| Environmental DNA technology is an emerging technology that has been used in research on biodiversity,invasive species monitoring,and rare species discovery in various ecosystems.This thesis compares the extraction efficiency and purification efficiency of environmental DNA from mangrove soil through a variety of methods;the complete mitochondrial genome sequence of three species of Clam family has been completed.In order to improve the macromolecular barcode database,this research has successively supplemented mangrove macrobenthos Molecular barcode data.In addition,we used Pac Bio’s third-generation sequencing technology to determine the full-length transcriptome of the dominant benthic mangrove clam(Gelonia erosa)in Guangxi mangroves and analyzed it to provide basic data for studying its biological characteristics.The results of this study are as follows:(1)Extracting high-quality,high-quantity and wide-coverage environmental DNA is the first step to establish the effectiveness of mangrove biodiversity research results based on environmental DNA technology.This study compared six methods for extracting total DNA from mangrove soil and two methods for DNA purification;PCR amplification was performed with a number of pairs of universal primers for benthic invertebrates,bacteria,fungi,plants,nematodes,etc.,to evaluate different methods Species coverage for extraction and purification of environmental DNA.The results showed that there were significant differences in DNA extraction efficiency between different extraction methods,and there were also differences in the yield of DNA after purification by the two methods,but the quality of DNA had no significant effect.The PCR amplification results showed that the DNA extracted by the six methods ccould be amplified with the primers of bacteria and invertebrates to obtain the target gene fragments.When amplifying the mat K general primers corresponding to plants,the DNA extracted by DNeasy Power Soil kit had the best effect.The DNA of mangrove soils from different sampling points differed greatly when amplifying the corresponding primers of fungi,which was presumably related to the soil particle size.(2)In this thesis,the complete mitochondrial genomes of three molluscs in the Bivalvia family Clamidae were shown by Novoseq 6000 sequencing,namely: mangrove clam(Gelonia erosa),river clam(Corbicula fluminea),and groove fairy clam(Cyrenobatissa subsulcata),the length of the complete mitochondrial genome of the mangrove clam was 15041 bp,including 36 genes,including 12 protein coding genes,22 transfer RNAs and 2 ribose RNAs(12S and 16S),showing a typical ocean The mitochondrial gene composition of bivalves,the overall base composition A was 21.16%,T is 46.11%,C is 7.73%,and G is 25%.The length of the complete mitochondrial genome of C.vulgaris was 17910 bp,and the overall base composition A was 26.1%,T is 47.71%,C was 9.09%,and G was 21.57%.The length of the complete mitochondrial genome of Clam clam was 18074 bp,and the total base composition Composition A was 26.08%,T was 42.9%,C was 9.11%,and G was 21.9%.The complete mitochondrial genome of the two species of river clam and concave fairy clam includes 37 genes,of which 13 were protein-coding genes,22 One transfer RNA and two ribose RNAs(12S and 16S)had one more protein-coding gene ATP8 than mangrove clams.Consistent with mangrove clams,both also showed A+T preference.(3)The mangrove clam full-length transcriptome was determined based on Pac Bio’s third-generation sequencing technology.The sequencing results were subjected to SSR analysis,CDS prediction,Lnc RNA prediction,transcript function annotation,and transcript quantification.The results showed that the third-generation sequencing obtained 9.36 Gb Clean data,215,636 ROIs,and87310 full-length non-chimeric sequences.The full-length sequence was clustered using the RS＿Iso Seq module of SMRT Analysis software,and a total of 31881 identical transcript sequences were obtained.After using non-full-length sequence correction,24491 high-quality full-length transcript sequences and 7390 low-quality full-length transcript sequences were obtained,and 7390 low-quality sequences were corrected using second-generation data.In this study,22749 transcripts were obtained,9615 SSRs,14496 complete CDS regions,and 3870 Lnc RNAs were predicted. |
