Paleopolyploidies and Genomic Fractionation in Major Eudicot Clades.

Eudicots account for ~75% of living angiosperms, containing important food and energy crops. Recently, high-quality genome sequences of several eudicots including Aquilegia coerulea and Nelumbo nucifera have become available, providing an opportunity to investigate the early evolutionary characteristics of eudicots. We performed genomic hierarchical and event-related alignments to infer homology within and between representative species of eudicots. The results provide strong evidence for multiple independent polyploidization events during the early diversification of eudicots, three of which are likely to be allopolyploids: The core eudicot-common hexaploidy (ECH), Nelumbo-specific tetraploidy (NST), and Ranunculales-common tetraploidy (RCT). Using different genomes as references, we constructed genomic alignment to list the orthologous and paralogous genes produced by polyploidization and speciation. This could provide a fundamental framework for studying other eudicot genomes and gene(s) evolution. Further, we revealed significantly divergent evolutionary rates among these species. By performing evolutionary rate correction, we dated RCT to be ~118-134 million years ago (Mya), after Ranunculales diverged with core eudicots at ~123-139 Mya. Moreover, we characterized genomic fractionation resulting from gene loss and retention after polyploidizations. Notably, we revealed a high degree of divergence between subgenomes. In particular, synonymous nucleotide substitutions at synonymous sites (Ks) and phylogenomic analyses implied that A. coerulea might provide the subgenome(s) for the gamma-hexaploid hybridization.

Triplication is the main evolutionary driving force of NLP transcription factor family in Chinese cabbage and related species.

The NODULE-INCEPTION-like protein (NLP) is a plant-specific transcription factor (TF) family that plays an important role in both signal transduction and nitrate assimilation. However, the NLP gene family in Chinese cabbage (Brassica rapa) has yet to be studied. Here we identified 17, 16, and 32 NLP genes in Chinese cabbage, Brassica oleracea, and Brassica napus, respectively. We found that duplication of those NLP genes almost always originated from genome-wide duplication events. Further analysis (using Arabidopsis as a reference) revealed that the NLP family in Chinese cabbage and B. oleracea was characterized by direct expansion caused by whole-genome duplication. By contrast, indirect expansion characterized B. napus, which arose from hybridization and fusion of the two species. In addition, phylogenetic and homology analyses showed that the Brassica NLP gene family has been highly conserved in evolution. Finally, we also identified optimal codons for four studied species. Altogether, through comparative genome analysis methods, we presented compelling evidence that triplication is the main driving force for the NLP TF family's evolution in Chinese cabbage and related Brassica plants, a process evidently highly conserved. This work will help in better understanding the impact of genome-wide duplication on gene families of plants.

Illegitimate Recombination between Duplicated Genes Generated from Recursive Polyploidizations Accelerated the Divergence of the Genus Arachis.

The peanut (Arachis hypogaea L.) is the leading oil and food crop among the legume family. Extensive duplicate gene pairs generated from recursive polyploidizations with high sequence similarity could result from gene conversion, caused by illegitimate DNA recombination. Here, through synteny-based comparisons of two diploid and three tetraploid peanut genomes, we identified the duplicated genes generated from legume common tetraploidy (LCT) and peanut recent allo-tetraploidy (PRT) within genomes. In each peanut genome (or subgenomes), we inferred that 6.8-13.1% of LCT-related and 11.3-16.5% of PRT-related duplicates were affected by gene conversion, in which the LCT-related duplicates were the most affected by partial gene conversion, whereas the PRT-related duplicates were the most affected by whole gene conversion. Notably, we observed the conversion between duplicates as the long-lasting contribution of polyploidizations accelerated the divergence of different Arachis genomes. Moreover, we found that the converted duplicates are unevenly distributed across the chromosomes and are more often near the ends of the chromosomes in each genome. We also confirmed that well-preserved homoeologous chromosome regions may facilitate duplicates' conversion. In addition, we found that these biological functions contain a higher number of preferentially converted genes, such as catalytic activity-related genes. We identified specific domains that are involved in converted genes, implying that conversions are associated with important traits of peanut growth and development.

Polyploidy Index and Its Implications for the Evolution of Polyploids.

Polyploidy has contributed to the divergence and domestication of plants; however, estimation of the relative roles that different types of polyploidy have played during evolution has been difficult. Unbalanced and balanced gene removal was previously related to allopolyploidies and autopolyploidies, respectively. Here, to infer the types of polyploidies and evaluate their evolutionary effects, we devised a statistic, the Polyploidy-index or P-index, to characterize the degree of divergence between subgenomes of a polyploidy, to find whether there has been a balanced or unbalanced gene removal from the homoeologous regions. Based on a P-index threshold of 0.3 that distinguishes between known or previously inferred allo- or autopolyploidies, we found that 87.5% of 24 angiosperm paleo-polyploidies were likely produced by allopolyploidizations, responsible for establishment of major tribes such as Poaceae and Fabaceae, and large groups such as monocots and eudicots. These findings suggest that >99.7% of plant genomes likely derived directly from allopolyploidies, with autopolyploidies responsible for the establishment of only a few small genera, including Glycine, Malus, and Populus, each containing tens of species. Overall, these findings show that polyploids with high divergence between subgenomes (presumably allopolyploids) established the major plant groups, possibly through secondary contact between previously isolated populations and hybrid vigor associated with their re-joining.

Reconstruction of evolutionary trajectories of chromosomes unraveled independent genomic repatterning between Triticeae and Brachypodium.

BACKGROUND: After polyploidization, a genome may experience large-scale genome-repatterning, featuring wide-spread DNA rearrangement and loss, and often chromosome number reduction. Grasses share a common tetraploidization, after which the originally doubled chromosome numbers reduced to different chromosome numbers among them. A telomere-centric reduction model was proposed previously to explain chromosome number reduction. With Brachpodium as an intermediate linking different major lineages of grasses and a model plant of the Pooideae plants, we wonder whether it mediated the evolution from ancestral grass karyotype to Triticeae karyotype. RESULTS: By inferring the homology among Triticeae, rice, and Brachpodium chromosomes, we reconstructed the evolutionary trajectories of the Triticeae chromosomes. By performing comparative genomics analysis with rice as a reference, we reconstructed the evolutionary trajectories of Pooideae plants, including Ae. Tauschii (2n = 14, DD), barley (2n = 14), Triticum turgidum (2n = 4x = 28, AABB), and Brachypodium (2n = 10). Their extant Pooidea and Brachypodium chromosomes were independently produced after sequential nested chromosome fusions in the last tens of millions of years, respectively, after their split from rice. More frequently than would be expected by chance, in Brachypodium, the 'invading' and 'invaded' chromosomes are homoeologs, originating from duplication of a common ancestral chromosome, that is, with more extensive DNA-level correspondence to one another than random chromosomes, nested chromosome fusion events between homoeologs account for three of seven cases in Brachypodium (P-value≈0.00078). However, this phenomenon was not observed during the formation of other Pooideae chromosomes. CONCLUSIONS: Notably, we found that the Brachypodium chromosomes formed through exclusively distinctive trajectories from those of Pooideae plants, and were well explained by the telomere-centric model. Our work will contribute to understanding the structural and functional innovation of chromosomes in different Pooideae lineages and beyond.

Involvement of jasmonic acid, ethylene and salicylic acid signaling pathways behind the systemic resistance induced by Trichoderma longibrachiatum H9 in cucumber.

BACKGROUND: Trichoderma spp. are effective biocontrol agents for many plant pathogens, thus the mechanism of Trichoderma-induced plant resistance is not fully understood. In this study, a novel Trichoderma strain was identified, which could promote plant growth and reduce the disease index of gray mold caused by Botrytis cinerea in cucumber. To assess the impact of Trichoderma inoculation on the plant response, a multi-omics approach was performed in the Trichoderma-inoculated cucumber plants through the analyses of the plant transcriptome, proteome, and phytohormone content. RESULTS: A novel Trichoderma strain was identified by morphological and molecular analysis, here named T. longibrachiatum H9. Inoculation of T. longibrachiatum H9 to cucumber roots promoted plant growth in terms of root length, plant height, and fresh weight. Root colonization of T. longibrachiatum H9 in the outer layer of epidermis significantly inhibited the foliar pathogen B. cinerea infection in cucumber. The plant transcriptome and proteome analyses indicated that a large number of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified in cucumber plants 96 h post T. longibrachiatum H9 inoculation. Up-regulated DEGs and DEPs were mainly associated with defense/stress processes, secondary metabolism, and phytohormone synthesis and signaling, including jasmonic acid (JA), ethylene (ET) and salicylic acid (SA), in the T. longibrachiatum H9-inoculated cucumber plants in comparison to untreated plants. Moreover, the JA and SA contents significantly increased in cucumber plants with T. longibrachiatum H9 inoculation. CONCLUSIONS: Application of T. longibrachiatum H9 to the roots of cucumber plants effectively promoted plant growth and significantly reduced the disease index of gray mold caused by B. cinerea. The analyses of the plant transcriptome, proteome and phytohormone content demonstrated that T. longibrachiatum H9 mediated plant systemic resistance to B. cinerea challenge through the activation of signaling pathways associated with the phytohormones JA/ET and SA in cucumber.

Recursive Paleohexaploidization Shaped the Durian Genome.

The durian (Durio zibethinus) genome has recently become available, and analysis of this genome reveals two paleopolyploidization events previously inferred as shared with cotton (Gossypium spp.). Here, we reanalyzed the durian genome in comparison with other well-characterized genomes. We found that durian and cotton were actually affected by different polyploidization events: hexaploidization in durian ∼19-21 million years ago (mya) and decaploidization in cotton ∼13-14 mya. Previous interpretations of shared polyploidization events may have resulted from the elevated evolutionary rates in cotton genes due to the decaploidization and insufficient consideration of the complexity of plant genomes. The decaploidization elevated evolutionary rates of cotton genes by ∼64% compared to durian and explained a previous ∼4-fold over dating of the event. In contrast, the hexaploidization in durian did not prominently elevate gene evolutionary rates, likely due to its long generation time. Moreover, divergent evolutionary rates probably explain 98.4% of reconstructed phylogenetic trees of homologous genes being incongruent with expected topology. The findings provide further insight into the roles played by polypoidization in the evolution of genomes and genes, and they suggest revisiting existing reconstructed phylogenetic trees.

Two Likely Auto-Tetraploidization Events Shaped Kiwifruit Genome and Contributed to Establishment of the Actinidiaceae Family.

The genome of kiwifruit (Actinidia chinensis) was sequenced previously, the first in the Actinidiaceae family. It was shown to have been affected by polyploidization events, the nature of which has been elusive. Here, we performed a reanalysis of the genome and found clear evidence of 2 tetraploidization events, with one occurring ∼50-57 million years ago (Mya) and the other ∼18-20 Mya. Two subgenomes produced by each event have been under balanced fractionation. Moreover, genes were revealed to express in a balanced way between duplicated copies of chromosomes. Besides, lowered evolutionary rates of kiwifruit genes were observed. These findings could be explained by the likely auto-tetraploidization nature of the polyploidization events. Besides, we found that polyploidy contributed to the expansion of key functional genes, e.g., vitamin C biosynthesis genes. The present work also provided an important comparative genomics resource in the Actinidiaceae and related families.

Comprehensive analyses of the BES1 gene family in Brassica napus and examination of their evolutionary pattern in representative species.

BACKGROUND: The BES1 gene family, an important class of plant-specific transcription factors, play key roles in the BR signal pathway in plants, regulating various development processes. Until now, there has been no comprehensive analysis of the BES1 gene family in Brassica napus, and a cross-genome exploration of their origin, copy number changes, and functional innovation in plants was also not available. RESULTS: We identified 28 BES1 genes in B. napus from its two subgenomes (AA and CC). We found that 71.43% of them were duplicated in the tetraploidization, and their gene expression showed a prominent subgenome bias in the roots. Additionally, we identified 104 BES1 genes in another 18 representative angiosperms and performed a comparative analysis with B. napus, including evolutionary trajectory, gene duplication, positive selection, and expression pattern. Exploiting the available genome datasets, we performed a large-scale analysis across plants and algae suggested that the BES1 gene family could have originated from group F, expanding to form other groups (A to E) by duplicating or alternatively deleting some domains. We detected an additional domain containing M4 to M8 in exclusively groups F1 and F2. We found evidence that whole-genome duplication (WGD) contributed the most to the expansion of this gene family among examined dicots, while dispersed duplication contributed the most to expansion in certain monocots. Moreover, we inferred that positive selection might have occurred on major phylogenetic nodes during the evolution of plants. CONCLUSIONS: Grossly, a cross-genome comparative analysis of the BES1 genes in B. napus and other species sheds light on understanding its copy number expansion, natural selection, and functional innovation.

An Overlooked Paleotetraploidization in Cucurbitaceae.

Cucurbitaceae plants are of considerable biological and economic importance, and genomes of cucumber, watermelon, and melon have been sequenced. However, a comparative genomics exploration of their genome structures and evolution has not been available. Here, we aimed at performing a hierarchical inference of genomic homology resulted from recursive paleopolyploidizations. Unexpectedly, we found that, shortly after a core-eudicot-common hexaploidy, a cucurbit-common tetraploidization (CCT) occurred, overlooked by previous reports. Moreover, we characterized gene loss (and retention) after these respective events, which were significantly unbalanced between inferred subgenomes, and between plants after their split. The inference of a dominant subgenome and a sensitive one suggested an allotetraploid nature of the CCT. Besides, we found divergent evolutionary rates among cucurbits, and after doing rate correction, we dated the CCT to be 90-102 Ma, likely common to all Cucurbitaceae plants, showing its important role in the establishment of the plant family.

Alignment of Common Wheat and Other Grass Genomes Establishes a Comparative Genomics Research Platform.

Grass genomes are complicated structures as they share a common tetraploidization, and particular genomes have been further affected by extra polyploidizations. These events and the following genomic re-patternings have resulted in a complex, interweaving gene homology both within a genome, and between genomes. Accurately deciphering the structure of these complicated plant genomes would help us better understand their compositional and functional evolution at multiple scales. Here, we build on our previous research by performing a hierarchical alignment of the common wheat genome vis-à-vis eight other sequenced grass genomes with most up-to-date assemblies, and annotations. With this data, we constructed a list of the homologous genes, and then, in a layer-by-layer process, separated their orthology, and paralogy that were established by speciations and recursive polyploidizations, respectively. Compared with the other grasses, the far fewer collinear outparalogous genes within each of three subgenomes of common wheat suggest that homoeologous recombination, and genomic fractionation should have occurred after its formation. In sum, this work contributes to the establishment of an important and timely comparative genomics platform for researchers in the grass community and possibly beyond. Homologous gene list can be found in Supplemental material.

Two Highly Similar Poplar Paleo-subgenomes Suggest an Autotetraploid Ancestor of Salicaceae Plants.

As a model plant to study perennial trees in the Salicaceae family, the poplar (Populus trichocarpa) genome was sequenced, revealing recurrent paleo-polyploidizations during its evolution. A comparative and hierarchical alignment of its genome to a well-selected reference genome would help us better understand poplar's genome structure and gene family evolution. Here, by adopting the relatively simpler grape (Vitis vinifera) genome as reference, and by inferring both intra- and inter-genomic gene collinearity, we produced a united alignment of these two genomes and hierarchically distinguished the layers of paralogous and orthologous genes, as related to recursive polyploidizations and speciation. We uncovered homologous blocks in the grape and poplar genomes and also between them. Moreover, we characterized the genes missing and found that poplar had two considerably similar subgenomes (≤0.05 difference in gene deletion) produced by the Salicaceae-common tetraploidization, suggesting its autotetraploid nature. Taken together, this work provides a timely and valuable dataset of orthologous and paralogous genes for further study of the genome structure and functional evolution of poplar and other Salicaceae plants.

Hierarchically Aligning 10 Legume Genomes Establishes a Family-Level Genomics Platform.

Mainly due to their economic importance, genomes of 10 legumes, including soybean (Glycine max), wild peanut (Arachis duranensis and Arachis ipaensis), and barrel medic (Medicago truncatula), have been sequenced. However, a family-level comparative genomics analysis has been unavailable. With grape (Vitis vinifera) and selected legume genomes as outgroups, we managed to perform a hierarchical and event-related alignment of these genomes and deconvoluted layers of homologous regions produced by ancestral polyploidizations or speciations. Consequently, we illustrated genomic fractionation characterized by widespread gene losses after the polyploidizations. Notably, high similarity in gene retention between recently duplicated chromosomes in soybean supported the likely autopolyploidy nature of its tetraploid ancestor. Moreover, although most gene losses were nearly random, largely but not fully described by geometric distribution, we showed that polyploidization contributed divergently to the copy number variation of important gene families. Besides, we showed significantly divergent evolutionary levels among legumes and, by performing synonymous nucleotide substitutions at synonymous sites correction, redated major evolutionary events during their expansion. This effort laid a solid foundation for further genomics exploration in the legume research community and beyond. We describe only a tiny fraction of legume comparative genomics analysis that we performed; more information was stored in the newly constructed Legume Comparative Genomics Research Platform (www.legumegrp.org).

Origination, Expansion, Evolutionary Trajectory, and Expression Bias of AP2/ERF Superfamily in Brassica napus.

The AP2/ERF superfamily, one of the most important transcription factor families, plays crucial roles in response to biotic and abiotic stresses. So far, a comprehensive evolutionary inference of its origination and expansion has not been available. Here, we identified 515 AP2/ERF genes in B. napus, a neo-tetraploid forming ~7500 years ago, and found that 82.14% of them were duplicated in the tetraploidization. A prominent subgenome bias was revealed in gene expression, tissue-specific, and gene conversion. Moreover, a large-scale analysis across plants and alga suggested that this superfamily could have been originated from AP2 family, expanding to form other families (ERF, and RAV). This process was accompanied by duplicating and/or alternative deleting AP2 domain, intragenic domain sequence conversion, and/or by acquiring other domains, resulting in copy number variations, alternatively contributing to functional innovation. We found that significant positive selection occurred at certain critical nodes during the evolution of land plants, possibly responding to changing environment. In conclusion, the present research revealed origination, functional innovation, and evolutionary trajectory of the AP2/ERF superfamily, contributing to understanding their roles in plant stress tolerance.