garden pools (Saini et al. 2022).However, its inclusion in the culture system, conservation in native habitats, and ornamental trade require a thorough evaluation of its genetic distinctiveness and diversity.Mitochondrial gene sequences are widely used to determine species diversity and evolutionary divergence among species.Therefore, the present study was conducted to generate the complete mitogenome sequence of L. rajasthanicus to decipher its structural characteristics and establish the phylogenetic relationship with closely related species.

Materials and methods
The live specimen of the golden variant of L. rajasthanicus was obtained from the culture pond of the College of Fisheries, Maharana Pratap University of Agriculture & Technology, Udaipur, Rajasthan, India (24.5788N 73.7067E) in the month of November 2022 (Figure 1).The fish specimen was identified as per the taxonomic keys provided by Talwar and Jhingran (1991), Jayaram (2010), andLal et al. (2015).The present study was approved by the Institutional Level Animal Ethics Committee of the College of Fisheries (Bihar Animal Sciences University), Kishanganj, India.The voucher specimen, along with tissue and DNA samples, is deposited in the repository of the Fisheries Resource Management Department of the College of Fisheries (Bihar Animal Sciences University), Kishanganj, Bihar, India, with accession number COF/1331/LR_01 (contact person: Rupam Samanta, rupamsamanta328@gmail.com).The total mitochondrial DNA was isolated from muscle tissue using the Alexgen DNA isolation kit as per the manufacturer's instructions.The complete mitochondrial DNA sequencing was performed using Illumina's high-throughput NovaSeq6000 with 2 � 150 bp paired-end chemistry.Details of the sequence depth and coverage map of the mitochondrial genome sequence generated in the present study are provided as supplementary material.A total of 11,913,402 reads generated (SRA accession no.SRR24210320) were de novo assembled into scaffolds using SPADes 3.15.4.A total of 1325 contigs were obtained, and the mitogenome was annotated with the help of MitoAnnotator (Zhu et al. 2023).MitoAnnotator allowed annotation of proteins, non-coding RNAs (tRNAs and rRNAs), and d-loop using blastx and blastn against known annotated mitochondrial proteins and rRNA genes, respectively.The maximum-likelihood (ML) phylogenetic tree was constructed based on the JTT matrix-based model (Jones et al. 1992) with the help of MEGA ver.11.0.13(Tamura et al. 2021), using a mitochondrial protein sequence dataset of all the 13 mitochondrial protein-coding genes (PCGs) of 21 closely related species and one outgroup species Tor putitora downloaded from the NCBI database (accession number AP011326).The initial tree(s) for the heuristic search were obtained automatically by applying the Neighbor-Joining and BioNJ algorithms  to an estimated pair-wise distance matrix using the JTT model and then selecting the topology with a superior log likelihood value (-16516.24).Pairwise genetic divergence was also estimated using the Poisson correction model (Zuckerkandl and Pauling 1965).In this analysis, all ambiguous positions were removed for each sequence pair and a total of 3794 positions were used in the final dataset.

Results
The complete mitogenome of L. rajasthanicus is 16,738 bp in length (GenBank accession no.OQ834146, Bio Project accession no.PRJNA957111), comprising 13 PCGs, 22 tRNA genes, two rRNA genes, and a 1069 bp long control region, or Dloop (Figure 2).The majority of genes were found on the H strand, except ND6, tRNA Glu , tRNA Pro , tRNA Gln , tRNA Ala , tRNA Asn , tRNA Cys , tRNA Tyr , and tRNA Ser , which were encoded on the L strand.ATP6, ND2, and ND5 were found most divergent among all the PCGs.The GC% of the mitogenome of the studied species was found to be 42.76.D-loop was found highly A þ T rich with 62.96% AT content.Two simple sequence repeats (SSRs) were also identified in the studied mitogenome.The ML phylogenetic tree (Figure 3) showed a distinct node of L. rajasthanicus supported by a high bootstrap value (99) and L. rajasthanicus had the closest relationship with L. dussumieri followed by L. fimbriatus and L. gonius but a distant relation with L. boggut.The pairwise evolutionary divergence was found to be minimum with L. dussumieri (0.13%) and maximum with L. lineatus (2.59%) whereas divergence with phenotypically closer L. boggut was estimated as 1.60%.

Figure 1 .
Figure 1.Image of voucher specimen of Labeo rajasthanicus with specimen repository accession number COF/1331/LR_01 (source of image: original image taken by author Mamta Singh).

Figure 2 .
Figure 2. Map of Labeo rajasthanicus mitochondrial genome of 16,738 bp representing 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a 1069 bp long control region or D-loop.