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Molecular Identi Cation of Commercial Fish Maws Using Mtdna 16S Rrna Nucleotide Sequences

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Molecular Identi Cation of Commercial Fish Maws Using Mtdna 16S Rrna Nucleotide Sequences Molecular Identication of Commercial Fish Maws using mtDNA 16S rRNA Nucleotide Sequences Jinju Zhang Jinan University Qiuting Deng Jinan University Nanxin Li Jinan University Xindan Liu Jinan University Menghua Wu Jinan University Zhiguo Ma Jinan University Ying Zhang ( [email protected] ) Jinan University Hui Cao Jinan University Research Keywords: Fish maw, Swim bladder, mtDNA, 16S rRNA gene, DNA sequencing, Origin identication Posted Date: May 29th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-31129/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/19 Abstract Background: Fish maws (dried swim bladders of sh) have long been used as a medicinal material and valuable tonic food which are very popular in Southeast Asia. It is dicult to identify the original species of sh maws sold on the market due to a lack of taxonomic characteristics. The present study aims to investigate the origin of commercial sh maws using DNA sequencing based on a partial sequence of the mtDNA 16S rRNA gene. Methods: Mitochondrial genomic DNA was extracted from a total of 44 individual sh maw samples collected from different markets by a QIAamp® DNA Mini Kit. The fragments of the 16S rRNA gene were amplied by PCR and bidirectionally sequenced using an ABI 3730 genetic analyser. Sequence assembly was performed with DNASTAR SeqMan software, and the resulting sequence was compared with reference sequences in GenBank using BLAST. Interspecic divergence and intraspecic variation were calculated by the Kimura 2-parameter model. Sample sequences were clustered in MEGA 6 using a neighbour-joining tree. Results: Forty-two of 44 sh maw samples were sequenced successfully. Fourteen taxa matched known sh species in the DNA sequence database, and 10 of them were supported by high homogeneity (99- 100%). According to the results, 88% of the total samples belonged to family Sciaenidae. The clustering and genetic distance results, including in-group divergence and out-group divergence, indicated confidence in species identifications. Moreover, there was no strict correspondence between the commercial names provided by traders and the origin of the sh maws. This study also found a probable correlation between the molecular characteristics and morphological features of sh maws from croakers and non-croakers. Conclusion: In this study, DNA sequencing based on mtDNA 16S rRNA was carried out successfully to identify the origin of ~95% of the samples. This result indicated that 16S rRNA is a suitable barcode for identifying sh maws. The identication results allowed us to learn more about the sh species available in the sh maw market in Guangdong-Hongkong-Macao Great Bay. In addition, the association between trade category and species found in this study provides both consumers and merchants with an important reference for identifying the origin of sh maws. Background Fish maws have long been used as a luxury seafood and medicinal tonics in Southeast Asia, particularly in southern China [1]. Currently, sh maws from dried swim bladders have many pharmacological activities, such as enhancing immunity, learning and memory, promoting postpartum recovery and haemostasis, removing free radicals, anti-oxidant, anti-constipation, anti-fatigue, and aiding in cutaneous wound healing [2–6]. According to Chinese Crude Drugs Standards in Shandong, Hunan and Beijing, sh maws from family Sciaenidae (such as Larimichthys crocea, Pseudosciaena polyactis and Acipenser sinensis) and Page 2/19 Acipenseridae (such as Husodaurius georgi and Huso dauricus) are used as medicinal materials [7–9]. Other sh maws can be used only as foodstuffs. Family Sciaenidae (croakers) is a major taxon available in the medical sh maw market since most of the sh from family Acipenseridae are endangered and rarely observed. However, sh maws from non-croaker species such as eel, perch, catsh and carp are also sold on the market to meet the increasing demand [10]. Although sh maws have become an important food and traditional Chinese medicine, little information on the species to which they belong is available due to inecient and inaccurate species identication. In most cases, not only the sh species but also the market name and price of sh maws is determined by traders based on ambiguous morphological characteristics. Identication depends on a trader’s individual experience because there is no systematic approach with which to distinguish sh maws based on their morphological features [10]. In addition, for the purpose of long-term preservation, fresh swim bladders are placed in the sun and gradually turned to dry when making sh maws. This processing may destroy the morphological characteristics of sh maws. Before exposure to sunlight, some thick and large swim bladders are cut longitudinally to facilitate drying, which leads to the further removal of taxonomic features [11–12]. Therefore, it is dicult to accurately trace the sh species to which a maw belongs based on its shape, texture, size, thickness or other morphological characteristic [13]. This means that the same sh species may be sold under different names and at various prices due to the confusing relationships between species, names and prices [14]. Consumers also face a risk of paying very different prices for products of the same quality. DNA barcoding based on the mitochondrial 16S rRNA gene is a good alternative method to identify the species of sh products when morphological characteristic identication is dicult to obtain [15–16]. Given its high nucleotide divergence, the 16S rRNA gene has been used to identify and describe the phylogenetic relationships of sh species in family Sciaenidae [17–18]. The aim of this study is to identify the species of the main commercial sh maws using a fragment of the 16S rRNA gene and to explore the possible relationship between species and morphological characteristics. This work helps us better understand the sh species available in sh maw markets and provides an important reference for producers, merchants and consumers in sh maw species identication. Methods Chemicals, reagents and materials A QIAamp® DNA Mini Kit for DNA extraction was purchased from QIAGEN Co., Ltd. (Hilden, Germany). PrimeSTAR® HS DNA Polymerase was obtained from Takara Biomedical Technology Co., Ltd. (Osaka, Japan). Other reagents, including GoldView nucleic acid stains, 6 × loading buffer and DL2000 DNA marker, were purchased from TsingKe Biotech Co., Ltd. (Guangzhou, China). Forty-four commercial sh maw samples were purchased from some medicinal material markets (Guangdong, China). The trade name and production area of the sh maws were indicated by traders Page 3/19 (Table 1). Voucher specimens were deposited at Lingnan Branch of National Engineering Research Center for Modernization of Traditional Chinese Medicine. Page 4/19 Table 1 Sample information and the results of species identication Sample Trade name (In Production Species matched Percentage GenBank code Chinese) area identity (%) accession no. S1 Zhizhu jiao Malaysia Bahaba 95% JX232404.1 taipingensis S2 Zhizhu jiao Vietnam Boesemania 99% MH699839.1 microlepis S3 Zhizhu jiao Malaysia Bahaba 95% JX232404.1 taipingensis S4 Douhu jiao England Leptomelanosoma 99% MF593470.1 indicum S5 Douhu jiao Thailand Leptomelanosoma 99% MF593470.1 indicum S6 Douhu jiao Pakistan Leptomelanosoma 100% MF593470.1 indicum S7 Douhu jiao Australia / a / a / a S8 Hulu jiao Vietnam Sciades parkeri 100% KM508072.1 S9 Guanggong jiao India Protonibea 99% KY117237.1 diacanthus S10 Guanggong jiao / b Protonibea 100% KY117237.1 diacanthus S11 Guanggong jiao Guangxi, Cynoscion acoupa 99% KM508065.1 China S12 Baihua jiao Guangdong, Chrysochir aureus 99% JQ692068.1 China S13 Baihua jiao Malaysia Dendrophysa 94% KT630300.1 russelii S14 Manyu jiao South Genypterus 100% GU946612.1 Africa capensis S15 Manyu jiao Guangdong, Muraenesox 97% KX426277.1 China cinereus S16 Shiban jiao India Ompok pabda 96% GQ469559.1 Production area was claimed by the trader a Failure in species identication b No production area information was provided by the trader Page 5/19 Sample Trade name (In Production Species matched Percentage GenBank code Chinese) area identity (%) accession no. S17 Beihai jiao Guangxi, Cynoscion acoupa 100% KM508065.1 China S18 Beihai jiao Guangxi, Cynoscion acoupa 100% KM508065.1 China S19 Beihai jiao Guangxi, Cynoscion acoupa 99% KM508065.1 China S20 Chizui jiao Guangdong, Protonibea 99% KY117237.1 China diacanthus S21 Chizui jiao Guangdong, Protonibea 99% KY117237.1 China diacanthus S22 Chizui jiao Guangdong, Protonibea 99% KY117237.1 China diacanthus S23 Chizui jiao Guangdong, Protonibea 99% KY117237.1 China diacanthus S24 Chizui jiao Thailand Protonibea 99% KJ643927.1 diacanthus S25 Chizui jiao Guangdong, Protonibea 99% KY117237.1 China diacanthus S26 Chizui jiao South Protonibea 100% KY117237.1 China Sea diacanthus S27 Chizui jiao South Protonibea 100% KY117237.1 China Sea diacanthus S28 Jixin jiao Guangdong, Diodon liturosus 99% MG544189.1 China S29 Xiaohua jiao Zhejiang, Lates niloticus 100% KY213963.1 China S30 Huanghua jiao Guangdong, Lates niloticus 100% KY213963.1 China S31 Huanghua jiao Australia Lates niloticus 99% KY213963.1 S32 Huanghua jiao Guangxi, Lates niloticus 99% KY213963.1 China Production area was claimed by the trader a Failure in species identication b No production area information was provided by the trader Page 6/19 Sample Trade name (In Production Species matched Percentage GenBank code Chinese) area identity
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