Survey Of Bacteriophage Genetic Diversity In Wetland Sediments In Northeast China

Viruses are the most abundant biological entities on the planet,and play a significant role in ecological process.Bacteriophages are the most abundant viruses in natural condition.In recent years,with the development of molecular biological techniques,the study of the genetic diversity of environmental viruses has also gradually increased.Many studies about genetic diversity of some families of environmental viruses have showed that there are many unknown viral clusters in the environment and the distribution of viral genetics has distinct geographical features.International studies on viral ecology in environments mainly focus on marine,freshwater lakes,paddy field and upland black soil,however,the natural wetlands have been poorly studied.The wetland is considered as the “gold gene bank” in nature,welands have the characteristics of both aquatic and terrestrial ecosystems,and well-known as "the kidney of the earth".Northeast China,one of the major wetland distribution areas has many types of wetland resources.In this study,we surveyed the diversity and distribution of bacteriophage based on three biomarker genes g23,DNA pol and phoH found in wetland sediments in Northeast China by using culture-independent PCR,cloning and Sanger sequencing methods.The main results are as follows:(1)Two hundred and sixty two different T4-type bacteriophage g23 clones were obtained from six wetland sediment samples in northeast China.The identities at the amino acid level within the g23 in this study ranged from 59% to 99%,and these g23 clones were distributed into thirteen clusters(Clusters I~ XIII).By constructing a phylogenetic tree and combining UniFrac analysis,the several new g23 gene clusters were found in this study.The study also found that the distribution of bacteriophage in wetland ecosystems is distinctly different from that in upland black soils,but similar to that in aquatic ecosystems such as oceans and lakes and paddy ecosystems.In addition,the variation of T4-type bacteriophage in wetland sediments is higher than in other ecosystems.(2)Sixty six cyanopodovirus DNA pol clones were obtained from two costal wetland sediment samples in northeast China,the identities at the amino acid level within the DNA pol in this study ranged from 80% to 99%,these clones were distributed into six clusters,the distribution patterns of clones into different clusters were significantly differed btween two wetlands,which suggested that cyanophage pol assemblages in wetland sediments are spatially distributed.Simultaneously,the clones from the wetland sediments were alone or overlap with the pol sequences from Chesapeake Bay,and four new wetland unique groups of cyanopodoviruses(Wetland Clusters 1~4)were observed.Non-metric multidimensional scaling analysis of all DNA pol sequences from various environments indicated that cyanopodovirus communities of coastal wetlands are in the intermediate position between marine water environments and terrestrial freshwater environments,and the cyanopodovirus communities in wetland sediments were similar to those in marine and coastal waters,but distinct from those in lake freshwater and paddy fields.(3)At least fifty eight bacteriophage phoH clones were obtained from four wetland sediments of northeast China.The identities at the amino acid level within the phoH in this study ranged from 48% to 100%.Nearly 16.67% of phoH clones in wetland sediments were novel,with the highest identity of ? 75% with known sequences.These clones were distributed into Group 2,3,4 and 6,also,two subgroups(Group 2c and Group 4c)were formed exclusively in this study,which indicated novel bacteriophage phoH groups exist in the wetland environment.Phylogenetic analysis found that about 86% of the bacteriophage phoH clones was identified to be derived from the cyanobacterial virus.Non-metric multidimensional scaling analysis indicated that the bacteriophage phoH assemblages obtained from Yalujiangkou were similar to those in marine environments,the bacteriophage phoH assemblages obtained from Xingkaihu were similar to those in paddy fields,thus the bacteriophage phoH assemblages obtained from two wetlands were distinctly different.In this study,it's found that phoH can be used as an effective marker gene to study phage diversity in different environments.In conclustion,three kinds of molecular marker genes were used as target genes,and different marker gene sequences that characterize the genetic diversity of bacteriophages were obtained from typical wetland sediments in Northeast China.It was found that the T4-type bacteriophage g23 gene assemblages were distinctly different among different types of wetland environments.Four unique cyanophage DNA pol gene groups and two unique bacteriophage phoH subgroups were formed in wetland ecosystem.In summary,the bacteriophage communities in wetland sediments were between aquatic and terrestrial ecosystem,and similar to marine and lake,but distinct to terrestrial ecosystem.Especially,the bacteriophage communities in coastal wetland were distinct to those in lake wetland and swamp wetland,but similar to marine ecosystem.Thus,it may provide further data support for further elucidation of phage functions in wetland ecosystems.