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鱼类 环境DNA metabarcoding片段的近缘物种识别差异

陈治 马春来 叶乐 杨超杰 王海山

陈治,马春来,叶乐,等. 鱼类 环境DNA metabarcoding片段的近缘物种识别差异[J]. 海洋学报,2022,44(9):1–15 doi: 10.12284/hyxb2022150
引用本文: 陈治,马春来,叶乐,等. 鱼类 环境DNA metabarcoding片段的近缘物种识别差异[J]. 海洋学报,2022,44(9):1–15 doi: 10.12284/hyxb2022150
Chen Zhi,Ma Chunlai,Ye Le, et al. Differences of eDNA metabarcoding fragments in relative fish species resolution[J]. Haiyang Xuebao,2022, 44(9):1–15 doi: 10.12284/hyxb2022150
Citation: Chen Zhi,Ma Chunlai,Ye Le, et al. Differences of eDNA metabarcoding fragments in relative fish species resolution[J]. Haiyang Xuebao,2022, 44(9):1–15 doi: 10.12284/hyxb2022150

鱼类 环境DNA metabarcoding片段的近缘物种识别差异

doi: 10.12284/hyxb2022150
基金项目: 国家自然科学基金(32002389);海南省自然科学基金(422RC717);海南热带海洋学院引进人才科研启动资助项目(RHDRC201907)。
详细信息
    作者简介:

    陈治(1990-),男,讲师,博士,主要从事海洋鱼类分类学及其资源保护研究。E-mail: change@139.com

    通讯作者:

    王海山(1983-),男,讲师,博士,主要从事海洋生物多样性及其系统地理学研究。E-mail: jazz-123@163.com

  • 中图分类号: S931

Differences of eDNA metabarcoding fragments in relative fish species resolution

  • 摘要: 已知的鱼类环境DNA(eDNA)metabarcoding片段均未被针对性考察其对近缘物种的适用性,实际使用过程中存在“物种丢失”风险。为筛选出物种识别率最高的片段,本研究比较了15个主流片段对106属(共935种)鱼类的识别差异。研究结果如下:(1)蛋白质编码基因(COI,片段15)的物种识别率最高,但其引物通用性最差;片段09、片段11、片段07、片段03、片段12的引物序列总平均遗传距离也较大,均存在eDNA低效扩增的风险;(2)片段长度影响物种识别率,核糖体基因中片段05、片段06、片段01、片段02及片段13的物种识别率较高;(3)非度量多维尺度分析(NMDS)显示,不同基因、同一基因不同片段的识别结果存在较大差异,应考虑多片段、多基因组合应用;片段01与片段02、片段05与片段06等在NMDS图上距离较近,存在相互替代性;(4)物种类群影响识别结果,eDNA研究仍需要进一步开发高识别率片段。综合物种识别率、引物通用性、NMDS分析等多方面因素,本研究推荐2×150 bp测序平台使用片段01(MiFish-U)、2×250 bp测序平台使用片段05(Ac12S),辅以片段13(Vert-16S-eDNA)等进行近缘鱼类多样性调查。本研究旨在为提高鱼类eDNA调查结果准确性提供一定技术支撑。
  • 图  1  本研究中15个鱼类eDNA metabarcoding片段和对应引物在目标基因上的位置

    以花鳗鲡(Anguilla marmorata)线粒体基因序列(GenBank登录号:NC006540)为标注模板

    Fig.  1  Locations of the 15 fish eDNA metabarcoding fragments and primer pairs on the target mitochondrial genes

    Sequence of the marbled eel (Anguilla marmorata) (GenBank accession number: NC006540) was used as template

    图  2  本研究中15个鱼类eDNA metabarcoding片段的物种识别率及对应引物序列总平均遗传距离

    Fig.  2  Fish species resolution rates of 15 environmental DNA metabarcoding fragments and overall mean distances of primer pairs in this study

    图  3  本研究中106属鱼类的物种识别率

    Fig.  3  Fish species resolution rates of 106 genera in this study

    图  4  不同metabarcoding片段的NMDS分析

    F01表示片段01,其余依次类推

    Fig.  4  Analysis of non-metric multidimensional scaling for different metabarcoding fragments

    F01 represents fragment 01, and the rest are followed by analogy

    表  1  本研究中15个鱼类eDNA metabarcoding片段简介

    Tab.  1  Summary of 15 fish eDNA metabarcoding fragments analyzed in present study

    metabarcoding片段对应引物名称目标基因长度/bp引物序列(5'-3'设计者
    片段01MiFish-U12S169For-GTCGGTAAAACTCGTGCCAGCMiya等[4]
    Rev-CATAGTGGGGTATCTAATCCCAGTTTG
    片段02Tele0212S165For-AAACTCGTGCCAGCCACCTaberlet等[16]
    Rev-GGGTATCTAATCCCAGTTTG
    片段03Am12S12S242For-AGCCACCGCGGTTATACGEvans等[17]
    Rev-CAAGTCCTTTGGGTTTTAAGC
    片段0412S-V512S107For-ACTGGGATTAGATACCCCRiaz等[18]
    Rev-TAGAACAGGCTCCTCTAG
    片段05Ac12S12S392For-ACTGGGATTAGATACCCCACTATGEvans等[17]
    Rev-GAGAGTGACGGGCGGTGT
    片段06AcMDB0712S282For-GCCTATATACCGCCGTCGBylemans等[19]
    Rev-GTACACTTACCATGTTACGACTT
    片段07NeoFish_312S196For-CGCCGTCGCAAGCTTACCCTMilan等[20]
    Rev-AGTGACGGGCGGTGTGTGC
    片段08Tele0112S64For-ACACCGCCCGTCACTCTValentini等[8]
    Rev-CTTCCGGTACACTTACCATG
    片段09Ac16S16S339For-CCTTTTGCATCATGATTTAGCEvans等[[17]
    Rev-CAGGTGGCTGCTTTTAGGC
    片段10L2513/H271416S206For-GCCTGTTTACCAAAAACATCACKitano等[21]
    Rev-CTCCATAGGGTCTTCTCGTCTT
    片段11Fish16S16S84For-CGAGAAGACCCTWTGGAGCTTIAGShaw等[22]
    Rev-GGTCGCCCCAACCRAAG
    片段12Ve16S16S335For-CGAGAAGACCCTATGGAGCTTAEvans等[17]
    Rev-AATCGTTGAACAAACGAACC
    片段13Vert-16S-eDNA16S284For-AGACGAGAAGACCCYDTGGAGCTTVences等[5]
    Rev-GATCCAACATCGAGGTCGTAA
    片段14Fish16S F/D-2R16S222For-GACCCTATGGAGCTTTAGACDiBattista等[23]
    Rev-CGCTGTTATCCCTADRGTAACT
    片段15PS1COI199For-ACCTGCCTGCCGTATTTGGYGCYTGRGCCGGRATAGTBalasingham等[6]
    Rev-ACGCCACCGAGCCARAARCTYATRTTRTTYATTCG
    注:片段长度以花鳗鲡(Anguilla marmorata)(GenBank登录号:NC006540)为参考。
    下载: 导出CSV

    表  2  metabarcoding片段对各类群的识别率

    Tab.  2  The species resolution rates of metabarcoding fragments for different groups

    物种类群物种数识别率/%
    片段 01片段 02片段 03片段 04片段 05片段 06片段 07片段 08片段 09片段 10片段 11片段12片段 13片段 14片段15全部片段
    鱊属 Acheilognathus1471.4371.4385.7171.4385.7185.7171.4357.1471.4385.7157.1485.7185.7171.4310077.14±11.83
    鲟属 Acipenser1513.332026.6713.3346.6733.33406.6733.33033.336.6713.336.6773.3324.44± 19.46
    光唇鱼属 Acrossocheilus1656.255062.55087.57562.56.2568.7543.755062.55043.7510057.92± 21.45
    黑头鱼属 Alepocephalus757.1414.2928.57028.5728.5728.57028.5714.2914.2928.5742.8628.5710029.52± 24.43
    西鲱属 Alosa65000033.3333.3333.330066.6716.6716.6716.6716.6710025.56± 28.78
    双锯鱼属 Amphiprion1070506020605030106060308060504048.67± 19.22
    鳗鲡属 Anguilla1729.4135.2964.71076.4770.5976.4741.1864.7147.0635.2976.4764.7152.9488.2454.9± 23.5
    细鲫属 Aphyocypris560606020604040010080601008010010064± 30.43
    旗鳉属 Aphyosemion810010010010010010010012.510010010010010010010094.17± 22.59
    深海鳐属 Bathyraja812.5255012.52512.512.5012.512.50252537.55020.83±15.43
    鲫属 Carassius5000060202006040040006020±25.07
    真鲨属 Carcharhinus1464.2971.4310078.571001001005085.7135.7114.295064.2971.4310072.38±26.57
    刺尻鱼属 Centropyge16757587.531.2562.556.2562.512.587.537.50505062.510056.67±27.8
    九棘鲈属 Cephalopholis5100100100401006060040406010010010010073.33±32.66
    鳢属 Channa862.562.562.562.562.562.562.5257575757575755064.17±13.25
    红鳍鲌属 Chanodichthys520202020606000100600100606010045.33±36.62
    燕鳐属 Cheilopogon837.537.5250255050062.52512.512.52512.57530±22.06
    鲮属 Cirrhinus56060604010010010006060206040406057.33±28.15
    胡子鲇属 Clarias666.6716.6733.3333.3383.3333.3333.33066.6733.33066.6766.6733.3310044.44±29.32
    花鳅属 Cobitis1181.8281.8281.8281.8272.7381.8281.829.0963.6481.8263.6481.8281.8281.8210075.15±20.17
    鲚属 Coilia616.67505016.6766.6710066.6705066.6733.3333.3316.6716.6710045.56±30.52
    白鲑属 Coregonus147.147.140021.4335.7121.437.140000007.147.14±10.8
    兵鲶属 Corydoras633.3333.3333.3316.67505066.6716.67505016.6700066.6732.22±23.12
    杜父鱼属 Cottus1250751005083.335041.678.3383.3383.3341.6783.3366.6710010067.78±26.7
    舌鳎属 Cynoglossus1210010010083.3310010010083.3310083.3383.3383.3383.3383.3310092.22±8.61
    长吻鳐属 Dipturus8012.537.512.55037.537.505012.5025252562.525.83±19.75
    盘属 Discogobio5404060406040600204004060406040±20
    塘鳢属 Eleotris666.6766.67100100100100100010010066.6766.6766.6766.6710080±27.6
    Enteromius5100100100100100100100401001001001008010010094.67±15.98
    石斑鱼属 Epinephelus2290.9190.9190.915090.9190.9190.9136.3672.7390.9122.7381.8268.1863.6490.9174.85±22.52
    扁鳉属 Epiplatys771.4371.4371.4371.4371.4371.4371.43071.4371.4371.4371.4371.4371.4371.4366.67±18.44
    镖鲈属 Etheostoma1010010010010010010010040100709010010010010093.33±16.76
    底鳄鳉属 Fundulopanchax5100100100100100100100201001006010010010010092±22.42
    底鳉属 Fundulus560606020100100100010010010010010010010080±32.95
    南乳鱼属 Galaxias5100100100601001001000100606010010010010085.33±28.75
    墨头鱼属 Garra101001001005010010010030801005010010010010087.33±23.74
    骨尾鱼属 Gila540206020604040060602060606010046.67±24.69
    露齿鲨属 Glyphis82512.525037.537.537.50252525252537.52524.17±12.01
    纹胸属 Glyptothorax1118.1818.1863.6418.1836.3627.2736.369.0954.559.0927.2754.5554.5563.6481.8238.18±22.59
    颌须属 Gnathopogon633.3366.6710010010066.6766.6701005016.6733.3333.3333.3366.6757.78±32.65
    鳅鮀属 Gobiobotia61005010050100100100066.6766.6716.6783.3383.3366.6710072.22±31.91
    裸鲤属 Gymnocypris837.537.537.5012.512.50012.537.512.512.512.502516.67±14.69
    海猪鱼属 Halichoeres51001001001001001001002010010010010010010010094.67±20.66
    鳠属 Hemibagrus56060608060406006060206060606053.33±19.52
    䱻属 Hemibarbus742.8642.8642.8628.5742.8628.5714.29042.8628.5714.2942.8642.8642.8610037.14±22.13
    海马属 Hippocampus1888.8988.8988.8955.5666.6766.6761.1127.7810077.7855.5610010010010078.52±22.11
    刺蝶鱼属 Holacanthus54040604060606004020204040406041.33±17.67
    下鱵属 Hyporhamphus51001001001001001001004010010010010010010010096±15.49
    野鲮属 Labeo2070708035908075106030255540508056.67±24.1
    兔头鲀属 Lagocephalus81001001007510010010025100757562.57562.510083.33±21.99
    太阳鱼属 Lepomis610010010050100100100010010066.6710010010010087.78±28.5
    薄鳅属 Leptobotia65016.6766.675066.675033.33033.3316.675033.3333.3333.335038.89±18.55
    䱀属 Liobagrus966.6777.7877.7811.1177.7855.5666.67033.3333.3333.3310010010077.7860.74±31.95
    笛鲷属 Lutjanus1384.6284.6210010010010084.6223.0810069.2330.7710010010010085.13±25.42
    狼绵鳚属 Lycodes5606060406060600100402060606010056±25.3
    虹银汉鱼属 Melanotaenia610010050066.6733.3333.33066.6705033.3310066.6710053.33±36.84
    小鳔属 Microphysogobio1464.2964.2985.7164.2971.435035.717.1485.7135.7135.7157.1471.4357.1410059.05±23.76
    泥鳅属 Misgurnus510010010020606040060600601006010061.33±35.02
    假鳃鳉属 Nothobranchius810010010010010010010012.510010010010010010010094.17±22.59
    美洲鱥属 Notropis1010080100601001008010100607010010010010084±25.58
    沙塘鳢属 Odontobutis610010010010010010066.6733.3310010010010010010010093.33±18.69
    太平洋鲑属 Oncorhynchus837.562.510037.51007575037.50050505010051.67±34.68
    白甲鱼属 Onychostoma1181.8263.6445.4518.1863.6463.6454.5527.2754.5572.73081.8254.5545.4563.6452.73±22.85
    马口鱼属 Opsariichthys510012010040606020060604010010010010070.67±35.35
    罗非鱼属 Oreochromis966.6766.6766.6733.3344.4411.1144.44055.5622.2222.2244.4433.3322.2277.7840.74±22.88
    青鳉属 Oryzias10808080808080808010010010010010010010089.33±10.33
    纹唇鱼属 Osteochilus5801001008010010010001001008010010010010089.33±26.04
    鲳属 Pampus728.5757.1428.5714.2928.5728.5728.57028.5728.5714.2914.2914.2914.2942.8624.76±13.73
    属 Pareuchiloglanis56060602060100600601006060606010061.33±26.69
    鱥属 Phoxinus710010010057.1410071.4371.43071.4371.4314.2910010010010077.14±32.31
    花鳉属 Poecilia520202020602040202020202020206026.67±14.47
    多鳍鱼属 Polypterus1010010010010010010010040100806010010010010092±18.21
    原唇齿脂鲤属 Prochilodus520202002004000004040402017.33±16.68
    拟鲿属 Pseudobagrus1241.6758.337533.3358.3358.3358.3316.6766.678.338.3333.33252583.3343.33±24.03
    拟腹吸鳅属 Pseudogastromyzon933.3355.5655.56055.5655.5655.56077.7855.5644.4410077.7855.5677.7853.34±26.96
    多刺鱼属 Pungitius771.4310010028.5710057.1457.14028.5728.57071.4371.4342.8610057.14±34.99
    鳐属 Raja616.6716.6733.3316.6766.6716.6716.6705066.6716.6766.6766.6766.6766.6738.89±25.72
    波鱼属 Rasbora710010010071.4310071.4310014.2971.4310057.1471.4371.4371.4310080±24.03
    吻鰕虎鱼属 Rhinogobius5100100100100100100100010010010010010010010093.33±25.82
    鳑鲏属 Rhodeus10808080308080602010080501001008010074.67±24.75
    红点鲑属 Salvelinus128.338.33258.33252525016.678.3308.3308.3358.3315±15.17
    鳈属 Sarcocheilichthys955.5644.4455.5611.1155.5655.5633.33055.5633.33044.4433.3344.4455.5638.52±20.08
    沙丁鱼属 Sardinella610010010066.671005066.6733.335066.6766.6710010066.6710077.78±23.29
    蛇属 Saurogobio710010010071.4310071.4371.43071.4371.4314.2910010010010078.1±31.88
    南鳅属 Schistura1110010010010010010010054.5510010063.6410010010010094.55±14.5
    裸裂尻鱼属 Schizopygopsis714.2914.2914.29014.2928.5714.29028.570042.8642.8642.8671.4321.91±20.82
    裂腹鱼属 Schizothorax2611.5411.5415.387.6923.0823.0811.543.8515.387.69011.543.8511.5430.7712.56±8.15
    马鲛鱼属 Scomberomorus666.6766.6766.6733.3366.6766.6766.6733.3310066.6710066.6766.6766.6710068.89±19.79
    平鲉属 Sebastes1931.5831.5831.5810.5336.8447.3726.32010.5315.79026.3215.7915.7968.4224.56±18.09
    瓢鳍虾虎鱼属 Sicyopterus1833.3333.3344.4422.225066.675033.3383.3333.3333.3366.6755.5666.6788.8950.74±20.13
    篮子鱼属 Siganus633.3333.3333.3316.6710066.6766.67066.6716.67016.670033.3332.22±30.52
    鱚属 Sillago61001001001001001001005010010066.6710010010010094.44±15
    鲶属 Silurus666.6766.6766.675010010066.6716.6766.67505066.6766.6710010068.89±23.46
    鳜属 Siniperca757.1442.8610028.5742.8628.5728.57042.8628.5714.2914.2914.2914.2928.5732.38±23.82
    金线鲃属 Sinocyclocheilus1485.7185.7185.7135.7178.5778.5764.2928.575057.1421.4364.2957.1478.5785.7163.81±21.84
    银属 Squalidus742.8628.5757.1442.8610057.1457.1414.2957.1428.5714.2942.8628.5728.5710046.67±26.16
    扁鲨属 Squatina560606020606060020404060606010050.67±23.74
    疯鲿属 Tachysurus87575752510010010007537.507550507560.83±33.03
    东方鲀属 Takifugu1606.256.256.2537.52512.512.512.518.756.2531.252531.2562.519.58±16.34
    田中鳑鲏属 Tanakia610010010010010010010016.6710010033.3310010010010090±26.58
    鲀属 Tetraodon51001001006060606001001000100606010070.67±34.53
    棱鳀属 Thryssa610010010066.67100100100010010010010010010010091.11±26.63
    金枪鱼属 Thunnus9011.1111.110000000000001.48±3.91
    茴鱼属 Thymallus10202040030502003020102030106024±16.82
    结鱼属 Tor771.4371.43100071.4342.8657.14028.5757.1428.5771.4357.1428.5710052.38±30.86
    高原鳅属 Triplophysa2147.6247.6280.9542.8671.4371.4361.94.7666.6752.3819.0547.6247.6247.6280.9552.7±21.1
    全部物种93562.3±31.8461.95±32.5468.9±30.8142.92±33.4671.62±27.364.34±29.1360.89±29.4811.79±17.7964.6±30.6554.58±32.2836.71±32.2764.2±31.861.6±32.3559.71±32.7882.76±24.66-
    下载: 导出CSV
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  • 收稿日期:  2022-02-17
  • 修回日期:  2022-03-25
  • 网络出版日期:  2022-04-24

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