Analysis Of Cyanophage Genetic Diversity And Viral Metagenomics In Paddy Waters In Northeast China

Virus,entity of simple biological structure,enormous quantity and the extensive distribution range,plays important ecological roles in the nature.The previous studies on the genetic diversity of some environmental virus families fully indicate that there are many unknown virus groups existing in various environments,and viral genetic distribution has distinct geographical features.International studies on viral ecology in aquatic environments mainly focus on the ocean and lake ecosystems,but the paddy fields that with the characteristics of both aquatic and terrestrial ecosystems.In this study,we surveyed the diversity and distribution of cyanophage based on biomarker genes psbA,DNA pol and phoH found in paddy waters in Northeast China using culture-independent PCR and Sanger sequencing methods.Besides,we surveyed the composition of viral genomes in paddy waters in Northeast China by metagenomics technology(high-throughput sequencing technology).The results were as follows:1.We isolated 152 cyanophage psbA clones from two paddy waters of northeast China.We found that although the majority of psbA sequences observed in this study had relative high similarity with those from Japanese paddy waters,two(PFW-1a and PFW-2)and(PW-1~PW-7)seven specific cyanophage psbA groups were constructed in JPWs and this study,respectively,implying that cyanophage psbA assemblages in paddy waters,to some extent are different between two countries.In addition,we also found that the majority of psbA clones from paddy waters of both countries fell into the subcluster α-2,suggesting that the distribution of cyanophage psbA genes in paddy waters is narrow,but far away from those from environmental freshwater and seawater.2.We isolated 532 bacteriophage DNA pol clones from four paddy waters of northeast China.Based on the overall architecture of the phylogenetic tree and the closest relatives of those DNA pol clones,a total of 438 DNA pol clones were identified as cyanopodoviruses.The clones from the paddy waters formed nine unique groups of cyanopodoviruses either exclusively or with clones from East Lake in China(Subclusters α-1 to α-8 and Cluster β).None of the clones from open oceans or coastal waters fell into these unique groups.Additionally,the distribution proportions of the clones in different cyanopodovirus groups varied among paddy water samples,which suggested that the cyanopodovirus compositions were spatially distributed in the paddy fields.The comparison of clone libraries in different studies indicated that the diversity of cyanopodoviruses in paddy waters was comparable to the diversity in the open oceans but was less than the diversity in the coastal estuary of Chesapeake Bay.Non-metric multidimensional scaling analysis indicated that the cyanopodovirus communities in paddy waters were similar to those in lake freshwater but distinct from the communities in marine and coastal waters.3.We isolated 424 bacteriophage phoH clones from four paddy waters of northeast China.The majority of phoH sequences in paddy waters were novel,with the highest identity of ≤70% with phoH sequences found in database.Four unique groups(Group α,Group β,Group γ and Group δ)and seven new subgroups(Group 2b,Group 3d,Group 3e,Group 6a,Group 6b,Group 6c and Group 6d)were formed exclusively with the clones from the paddy waters,suggesting novel phage phoH groups exist in the paddy ecosystem.Additionally,the distribution proportions of phoH clones in different groups differed not only between marine waters and paddy waters but also among paddy water samples,suggesting the phage community is biogeographically distributed.Furthermore,non-metric multidimensional scaling analysis indicated that phage phoH assemblages in paddy waters were distinct from those in marine waters.Based on the overall architecture of the phylogenetic tree and the closest relatives of those phoH clones,approximately 90%(379/424)of phoH clones that phylogenetically distributed into Cluster I or Group 1,2,3,6,α and β might originate from cyanophages.4.We obtained 5582,13325 and 5582 assembled sequences(contigs)in viral metagenomes of paddy waters from Jiansanjiang,Mudanjiang and Yanjiagang,respectively.Analysis showed that there were 50%,40% and 20% of the metagenomic sequences from samples Jiansanjiang,Mudanjiang and Yanjiagang belonged to viruses,respectively.Among those sequences,5 five families of ssDNA viruses and 1 family of dsDNA virus were identified with Microviridae and Circoviridae of ssDNA viruses being the highest and second highest.There were 24,25 and 27 categories of functional genes being assigned to samples Jiansanjiang,Mudanjiang and Yanjiagng,respectively.Among the “phages and prophages”,functional genes encoding phage capsid proteins and r1t-like streptococcal phage proteins had the highest and second highest abundance.Furthermore,comparison of the three viral genomes in this study showed that there was significant difference on the abundance of virus families and virus-related functional genes but not on the catagories.In conclusion,we obtained thousands of different molecular marker gene sequences targeting on the genetic diversity of cyanophages in this study.Seven novel paddy-specific cyanophage psbA groups were found,and the distribution of cyanophage psbA genes in paddy waters was narrow.Nine unique cyanophage DNA pol genes groups and four unique bacteriophage/cyanophage phoH groups were formed and geographically distributed in paddy environment.Overall,the cyanophage communities in paddy waters were similar to those in lake but distinct from the communities in marine.Moreover,the analysis of viral metagenomics showed that there was significant difference on the abundance of virus families and virus-related functional genes but not on the catagories in paddy environment.