Increased expression diversity buffers the loss of adaptive potential caused by reduction of genetic diversity in new unfavourable environments.

Mechanisms underlying adaptation to rapid environmental change are issues in evolutionary biology. It is widely accepted that reduction in genetic diversity when suddenly exposed to an unfavourable environment limits the adaptive potential of populations. With growing empirical evidence that expression diversity is likely to increase in the new environment, the role that expression diversity plays in adaptation needs to be theorized. Here, we first established a negative exponential relationship between expression diversity and genetic diversity using a phenomenological differential equation. We then derived a complex trade-off relationship between the changes of expression and genetic diversity, which followed a combination of exponential functions. Furthermore, we found the increase in expression diversity could buffer the loss of adaptive potential as genetic diversity decreased to a certain extent. These theoretical deductions were validated by transcriptomic data of Miscanthus lutarioriparius grown in two experimental fields and supported by good fit and random simulation. These results suggest that increased expression diversity may compensate the loss of genetic diversity and allow the populations to maintain a certain level of phenotypic variation to cope with sudden environmental change. This may buffer the quick diminishing of adaptive potential and consequently increases the change of adaptation to the new environment.

Coexpression network revealing the plasticity and robustness of population transcriptome during the initial stage of domesticating energy crop Miscanthus lutarioriparius.

KEY MESSAGE: Coexpression network revealing genes with Co-variation Expression pattern (CE) and those with Top rank of Expression fold change (TE) played different roles in responding to new environment of Miscanthus lutarioriparius. Variation in gene expression level, the product of genetic and/or environmental perturbation, determines the robustness-to-plasticity spectrum of a phenotype in plants. Understanding how expression variation of plant population response to a new field is crucial to domesticate energy crops. Weighted Gene Coexpression Network Analysis (WGCNA) was used to explore the patterns of expression variation based on 72 Miscanthus lutarioriparius transcriptomes from two contrasting environments, one near the native habitat and the other in one harsh domesticating region. The 932 genes with Co-variation Expression pattern (CE) and other 932 genes with Top rank of Expression fold change (TE) were identified and the former were strongly associated with the water use efficiency (r ≥ 0.55, P ≤ 10-7). Functional enrichment of CE genes were related to three organelles, which well matched the annotation of twelve motifs identified from their conserved noncoding sequence; while TE genes were mostly related to biotic and/or abiotic stress. The expression robustness of CE genes with high genetic diversity kept relatively stable between environments while the harsh environment reduced the expression robustness of TE genes with low genetic diversity. The expression plasticity of CE genes was increased less than that of TE genes. These results suggested that expression variation of CE genes and TE genes could account for the robustness and plasticity of acclimation ability of Miscanthus, respectively. The patterns of expression variation revealed by transcriptomic network would shed new light on breeding and domestication of energy crops.

Systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change.

BACKGROUND: Long non-coding RNA (lncRNA) is a class of non-coding RNA with important regulatory roles in biological process of organisms. The systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change are still poorly understood. Here we identified 17,610 lncRNAs and calculated their expression levels based on RNA-seq of 80 individuals of Miscanthus lutarioriparius from two environments, the nearly native habitats and transplanted field, respectively. RESULTS: LncRNAs had significantly higher expression diversity and lower expression frequency in population than protein coding mRNAs in both environments, which suggested that lncRNAs may experience more relaxed selection or divergent evolution in population compared with protein coding RNAs. In addition, the increase of expression diversity for lncRNAs was always significantly higher and the magnitude of fold change of expression in new stress environment was significantly larger than protein-coding mRNAs. These results suggested that lncRNAs may be more sensitive to environmental change than protein-coding mRNAs. Analysis of environment-robust and environment-specific lncRNA-mRNA co-expression network between two environments revealed the characterization of lncRNAs in response to environmental change. Furthermore, candidate lncRNAs contributing to water use efficiency (WUE) identified based on the WUE-lncRNA-mRNA co-expression network suggested the roles of lncRNAs in response to environmental change. CONCLUSION: Our study provided a comprehensive understanding of expression characterization of lncRNAs in population for M. lutarioriparius under field condition, which would be useful to explore the roles of lncRNAs and could accelerate the process of adaptation in new environment for many plants.

Population transcriptomic characterization of the genetic and expression variation of a candidate progenitor of Miscanthus energy crops.

The use of transcriptome data in the study of the population genetics of a species can capture faint signals of both genetic variation and expression variation and can provide a broad picture of a species' genomic response to environmental conditions. In this study, we characterized the genetic and expression diversity of Miscanthus lutarioriparius by comparing more than 16,225 transcripts obtained from 78 individuals, belonging to 10 populations distributed across the species' entire geographic range. We only observed a low level of nucleotide diversity (π = 0.000434) among the transcriptome data of these populations, which is consistent with highly conserved sequences of functional elements and protein-coding genes captured with this method. Tests of population divergence using the transcriptome data were consistent with previous microsatellite data but proved to be more sensitive, particularly if gene expression variation was considered as well. For example, the analysis of expression data showed that genes involved in photosynthetic processes and responses to temperature or reactive oxygen species stimuli were significantly enriched in certain populations. This differential gene expression was primarily observed among populations and not within populations. Interestingly, nucleotide diversity was significantly negatively correlated with expression diversity within populations, while this correlation was positive among populations. This suggests that genetic and expression variation play separate roles in adaptation and population persistence. Combining analyses of genetic and gene expression variation represents a promising approach for studying the population genetics of wild species and may uncover both adaptive and nonadaptive processes.

An-1 encodes a basic helix-loop-helix protein that regulates awn development, grain size, and grain number in rice.

Long awns are important for seed dispersal in wild rice (Oryza rufipogon), but are absent in cultivated rice (Oryza sativa). The genetic mechanism involved in loss-of-awn in cultivated rice remains unknown. We report here the molecular cloning of a major quantitative trait locus, An-1, which regulates long awn formation in O. rufipogon. An-1 encodes a basic helix-loop-helix protein, which regulates cell division. The nearly-isogenic line (NIL-An-1) carrying a wild allele An-1 in the genetic background of the awnless indica Guangluai4 produces long awns and longer grains, but significantly fewer grains per panicle compared with Guangluai4. Transgenic studies confirmed that An-1 positively regulates awn elongation, but negatively regulates grain number per panicle. Genetic variations in the An-1 locus were found to be associated with awn loss in cultivated rice. Population genetic analysis of wild and cultivated rice showed a significant reduction in nucleotide diversity of the An-1 locus in rice cultivars, suggesting that the An-1 locus was a major target for artificial selection. Thus, we propose that awn loss was favored and strongly selected by humans, as genetic variations at the An-1 locus that cause awn loss would increase grain numbers and subsequently improve grain yield in cultivated rice.

Transcriptome-wide characterization of candidate genes for improving the water use efficiency of energy crops grown on semiarid land.

Understanding the genetic basis of water use efficiency (WUE) and its roles in plant adaptation to a drought environment is essential for the production of second-generation energy crops in water-deficit marginal land. In this study, RNA-Seq and WUE measurements were performed for 78 individuals of Miscanthus lutarioriparius grown in two common gardens, one located in warm and wet Central China near the native habitats of the species and the other located in the semiarid Loess Plateau, the domestication site of the energy crop. The field measurements showed that WUE of M. lutarioriparius in the semiarid location was significantly higher than that in the wet location. A matrix correlation analysis was conducted between gene expression levels and WUE to identify candidate genes involved in the improvement of WUE from the native to the domestication site. A total of 48 candidate genes were identified and assigned to functional categories, including photosynthesis, stomatal regulation, protein metabolism, and abiotic stress responses. Of these genes, nearly 73% were up-regulated in the semiarid site. It was also found that the relatively high expression variation of the WUE-related genes was affected to a larger extent by environment than by genetic variation. The study demonstrates that transcriptome-wide correlation between physiological phenotypes and expression levels offers an effective means for identifying candidate genes involved in the adaptation to environmental changes.

Genetic control of seed shattering in rice by the APETALA2 transcription factor shattering abortion1.

Seed shattering is an important agricultural trait in crop domestication. SH4 (for grain shattering quantitative trait locus on chromosome 4) and qSH1 (for quantitative trait locus of seed shattering on chromosome 1) genes have been identified as required for reduced seed shattering during rice (Oryza sativa) domestication. However, the regulatory pathways of seed shattering in rice remain unknown. Here, we identified a seed shattering abortion1 (shat1) mutant in a wild rice introgression line. The SHAT1 gene, which encodes an APETALA2 transcription factor, is required for seed shattering through specifying abscission zone (AZ) development in rice. Genetic analyses revealed that the expression of SHAT1 in AZ was positively regulated by the trihelix transcription factor SH4. We also identified a frameshift mutant of SH4 that completely eliminated AZs and showed nonshattering. Our results suggest a genetic model in which the persistent and concentrated expression of active SHAT1 and SH4 in the AZ during early spikelet developmental stages is required for conferring AZ identification. qSH1 functioned downstream of SHAT1 and SH4, through maintaining SHAT1 and SH4 expression in AZ, thus promoting AZ differentiation.

Population transcriptomics reveals a potentially positive role of expression diversity in adaptation.

While it is widely accepted that genetic diversity determines the potential of adaptation, the role that gene expression variation plays in adaptation remains poorly known. Here we show that gene expression diversity could have played a positive role in the adaptation of Miscanthus lutarioriparius. RNA-seq was conducted for 80 individuals of the species, with half planted in the energy crop domestication site and the other half planted in the control site near native habitats. A leaf reference transcriptome consisting of 18,503 high-quality transcripts was obtained using a pipeline developed for de novo assembling with population RNA-seq data. The population structure and genetic diversity of M. lutarioriparius were estimated based on 30,609 genic single nucleotide polymorphisms. Population expression (Ep ) and expression diversity (Ed ) were defined to measure the average level and the magnitude of variation of a gene expression in the population, respectively. It was found that expression diversity increased while genetic diversity decreased after the species was transplanted from the native habitats to the harsh domestication site, especially for genes involved in abiotic stress resistance, histone methylation, and biomass synthesis under water limitation. The increased expression diversity could have enriched phenotypic variation directly subject to selections in the new environment.

Multiple species of wild tree peonies gave rise to the 'king of flowers', Paeonia suffruticosa Andrews.

The origin of cultivated tree peonies, known as the 'king of flowers' in China for more than 1000 years, has attracted considerable interest, but remained unsolved. Here, we conducted phylogenetic analyses of explicitly sampled traditional cultivars of tree peonies and all wild species from the shrubby section Moutan of the genus Paeonia based on sequences of 14 fast-evolved chloroplast regions and 25 presumably single-copy nuclear markers identified from RNA-seq data. The phylogeny of the wild species inferred from the nuclear markers was fully resolved and largely congruent with morphology and classification. The incongruence between the nuclear and chloroplast trees suggested that there had been gene flow between the wild species. The comparison of nuclear and chloroplast phylogenies including cultivars showed that the cultivated tree peonies originated from homoploid hybridization among five wild species. Since the origin, thousands of cultivated varieties have spread worldwide, whereas four parental species are currently endangered or on the verge of extinction. The documentation of extensive homoploid hybridization involved in tree peony domestication provides new insights into the mechanisms underlying the origins of garden ornamentals and the way of preserving natural genetic resources through domestication.

Hollow fiber-based Fabry-Perot cavity for liquid surface tension measurement.

A Fabry-Perot (FP) cavity, formed by a single-mode fiber (SMF) fusion spliced with a section of hollow fiber, was proposed and experimentally used as a sensor for liquid surface tension measurement. Due to the FP cavity length changing with different liquids, the surface tension can be measured by monitoring the interference wavelength spacing. In our experiment, five kinds of liquids were measured, and the relationship between wavelength spacing and liquid surface tension was obtained. The experimental results show that the achieved sensitivity to liquid surface tension is 30.96 nm/(N/m), and the maximum error is 5.43%.

Genetic control of a transition from black to straw-white seed hull in rice domestication.

The genetic mechanism involved in a transition from the black-colored seed hull of the ancestral wild rice (Oryza rufipogon and Oryza nivara) to the straw-white seed hull of cultivated rice (Oryza sativa) during grain ripening remains unknown. We report that the black hull of O. rufipogon was controlled by the Black hull4 (Bh4) gene, which was fine-mapped to an 8.8-kb region on rice chromosome 4 using a cross between O. rufipogon W1943 (black hull) and O. sativa indica cv Guangluai 4 (straw-white hull). Bh4 encodes an amino acid transporter. A 22-bp deletion within exon 3 of the bh4 variant disrupted the Bh4 function, leading to the straw-white hull in cultivated rice. Transgenic study indicated that Bh4 could restore the black pigment on hulls in cv Guangluai 4 and Kasalath. Bh4 sequence alignment of all taxa with the outgroup Oryza barthii showed that the wild rice maintained comparable levels of nucleotide diversity that were about 70 times higher than those in the cultivated rice. The results from the maximum likelihood Hudson-Kreitman-Aguade test suggested that the significant reduction in nucleotide diversity in rice cultivars could be caused by artificial selection. We propose that the straw-white hull was selected as an important visual phenotype of nonshattered grains during rice domestication.

Study on Combustion and Emissions of a Combined Injection Spark Ignition Engine with Natural Gas Direct Injection Plus Ethanol Port Injection under Lean-Burn Conditions.

Conventional ethanol spark ignition (SI) engines have poor fuel atomization and mixture formation. The objective of this paper is to improve the combustion and emission performance of ethanol SI engines under lean-burn conditions through the dual-injection mode with ethanol port injection and compressed natural gas (CNG) direct injection (CDI+EPI). This paper studies the engine performance at 1500 rpm under five CNG direct injection ratios (CDIr) and five excess air ratios (λ). The results show that as the CDIr increases under lean-burn conditions, the following occurs: the minimum advance for best torque (MBT), the coefficient of variation (CoVIMEP), and CO and HC emissions decrease; the crankshaft rotation or time with cumulative heat release rate ranging from 10% to 90% (CA 10-90) and NOx emissions first decrease and then increase; and torque, peak in-cylinder pressure (Pmax), and the λ limit first increase and then decrease. The larger the CDIr is, the less influence λ has on the MBT. When CDIr = 15%, the CoVIMEP can be effectively reduced, the engine can still work stably in all lean-burn conditions, and the λ limit will reach the maximum value of 1.73, 19.31% higher than that of the original engine (CDIr = 0). When λ = 1.1, CO emissions decrease the most and HC emissions decrease the least. At this time, CO and HC emissions decrease by 1.56 vol % and 30 ppm, respectively, on average for every 0.1 decrease in λ. For CA 10-90, torque, and Pmax, λ = 1.1, 15% CDI, and 85% EPI is the optimal combination under lean-burn conditions. When CDIr ≥ 15%, NOx emissions are at an ideal level. Under lean-burn conditions, direct-injection CNG can form a good stratified natural gas/ethanol mixture in the cylinder, effectively improving the engine's power and stability and reducing emissions. The λ = 1.1, 15% CDI, 85% EPI combination provides a cutting-edge and outstanding solution for a natural gas/ethanol combined injection SI engine.

Development of energy plants from hybrids between Miscanthus sacchariflorus and M. lutarioriparius grown on reclaimed mine land in the Loess Plateau of China.

Miscanthus, a promising bioenergy plant, has a high biomass yield with high cellulose content suitable for biofuel production. However, harsh climatic and poor soil conditions, such as barren lands or abandoned mines, pose a challenge to the survival and yield of Miscanthus feedstock on the marginal land. The selection from the interspecific hybrids of Miscanthus might combine high survival rates and high yield, which benefits energy crop development in multi-stressful environments. A total of 113 F1 hybrids between Miscanthus sacchariflorus and M. lutarioriparius together with the parents were planted and evaluated for multiple morphological and physiological traits on the mine land of the Loess Plateau of China. The majority of hybrids had higher establishment rates than M. sacchariflorus while M. lutarioriparius failed to survive for the first winter. Nearly all hybrid genotypes outperformed M. lutarioriparius for yield-related traits including plant height, tiller number, tiller diameter, and leaf area. The average biomass of the hybrids was 20 times higher than that of surviving parent, M. sacchariflorus. Furthermore, the photosynthetic rates and water use efficiency of the hybrids were both significantly higher than those of the parents, which might be partly responsible for their higher yield. A total of 29 hybrids with outstanding traits related to yield and stress tolerance were identified as candidates. The study investigated for the first time the hybrids between local individuals of M. sacchariflorus and high-biomass M. lutarioriparius, suggesting that this could be an effective approach for high-yield energy crop development on vast of marginal lands.

Transcriptomic Characterization of Miscanthus sacchariflorus × M. lutarioriparius and Its Implications for Energy Crop Development in the Semiarid Mine Area.

Miscanthus interspecific hybrids have been proved to have better adaptability in marginal lands than their parents. Miscanthus sacchariflorus and Miscanthus lutarioriparius were used as the parents to develop hybrids. We performed the transcriptome for 110 F1 hybrids of Miscanthus sacchariflorus × Miscanthus lutarioriparius and their parents that had been established on the Loess Plateau mine area, to estimate the population's genetic expression variation, and illuminate the adaptive mechanism of the F1 population. The result speculated that the F1 population has mainly inherited the stress response metabolic pathway of its female parent (M. sacchariflorus), which may be responsible for its higher environmental adaptability and biomass yield compared with male parents. Based on PopART, we assembled a leaf reference transcriptome for M. sacchariflorus (LRTMS) and obtained 8116 high-quality transcripts. When we analyze the differential expression of genes between F1 population and parent, 39 and 56 differentially expressed genes were screened out in the female parent and male parent, respectively. The enrichment analysis showed that pathways of carbohydrate metabolism, lipid metabolism, biosynthesis of secondary metabolites and circadian rhythm-plant played a key role in resisting the harsh environment. The carbohydrate metabolism and lipid metabolism were also significantly enriched, and the synthesis of these substances facilitated the yield. The results provided an insight into breeding Miscanthus hybrids more suited to the harsh environment of the Loess Plateau.

Genetics and phylogenetics of rice domestication.

With genetically divergent cultivars and ecologically distinct wild progenitors, rice has posed a great challenge to the genetic and phylogenetic studies of the origin and evolution of crop species. A growing body of phylogenetic evidence suggested that the diverged genomic backgrounds of indica and japonica rice cultivars were derived independently from genetically distinct wild populations. However, a domestication gene, sh4, which was responsible for the reduction of grain shattering, seems to have originated only once, and it is now fixed in both cultivars. Two models have been proposed to reconcile these data. Whereas the 'combination model' emphasizes the importance of early introgression between independently domesticated cultivars, the 'snowballing model' emphasizes the importance of introgression from local populations of wild species into an ancestral domesticated population. In either case, the domestication of rice was a dynamic process.

Mapping 49 quantitative trait loci at high resolution through sequencing-based genotyping of rice recombinant inbred lines.

Mapping chromosome regions responsible for quantitative phenotypic variation in recombinant populations provides an effective means to characterize the genetic basis of complex traits. We conducted a quantitative trait loci (QTL) analysis of 150 rice recombinant inbred lines (RILs) derived from a cross between two cultivars, Oryza sativa ssp. indica cv. 93-11 and Oryza sativa ssp. japonica cv. Nipponbare. The RILs were genotyped through next-generation sequencing, which accurately determined the recombination breakpoints and provided a new type of genetic markers, recombination bins, for QTL analysis. We detected 49 QTL with phenotypic effect ranging from 3.2 to 46.0% for 14 agronomics traits. Five QTL of relatively large effect (14.6-46.0%) were located on small genomic regions, where strong candidate genes were found. The analysis using sequencing-based genotyping thus offers a powerful solution to map QTL with high resolution. Moreover, the RILs developed in this study serve as an excellent system for mapping and studying genetic basis of agricultural and biological traits of rice.

Toward the domestication of lignocellulosic energy crops: learning from food crop domestication.

Domestication of cereal crops has provided a stable source of food for thousands of years. The extent to which lignocellulosic crops will contribute to the world's renewable energy depends largely on how the new crops will be domesticated. Growing miscanthus as biofuel feedstocks on marginal and degraded land in northern and northwestern China offers an example for developing theoretical framework and practical strategies for energy crop domestication. The domestication should incorporate the highest possible genetic diversity from wild species, focus on the improvement of drought and cold tolerance especially in the stage of crop establishment, increase the efficiencies of water and nutrient uses and photosynthesis, adjust vegetative growing season according to local temperature and precipitation, and reduce or prevent seed production. Positive ecological effects on soil conservation, landscape restoration, carbon sequestration, and hydrological cycles should be maximized, while negative impact on biodiversity needs to be minimized. With the development of other sources of renewable energy, the role of lignocellulosic crops may evolve from primarily energy production to increasingly ecological restoration and biomaterial development. The integration of this new cropping system into the existing agriculture may open a new avenue to the long-term sustainability of our society.

Water Use Efficiency and Stress Tolerance of the Potential Energy Crop Miscanthus lutarioriparius Grown on the Loess Plateau of China.

As a potential energy crop with high biomass yield, Miscanthus lutarioriparius (M. lutarioriparius), endemic to the Long River Range in central China, needs to be investigated for its acclimation to stressful climatic and soil conditions often found on the marginal land. In this study, traits related to acclimation and yield, including survival rates, plant height (PH), stem diameter (SD), tiller number (TN), water use efficiency (WUE), and photosynthetic rates (A), were examined for 41 M. lutarioriparius populations that transplanted to the arid and cold Loess Plateau of China. The results showed that the average survival rate of M. lutarioriparius populations was only 4.16% over the first winter but the overwinter rate increased to 35.03% after the second winter, suggesting that plants having survived the first winter could have acclaimed to the low temperature. The strikingly high survival rates over the second winter were found to be 95.83% and 80.85%, respectively, for HG18 and HG39 populations. These populations might be especially valuable for the selection of energy crops for such an area. Those individuals surviving for the two consecutive winters showed significantly higher WUE than those measured after the first winter. The high WUE and low stomatal conductance (gs) observed in survived individuals could have been responsible for their acclimation to this new and harsh environment. A total of 61 individuals with productive growth traits and strong resistance to cold and drought were identified for further energy crop development. This study showed that the variation of M. lutarioriparius held great potential for developing energy crops following continuous field selection.

Chromosome-scale assembly and analysis of biomass crop Miscanthus lutarioriparius genome.

Miscanthus, a rhizomatous perennial plant, has great potential for bioenergy production for its high biomass and stress tolerance. We report a chromosome-scale assembly of Miscanthus lutarioriparius genome by combining Oxford Nanopore sequencing and Hi-C technologies. The 2.07-Gb assembly covers 96.64% of the genome, with contig N50 of 1.71 Mb. The centromere and telomere sequences are assembled for all 19 chromosomes and chromosome 10, respectively. Allotetraploid origin of the M. lutarioriparius is confirmed using centromeric satellite repeats. The tetraploid genome structure and several chromosomal rearrangements relative to sorghum are clearly demonstrated. Tandem duplicate genes of M. lutarioriparius are functional enriched not only in terms related to stress response, but cell wall biosynthesis. Gene families related to disease resistance, cell wall biosynthesis and metal ion transport are greatly expanded and evolved. The expansion of these families may be an important genomic basis for the enhancement of remarkable traits of M. lutarioriparius.

Selection on grain shattering genes and rates of rice domestication.

Molecular cloning of major quantitative trait loci (QTLs) responsible for the reduction of rice grain shattering, a hallmark of cereal domestication, provided opportunities for in-depth investigation of domestication processes. Here, we studied nucleotide variation at the shattering loci, sh4 and qSH1, for cultivated rice, Oryza sativa ssp. indica and Oryza sativa ssp. japonica, and the wild progenitors, Oryza nivara andOryza rufipogon. The nonshattering sh4 allele was fixed in all rice cultivars, with levels of sequence polymorphism significantly reduced in both indica and japonica cultivars relative to the wild progenitors. The sh4 phylogeny together with the neutrality tests and coalescent simulations suggested that sh4 had a single origin and was fixed by artificial selection during the domestication of rice. Selection on qSH1 was not detected in indica and remained unclear in japonica. Selection on sh4 could be strong enough to have driven its fixation in a population of cultivated rice within a period of c. 100 yr. The slow fixation of the nonshattering phenotype observed at the archeological sites might be a result of relatively weak selection on mutations other than sh4 in early rice cultivation. The fixation of sh4 could have been achieved later through strong selection for the optimal phenotype.