Research And Development Of Nuclear DNA Molecular Markers In Freshwater Crabs Of The Family Crabidae And Their Application In Phylogenetic Studies

Molecular marker is based on genomic DNA contains abundant polymorphism and developed can directly reflect the individual differences in DNA level of a new type of genetic marker,it is following the morphological markers,cytological markers,biochemical markers,the most reliable genetic markers.With the development of molecular biology techniques,analysis speed of the new generation sequencing technology faster,lower cost,the greater the amount of information,which makes the research of the DNA molecular marker is very active,and the development of nuclear DNA markers is a hot research in the field of molecular biology,compared the nuclear ribosomal DNA and mitochondrial DNA markers,rich evolutionary information contains the nuclear protein encoding gene,has a wide range of evolutionary rates in different taxa,can provide molecular system effective signal information in invertebrate taxa nuclear DNA markers are increasingly being developed and utilized,provides a powerful tool for the study of the species phylogeny and classification system.China is the most species-rich country for the primary freshwater crabs in the world,with 302 recorded species and subspecies(42 in Taiwan),43genera and 2 families distributed across tropical,subtropical and warm temperate areas.The Chinese freshwater crab fauna is dominated by the Potamidae,which all belong to Potamiscinae.The extraordinary species richness of the Potamindae,which means 42 genera and 276 species and subspecies,along with the limited dispersal ability,makes it an ideal model for research on diversification of primary freshwater crabs across China and even the East Asian continent and its driven factors.Due to the lack of decapod genomic information,only nuclear DNA markers have been developed and is limited by,so the Decapoda genomic information to further enrich,will help us to develop and use more nuclear DNA molecular markers;and the system crabs multi-core DNA molecular marker of the subfamily China of freshwater crab a relationship study deficiency can effectively compensate for the freshwater crab species classification and identification with traditional morphology based classification,and based on a single gene or mitochondrial marker gene phylogenetic tree support degree is not high,and lay a solid foundation for the further study of nearly every genus within the subfamily crabs and the relationship between species.This study is based on extensive taxon sampling,the sampling area covers the entire China subfamily in the distribution area,a total of 27 sampling points,41 genera,58 species of freshwater crab(crab subfamily known genera accounted for nearly 57%),with a new generation of sequencing technology to design the database of two batch sequencing method of PCR products rapid determination of gene sequences,with 64 nuclear DNA markers reconstructed the phylogenetic relationships within the subfamily in freshwater crab,to further explore the China diversity of the subfamily evolution near freshwater crab.The paper is divided into four chapters,and the main results and conclusions are obtained.(1)We used the Survey genome and transcriptome data of Yangtze river crab to search for genes.We predicted 637 genes,which are single exon genes,and these genes are single copy genes.(2)Sinopotamon yangtsekiense Survey genome data search based on screened were applied to potamidae(64),non quasi potamonautes(59),Sinopotamon(52)nuclear DNA markers.(3)Based on the next-generation sequencing technology,we designed two methods for database sequencing of batch PCR amplification products.On the basis of saving a lot of time and cost,we got 64 nuclear DNA molecular markers in 2 families,41 genera and 58 species in China.The total length of the 64 nuclear DNA markers reached 441 10bp,and the average gene mutation rate was 24.4%.(4)With 64 nuclear protein encoding gene sequence China rebuilt the relationship between the freshwater crab crab near subfamily system.The results showed that nearly crabs were divided into 4 subfamilies of main branches of molecular phylogeny of the maximum likelihood method and Bayesian method based on the distribution of species in Hengduan Mountains formed two branches outside the Hengduan Mountains near the freshwater crab crab subfamily is a monophyletic group,the branches and distribution of species in Hengduan Mountains each sister group relationship.