Construction Of A Database For Plant Miniature Inverted-repeat Transposable Element And Genetic Cloning Of Two Genes Conferring Symptomless To Tobacco Mosaic Virus In Tobacco

Miniature inverted-repeat transposable element(MITE) are short and non-autonomous transposable elements that have a structure resembling that of DNA transposons, with a typical terminal inverted repeats(TIR) flanked by target site duplications(TSD). Unlike the DNA transposons, MITE had a high copy number within the genome and are usually preferentially inserted into or near genes, provoking a hypothesis that MITEs might have an important role in gene regulation and might be driver for the genome evolution and species diversification. Although numerous progresses had been made on the study of MITE, the process of study had been greatly hampered due to the lack of open resources that would allow the researchers to carry out MITE studies in large-scale. Here, we report the de novo identification of MITE sequences from 41 sequenced plant genomes, using MITE Digger, MITE-Hunter and/or Repetitive Sequence with Precise Boundaries(RSPB). To ensure MITE annotation accuracy, at least one representative MITE sequence for each MITE family was manually validated. Combined with the MITE identified previously from the rice genome, more than 2.3 million MITEs classified into 3,527 MITE families were obtained. The number of MITEs and MITE families across species varies, but, in general, higher plants contain more MITEs and MITE families than lower plants. The number of MITE sequences in a genome is significantly correlated with genome size, suggesting an evolutionary role of MITE in genome evolution. We also identified 10 potentially active MITE groups with at least 20 identical copies in a genome. A series of databases(plant MITE database, P-MITE), available on line http://pmite.hzau.edu.cn/, were constructed to host all the sequences identified from the 41 plant genomes. The database has implemented many functions including sequence browsing, searching and downloading. The database had been proved valuable resources for the study of MITE origin, amplification and roles of MITE on gene regulation and genome evolutionBulk segregant analysis(BSA) is a technique that could be used to identify molecular markers linked with target genes. Combining RNA-seq with BSA allows one to rapidly identify genomic loci that are responsible for the phenotype. Although many methods have been developed for such analysis, none of them could be used in the absence of a good reference genome. We here report snpMapper, a pipeline for the analysis of BSA RNA-seq data. We apply the pipeline in the analysis of RNA-seq data derived from single-gene mapping population, multiple-gene mapping population and RNA-seq data from a BC2 population of a non-model species which does not have a good reference genome. The results indicated that in all the three cases, snpMapper could successfully identify a short genomic interval where target genes are mapped or molecular markers that are highly linked with the target genes. We thus concluded that the pipeline could be used for the linkage analysis and accelerate the process of gene cloning.Efficient multiplication of tobamovirus requires the interaction between virus and the host factors. However, information regarding such host factors is limited. Here we report the map-based cloning of two genes from allotetraploid Nicotiana tabacum, NtTOM2A-Ntom and Nt TOM2A-Nsyl, which are required for efficient multiplication of tobacco mosaic virus(TMV) in tobacco. Genetic analysis shows that the two genes are homoeologous, located on homoeologous chromosomes of the two subgenomes. Simultaneously mutations of the two genes results in the poor accumulation of TMV in tobacco, and consequently tobacco had a symptomless phenotype. qRT-PCR analysis shows that Nt TOM2 As are expressed in all tissues with a higher expression level in root and expression of NtTOM2 As did not change after TMV innoculation. We construct a near isogenic line(NIL) for NtTOM2A-Nsyl and perform an RNA-seq analysis. Transformation of TOM2 A orthologs from 11 different species shows that all the TOM2 A homologs retained the ability to support TMV multiplication.We silenced the expression of SlTOM2 A in tomato and identified 8 trangenic lines in which the expression of SlTOM2 A was significant reduced. These transgenic lines will be used to study whether silencing of SlTOM2 A could results in low accumulation of TMV in tomato and thus provide a new strategy to develop TMV-resistant crops.