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Complete Mitochondrial Genome of Cultellus Attenuatus and Its Phylogenetic Implications

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Complete Mitochondrial Genome of Cultellus Attenuatus and Its Phylogenetic Implications Complete mitochondrial genome of Cultellus attenuatus and its phylogenetic implications Haikun Li ( [email protected] ) Ocean University of China Fisheries College Ruihai Yu Ocean University of China Fisheries College Peizhen Ma Institute of Oceanology Chinese Academy of Sciences Chunhua Li Ocean University of China Fisheries College Short Report Keywords: Cultellus attenuates, M itochondrial gene, Phylogeny, Heterodonta Posted Date: September 3rd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-868770/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Complete mitochondrial genome of Cultellus attenuatus and its phylogenetic implications 1 1 2 1 1 Haikun Li , Ruihai Yu , Peizhen Ma , Chunhua Li 1 2 Key Laboratory of Mariculture, Ministry of Education, Ocean University 3 of China, 5 Yushan Road, Qingdao 266003, China 2 4 Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, 5 Qingdao 266071, China 6 Corresponding author 7 Ruihai Yu, Key Laboratory of Mariculture, Ministry of Education, Ocean 8 University of China, Qingdao 266003, China. 9 Email: [email protected] 10 Acknowledgments 11 This work was supported by the grants from National Natural Science 12 Foundation of China (31172403), Science and Technology Innovation 13 Commission Project of Shenzhen, China (CXY201106270024A). 1 14 Abstract 15 The complete mitochondrial genome of Cultellus attenuates, a new 16 aquaculture species, was sequenced and compared with mitogenomes 17 from seven species of Heterodonta bivalve mollusk in the gene bank. The 18 mitochondrial genome of C. attenuatus is 16888bp in length and contains 19 36 genes, including 12 protein-coding genes, 2 ribosomal RNAs and 22 20 transfer RNAs, and all genes are encoded on the same strand. In 21 comparison with C. attenuates, the mitochondrial genes of the 22 Sinonovacula constricta from the same family were not rearranged, but 23 those of six other species from different family were rearranged to 24 different degrees. The largest non-coding region of C. attenuatus is 25 1173bp in length and with the A + T content of 68.24%, located between 26 nad2 and trnK. The results of phylogenetic analysis show that the C. 27 attenuates and the S. constricta belonging to Cultellidae cluster into one 28 branch while two species of Solenidae (Solen grandis and Solen strictus) 29 are clustering as their sister taxon. These data not only contribute to the 30 understanding of the phylogenetic relationship of the Heterodonta, but 31 also serve as a resource for the development of the genetic markers in 32 aquaculture. 33 Keywords Cultellus attenuates · Mitochondrial gene · Phylogeny · 34 Heterodonta 2 35 Introduction 36 Most animals’ mitochondrial DNA is a closed circular molecule 37 independent of the nuclear DNA, ranging in size from 14 to 17 kb. It 38 contains 37 genes including 13 proteins, 2 ribosomal RNAs, and 22 [1] 39 transfer RNAs .The mitochondrial genes are quite conservative in 40 replication, featuring matrilineal inheritance, no rearrangement and high [2] 41 substitution rates . Many characteristics make mitochondrial genes in 42 value and have has become a powerful source in genetic evolution, [3-5] 43 phylogeographic analysis, and species identification . 44 In bivalve mollusk, Mytilus edulis was the first to obtain complete [6] 45 mitochondrial genetic information , and subsequently mitochondrial [7] [8] 46 genes were obtained in other categories, including oysters , scallops , [9] [10] 47 clam and razor shells . Although mitochondrial genetic resources of 48 bivalve are increasingly being developed, it is only limited to some 49 species with high economic value. For niche classes, the molecular [11] 50 information is scarce . In the species of Cultellidae family, only the 51 mitochondrial genes of Sinonovacula constricta have been determined 52 while those of the other species including Cultellus attenuatus have been 53 not, which has been very unfavorable for the phylogenetic research in this [12,13] 54 family . 55 C. attenuatus mainly inhabits the shallow sea area of the subtidal zones, 56 and can be found in Japan, South Korea, the Philippines and in China 3 [14] 57 (mainly from Liaoning in the north to Taiwan in the south) . C. 58 attenuatus has high edible value due to its nutritional composition of low 59 fat and high protein, which makes it more and more popular among [15] 60 Chinese consumers . In this way, the high market value of C. 61 attenuatus has makes its artificial catching gradually increase while, the [14] 62 quantity of wild resources ones decrease . In china, artificial breeding 63 of C. attenuatus has been carried out in an attempt to restore the 64 diminishing wild resources by introducing hatchery-produced seed. The 65 study of mitochondrial gene can be applied to produce genetic markers 66 that can monitor the restoration of aquatic animal stock, which is quite [3,16-17] 67 helpful for the restoration and conservation of wild population . 68 Therefore, it is necessary to obtain the mitochondrial gene sequence of C. 69 attenuates. However, no relevant reports have been reported so far. In this 70 study, we obtained and analyzed the entire mitochondrial gene of C. 71 attenuates, and compared the mitochondrial gene with other family 72 species from the Heterodonta. The data not only contribute to our 73 understanding of the phylogenetic status, but also serve as a source for 74 the development of useful genetic markers in aquaculture. 75 Materials and methods 76 Sample collection and DNA extraction 4 77 The lived C. attenuatus were collected from Bohai Bay (Dongying City, 78 Shandong Province, China). The total genomic DNA was extracted from 79 adductor muscle of an individual C. attenuatus by a DNA extraction kit. 80 PCR and DNA sequencing 81 The PCR amplification of cox1 and rrnL gene was performed with the [13] 82 primer set of LCO1490/HCO2198 and rrnLAR-L/rrnLAR-H , 83 respectively. PCR was performed in 50 μl volumes containing 0.5 units 84 Taq polymerase (Takara), 1×PCR buffer, 0.2mM of each dNTP, 1 μM of 85 each primer, 1.5 mM of MgCl2 and50 ng of genomic DNA. The PCR 86 cycle was as follows: 94◦ C for 2 min, then 35 cycles of 94ºC for 30s, 40 s 87 at 52ºC and 1 min at 72ºC, followed by a final extension at 72ºC for 5 88 min. 89 Based on the above two sequences, two sets of Long-PCR primers LB-L/ [13] 90 LB-H and LC-L/LC-L were used for long-PCR amplification . 91 Long-PCR was performed in 25 μL volumes containing 1×GC buffer I, 92 1.25 U of LATaq (Takara), 0.5 mM of each dNTP, 0.4μM of each primer 93 and 50 ng of genomic DNA. The PCR conditions were as follows: 94ºC 94 for 2 min, 30 cycles of 94ºC for 20s, 1 min/kb at 68ºC and a final 95 extension at 72ºC for 10 min. The PCR products were sequenced by 96 Personal Biotechnology Co. Ltd. Qingdao. 97 Analysis of sequence data 5 98 The protein-coding and ribosomal RNA genes were identified by their 99 similarity to published gene sequences through BLAST searches 100 (http://www.ncbi.nlm.nih.gov/BLAST/). The transfer RNA were [18] [19] 101 identified by tRNAscan-SE v.1.21 and DOGMA using invertebrate 102 mitochondrial genetic code. CGView was used to map the whole [20] 103 mitochondrial genome circle . 104 Phylogenetic analysis 105 To clearly show the phylogenetic relationship of the Heterodonta, the 106 mitochondrial gene sequences of 18 species from the Heterodonta were 107 obtained from the gene bank. The Chlamys farreri and Mimachlamys 108 nobilis were used as distinct outgroups. The amino acid sequences of 12 109 proteins (except atp8) were adopted for phylogenetic analysis. The amino 110 acid sequences of 12 mitochondrial proteins were aligned by MEAG 7.0. [21] 111 . The phylogenetic relationships of heterodont bivalves were 112 reconstructed by the Neighbor-Joining (NJ)，and 1000 bootstraps were 113 used to estimate. 114 Results and discussion 115 Genome organization and nucleotide composition 116 The mitochondrial genome of C. attenuates is 16,888 bp in length and 117 contains 12 protein-coding genes, 22 transfer RNA genes, and 2 118 ribosomal RNA genes (Fig. 1). Compared with the genome size of the 6 119 sequenced mollusk mtDNAs, the mitochondrial genome size is within the 120 normal range. Besides, four overlaps were detected in mitochondrial 121 genome, with the size of 1bp, 6bp, 1bp and 3bp respectively, among 122 which the overlap between trnE and trnS2 was the largest (Table 1). Of 123 the whole mitogenome of C. attenuates, its A+T content is 66.46% (Table 124 2), comparable to that of S. constricta (67.00%) and Solen grandis 125 (64.84%). In addition, the mitochondrial gene length of C. attenuates is [22] 126 376bp shorter than that of S. constricta in the same family , while [10] 127 104bp longer than that of S. grandis in the different family . 128 Protein-coding genes and codon usage 129 The mitochondria of C. attenuates contain 12 protein-coding genes, 130 which, range in size from 291 bp to 1761 bp and, with a total length of 131 11470bp, accounting for 67.92% of the total length of the genes (Table 1). 132 There are two starting codons in these 12 protein-coding genes, ATG for 133 7 protein-coding genes (cox2, cox3, cob, atp6, nad2, nad3 and nad6), 134 ATT for 3 protein-coding genes (cox1, nad4 and nad5) and ATA for 2 135 protein-coding genes (nad41 and nad1), which differs from the initial 136 codon composition of the protein-coding genes in mtDNA of S. grandis.
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