Kai Song

Covariation in Plant Functional Traits and Soil Fertility within Two Species-Rich Forests

The distribution of plant species along environmental gradients is expected to be predictable based on organismal function. Plant functional trait research has shown that trait values generally vary predictably along broad-scale climatic and soil gradients. This work has also demonstrated that at any one point along these gradients there is a large amount of interspecific trait variation. The present research proposes that this variation may be explained by the local-scale sorting of traits along soil fertility and acidity axes. Specifically, we predicted that trait values associated with high resource acquisition and growth rates would be found on soils that are more fertile and less acidic. We tested the expected relationships at the species-level and quadrat-level (20×20 m) using two large forest plots in Panama and China that contain over 450 species combined. Predicted relationships between leaf area and wood density and soil fertility were supported in some instances, but the majority of the predicted relationships were rejected. Alternative resource axes, such as light gradients, therefore likely play a larger role in determining the interspecific variability in plant functional traits in the two forests studied.

Bias and Correction in RNA-seq Data for Marine Species

RNA-seq is a recently developed approach widely used for transcriptome profiling in biological analyses that use next-generation sequencing technologies. Accurate estimation of gene expression levels is critical for answering biological questions. Here, we show that the commonly used measure of gene expression levels, fragments per kilobase of transcript per million mapped reads (FPKM), is biased in transcript length, GC content, and dinucleotide frequencies in the RNA-seq analysis of marine species. We used a generalized linear model to correct the observed biases of FPKM. We used RNA-seq data sets from eight species obtained by different sequencing methods to evaluate the correction methods. Our work contributes to the understanding of potential technical artifacts in RNA-seq experiments for marine species, and presents a means by which more accurate gene expression measures can be obtained.

Candidate Gene Polymorphisms and their Association with Glycogen Content in the Pacific Oyster Crassostrea gigas.

Background The Pacific oyster Crassostrea gigas is an important cultivated shellfish that is rich in nutrients. It contains high levels of glycogen, which is of high nutritional value. To investigate the genetic basis of this high glycogen content and its variation, we conducted a candidate gene association analysis using a wild population, and confirmed our results using an independent population, via targeted gene resequencing and mRNA expression analysis. Results We validated 295 SNPs in the 90 candidate genes surveyed for association with glycogen content, 86 of were ultimately genotyped in all 144 experimental individuals from Jiaonan (JN). In addition, 732 SNPs were genotyped via targeted gene resequencing. Two SNPs (Cg_SNP_TY202 and Cg_SNP_3021) in Cg_GD1 (glycogen debranching enzyme) and one SNP (Cg_SNP_4) in Cg_GP1 (glycogen phosphorylase) were identified as being associated with glycogen content. The glycogen content of individuals with genotypes TT and TC in Cg_SNP_TY202 was higher than that of individuals with genotype CC. The transcript abundance of both glycogen-associated genes was differentially expressed in high glycogen content and low glycogen content individuals. Conclusions This study identified three polymorphisms in two genes associated with oyster glycogen content, via candidate gene association analysis. The transcript abundance differences in Cg_GD1 and Cg_GP1 between low- and the high-glycogen content individuals suggests that it is possible that transcript regulation is mediated by variations of Cg_SNP_TY202, Cg_SNP_3021, and Cg_SNP_4. These findings will not only provide insights into the genetic basis of oyster quality, but also promote research into the molecular breeding of oysters.

The association between DNA methylation and exon expression in the Pacific oyster Crassostrea gigas

Background DNA methylation is one of the most important epigenetic modifications of eukaryotic genomes and is believed to play integral roles in diverse biological processes. Although DNA methylation has been well studied in mammals, data are limited in invertebrates, particularly Mollusca. The Pacific oyster Crassostrea gigas is an emerging genetic model for functional analysis of DNA methylation in Mollusca. Recent studies have shown that there is a positive association between methylation status and gene expression in C. gigas; however, whether this association exists at the exon level remains to be determined. Results In this study, we characterized the genome-wide methylation pattern across two different tissues of C. gigas and found that methylated genes are expressed in more tissues and development stages than unmethylated genes. Furthermore, we found that different types of exons had different methylation levels, with the lowest methylation levels in the first exons, followed by the last exons, and the internal exons. We found that the exons included in the gene transcript contained significantly higher DNA methylation levels than skipped exons. We observed that the DNA methylation levels increased slowly after the start sites and end sites of exons seperately, and then decreased quickly towards the middle sites of exons. We also found that methylated exons were significantly longer than unmethylated exons. Conclusion This study constitutes the first genome-wide analysis to show an association between exon-level DNA methylation and mRNA expression in the oyster. Our findings suggest that exon-level DNA methylation may play a role in the construction of alternative splicing by positively influencing exon inclusion during transcription.

Temperature, energy metabolism, and adaptive divergence in two oyster subspecies

Abstract Comparisons of related species that have diverse spatial distributions provide an efficient way to investigate adaptive evolution in face of increasing global warming. The oyster subjected to high environmental selections is a model species as sessile marine invertebrate. This study aimed to detect the adaptive divergence of energy metabolism in two oyster subspecies from the genus Crassostrea—C. gigas gigas and C. gigas angulata—which are broadly distributed along the northern and southern coasts of China, respectively. We examined the effects of acute thermal stress on energy metabolism in two oyster subspecies after being common gardened for one generation in identical conditions. Thermal responses were assessed by incorporating physiological, molecular, and genomic approaches. Southern oysters exhibited higher fluctuations in metabolic rate, activities of key energetic enzymes, and levels of thermally induced gene expression than northern oysters. For genes involved in energy metabolism, the former displayed higher basal levels of gene expression and a more pronounced downregulation of thermally induced expression, while the later exhibited lower basal levels and a less pronounced downregulation of gene expression. Contrary expression pattern was observed in oxidative stress gene. Besides, energy metabolic tradeoffs were detected in both subspecies. Furthermore, the genetic divergence of a nonsynonymous SNP (SOD‐132) and five synonymous SNPs in other genes was identified and validated in these two subspecies, which possibly affects downstream functions and explains the aforementioned phenotypic variations. Our study demonstrates that differentiations in energy metabolism underlie the plasticity of adaptive divergence in two oyster subspecies and suggest C. gigas angulata with moderate phenotypic plasticity has higher adaptive potential to cope with exacerbated global warming.

Coverage recommendation for genotyping analysis of highly heterologous species using next-generation sequencing technology

Next-generation sequencing (NGS) technology is being applied to an increasing number of non-model species and has been used as the primary approach for accurate genotyping in genetic and evolutionary studies. However, inferring genotypes from sequencing data is challenging, particularly for organisms with a high degree of heterozygosity. This is because genotype calls from sequencing data are often inaccurate due to low sequencing coverage, and if this is not accounted for, genotype uncertainty can lead to serious bias in downstream analyses, such as quantitative trait locus mapping and genome-wide association studies. Here, we used high-coverage reference data sets from Crassostrea gigas to simulate sequencing data with different coverage, and we evaluate the influence of genotype calling rate and accuracy as a function of coverage. Having initially identified the appropriate parameter settings for filtering to ensure genotype accuracy, we used two different single-nucleotide polymorphism (SNP) calling pipelines, single-sample and multi-sample. We found that a coverage of 15× was suitable for obtaining sufficient numbers of SNPs with high accuracy. Our work provides guidelines for the selection of sequence coverage when using NGS to investigate species with a high degree of heterozygosity and rapid decay of linkage disequilibrium.

Draft genome and SNPs associated with carotenoid accumulation in adductor muscles of bay scallop (Argopecten irradians)

Carotenoids are commonly deposited in the gonads of marine bivalves but rarely in their adductor muscles. An orange-adductor variant was identified in our breeding program for the bay scallop Argopecten irradians. In the present study, bay scallop genome survey sequencing was conducted, followed by genotyping by sequencing (GBS)-based case-control association analysis in a selfing family that exhibited segregation in adductor color. K-mer analysis (K=17) revealed that the bay scallop genome is about 990 Mb in length. De novo assembly produced 217,310 scaffold sequences, which provided 72.1% coverage of the whole genome and covered 72,187 transcripts, thereby yielding the most informative sequence resource for bay scallop to date. The average carotenoid content of the orange-adductor progenies was significantly higher than that of the white-adductor progenies. Thus, 20 individuals of each subgroup were sampled for case-control analysis. As many as 15,224 heterozygous loci were identified in the parent, among which 9280 were genotyped in at least 10 individuals of each of the two sub-groups. Association analysis indicated that 126 SNPs were associated with carotenoid accumulation in the adductor muscle and that 88 of these were significantly enriched on 28 scaffolds (FDR controlled P < 0.05). The SNPs and genes located on these scaffolds can serve as valuable candidates for further research into the mechanisms by which marine bivalves accumulate carotenoids in their adductor muscles.

Divergence and plasticity shape adaptive potential of the Pacific oyster

The interplay between divergence and phenotypic plasticity is critical to our understanding of a species’ adaptive potential under rapid climate changes. We investigated divergence and plasticity in natural populations of the Pacific oyster Crassostrea gigas with a congeneric oyster Crassostrea angulata from southern China used as an outgroup. Genome re-sequencing of 371 oysters revealed unexpected genetic divergence in a small area that coincided with phenotypic divergence in growth, physiology, heat tolerance and gene expression across environmental gradients. These findings suggest that selection and local adaptation are pervasive and, together with limited gene flow, influence population structure. Genes showing sequence differentiation between populations also diverged in transcriptional response to heat stress. Plasticity in gene expression is positively correlated with evolved divergence, indicating that plasticity is adaptive and favoured by organisms under dynamic environments. Divergence in heat tolerance—partly through acetylation-mediated energy depression—implies differentiation in adaptive potential. Trade-offs between growth and survival may play an important role in local adaptation of oysters and other marine invertebrates.Adaptive differentiation is poorly understood in marine systems. Here, the authors combine genome sequencing with gene expression and physiology to show that genetic divergence and plasticity contribute to local adaptation of Pacific oyster populations.

Relationship Among Intron Length, Gene Expression, and Nucleotide Diversity in the Pacific Oyster Crassostrea gigas

Crassostrea gigas is a model mollusk, but its genetic features have not been studied comprehensively. In this study, we used whole-genome resequencing data to identify and characterize nucleotide diversity and population recombination rate in a diverse collection of 21 C. gigas samples. Our analyses revealed that C. gigas harbors both extremely high genetic diversity and recombination rates across the whole genome as compared with those of the other taxa. The noncoding regions, introns, intergenic spacers, and untranslated regions (UTRs) showed a lower level diversity than the synonymous sites. The larger introns tended to have lower diversity. Moreover, we found a negative association of the non-synonymous diversity with gene expression, which suggested that purifying selection played an important role in shaping genetic diversity. The nucleotide diversity at the 100- and 50-kb levels was positively correlated with population recombination rates, which was expected if the diversity was shaped by purifying selection or hitchhiking of advantageous mutants. Our work gives a general picture of the oyster’s polymorphism pattern and its association with recombination rates.

Transcriptomics and Fitness Data Reveal Adaptive Plasticity of Thermal Tolerance in Oysters Inhabiting Different Tidal Zones

Fine-scale adaptive evolution is always constrained by strong gene flow at vertical level in marine organisms. Rapid environmental fluctuations and phenotypic plasticity through optimization of fitness-related traits in organisms play important roles in shaping intraspecific divergence. The coastal systems experience strong variations in multiple abiotic environmental factors, especially the temperature. We used a typical intertidal species, Pacific oyster (Crassostrea gigas), to investigate the interaction between plasticity and adaptive evolution. We collected intertidal and subtidal oysters from two ecological niches and carried out common garden experiments for one generation. We identified fine-scale vertical adaptive divergence between intertidal and subtidal F1 progeny at both sites, based on different hierarchical phenotypes, including morphological, physiological, and molecular traits. We further quantified the global plasticity to thermal stress through transcriptomic analysis. The intertidal oysters exhibited slow growth rate. However, they showed high survival and metabolic rates under heat stress, indicating vertically fine-scale phenotypic adaptive mechanisms and evolutionary trade-offs between growth and thermal tolerance. Transcriptomic analysis confirmed that the intertidal oysters have evolved high plasticity. The genes were classified into three types: evolutionarily divergent, concordantly plastic, and adaptive plastic genes. The evolved divergence between intertidal and subtidal oysters for these gene sets showed a significant positive correlation with plastic changes of subtidal populations in response to high temperature. Furthermore, the intertidal oysters exhibited delayed large-scale increase in expressional plasticity than that in subtidal counterparts. The same direction between plasticity and selection suggests that the oysters have evolved adaptive plasticity. This implies that adaptive plasticity facilitates the oyster to adapt to severe intertidal zones. The oysters exposed to strong environmental variability are thermal tolerant and have high adaptive potential to face the current global warming. Our findings will not only provide new insights into the significant role of plasticity in adaptive evolution that can be extended to other marine invertebrates, but also provide basic information for oyster resources conservation and reef reestablishment.