Li-Zhi Gao

Thirteen Camellia chloroplast genome sequences determined by high-throughput sequencing: genome structure and phylogenetic relationships

BackgroundCamellia is an economically and phylogenetically important genus in the family Theaceae. Owing to numerous hybridization and polyploidization, it is taxonomically and phylogenetically ranked as one of the most challengingly difficult taxa in plants. Sequence comparisons of chloroplast (cp) genomes are of great interest to provide a robust evidence for taxonomic studies, species identification and understanding mechanisms that underlie the evolution of the Camellia species.ResultsThe eight complete cp genomes and five draft cp genome sequences of Camellia species were determined using Illumina sequencing technology via a combined strategy of de novo and reference-guided assembly. The Camellia cp genomes exhibited typical circular structure that was rather conserved in genomic structure and the synteny of gene order. Differences of repeat sequences, simple sequence repeats, indels and substitutions were further examined among five complete cp genomes, representing a wide phylogenetic diversity in the genus. A total of fifteen molecular markers were identified with more than 1.5% sequence divergence that may be useful for further phylogenetic analysis and species identification of Camellia. Our results showed that, rather than functional constrains, it is the regional constraints that strongly affect sequence evolution of the cp genomes. In a substantial improvement over prior studies, evolutionary relationships of the section Thea were determined on basis of phylogenomic analyses of cp genome sequences.ConclusionsDespite a high degree of conservation between the Camellia cp genomes, sequence variation among species could still be detected, representing a wide phylogenetic diversity in the genus. Furthermore, phylogenomic analysis was conducted using 18 complete cp genomes and 5 draft cp genome sequences of Camellia species. Our results support Chang’s taxonomical treatment that C. pubicosta may be classified into sect. Thea, and indicate that taxonomical value of the number of ovaries should be reconsidered when classifying the Camellia species. The availability of these cp genomes provides valuable genetic information for accurately identifying species, clarifying taxonomy and reconstructing the phylogeny of the genus Camellia.

An Improved Chloroplast DNA Extraction Procedure for Whole Plastid Genome Sequencing

Background Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs. Methodology/Principal Findings We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method. Conclusion Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

Rapid diversification of five Oryza AA genomes associated with rice adaptation

Significance Asian rice (Oryza sativa) is among the world’s most important crops. The genus Oryza has become a model for the study of plant genome structure, function, and evolution. We have undertaken de novo, full-genome sequence analysis of five diploid AA-genome species that are closely related to O. sativa. These species are native to quite different environments, representing four continents, thus exhibiting very different adaptations. Our studies identify specific genetic changes, in both gene copy number and the degree of diversifying natural selection, that indicate specific genes responsible for these adaptations, particularly in genes related to defense against pathogens and reproductive diversification. This genome discovery and comparative analysis provide a powerful tool for future Oryza study and rice improvement. Comparative genomic analyses among closely related species can greatly enhance our understanding of plant gene and genome evolution. We report de novo-assembled AA-genome sequences for Oryza nivara, Oryza glaberrima, Oryza barthii, Oryza glumaepatula, and Oryza meridionalis. Our analyses reveal massive levels of genomic structural variation, including segmental duplication and rapid gene family turnover, with particularly high instability in defense-related genes. We show, on a genomic scale, how lineage-specific expansion or contraction of gene families has led to their morphological and reproductive diversification, thus enlightening the evolutionary process of speciation and adaptation. Despite strong purifying selective pressures on most Oryza genes, we documented a large number of positively selected genes, especially those genes involved in flower development, reproduction, and resistance-related processes. These diversifying genes are expected to have played key roles in adaptations to their ecological niches in Asia, South America, Africa and Australia. Extensive variation in noncoding RNA gene numbers, function enrichment, and rates of sequence divergence might also help account for the different genetic adaptations of these rice species. Collectively, these resources provide new opportunities for evolutionary genomics, numerous insights into recent speciation, a valuable database of functional variation for crop improvement, and tools for efficient conservation of wild rice germplasm.

Prevalent Role of Gene Features in Determining Evolutionary Fates of Whole-Genome Duplication Duplicated Genes in Flowering Plants

Some gene features, including evolution rate and gene structural complexity, are highly correlated to a gene’s evolutionary fate after whole-genome duplication. The evolution of genes and genomes after polyploidization has been the subject of extensive studies in evolutionary biology and plant sciences. While a significant number of duplicated genes are rapidly removed during a process called fractionation, which operates after the whole-genome duplication (WGD), another considerable number of genes are retained preferentially, leading to the phenomenon of biased gene retention. However, the evolutionary mechanisms underlying gene retention after WGD remain largely unknown. Through genome-wide analyses of sequence and functional data, we comprehensively investigated the relationships between gene features and the retention probability of duplicated genes after WGDs in six plant genomes, Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa), soybean (Glycine max), rice (Oryza sativa), sorghum (Sorghum bicolor), and maize (Zea mays). The results showed that multiple gene features were correlated with the probability of gene retention. Using a logistic regression model based on principal component analysis, we resolved evolutionary rate, structural complexity, and GC3 content as the three major contributors to gene retention. Cluster analysis of these features further classified retained genes into three distinct groups in terms of gene features and evolutionary behaviors. Type I genes are more prone to be selected by dosage balance; type II genes are possibly subject to subfunctionalization; and type III genes may serve as potential targets for neofunctionalization. This study highlights that gene features are able to act jointly as primary forces when determining the retention and evolution of WGD-derived duplicated genes in flowering plants. These findings thus may help to provide a resolution to the debate on different evolutionary models of gene fates after WGDs.

Autotetraploid rice methylome analysis reveals methylation variation of transposable elements and their effects on gene expression

Significance Whole genome duplication (WGD) has long been recognized as a major force in angiosperm evolution. DNA methylation variation is known to be involved in polyploidization events. We synthesized autotetraploid rice that may rule out disturbances from hybridization and investigated the scope and scale of DNA methylation variation in response to WGD in neopolyploid rice. We found that WGD prompts increased methylation in class II transposable elements, which then suppress the genome-wide expression level of nearby genes. Hypermethylation of these transposable elements inhibits their transposition, stabilizing the integrity of chromosomes, and decreases nearby gene expression, potentially reducing deleterious genome-dosage effects. Our results indicate that DNA methylation plays an important role in assisting plant neopolyploids rapidly adapt to WGD. Polyploidy, or whole-genome duplication (WGD), serves as a key innovation in plant evolution and is an important genomic feature for all eukaryotes. Neopolyploids have to overcome difficulties in meiosis, genomic alterations, changes of gene expression, and epigenomic reorganization. However, the underlying mechanisms for these processes are poorly understood. One of the most interesting aspects is that genome doubling events increase the dosage of all genes. Unlike allopolyploids entangled by both hybridization and polyploidization, autopolyploids, especially artificial lines, in relatively uniform genetic background offer a model system to understand mechanisms of genome-dosage effects. To investigate DNA methylation effects in response to WGD rather than hybridization, we produced autotetraploid rice with its diploid donor, Oryza sativa ssp. indica cv. Aijiaonante, both of which were independently self-pollinated over 48 generations, and generated and compared their comprehensive transcriptomes, base pair-resolution methylomes, and siRNAomes. DNA methylation variation of transposable elements (TEs) was observed as widespread in autotetraploid rice, in which hypermethylation of class II DNA transposons was predominantly noted in CHG and CHH contexts. This was accompanied by changes of 24-nt siRNA abundance, indicating the role of the RNA-directed DNA methylation pathway. Our results showed that the increased methylation state of class II TEs may suppress the expression of neighboring genes in autotetraploid rice that has obtained double alleles, leading to no significant differences in transcriptome alterations for most genes from its diploid donor. Collectively, our findings suggest that chromosome doubling induces methylation variation in TEs that affect gene expression and may become a “genome shock” response factor to help neoautopolyploids adapt to genome-dosage effects.

Genome Size Variation among and within Camellia Species by Using Flow Cytometric Analysis

Background The genus Camellia, belonging to the family Theaceae, is economically important group in flowering plants. Frequent interspecific hybridization together with polyploidization has made them become taxonomically “difficult taxa”. The DNA content is often used to measure genome size variation and has largely advanced our understanding of plant evolution and genome variation. The goals of this study were to investigate patterns of interspecific and intraspecific variation of DNA contents and further explore genome size evolution in a phylogenetic context of the genus. Methodology/Principal Findings The DNA amount in the genus was determined by using propidium iodide flow cytometry analysis for a total of 139 individual plants representing almost all sections of the two subgenera, Camellia and Thea. An improved WPB buffer was proven to be suitable for the Camellia species, which was able to counteract the negative effects of secondary metabolite and generated high-quality results with low coefficient of variation values (CV) <5%. Our results showed trivial effects on different tissues of flowers, leaves and buds as well as cytosolic compounds on the estimation of DNA amount. The DNA content of C. sinensis var. assamica was estimated to be 1C = 3.01 pg by flow cytometric analysis, which is equal to a genome size of about 2940 Mb. Conclusion Intraspecific and interspecific variations were observed in the genus Camellia, and as expected, the latter was larger than the former. Our study suggests a directional trend of increasing genome size in the genus Camellia probably owing to the frequent polyploidization events.

Plastid Genome Sequence of a Wild Woody Oil Species, Prinsepia utilis, Provides Insights into Evolutionary and Mutational Patterns of Rosaceae Chloroplast Genomes

Background Prinsepia utilis Royle is a wild woody oil species of Rosaceae that yields edible oil which has been proved to possess particular benefits for human health and medical therapy. However, the lack of bred varieties has largely impeded exploiting immense potentials for high quality of its seed oil. It is urgently needed to enlarge the knowledge of genetic basis of the species and develop genetic markers to enhance modern breeding programs. Results Here we reported the complete chloroplast (cp) genome of 156,328 bp. Comparative cp sequence analyses of P . utilis along with other four Rosaceae species resulted in similar genome structures, gene orders, and gene contents. Contraction/expansion of inverted repeat regions (IRs) explained part of the length variation in the Rosaceae cp genomes. Genome sequence alignments revealed that nucleotide diversity was associated with AT content, and large single copy regions (LSC) and small single copy regions (SSC) harbored higher sequence variations in both coding and non-coding regions than IRs. Simple sequence repeats (SSRs) were detected in the P . utilis and compared with those of the other four Rosaceae cp genomes. Almost all the SSR loci were composed of A or T, therefore it might contribute to the A-T richness of cp genomes and be associated with AT biased sequence variation. Among all the protein-coding genes, ycf1 showed the highest sequence divergence, indicating that it could accomplish the discrimination of species within Rosaceae as well as within angiosperms better than other genes. Conclusions With the addition of this new sequenced cp genome, high nucleotide substitution rate and abundant deletions/insertions were observed, suggesting a greater genomic dynamics than previously explored in Rosaceae. The availability of the complete cp genome of P . utilis will provide chloroplast markers and genetic information to better enhance the conservation and utilization of this woody oil plant.

Transcriptome Analysis of the Oil-Rich Tea Plant, Camellia oleifera, Reveals Candidate Genes Related to Lipid Metabolism

Background Rapidly driven by the need for developing sustainable sources of nutritionally important fatty acids and the rising concerns about environmental impacts after using fossil oil, oil-plants have received increasing awareness nowadays. As an important oil-rich plant in China, Camellia oleifera has played a vital role in providing nutritional applications, biofuel productions and chemical feedstocks. However, the lack of C. oleifera genome sequences and little genetic information have largely hampered the urgent needs for efficient utilization of the abundant germplasms towards modern breeding efforts of this woody oil-plant. Results Here, using the 454 GS-FLX sequencing platform, we generated approximately 600,000 RNA-Seq reads from four tissues of C. oleifera. These reads were trimmed and assembled into 104,842 non-redundant putative transcripts with a total length of ∼38.9 Mb, representing more than 218-fold of all the C. oleifera sequences currently deposited in the GenBank (as of March 2014). Based on the BLAST similarity searches, nearly 42.6% transcripts could be annotated with known genes, conserved domains, or Gene Ontology (GO) terms. Comparisons with the cultivated tea tree, C. sinensis, identified 3,022 pairs of orthologs, of which 211 exhibited the evidence under positive selection. Pathway analysis detected the majority of genes potentially related to lipid metabolism. Evolutionary analysis of omega-6 fatty acid desaturase (FAD2) genes among 20 oil-plants unexpectedly suggests that a parallel evolution may occur between C. oleifera and Olea oleifera. Additionally, more than 2,300 simple sequence repeats (SSRs) and 20,200 single-nucleotide polymorphisms (SNPs) were detected in the C. oleifera transcriptome. Conclusions The generated transcriptome represents a considerable increase in the number of sequences deposited in the public databases, providing an unprecedented opportunity to discover all related-genes associated with lipid metabolic pathway in C. oleifera. It will greatly enhance the generation of new varieties of C. oleifera with increased yields and high quality.

Contradiction between plastid gene transcription and function due to complex posttranscriptional splicing: an exemplary study of ycf15 function and evolution in angiosperms.

Plant chloroplast genes are usually co-transcribed while its posttranscriptional splicing is fairly complex and remains largely unsolved. On basis of sequencing the three complete Camellia (Theaceae) chloroplast genomes for the first time, we comprehensively analyzed the evolutionary patterns of ycf15, a plastid gene quite paradoxical in terms of its function and evolution, along the inferred angiosperm phylogeny. Although many species in separate lineages including the three species reported here contained an intact ycf15 gene in their chloroplast genomes, the phylogenetic mixture of both intact and obviously disabled ycf15 genes imply that they are all non-functional. Both intracellular gene transfer (IGT) and horizontal gene transfer (HGT) failed to explain such distributional anomalies. While, transcriptome analyses revealed that ycf15 was transcribed as precursor polycistronic transcript which contained ycf2, ycf15 and antisense trnL-CAA. The transcriptome assembly was surprisingly found to cover near the complete Camellia chloroplast genome. Many non-coding regions including pseudogenes were mapped by multiple transcripts, indicating the generality of pseudogene transcriptions. Our results suggest that plastid DNA posttranscriptional splicing may involve complex cleavage of non-functional genes.

Evolution of microRNA genes in Oryza sativa and Arabidopsis thaliana: an update of the inverted duplication model.

The origin and evolution of microRNA (miRNA) genes, which are of significance in tuning and buffering gene expressions in a number of critical cellular processes, have long attracted evolutionary biologists. However, genome-wide perspectives on their origins, potential mechanisms of their de novo generation and subsequent evolution remain largely unsolved in flowering plants. Here, genome-wide analyses of Oryza sativa and Arabidopsis thaliana revealed apparently divergent patterns of miRNA gene origins. A large proportion of miRNA genes in O. sativa were TE-related and MITE-related miRNAs in particular, whereas the fraction of these miRNA genes much decreased in A. thaliana. Our results show that the majority of TE-related and pseudogene-related miRNA genes have originated through inverted duplication instead of segmental or tandem duplication events. Based on the presented findings, we hypothesize and illustrate the four likely molecular mechanisms to de novo generate novel miRNA genes from TEs and pseudogenes. Our rice genome analysis demonstrates that non-MITEs and MITEs mediated inverted duplications have played different roles in de novo generating miRNA genes. It is confirmed that the previously proposed inverted duplication model may give explanations for non-MITEs mediated duplication events. However, many other miRNA genes, known from the earlier proposed model, were rather arisen from MITE transpositions into target genes to yield binding sites. We further investigated evolutionary processes spawned from de novo generated to maturely-formed miRNA genes and their regulatory systems. We found that miRNAs increase the tunability of some gene regulatory systems with low gene copy numbers. The results also suggest that gene balance effects may have largely contributed to the evolution of miRNA regulatory systems.