| Accurate species identification is important to investigate fish early life history stage and evaluate annual supplement investigation on the fish resources. In this regard, this study adopted the DNA barcoding approach, selected Cyt b gene as a marker gene, selected the appropriate universal primers for PCR amplification, sequenced the resulting fragments amplified and analyzes the sequence to make identification species of drifting eggs and adhesive fish eggs collected in Jiangjin section of the upper Yangtze River in order. To establish a rapid and accurate molecular identification method for fish early life resources. Meanwhile, we investigated and assessed plankton and water quality in artificial fish nest sites for inhabit restoring. The main research results are as follows:1. Established the identification method for fishes of early life history based on DNA barcoding approachThe key step in the identification of fish eggs based on DNA barcoding technology is eggs DNA extraction, selecting universal primers, PCR amplification, DNA sequencing and sequence analysis. About the problem, what was compared is the effect of three kinds of single eggs DNA extraction methods and three pairs of universal primers amplification and the accuracy of the sequencing results in our study. Finally we selected the quick method of DNA extraction of fish eggs as the single eggs DNA extraction method, universal primer H15149 / L14735 to amplify the length of 464 bp DNA fragment on Cyt b gene. Then without the purified PCR products was sequenced. Then we used the sequence to blast in the NCBI database for species identification. It can be considered the same species of fish with more than 98% similarity. 2. Species identification of adhesive fish eggs in artificial fish nest in Jiangjin section of the upper Yangtze River in 2014We collected adhesive fish eggs in three artificial fish spawning nests at Shunjiang, Zhuyang and Longhua in Jiangjin section of the upper Yangtze River on April 29 th and May 11 th, 2014. We adopted the above molecular identification techniques to identified 147 samples all collected. The result showed that fish eggs both are Carassius auratus and Cyprinus carpio in three sampling sites. There were 16 Carassius auratus accounting for 80% and 4 Cyprinus carpio accounting for 20% of 20 samples at Shunjiang. There were 20 Carassius auratus accounting for 23.26% and 66 Cyprinus carpio accounting for 76.74% of 86 samples at Zhuyang. There were 31 Carassius auratus accounting for 73.81% and 11 Cyprinus carpio accounting for 26.19% of 42 samples in Longhua. 3. Species identification of adhesive fish eggs in artificial fish nest in Jiangjin section of the upper Yangtze River in 2015We collected adhesive fish eggs in four artificial fish spawning nests Shunjiang, Zhuyang, Erlangshi and Shima in Jiangjin section of the upper Yangtze River on March 26 th, April 15 th and May13 th, 2015. In all we have collected randomly 1989 samples on. 468 samples were identified species by the above molecular identification techniques, including 400 fish eggs and 68 larval fish. The result showed that fish eggs both are Carassius auratus and Cyprinus carpio.And larval fish includes 11 kinds, such as Rhinogobius leavelli, Gnathopogon imberbis, Rhodeus ocellatus, Sarcocheilichthys sinensis, Acheilognathus macropterus, Neosalanx taihuensis, Pseudorasbora parva, Gambusia affinis, and Gnathopogon herzensteini. 4. Species identification of drifting eggs in Jiangjin section of the upper Yangtze River 2015We collected randomly 107 samples with conical net from May 20 th to June 9th. All samples were identified species by the above molecular identification techniques which result showed that there were 14 kinds of fish eggs. There were 2 Hypophthalmichthys molitrixaccounting for 1.85%, 2 Jinshaia sinensisaccounting for 1.85%, 19 Rhinogobio typusaccounting for 17.59%, 1 Rhinogobius giurinus accounting for 0.93%, 36 Gobiobotia filifer accounting for 33.33%, 5 Coreius heterokon accounting for 4.63%, 22 Pseudolaubuca engraulis accounting for 20.37%, 7 Saurogobio gymnocheilus accounting for 6.48%, 4 Ctenopharyngodon idellus accounting for 3.70%, 5 Squalidus argentatus accounting for 4.63%, 1 Botia superciliaris 0.93%, 1 Pseudolaubuca sinensis accounting for 0.93%, 1 Xenophysogobio boulengeri accounting for 0.93% and 1 Lepturichthys fimbriata accounting for 0.93%。 5. The survey of plankton and water quality at artificial fish nest sites in Jiangjin section of the upper Yangtze River in 2015To realize the ecological environment of adhesive fish eggs spawning site, we investigated aquatic organisms and water environment from March to May in 2015. We have identified 6 phyla and 147 species of phytoplankton and 3 phyla and 19 species zooplankton from qualitative and quantitative samples at Shunjiang. A total of 6 phyla and 160 species of phytoplankton and 3 phyla and 19 species zooplankton were identified Zhuyang. A total of 6 phyla and 116 species of phytoplankton and 3 phyla and 52 species zooplankton were identified in Erlangshi. A total of 6 phyla and 96 species of phytoplankton and 3 phyla and 32 species zooplankton were identified at Shima. Based on the species and quantity of phytoplankton, Water quality was conducted with the biotic diversity index(Shannon-Weiner, Margalef and Pielou index). Shannon – Wiener, Margalef and Pielou index ranged from 1.663 to 3.342, from 5.438 to 10.135 and from 0.470 to 0.826 respectively. The result showed that the water were clean or polluted lightly, except Erlangshi was polluted moderately in May. |
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