The Whole Genome Sequencing,Assembly And Comparative Genomic Research Of Plant Organelle

Complete organellar genome sequences (chloroplasts and mitochondria) provide valuable resources and information for studying plant molecular ecology and evolution. As high-throughput sequencing technology advances, it becomes the norm that a shotgun approach is used to obtain complete genome sequences. However, associated techniques are often cumbersome, time-consuming, and difficult, because true organellar DNA is difficult to separate efficiently from nuclear copies。Therefore, to assemble organellar sequences from the whole genome, shotgun reads are inevitable.We report here a new, rapid procedure for plant chloroplast and mitochondrial genome sequencing and assembly using the Roche/454GS FLX platform. Plant cells can contain multiple copies of the organellar genomes, and there is a significant correlation between the depth of sequence reads in contigs and the number of copies of the genome. We retrospectively extracted assembled contigs of either chloroplast or mitochondrial sequences from the whole genome shotgun data. Moreover, the contig connection graph property of Newbler (454platform-specific sequence assembler) ensures an efficient final assembly. This procedure does not need to isolate organellar DNA from the mixture of nuclear and organellar DNA for sequencing, and almost all of the sequencing data can be used to the whole genome sequencing project of this plant. In addition, this procedure can well assemble the mitochondrial genome with multipartite structures, which cannot be solved with traditional method. Using this procedure, we completely assembled both chloroplast and mitochondrial genomes of a resurrection plant, Boea hygrometrica and the Hassawi rice and its hybrid, which provided the effect and validation of this procedure.The complete nucleotide sequences of the chloroplast (cp) and mitochondrial (mt) genomes of resurrection plant Boea hygrometrica (Bh, Gesneriaceae) have been determined with the lengths of153,493bp and510,519bp, respectively. The smaller chloroplast genome contains more genes (147) with a72%coding sequence, and the larger mitochondrial genome has less genes (65) with a coding faction of12%. Similar to other seed plants, the Bh cp genome has a typical quadripartite organization with a conserved gene in each region. The Bh mt genome has three recombinant sequence repeats of222bp,843bp, and1474bp in length, which divide the genome into a single master circle (MC) and four isomeric molecules. Compared to other angiosperms, one remarkable feature of the Bh mt genome is the frequent transfer of genetic material from the cp genome during recent Bh evolution. We also analyzed organellar genome evolution in general regarding genome features as well as compositional dynamics of sequence and gene structure/organization, providing clues for the understanding of the evolution of organellar genomes in plants. The cp-derived sequences including tRNAs found in angiosperm mt genomes support the conclusion that frequent gene transfer events may have begun early in the land plant lineage.Hassawi rice (Oryza sativa L.) is a landrace adapted to the climate of Saudi Arabia, characterized by its strong resistance to soil salinity and drought. Using high quality sequencing reads extracted from raw data of a whole genome sequencing project, we assembled both chloroplast (cp) and mitochondrial (mt) genomes of the wild-type Hassawi rice (Hassawi-1) and its dwarf hybrid (Hassawi-2). We discovered16InDels (insertions and deletions) but no SNP (single nucleotide polymorphism) is present between the two Hassawi cp genomes. We identified48InDels and26SNPs in the two Hassawi mt genomes and a new type of sequence variation, termed reverse complementary variation (RCV) in the rice cp genomes. There are two and five RCVs identified in Hassawi-1when compared to93-11(indica) and Nipponbare (japonica), respectively. Microsatellite sequence analysis showed there are more SSRs in the genie regions of both cp and mt genomes in the Hassawi rice than in the other rice varieties. There are also large repeats in the Hassawi mt genomes, with the longest length of96,168bp and96,165bp in Hassawi-1and Hassawi-2, respectively. We believe that frequent DNA rearrangement in the Hassawi mt and cp genomes indicate ongoing dynamic processes to reach genetic stability under strong environmental pressures. Based on sequence variation analysis and the breeding history, we predicted that both Hassawi-1and Hassawi-2originated from the Indonesian variety Peta since genetic diversity between the two Hassawi cultivars is very low albeit an unknown historic origin of the wild-type Hassawi rice. Understanding the inheritance of the wild-type Hassawi and its hybrid provides important genetic information for their future breeding as well as genetic and molecular studies.