Activation and negative feedback regulation of SlHY5 transcription by the SlBBX20/21-SlHY5 transcription factor module in UV-B signaling.

In tomato (Solanum lycopersicum) and other plants, the photoreceptor UV-RESISTANCE LOCUS 8 regulates plant UV-B photomorphogenesis by modulating the transcription of many genes, the majority of which depends on the transcription factor ELONGATED HYPOCOTYL 5 (HY5). HY5 transcription is induced and then rapidly attenuated by UV-B. However, neither the transcription factors that activate HY5 transcription nor the mechanism for its attenuation during UV-B signaling is known. Here, we report that the tomato B-BOX (BBX) transcription factors SlBBX20 and SlBBX21 interact with SlHY5 and bind to the SlHY5 promoter to activate its transcription. UV-B-induced SlHY5 expression and SlHY5-controlled UV-B responses are normal in slbbx20 and slbbx21 single mutants, but strongly compromised in the slbbx20 slbbx21 double mutant. Surprisingly, UV-B responses are also compromised in lines overexpressing SlBBX20 or SlBBX21. Both SlHY5 and SlBBX20 bind to G-box1 in the SlHY5 promoter. SlHY5 outcompetes SlBBX20 for binding to the SlHY5 promoter in vitro, and inhibits the association of SlBBX20 with the SlHY5 promoter in vivo. Overexpressing 35S:SlHY5-FLAG in the WT background inhibits UV-B-induced endogenous SlHY5 expression. Together, our results reveal the critical role of the SlBBX20/21-SlHY5 module in activating the expression of SlHY5, the gene product of which inhibits its own gene transcription under UV-B, forming an autoregulatory negative feedback loop that balances SlHY5 transcription in plants.

Parallel ddRAD and Genome Skimming Analyses Reveal a Radiative and Reticulate Evolutionary History of the Temperate Bamboos.

Rapid evolutionary radiations are among the most challenging phylogenetic problems, wherein different types of data (e.g., morphology and molecular) or genetic markers (e.g., nuclear and organelle) often yield inconsistent results. The tribe Arundinarieae, that is, the temperate bamboos, is a clade of tetraploid originated 22 Ma and subsequently radiated in East Asia. Previous studies of Arundinarieae have found conflicting relationships and/or low support. Here, we obtain nuclear markers from ddRAD data for 213 Arundinarieae taxa and parallel sampling of chloroplast genomes from genome skimming for 147 taxa. We first assess the feasibility of using ddRAD-seq data for phylogenetic estimates of paleopolyploid and rapidly radiated lineages, optimize clustering thresholds, and analysis workflow for orthology identification. Reference-based ddRAD data assembly approaches perform well and yield strongly supported relationships that are generally concordant with morphology-based taxonomy. We recover five major lineages, two of which are notable (the pachymorph and leptomorph lineages), in that they correspond with distinct rhizome morphologies. By contrast, the phylogeny from chloroplast genomes differed significantly. Based on multiple lines of evidence, the ddRAD tree is favored as the best species tree estimation for temperate bamboos. Using a time-calibrated ddRAD tree, we find that Arundinarieae diversified rapidly around the mid-Miocene corresponding with intensification of the East Asian monsoon and the evolution of key innovations including the leptomorph rhizomes. Our results provide a highly resolved phylogeny of Arundinarieae, shed new light on the radiation and reticulate evolutionary history of this tribe, and provide an empirical example for the study of recalcitrant plant radiations. [Arundinarieae; ddRAD; paleopolyploid; genome skimming; rapid diversification; incongruence.].

Pivotal roles of Tomato photoreceptor SlUVR8 in seedling development and UV-B stress tolerance.

UV RESISTANCE LOCUS 8 (UVR8) is a UV-B photoreceptor that regulates various aspects of plant photomorphogenesis. Physiological functions of UVR8 have been extensively investigated in Arabidopsis. However, functions of Tomato UVR8 (SlUVR8) are largely unknown. To analyze physiological functions of SlUVR8, we generated sluvr8 knock-out mutant lines with CRISPR-CAS9 gene editing approach. At seedling stage, SlUVR8 regulates hypocotyl elongation and anthocyanin accumulation under UV-B. Moreover, SlUVR8 regulates acclimation to low dose UV-B and promotes tolerance to elevated UV-B stress. These results revealed pivotal roles of SlUVR8 in the regulation of Tomato seedling development and UV-B stress tolerance. The manipulation of photoreceptor SlUVR8 may represent a powerful tool to improve Tomato plant performance in nature where high dose UV-B is present.

Resolving complicated relationships of the Panax bipinnatifidus complex in southwestern China by RAD-seq data.

The P. binpinnatifidus complex included most of the Panax species distributed in Sino-Himalaya regions except for P. pseudoginseng, P. stipuleanatus and P. notoginseng. However, the delimitation and identification of these taxa within the species complex are very difficult due to the existence of morphological intermediates, and their evolutionary relationships remain unresolved despite several studies have been carried out based on traditional DNA markers. The taxonomic uncertainty hinders the identification, conservation and exploration of these wild populations of Panax. To study this species complex, we employed ddRAD-seq data of these taxa from 18 different localities of southwestern China, using two RAD analysis pipelines, STACKS and pyRAD. Based on the results of phylogenetic analysis, the species complex was divided into four clades with high supports, which largely agreed with morphologically described species. Two clades, corresponding to P. vietnamensis and P. zingiberensis, respectively, were sister groups, indicating that these two species had a closer genetic relationship; the third clade was consisted of samples with bamboo-like rhizomes named as P. wangianus clade, and the fourth one with moniliform rhizomes was named as P. bipinnatifidus clade. The population genetic structure analysis and D-statistics test showed the localized admixture among these species, which indicated that introgression had occurred among the related lineages continuously distributed in southeastern Yunnan and adjacent regions.

ddRAD analyses reveal a credible phylogenetic relationship of the four main genera of Bambusa-Dendrocalamus-Gigantochloa complex (Poaceae: Bambusoideae).

The Bambusa-Dendrocalamus-Gigantochloa complex (BDG complex) is the most diversified and phylogenetically recalcitrant group of the paleotropical woody bamboos. Species of this complex occur in tropical and subtropical Asia and most of them are of great economic, cultural and ecological value. The lack of resolution achieved through the analyses of previous molecular datasets has long confounded its phylogenetic estimation and generic delimitation. Here, we adopted a ddRAD-seq strategy to investigate phylogenetic relationships of the four main genera (Bambusa, Dendrocalamus, Gigantochloa, and Melocalamus) in the BDG complex. A total of 102 species were sampled, and SNP data were generated. Both MP and ML analyses of the ddRAD-seq data resulted in a well-resolved topology with Gigantochloa and Melocalamus confirmed as monophyletic, and Melocalamus resolved as sister to the rest of the complex. Bambusa and Dendrocalamus were both resolved as paraphyletic. The phylogenetic relationships were mostly supported by morphological evidence including characters of the branch complement, rachilla, lodicules, filaments and stigma. We also generated and assembled complete plastid genomes of 48 representative species. There were conflicts between the plastome and the ddRAD topologies. Our study demonstrated that RAD-seq can be used to reconstruct evolutionary history of lineages such as the bamboos where ancient hybridization and polyploidy play a significant role. The four genera of the BDG complex have a complex evolutionary history which is likely a product of ancient introgression events.

Complementary Transcriptome and Proteome Analyses Provide Insight into the Floral Transition in Bamboo (Dendrocalamus latiflorus Munro).

Most woody bamboos bloom only once after long vegetative growth phases and die immediately afterwards. It is difficult, however, to determine the timing of the floral transition, as little information is available on the molecular mechanism of plant maturity in bamboos. To uncover the bamboo floral transition mechanism, its morpho-physiological characteristics, transcriptomes and large-scale quantitative proteomes were investigated in leaves which were collected at different stages during floral transition in a woody bamboo, Dendrocalamus latiflorus. We identified many flowering time-associated genes and the continued increase and decrease genes were screened as flowering biomarker genes (e.g., the MADS14 and bHLH13 genes). These different genes were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG). And the photoperiod pathways depending on the circadian rhythm may play an essential role in the bamboo floral transition. In addition, a total of 721 differently expressed proteins of leaves from the vegetative-to-reproductive stages were identified. Fifty-five genes were specifically differentially expressed at both the transcriptomic and proteomic levels, including genes related to photosynthesis and nucleotide sugar, which may be involved in the floral transition. This work provides insights into bamboo flowers and the management of forest breeding.

The C-terminal 17 amino acids of the photoreceptor UVR8 is involved in the fine-tuning of UV-B signaling.

Plant UV-B responses are mediated by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8). In response to UV-B irradiation, UVR8 homodimers dissociate into monomers that bind to the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). The interaction of the C27 domain in the C-terminal tail of UVR8 with the WD40 domain of COP1 is critical for UV-B signaling. However, the function of the last 17 amino acids (C17) of the C-terminus of UVR8, which are adjacent to C27, is unknown, although they are largely conserved in land plants. In this study, we established that Arabidopsis thaliana UVR8 C17 binds to full-length UVR8, but not to COP1, and reduces COP1 binding to the remaining portion of UVR8, including C27. We hypothesized that overexpression of C17 in a wild-type background would have a dominant negative effect on UVR8 activity; however, C17 overexpression caused strong silencing of endogenous UVR8, precluding a detailed analysis. We therefore generated YFP-UVR8N423 transgenic lines, in which C17 was deleted, to examine C17 function indirectly. YFP-UVR8N423 was more active than YFP-UVR8, suggesting that C17 inhibits UV-B signaling by attenuating binding between C27 and COP1. Our study reveals an inhibitory role for UVR8 C17 in fine-tuning UVR8-COP1 interactions during UV-B signaling.

Population structure of wild soybean (Glycine soja) based on SLAF-seq have implications for its conservation.

Background: Glycine soja Sieb. & Zucc. is the wild ancestor from which the important crop plant soybean was bred. G. soja provides important germplasm resources for the breeding and improvement of cultivated soybean crops, however the species is threatened by habitat loss and fragmentation, and is experiencing population declines across its natural range. Understanding the patterns of genetic diversity in G. soja populations can help to inform conservation practices. Methods: In this study, we analyzed the genetic diversity and differentiation of G. soja at different sites and investigated the gene flow within the species. We obtained 147 G. soja accessions collected from 16 locations across the natural range of the species from China, Korea and Japan. Samples were analyzed using SLAF-seq (Specific-Locus Amplified Fragment Sequencing). Results: We obtained a total of 56,489 highly consistent SNPs. Our results suggested that G. soja harbors relatively high diversity and that populations of this species are highly differentiated. The populations harboring high genetic diversity, especially KR, should be considered first when devising conservation plans for the protection of G. soja, and in situ protection should be adopted in KR. G. soja populations from the Yangtze River, the Korean peninsula and northeastern China have a close relationship, although these areas are geographically disconnected. Other populations from north China clustered together. Analysis of gene flow suggested that historical migrations of G. soja may have occurred from the south northwards across the East-Asia land-bridge, but not across north China. All G. soja populations could be divided into one of two lineages, and these two lineages should be treated separately when formulating protection policies.

Chromosome-scale genome assembly of Prunus pusilliflora provides novel insights into genome evolution, disease resistance, and dormancy release in Cerasus L.

Prunus pusilliflora is a wild cherry germplasm resource distributed mainly in Southwest China. Despite its ornamental and economic value, a high-quality assembled P. pusilliflora genome is unavailable, hindering our understanding of its genetic background, population diversity, and evolutionary processes. Here, we de novo assembled a chromosome-scale P. pusilliflora genome using Oxford Nanopore, Illumina, and chromosome conformation capture sequencing. The assembled genome size was 309.62 Mb, with 76 scaffolds anchored to eight pseudochromosomes. We predicted 33 035 protein-coding genes, functionally annotated 98.27% of them, and identified repetitive sequences covering 49.08% of the genome. We found that P. pusilliflora is closely related to Prunus serrulata and Prunus yedoensis, having diverged from them ~41.8 million years ago. A comparative genomic analysis revealed that P. pusilliflora has 643 expanded and 1128 contracted gene families. Furthermore, we found that P. pusilliflora is more resistant to Colletotrichum viniferum, Phytophthora capsici, and Pseudomonas syringae pv. tomato (Pst) DC3000 infections than cultivated Prunus avium. P. pusilliflora also has considerably more nucleotide-binding site-type resistance gene analogs than P. avium, which explains its stronger disease resistance. The cytochrome P450 and WRKY families of 263 and 61 proteins were divided into 42 and 8 subfamilies respectively in P. pusilliflora. Furthermore, 81 MADS-box genes were identified in P. pusilliflora, accompanying expansions of the SVP and AGL15 subfamilies and loss of the TM3 subfamily. Our assembly of a high-quality P. pusilliflora genome will be valuable for further research on cherries and molecular breeding.

Detection of UVR8 Homodimers and Monomers by Immunoblotting Analysis in Tomato.

The UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor mediating photomorphogenic responses to UV-B. UVR8 exists as homodimer in plants and UV-B induces dissociation of dimeric UVR8 into monomers to initiate responses. The monomer/dimer status of UVR8 is reversible and a dynamic photo-equilibrium is established in plants according to the ambient light conditions. Here we describe a method to detect UVR8 homodimer and monomer by immunoblotting method from tomato (Solanum lycopersicum) plants. The feature of this method is that protein samples are not boiled prior to loading on an SDS-PAGE gel, which allows the detection of UVR8 homodimer and monomers simultaneously with a single antibody.

Rose without prickle: genomic insights linked to moisture adaptation.

Prickles act against herbivores, pathogens or mechanical injury, while also preventing water loss. However, whether prickles have new function and the molecular genetics of prickle patterning remain poorly explored. Here, we generated a high-quality reference genome assembly for 'Basye's Thornless' (BT), a prickle-free cultivar of Rosa wichuraiana, to identify genetic elements related to stem prickle development. The BT genome harbors a high level of sequence diversity in itself and with cultivar 'Old Blush' (R. chinensis), a founder genotype in rose domestication. Inheritance of stem prickle density was determined and two QTL were identified. Differentially expressed genes in QTL were involved in water-related functions, suggesting that prickle density may hitchhike with adaptations to moist environments. While the prickle-related gene-regulatory-network (GRN) was highly conserved, the expression variation of key candidate genes was associated with prickle density. Our study provides fundamental resources and insights for genome evolution in the Rosaceae. Ongoing efforts on identification of the molecular bases for key rose traits may lead to improvements for horticultural markets.

The complete chloroplast genome sequence of Mangifera sylvatica Roxb. (Anacardiaceae) and its phylogenetic analysis.

In this study, we firstly reported the complete chloroplast (cp) genome sequences of the Mangifera sylvatica from Nanning, Guangxi province, China. The complete wild mango cp genome size is 158063 bp with a typical small single-copy region (SSC, 18340 bp), a large single-copy region (LSC, 87008 bp) and a pair of inverted repeats (IRs, 26379 bp and 26379 bp respectively). Out of 112 unique annotated genes in mango cp genome, 78 found to be protein coding, 30 to be tRNA and 4 rRNA genes. Besides, we found 51 microsatellite sequences (SSRs) in the cp genome. Sequence alignment and ML analysis of 29 full plastome data revealed M. sylvatica shares the closest relationship with cultivated mango (M. indica) and form a sister group with Rhus chinensis within Anacardiaceae.

Molecular Sex Identification in the Hardy Rubber Tree (Eucommia ulmoides Oliver) via ddRAD Markers.

Eucommia ulmoides, also known as the industrially and medicinally important hardy rubber tree, is the sole species of Eucommiaceae. Nevertheless, its dioecious property hinders sex recognition by traditional morphological observation at very early developmental stages, thus inhibiting breeding and economic cropping. In this study, double-digest restriction site-associated DNA sequencing (ddRAD-seq) was applied to screen sex-linked molecular markers for sex identification and investigation of the sex determination system in 20 male and female E. ulmoides individual plants, respectively. In consequence, five candidate male-specific loci but no female-specific loci were predicated among the 183,752 male and 147,122 female catalogue loci by bioinformatics analysis. Subsequent PCR (polymerase chain reaction) amplification and Sanger sequencing examinations were performed on another 24 individuals, 12 for each sex, from a separate population. One ideal sex-linked locus, MSL4, was identified among the five putative male-specific loci that were found using ddRAD data. MSL4 is 479 bp in length and highly conserved in all the male individuals, suggesting its feature of being stable and repeatable. Our results also indicated that the sex of E. ulmoides is likely determined genetically. In short, this study provides a consistent and reproducible ddRAD marker (MSL4) that is able to discriminate male from female seedlings in E. ulmoides, which will be valuable for rapid breeding practice and better commercial production of this economically important tree.

The development of a high-density genetic map significantly improves the quality of reference genome assemblies for rose.

Roses are important woody plants featuring a set of important traits that cannot be investigated in traditional model plants. Here, we used the restriction-site associated DNA sequencing (RAD-seq) technology to develop a high-density linkage map of the backcross progeny (BC1F1) between Rosa chinensis 'Old Blush' (OB) and R. wichuraiana 'Basyes' Thornless' (BT). We obtained 643.63 million pair-end reads and identified 139,834 polymorphic tags that were distributed uniformly in the rose genome. 2,213 reliable markers were assigned to seven linkage groups (LGs). The length of the genetic map was 1,027.425 cM in total with a mean distance of 0.96 cM per marker locus. This new linkage map allowed anchoring an extra of 1.21/23.14 Mb (12.18/44.52%) of the unassembled OB scaffolds to the seven reference pseudo-chromosomes, thus significantly improved the quality of assembly of OB reference genome. We demonstrate that, while this new linkage map shares high collinearity level with strawberry genome, it also features two chromosomal rearrangements, indicating its usefulness as a resource for understanding the evolutionary scenario among Rosaceae genomes. Together with the newly released genome sequences for OB, this linkage map will facilitate the identification of genetic components underpinning key agricultural and biological traits, hence should greatly advance the studies and breeding efforts of rose.

Genome Sequences Provide Insights into the Reticulate Origin and Unique Traits of Woody Bamboos.

Polyploidization is a major driver of speciation and its importance to plant evolution has been well recognized. Bamboos comprise one diploid herbaceous and three polyploid woody lineages, and are members of the only major subfamily in grasses that diversified in forests, with the woody members having a tree-like lignified culm. In this study, we generated four draft genome assemblies of major bamboo lineages with three different ploidy levels (diploid, tetraploid, and hexaploid). We also constructed a high-density genetic linkage map for a hexaploid species of bamboo, and used a linkage-map-based strategy for genome assembly and identification of subgenomes in polyploids. Further phylogenomic analyses using a large dataset of syntenic genes with expected copies based on ploidy levels revealed that woody bamboos originated subsequent to the divergence of the herbaceous bamboo lineage, and experienced complex reticulate evolution through three independent allopolyploid events involving four extinct diploid ancestors. A shared but distinct subgenome was identified in all polyploid forms, and the progenitor of this subgenome could have been critical in ancient polyploidizations and the origin of woody bamboos. Important genetic clues to the unique flowering behavior and woody trait in bamboos were also found. Taken together, our study provides significant insights into ancient reticulate evolution at the subgenome level in the absence of extant donor species, and offers a potential model scenario for broad-scale study of angiosperm origination by allopolyploidization.

A comparison of different methods for preserving plant molecular materials and the effect of degraded DNA on ddRAD sequencing.

Obtaining high-quality plant materials for experiments is challenging for many research projects. Therefore, it is of special importance to determine the best method for preserving biological macromolecules like DNA, which degrade over time. Although some research has demonstrated that DNA degradation has little effect on traditional molecular markers, the effects of DNA degradation on ddRAD-seq, a popular reduced-representation sequencing technology, have not been adequately investigated. In this study, we first chose six woody bamboo species (Bambusoideae, Poaceae) to explore appropriate methods for preserving molecular materials with two DNA extraction approaches. Then we sequenced twenty-one bamboos and examined the effects of DNA quality on data generation using the ddRAD-seq technique (MiddRAD-seq). Finally, we reconstructed phylogenies of twenty woody bamboo species. We found that the integrity of dry-powdered DNA was preserved longer than that of TE-dissolved DNA, regardless of whether the DNA was extracted by a modified CTAB protocol or DNAsecure plant kit. The ddRAD-seq data were robust, except when DNA was severely degraded. In addition, we resolved the phylogenetic positions of the sampled Phyllostachys spp. Our results suggest that dry-powdered DNA is the most appropriate preservation method for plant molecular materials. Furthermore, a moderate level of DNA degradation has little effect on reduced representation sequencing techniques represented by ddRAD-seq.

Using MiddRAD-seq data to develop polymorphic microsatellite markers for an endangered yew species.

Microsatellites are highly polymorphic markers which have been used in a wide range of genetic studies. In recent years, various sources of next-generation sequencing data have been used to develop new microsatellite loci, but compared with the more common shotgun genomic sequencing or transcriptome data, the potential utility of RAD-seq data for microsatellite ascertainment is comparatively under-used. In this study, we employed MiddRAD-seq data to develop polymorphic microsatellite loci for the endangered yew species Taxus florinii. Of 8,823,053 clean reads generated for ten individuals of a population, 94,851 (∼1%) contained microsatellite motifs. These corresponded to 2993 unique loci, of which 526 (∼18%) exhibited polymorphism. Of which, 237 were suitable for designing microsatellite primer pairs, and 128 loci were randomly selected for PCR validation and microsatellite screening. Out of the 128 primer pairs, 16 loci gave clear, reproducible patterns, and were then screened and characterized in 24 individuals from two populations. The total number of alleles per locus ranged from two to ten (mean = 4.875), and within-population expected heterozygosity from zero to 0.789 (mean = 0.530), indicating that these microsatellite loci will be useful for population genetics and speciation studies of T. florinii. This study represents one of few examples to mine polymorphic microsatellite loci from ddRAD data.

Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants.

BACKGROUND: The double digest restriction-site associated DNA sequencing technology (ddRAD-seq) is a reduced representation sequencing technology by sampling genome-wide enzyme loci developed on the basis of next-generation sequencing. ddRAD-seq has been widely applied to SNP marker development and genotyping on animals, especially on marine animals as the original ddRAD protocol is mainly built and trained based on animal data. However, wide application of ddRAD-seq technology in plant species has not been achieved so far. Here, we aim to develop an optimized ddRAD library preparation protocol be accessible to most angiosperm plant species without much startup pre-experiment and costs. RESULTS: We first tested several combinations of enzymes by in silico analysis of 23 plant species covering 17 families of angiosperm and 1 family of bryophyta and found AvaII + MspI enzyme pair produced consistently higher number of fragments in a broad range of plant species. Then we removed two purifying and one quantifying steps of the original protocol, replaced expensive consumables and apparatuses by conventional experimental apparatuses. Besides, we shortened P1 adapter from 37 to 25 bp and designed a new barcode-adapter system containing 20 pairs of barcodes of varying length. This is an optimized ddRAD strategy for angiosperm plants that is economical, time-saving and requires little technical expertise or investment in laboratory equipment. We refer to this simplified protocol as MiddRAD and we demonstrated the utility and flexibility of our approach by resolving phylogenetic relationships of two genera of woody bamboos (Dendrocalamus and Phyllostachys). Overall our results provide empirical evidence for using this method on different model and non-model plants to produce consistent data. CONCLUSIONS: As MiddRAD adopts an enzyme pair that works for a broad range of angiosperm plants, simplifies library constructing procedure and requires less DNA input, it will greatly facilitate designing a ddRAD project. Our optimization of this method may make ddRAD be widely used in fields of plant population genetics, phylogenetics, phylogeography and molecular breeding.