Research Of High-throughput Detection Of Rice Viral Disease In Hainan Winter-breeding Areas

Rice viral diseases cause serious damage to rice production.So far,10 rice viral diseases were reported in China.Hainan National Breeding and Multiplication region which is mainly consisted of Sanya,Ledong and Lingshui of Hainan Province,is the most important base for rice winter-breeding in China.At the same time,Hainan winter-breeding area is easy to spread rice diseases by gathering of germ plasm materials from all over the country.So it is very necessary to know the distribution of rice viruses in Hainan winter-breeding areas,and lay the foundation for further prevention and control of rice viral diseases.In recent years,the fast developing of next-generation sequencing methods has provids large-scale and high-throughput technologies for the detection of viruses.In this study,the distribution of rice virus in Hainan winter-breeding areas was surveyed by molecular techniques,small RNA sequencing and transcriptome sequencing methods simultaneously.We have conducted 6 batches of investigations of rice viral disease in Sanya,Lingshui,Ledong etc，from January 2014 to November 2015.The conclusions of this study are as follows:(I)Traditional molecular identification and field investigation showed that Rice ragged stunt virus(RRSV),Southern rice black-streaked dwarf virus(SRBSDV)and Rice grassy stunt virus(RGSV)were the three mainly rice viruses in Hainan winter-breeding areas,with the detection rates of 30.45%,7.61%and 0.51%,respectively.RRSV was the most severe and widely distributed virus,which has a detection rate up to 60%in some fields and caused total crop failures.Statistical result showed that rice viral diseases were more frequently detected in winter and spring than in summer and autumn.These findings would contribute to further exploration of rice viral diseases in Hainan winter-breeding areas.(II)Small RNA sequencing method was applied to detect rice viruses from three ricesamples collected in Hainan winter-breeding areas,including a healthy plant sample(JK),a virus-infected plant sample(Infect)and a mixed plant sample(Mix).Three rice viruses were identified by sequence alignment and assembly,including Rice ragged stunt virus,Rice yellow stunt virus and Southern rice black-streaked dwarf virus.Whole genome sequences of these three rice viruses were obtained by bioinformatics analysis.Among them,Rice yellow stunt virus was found in Hainan winter-breeding areas for the first time,which was not been detected by regular molecular method.RT-PCR primers were designed according to the genome sequence.The existence of Rice yellow stunt virus in Hainan winter-breeding areas was confirmed.Results show that small RNA sequencing is an efficient and rapid method for virus detection and genome assembly.(III)Transcriptome sequencing using Illumina HiSeq4000 Paired-end 150 sequencing strategy was applied to two pooled samples(NFR-A and NFR-B)using rice samples collected from the fifth and the sixth sampling times respectively.32Gb clean data was obtained respectively.A total 167,765 Unigenes were assembled from non-rice data,using de-novo assembly.BLAST analysis was performed against the GenBank Nt and Nr database.As a result,two contigs with very high identities to Rice tungro bacilliform virus and Oryza brachyantha geminivirus were obtained from BLAST Nt results;29 contigs with highly identify with viral amino acid from BLAST Nr results were also detected.In this study,multi-time,large-scale and high-throughput detections of rice viral diseases in Hainan winter-breeding areas were carried out by using traditional method and next-generation sequencing methods.Results showed that,transcriptome Sequencing and small RNA sequencing methods have many obvious advantages compared with regular methods.These new methods are especially useful for the identification of unknown and low titer plant viruses,and could be served as effective supplements to traditional detecting techniques.Furthermore,results indicating that a great variety of rice viral disease was existed in Hainan winter-breeding areas which makes new-developing methods very necessary for viruse detection.In general,results of this study could be leveraged for furtherdetection and prevention of rice viral diseases in Hainan winter-breeding areas.