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Background Super pangenomes, as complete genome sequencing at the genus level, have provided new insights into the speciation and evolution of functional genes. Genome size (GS) estimation is a critical first step. Although K-mer-based GS evaluators are applied extensively to guide genome assembly process and quality assessment, the results vary substantially with the tools and parameters used, presenting challenges for genus-level genome studies. Results Here, we investigated K-mer spectra from datasets of species with and without whole genome duplication, revealing that the trade-off in K-mer length amplified the signal of genomic characteristics related to repeat content or heterozygosity. Moreover, GS predictions were influenced by genomic heterozygosity and sequencing accuracy when different K-mer lengths were employed. In contrast, consistent GS predictions were obtained across all HiFi-based evaluations, demonstrating high accuracy of the derived limiting values from the regions of GS evaluation convergence during continuous variation of K. Unlike traditional methods that rely on single predictions, we introduced a closed-loop GS-estimating framework, that incorporates steady-value calculations, leveraging the continuity and accuracy of HiFi reads. Finally, we developed a high-performance pipeline, LVgs ( https://github.com/xingjianfeng100/LVgs ), by integrating FastK and GenomeScope 2.0. Conclusions The robustness and applicability of LVgs for genus-level species was demonstrated through its application to various diploid and polyploidy species.

The para rubber tree ( Hevea brasiliensis ) is the world’s sole commercial source of natural rubber, a vital industrial raw material. However, the narrow genetic diversity of this crop poses challenges for rubber breeding. Here, we generate high-quality de novo genome assemblies for three H. brasiliensis cultivars, two H. brasiliensis wild accessions, and three other Hevea species ( H. nitida , H. pauciflora , and H. benthamiana ). Through analyzing genomes of 94 Hevea accessions, we identify five distinct lineages that do not align with their previous species delineations. We discover multiple accessions with hybrid origins between these lineages, indicating incomplete reproductive isolation between them. Only two out of four wild lineages have been introduced to commercial rubber cultivars. Furthermore, we reveal that the rubber production traits emerged following the development of a large REF/SRPP gene cluster and its functional specialization in rubber-producing laticifers within this genus. These findings would enhance rubber breeding and benefit research communities. The para rubber tree is the world’s sole commercial source of natural rubber. Here, the authors assemble the pangenome based on five genomes of H. brasiliensis and three genomes of other Hevea species, and reveal species delineation and rubber trait evolution through phylogenomic analyses.

MicroRNAs (miRNAs) are widely involved in various aspects of plant growth and development. However, how miRNAs and their targets regulate natural rubber metabolism remains unclear in the rubber-producing dandelions, which are being developed as alternative commercial sources of natural rubber. Here, we combined small RNA sequencing, degradome sequencing, target gene prediction, and mRNA sequencing to identify miRNAs and their targets in two dandelion species, the high rubber-yielding Taraxacum kok-saghyz (Tk) and the low rubber-yielding T. spadiceum (Ts). A total of 142 miRNAs, including 108 known and 34 novel ones, were discovered, with 53 identified as differentially expressed (DE) between the latex of Tk and Ts. Degradome sequencing identified 145 targets corresponding to 74 miRNAs. TAPIR and psRNATarget, respectively, predicted 165 and 164 non-redundant targets for the 53 aforementioned DE miRNAs. Gene ontology (GO) enrichment analysis indicated the DE miRNAs and their targets might affect natural rubber production via regulating macromolecular biosynthesis and metabolism in latex. Four critical types of regulatory modules, including miR172-AP2/ERF, miR164-NAC, miR160-ARF, and miRN19-protein kinase, were identified and their interaction networks were constructed, indicating a potential involvement in natural rubber production. The findings and the large miRNA dataset presented here are beneficial to further deciphering the roles of miRNAs in the biosynthesis of natural rubber and medicinal metabolites in dandelion.

Sucrose-metabolizing enzymes in plant leaves have hitherto been investigated mainly in temperate plants, and rarely conducted in tandem with gene expression and sugar analysis. Here, we investigated the sugar content, gene expression, and the activity of sucrose-metabolizing enzymes in the leaves of , a tropical tree widely cultivated for natural rubber. Sucrose, fructose and glucose were the major sugars detected in leaves at four developmental stages (I to IV), with starch and quebrachitol as minor saccharides. Fructose and glucose contents increased until stage III, but decreased strongly at stage IV (mature leaves). On the other hand, sucrose increased continuously throughout leaf development. Activities of all sucrose-cleaving enzymes decreased markedly at maturation, consistent with transcript decline for most of their encoding genes. Activity of sucrose phosphate synthase (SPS) was low in spite of its high transcript levels at maturation. Hence, the high sucrose content in mature leaves was not due to increased sucrose-synthesizing activity, but more to the decline in sucrose cleavage. Gene expression and activities of sucrose-metabolizing enzymes in leaves showed striking differences compared with other plants. Unlike in most other species where vacuolar invertase predominates in sucrose cleavage in developing leaves, cytoplasmic invertase and sucrose synthase (cleavage direction) also featured prominently in . Whereas SPS is normally responsible for sucrose synthesis in plant leaves, sucrose synthase (synthesis direction) was comparable or higher than that of SPS in leaves. Mature leaves had an unusually high sucrose:starch ratio of about 11, the highest reported to date in plants.

Grasses possess basal and aerial axillary buds. Previous studies have largely focused on basal bud (tiller) formation but scarcely touched on aerial buds, which may lead to aerial branch development. Genotypes with and without aerial buds were identified in switchgrass (Panicum virgatum), a dedicated bioenergy crop. Bud development was characterized using scanning electron microscopy. Microarray, RNA-seq and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to identify regulators of bud formation. Gene function was characterized by down-regulation and overexpression. Overexpression of miR156 induced aerial bud formation in switchgrass. Various analyses revealed that SQUAMOSA PROMOTER BINDING PROTEIN LIKE4 (SPL4), one of the miR156 targets, directly regulated aerial axillary bud initiation. Down-regulation of SPL4 promoted aerial bud formation and increased basal buds, while overexpression of SPL4 seriously suppressed bud formation and tillering. RNA-seq and RT-qPCR identified potential downstream genes of SPL4. Unlike all previously reported genes acting as activators of basal bud initiation, SPL4 acts as a suppressor for the formation of both aerial and basal buds. The miR156-SPL4 module predominantly regulates aerial bud initiation and partially controls basal bud formation. Genetic manipulation of SPL4 led to altered plant architecture with increased branching, enhanced regrowth after cutting and improved biomass yield.

Background Sucrose (Suc), as the precursor molecule for rubber biosynthesis in Hevea brasiliensis , is transported via phloem-mediated long-distance transport from leaves to laticifers in trunk bark, where latex (cytoplasm of laticifers) is tapped for rubber. In our previous report, six Suc transporter (SUT) genes have been cloned in Hevea tree, among which HbSUT3 is verified to play an active role in Suc loading to the laticifers. In this study, another latex-abundant SUT isoform, HbSUT5 , with expressions only inferior to HbSUT3 was characterized especially for its roles in latex production. Results Both phylogenetic analysis and subcellular localization identify HbSUT5 as a tonoplast-localized SUT protein under the SUT4-clade (=type III). Suc uptake assay in baker’s yeast reveals HbSUT5 to be a typical Suc-H + symporter, but its high affinity for Suc (Km = 2.03 mM at pH 5.5) and the similar efficiency in transporting both Suc and maltose making it a peculiar SUT under the SUT4-clade. At the transcript level, HbSUT5 is abundantly and preferentially expressed in Hevea barks. The transcripts of HbSUT5 are conspicuously decreased both in Hevea latex and bark by two yield-stimulating treatments of tapping and ethephon, the patterns of which are contrary to HbSUT3. Under the ethephon treatment, the Suc level in latex cytosol decreases significantly, but that in latex lutoids (polydispersed vacuoles) changes little, suggesting a role of the decreased HbSUT5 expression in Suc compartmentalization in the lutoids and thus enhancing the Suc sink strength in laticifers. Conclusions Our findings provide insights into the roles of a vacuolar sucrose transporter, HbSUT5, in Suc exchange between lutoids and cytosol in rubber-producing laticifers.

In Hevea brasiliensis, an alkaline/neutral invertase (A/N‐Inv) is responsible for sucrose catabolism in latex (essentially the cytoplasm of rubber‐producing laticifers, the source of natural rubber) and implicated in rubber yield. However, neither the gene encoding this enzyme nor its molecular and biochemical properties have been well documented. Three Hevea A/N‐Inv genes, namely HbNIN1, 2 and 3, were first cloned and characterized in planta and in Escherichia coli. Cellular localizations of HbNIN2 mRNA and protein were probed. From latex, active A/N‐Inv proteins were purified, identified, and explored for enzymatic properties. HbNIN2 was identified as the major A/N‐Inv gene functioning in latex based on its functionality in E. coli, its latex‐predominant expression, the conspicuous localization of its mRNA and protein in the laticifers, and its expressional correlation with rubber yield. An active A/N‐Inv protein was partially purified from latex, and determined as HbNIN2. The enhancement of HbNIN2 enzymatic activity by pyridoxal is peculiar to A/N‐Invs in other plants. We conclude that HbNIN2, a cytosolic A/N‐Inv, is responsible for sucrose catabolism in rubber laticifers. The results contribute to the studies of sucrose catabolism in plants as a whole and natural rubber synthesis in particular.

Latex flow in Hevea brasiliensis (the Para rubber tree), the sole commercial source of natural rubber ( cis -1,4-polyisoprene, NR), renders it uniquely suited for the study of plant stress responses. Calcineurin B-like interacting protein kinases (CIPK) serving as calcium-sensor protein kinases react with calcineurin B-like proteins (CBL) to play crucial roles in hormone signaling transduction and response to abiotic stress in plant developmental processes. However, little is known about their functions in Hevea . In this study, a total of twelve CBL ( HbCBL ) and thirty CIPK ( HbCIPK ) genes were identified from the Hevea genome. Structure and phylogenetic analysis assigned these CIPKs to five groups and CBLs to four groups, and mapped onto fourteen of the eighteen Hevea chromosomes. RNA-seq and qPCR analysis showed that the expressions of HbCBL and HbCIPK genes varied in the seven Hevea tissues examined, i.e., latex (cytoplasm of rubber-producing laticifers), bark, leaf, root, seed, female flower, and male flower. The expressions of two HbCBL and sixteen HbCIPK genes showed upward trends during leaf development. Following ethylene yield stimulation and the latex tapping treatment, both practices invoking stress, the expression levels of most latex-expressed genes were significantly altered. Yeast two-hybrid test revealed interactions for multiple combinations of HbCBLs and HbCIPKs with substantial gene expression in latex or other Hevea tissues. However, all the HbCBL-HbCIPK complexes examined did not recruit HbSOS1 or AtSOS1 to form functional salt tolerance SOS pathway in yeast cells. Taken together, the results suggested a role of the Hevea CBL-CIPK network as a point of convergence for several different signaling pathways in growth, development, and stress responses in relation to latex production.

Increasing demand for natural rubber prompts studies into the mechanisms governing the productivity of rubber tree (Heveabrasiliensis). It is very interesting to notice that a rubber tree of clone PR107 in Yunnan, China is reported to yield more than 20 times higher than the average rubber tree. This super-high-yielding (SHY) rubber tree (designated as SY107), produced 4.12 kg of latex (cytoplasm of rubber producing laticifers, containing about 30% of rubber) per tapping, more than 7-fold higher than that of the control. This rubber tree is therefore a good material to study how the rubber production is regulated at a molecular aspect. A comprehensive cDNA-AFLP transcript profiling was performed on the latex of SY107 and its average counterparts by using the 384 selective primer pairs for two restriction enzyme combinations (ApoI/MseI and TaqI/MseI). A total of 746 differentially expressed (DE) transcript-derived fragments (TDFs) were identified, of which the expression patterns of 453 TDFs were further confirmed by RT-PCR. These RT-PCR confirmed TDFs represented 352 non-redundant genes, of which 215 had known or partially known functions and were grouped into 10 functional categories. The top three largest categories were transcription and protein synthesis (representing 24.7% of the total genes), defense and stress (15.3%), and primary and secondary metabolism (14.0%). Detailed analysis of the DE-genes suggests notable characteristics of SHY phenotype in improved sucrose loading capability, rubber biosynthesis-preferred sugar utilization, enhanced general metabolism and timely stress alleviation. However, the SHY phenotype has little correlation with rubber-biosynthesis pathway genes.

An amendment to this paper has been published and can be accessed via the original article.