| Mikania micrantha H.B.K. of Asteraceae has been listed as the first batch of alien invasive species in China. This species is such a highly hazardous weed that it has been listed as the world's 100 worst invasive alien species. M. micrantha is of the new world origin and is widely naturalized in the old world tropics. As a typical invasive weed, M. micrantha resulted in great ecological loss in China. The studies on M. micrantha could be of important practical significance and scientific value. In recent years, a large number of studies about M. micrantha have been reported, in which the fields such as the physiological ecology, community structure and ecological effects has been involved.However, the study of the genetic mechanisms of biological invasion is much less reported. With the development of next-generation high-throughput sequencing technology, it is possible to investigate the molecular mechanisms of biological invasions, through revealing the regulation of invasive expression and the molecular evolutionary process of "post-adaptation" on the basis of the genome data of invasive species, M. micrantha. However, the EST library construction and consequential sequencing on the genome level is based on the traditional Sanger methods which is laborious and cost a lot. With the advances of sequencing technology, a new generation of high-throughput sequencing technology which is more efficient and cost-effective has been used gradually in the field of invasive biology. However, de novo assembly of genome or transcriptome of non-model organisms is not only a difficult task but also a challenge considering only the short read length (75-90 nt) is available by the high-throughput sequencing technology nowadays. As an initial step towards understanding the molecular mechanisms by which plants become invasive, we present here the first transcriptome analysis for an invasive weed (i.e. Mikania micrantha) for which no prior genomic information was available. We further explored its molecular basis of invasion characteristics. This thesis consists of the following two parts:1. The transcriptome analysis of individual M. micrantha: The entire plant, including roots, was used for RNA extraction. Paired-end short read sequencing was done on one lane of the Illumina GAII system after reverse transcription. The analysis was based on the 75-nucloetide short reads data generated. A total of 51,782 unique sequences with an average length of 734 nt and a maximum length of 7,324 nt were assembled de novo based on 32 million filtered paired-end sequence reads for the transcriptome of an individual M. micrantha growing in the field. Seventy-three percent of the unique sequences showed significant similarity to existing proteins in the NCBI database, and 25,478 could be grouped based on gene ontology assignments. The sequences obtained in this study represent the first large-scale dataset of expressed genes in an invasive weed. Of particular interest are the sequences that share homology with genes involved in genome evolution, plasticity, secondary metabolism, and defense responses. This resource provides essential information to support the investigation of numerous questions regarding the invasive biology of M. micrantha and related species.2. Comparison of population genetic characteristics of source population and invasion population of M. micrantha: In this thesis, the population from Costa Rica in Central America (14 individuals) and population from Dongguan and Guangzhou (12 individuals) have been treated as the representatives of source population and invasion population of M. micrantha, respectively. The seedlings from different sources were cultured in the common garden for one month. Then we pooled two groups of individuals with equal amount of individual RNA respectively and sequenced the two RNA mixtures by Illumina GAII system. De novo assembling the transcriptome of population of M. micrantha and SNP identification were carried out on the basis of the reference sequence which comes from individual transcriptome obtained before. A total of 270,000 candidate SNP sites were screened from about 7.8 million loci based on the comparison results. The population genetic parameters such asθandπwere estimated for source population and invasion population of M. micrantha respectively (θ= 5.7‰,π= 4.1‰in population of Costa Rica,θ= 4.8‰,π= 3.0‰in population of Guangdong). The genetic diversity of population of Costa Rica was slightly higher than that of population of Guangdong. Our result also provided a wealth of SNP information available for the further research.The present study demonstrated a successful application of second-generation high-through sequencing technologies on the transcriptome analysis of non-model typical invasive species Mikania micrantha. The important population parameters such as the genetic diversity of populations and SNP loci were estimated based on the transcriptome analysis of M. micrantha Our study has revealed that next-generation DNA sequencing technologies can be used effectively for de novo sequencing of transcriptomes of non-model invasive species, making it possible to carry out rapid and low-cost sequencing for other important plant species. With these genomic approaches, it is possible to dissect the invasiveness-related genes and their expression and regulation patterns, identify invasive genotypes and hence understand plant invasiveness, which can be used to disassemble plant invasion mechanisms and define the evolutionary patterns during plant invasions. |
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