| 摘要: |
| 已知的鱼类环境DNA (environmental DNA, eDNA)宏条形码通用引物主要位于线粒体核糖体基因区，这些12S和16S引物存在部分近缘鱼类无法识别及参考序列不足等问题。本研究以海南岛淡水鱼类为调查对象，基于8目26科101属150种鱼类COⅠ序列，筛选出6个侧翼保守区；综合碱基变异、物种鉴定、eDNA降解及高通量测序读长需求，在4个侧翼保守区设计了26条引物，其中6条引物未达到Premier评分要求；72种海南淡水鱼类的首轮PCR结果显示，有11条引物的通用性较高，其中，PCR成功种数≥70且条带亮度均大于marker的引物有5条；次轮PCR结果显示，5条引物相互搭配产生的3 (正向) × 2 (反向)套引物组合的扩增成功率均为100% (72种/72种)，经PCR条带长度、亮度筛选后表现最优的引物组合为“HN-A-F4、HN-D-R3”(以下简称HN-COⅠ)。30个水样高通量测序结果显示，HN-COⅠ产生的待分析序列总数、鱼类序列总数、OTUs总数和鱼类OTUs总数分别为MiFish-U的0.77倍(8 919 976/11 532 126)、1.22倍(2 264 965/1 863 905)、0.85倍(406/477)和1.32倍(86/65)；HN-COⅠ产生的鱼类OTUs注释到种、属、科及科以上水平的占比分别为81.40%、11.63%和6.98%，MiFish-U则分别为81.54%、4.62%和13.85%。“引物+水样”的NMDS聚类图形成边界明显的2组(胁强系数=0.15)；HN-COⅠ的属内物种扩增子两两遗传距离最小值及平均值均分别是MiFish-U的1.23倍(0.006 9/0.005 6)和1.57倍(0.155 9/0.099 4)。室内6个密度组鲤鱼(Cyprinus carpio carpio)“生物量–拷贝数”线性回归方程的相关系数较低，HN-COⅠ及MiFish-U均无法准确反映鲤鱼的生物量。本研究筛选并比较了HN-COⅠ与MiFish-U的优劣，表明HN-COⅠ对海南岛淡水鱼类具有更高的靶向性，不仅在防止微生物、哺乳类等非目标生物eDNA污染方面有优势，而且更有利于海南岛淡水鱼类的检出和准确鉴定。 |
| 关键词: 海南岛 淡水鱼类 eDNA COⅠ 通用引物 |
| DOI：10.19663/j.issn2095-9869.20221107001 |
| 分类号: |
| 基金项目: |
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| Screening universal COⅠ primers for eDNA metabarcoding of freshwater fishes on Hainan Island |
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CHEN Zhi1,2, CAI Xingwei1, SHEN Zhixin3, ZHANG Qingfeng4, LI Fangyuan5, GU Yuan6, LI Gaojun7, ZHAO Guangjun8, WANG Zhenjiang9
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1.Hainan Academy of Ocean and Fisheries Sciences, Haikou 571126, China;2.Key Laboratory of Utilization and Conservation for Tropical Marine Bioresources, Ministry of Education,
Hainan Tropical Ocean University, Sanya 572022, China;3.Hainan Academy of Ocean and Fisheries Sciences, Haikou 571127, China;4.Hainan Academy of Ocean and Fisheries Sciences, Haikou 571128, China;5.Hainan Academy of Ocean and Fisheries Sciences, Haikou 571129, China;6.Hainan Academy of Ocean and Fisheries Sciences, Haikou 571130, China;7.Hainan Academy of Ocean and Fisheries Sciences, Haikou 571131, China;8.Hainan Academy of Ocean and Fisheries Sciences, Haikou 571132, China;9.Hainan Academy of Ocean and Fisheries Sciences, Haikou 571133, China
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| Abstract: |
| The known universal primers for environmental DNA (eDNA) metabarcoding of fish are mainly located in the mitochondrial ribosome gene regions. Not only are the reference sequences insufficient but also some relative fish species could not be identified using these 12S and 16S primers. In this study, the freshwater fishes of Hainan Island were selected to meet six investigation targets. (1) Six conserved regions were selected through the cytochrome c oxidase subunit I (COⅠ) sequences of 8 orders, 26 families, 101 genera, and 150 species; (2) Based on the base variation, species identification, eDNA degradation, and read length requirements of high-throughput sequencing, 26 primers were designed from four flanking conserved regions; however, six primers did not meet the Premier score requirements. (3) The first-round PCR results of 72 freshwater fish species on Hainan Island showed that 11 primers had high universality. Among them, there were 5 primers with more than 70 species successfully amplified and brighter PCR bands than marker. The second-round of PCR results showed that the amplification success rate of 3×2 (forward and reverse, respectively) primer combinations generated by the five primers was 100%, and the optimal primer combinations after PCR band length and brightness screening were "HN-A-F4, HN-D-R3" (hereinafter referred to as HN-COⅠ). (4) High-throughput sequencing results of 30 water samples showed that the total number of clean reads, fish sequences, operational taxonomic units (OTUs), and fish OTUs generated by HN-COⅠ were 0.77, 1.22, 0.85, and 1.32 times those of MiFish-U, respectively. (5) The proportion of fish OTUs annotated to species, genus, and family was 81.40%, 11.63%, and 6.98% for HN-COⅠ and 81.54%, 4.62%, and 13.85% for MiFish-U, respectively. The non-metric multidimensional scaling (NMDS) clustering pattern of "primer + water sample" formed two distinct groups with an obvious boundary (stress=0.15). The minimum and average values of pairwise genetic distances of species amplicons within genera of HN-COⅠ were 1.23 and 1.57 times those of MiFish-U, respectively. (6) The correlation coefficient of the linear regression equation of common carp biomass and sequence copies of the six indoor density groups was low. HN-COⅠ and MiFish-U could not accurately reflect the biomass of common carp. In this study, the advantages and disadvantages of HN-COⅠ were screened and compared with those of MiFish-U. The results showed that HN-COⅠ primers have a high targeting ability for freshwater fishes on Hainan Island. HN-COⅠ are advantageous in preventing eDNA contamination from non-target organisms, such as microorganisms and mammals, and is also conducive to the detection and accurate identification of freshwater fishes. |
| Key words: Hainan Island Freshwater fish Environmental DNA COⅠ Universal primers |
