Z. Jeffrey Chen

Genomewide Nonadditive Gene Regulation in Arabidopsis Allotetraploids

Polyploidy has occurred throughout the evolutionary history of all eukaryotes and is extremely common in plants. Reunification of the evolutionarily divergent genomes in allopolyploids creates regulatory incompatibilities that must be reconciled. Here we report genomewide gene expression analysis of Arabidopsis synthetic allotetraploids, using spotted 70-mer oligo-gene microarrays. We detected >15% transcriptome divergence between the progenitors, and 2105 and 1818 genes were highly expressed in Arabidopsis thaliana and A. arenosa, respectively. Approximately 5.2% (1362) and 5.6% (1469) genes displayed expression divergence from the midparent value (MPV) in two independently derived synthetic allotetraploids, suggesting nonadditive gene regulation following interspecific hybridization. Remarkably, the majority of nonadditively expressed genes in the allotetraploids also display expression changes between the parents, indicating that transcriptome divergence is reconciled during allopolyploid formation. Moreover, >65% of the nonadditively expressed genes in the allotetraploids are repressed, and >94% of the repressed genes in the allotetraploids match the genes that are expressed at higher levels in A. thaliana than in A. arenosa, consistent with the silencing of A. thaliana rRNA genes subjected to nucleolar dominance and with overall suppression of the A. thaliana phenotype in the synthetic allotetraploids and natural A. suecica. The nonadditive gene regulation is involved in various biological pathways, and the changes in gene expression are developmentally regulated. In contrast to the small effects of genome doubling on gene regulation in autotetraploids, the combination of two divergent genomes in allotetraploids by interspecific hybridization induces genomewide nonadditive gene regulation, providing a molecular basis for de novo variation and allopolyploid evolution.

Sequencing of allotetraploid cotton ( Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement

Upland cotton is a model for polyploid crop domestication and transgenic improvement. Here we sequenced the allotetraploid Gossypium hirsutum L. acc. TM-1 genome by integrating whole-genome shotgun reads, bacterial artificial chromosome (BAC)-end sequences and genotype-by-sequencing genetic maps. We assembled and annotated 32,032 A-subgenome genes and 34,402 D-subgenome genes. Structural rearrangements, gene loss, disrupted genes and sequence divergence were more common in the A subgenome than in the D subgenome, suggesting asymmetric evolution. However, no genome-wide expression dominance was found between the subgenomes. Genomic signatures of selection and domestication are associated with positively selected genes (PSGs) for fiber improvement in the A subgenome and for stress tolerance in the D subgenome. This draft genome sequence provides a resource for engineering superior cotton lines.

Altered circadian rhythms regulate growth vigour in hybrids and allopolyploids.

Segregating hybrids and stable allopolyploids display morphological vigour, and Arabidopsis allotetraploids are larger than the parents Arabidopsis thaliana and Arabidopsis arenosa—the mechanisms for this are unknown. Circadian clocks mediate metabolic pathways and increase fitness in animals and plants. Here we report that epigenetic modifications of the circadian clock genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and their reciprocal regulators TIMING OF CAB EXPRESSION 1 (TOC1) and GIGANTEA (GI) mediate expression changes in downstream genes and pathways. During the day, epigenetic repression of CCA1 and LHY induced the expression of TOC1, GI and downstream genes containing evening elements in chlorophyll and starch metabolic pathways in allotetraploids and F1 hybrids, which produced more chlorophyll and starch than the parents in the same environment. Mutations in cca1 and cca1 lhy and the daily repression of cca1 by RNA interference (RNAi) in TOC1::cca1(RNAi) transgenic plants increased the expression of downstream genes and increased chlorophyll and starch content, whereas constitutively expressing CCA1 or ectopically expressing TOC1::CCA1 had the opposite effect. The causal effects of CCA1 on output traits suggest that hybrids and allopolyploids gain advantages from the control of circadian-mediated physiological and metabolic pathways, leading to growth vigour and increased biomass.

Molecular mechanisms of polyploidy and hybrid vigor

Hybrids such as maize (Zea mays) or domestic dog (Canis lupus familiaris) grow bigger and stronger than their parents. This is also true for allopolyploids such as wheat (Triticum spp.) or frog (i.e. Xenopus and Silurana) that contain two or more sets of chromosomes from different species. The phenomenon, known as hybrid vigor or heterosis, was systematically characterized by Charles Darwin (1876). The rediscovery of heterosis in maize a century ago has revolutionized plant and animal breeding and production. Although genetic models for heterosis have been rigorously tested, the molecular bases remain elusive. Recent studies have determined the roles of nonadditive gene expression, small RNAs, and epigenetic regulation, including circadian-mediated metabolic pathways, in hybrid vigor, which could lead to better use and exploitation of the increased biomass and yield in hybrids and allopolyploids for food, feed, and biofuels.

Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids.

Small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs), control gene expression and epigenetic regulation. Although the roles of miRNAs and siRNAs have been extensively studied, their expression diversity and evolution in closely related species and interspecific hybrids are poorly understood. Here, we show comprehensive analyses of miRNA expression and siRNA distributions in two closely related species Arabidopsis thaliana and Arabidopsis arenosa, a natural allotetraploid Arabidopsis suecica, and two resynthesized allotetraploid lines (F1 and F7) derived from A. thaliana and A. arenosa. We found that repeat- and transposon-associated siRNAs were highly divergent between A. thaliana and A. arenosa. A. thaliana siRNA populations underwent rapid changes in F1 but were stably maintained in F7 and A. suecica. The correlation between siRNAs and nonadditive gene expression in allopolyploids is insignificant. In contrast, miRNA and tasiRNA sequences were conserved between species, but their expression patterns were highly variable between the allotetraploids and their progenitors. Many miRNAs tested were nonadditively expressed (deviating from the mid-parent value, MPV) in the allotetraploids and triggered unequal degradation of A. thaliana or A. arenosa targets. The data suggest that small RNAs produced during interspecific hybridization or polyploidization serve as a buffer against the genomic shock in interspecific hybrids and allopolyploids: Stable inheritance of repeat-associated siRNAs maintains chromatin and genome stability, whereas expression variation of miRNAs leads to changes in gene expression, growth vigor, and adaptation.

Toward Sequencing Cotton (Gossypium) Genomes

Despite rapidly decreasing costs and innovative technologies, sequencing of angiosperm genomes is not yet undertaken lightly. Generating larger amounts of sequence data more quickly does not address the difficulties of sequencing and assembling complex genomes de novo. The cotton ( Gossypium spp.)

Genomic and epigenetic insights into the molecular bases of heterosis

Heterosis, also known as hybrid vigour, is widespread in plants and animals, but the molecular bases for this phenomenon remain elusive. Recent studies in hybrids and allopolyploids using transcriptomic, proteomic, metabolomic, epigenomic and systems biology approaches have provided new insights. Emerging genomic and epigenetic perspectives suggest that heterosis arises from allelic interactions between parental genomes, leading to altered programming of genes that promote the growth, stress tolerance and fitness of hybrids. For example, epigenetic modifications of key regulatory genes in hybrids and allopolyploids can alter complex regulatory networks of physiology and metabolism, thus modulating biomass and leading to heterosis. The conceptual advances could help to improve plant and animal productivity through the manipulation of heterosis.

Genomic and Expression Plasticity of Polyploidy

Polyploidy or whole genome duplication (WGD) occurs throughout the evolutionary history of many plants and some animals, including crops such as wheat, cotton, and sugarcane. Recent studies have documented rapid and dynamic changes in genomic structure and gene expression in plant polyploids, which reflects genomic and functional plasticity of duplicate genes and genomes in plants. Common features of uniparental gene regulation and nonadditive gene expression in regulatory pathways responsive to growth, development, and stresses in many polyploids have led to the conclusion that epigenetic mechanisms including chromatin modifications and small RNAs play central roles in shaping molecular and phenotypic novelty that may be selected and domesticated in many polyploid plants and crops.

Evolution and Expression of Homeologous Loci in Tragopogon miscellus (Asteraceae), a Recent and Reciprocally Formed Allopolyploid

On both recent and ancient time scales, polyploidy (genome doubling) has been a significant evolutionary force in plants. Here, we examined multiple individuals from reciprocally formed populations of Tragopogon miscellus, an allotetraploid that formed repeatedly within the last 80 years from the diploids T. dubius and T. pratensis. Using cDNA–AFLPs followed by genomic and cDNA cleaved amplified polymorphic sequence (CAPS) analyses, we found differences in the evolution and expression of homeologous loci in T. miscellus. Fragment variation within T. miscellus, possibly attributable to reciprocal formation, comprised 0.6% of the cDNA–AFLP bands. Genomic and cDNA CAPS analyses of 10 candidate genes revealed that only one “transcript-derived fragment” (TDF44) showed differential expression of parental homeologs in T. miscellus; the T. pratensis homeolog was preferentially expressed by most polyploids in both populations. Most of the cDNA–AFLP polymorphisms apparently resulted from loss of parental fragments in the polyploids. Importantly, changes at the genomic level have occurred stochastically among individuals within the independently formed populations. Synthetic F1 hybrids between putative diploid progenitors are additive of their parental genomes, suggesting that polyploidization rather than hybridization induces genomic changes in Tragopogon.

Genome-wide analysis reveals rapid and dynamic changes in miRNA and siRNA sequence and expression during ovule and fiber development in allotetraploid cotton (Gossypium hirsutum L.)

BackgroundCotton fiber development undergoes rapid and dynamic changes in a single cell type, from fiber initiation, elongation, primary and secondary wall biosynthesis, to fiber maturation. Previous studies showed that cotton genes encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs) that mediate target gene regulation by mRNA degradation or translational repression.ResultsHere we sequenced and analyzed over 4 million small RNAs derived from fiber and non-fiber tissues in cotton. The 24-nucleotide small interfering RNAs (siRNAs) were more abundant and highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 31 miRNA families, including 27 conserved, 4 novel miRNA families and a candidate-novel miRNA, were identified in at least one of the cotton tissues examined. Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study. Sequencing, miRNA microarray, and small RNA blot analyses showed a trend of repression of miRNAs, including novel miRNAs, during ovule and fiber development, which correlated with upregulation of several target genes tested. Moreover, 223 targets of cotton miRNAs were predicted from the expressed sequence tags derived from cotton tissues, including ovules and fibers. The cotton miRNAs examined triggered cleavage in the predicted sites of the putative cotton targets in ovules and fibers.ConclusionsEnrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers. Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.