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KEY MESSAGE: Unreduced megagametophytes via second-division restitution were confirmed through heterozygosity analysis, and four candidate physical centromeres of rubber were located for the first time. The evaluation of maternal heterozygosity restitution (MHR) is vital in identifying the mechanism of 2n gametogenesis and assessing the utilization value of 2n gametes. In this study, three full-sib triploid populations were employed to evaluate the MHR of 2n female gametes of rubber tree clone GT1 and to confirm their genetic derivation. The 2n female gametes of GT1 were derived from second-division restitution (SDR) and transmitted more than half of the parental heterozygosity. In addition, low recombination frequency markers were developed, and four candidate physical centromeres of rubber tree were located for the first time. The confirmation that 2n female gametes of rubber tree clone GT1 are derived from SDR provides insights into the molecular mechanisms of 2n gametogenesis. In addition, the identified centromere location will aid in the development of centromeric markers for the rapid identification of the 2n gametogenesis mechanism.
Assuntos
Hevea , Triploidia , Hevea/genética , Diploide , Células Germinativas , Centrômero/genéticaRESUMO
The rubber tree (Hevea brasiliensis) is grown in tropical regions and is the major source of natural rubber. Using traditional breeding approaches, the latex yield has increased by sixfold in the last century. However, the underlying genetic basis of rubber yield improvement is largely unknown. Here, we present a high-quality, chromosome-level genome sequence of the wild rubber tree, the first report on selection signatures and a genome-wide association study (GWAS) of its yield traits. Population genomic analysis revealed a moderate population divergence between the Wickham clones and wild accessions. Interestingly, it is suggestive that H. brasiliensis and six relatives of the Hevea genus might belong to the same species. The selective sweep analysis found 361 obvious signatures in the domesticated clones associated with 245 genes. In a 15-year field trial, GWAS identified 155 marker-trait associations with latex yield, in which 326 candidate genes were found. Notably, six genes related to sugar transport and metabolism, and four genes related to ethylene biosynthesis and signalling are associated with latex yield. The homozygote frequencies of the causal nonsynonymous SNPs have been greatly increased under selection, which may have contributed to the fast latex yield improvement during the short domestication history. Our study provides insights into the genetic basis of the latex yield trait and has implications for genomic-assisted breeding by offering valuable resources in this new domesticated crop.
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Hevea , Borracha , Borracha/metabolismo , Hevea/genética , Hevea/metabolismo , Látex/metabolismo , Estudo de Associação Genômica Ampla , Melhoramento Vegetal , Genômica , Cromossomos/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genéticaRESUMO
The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 µM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.
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Small nucleolar RNAs (snoRNAs) are a class of conserved nuclear RNAs that play important roles in the modification of ribosomal RNAs (rRNAs) in plants. In rubber trees, rRNAs are run off with latex flow during tapping and need to be regenerated for maintaining the functions of the laticifer cells. SnoRNAs are expected to play essential roles in the regeneration of rRNAs. However, snoRNAs in the rubber tree have not been sufficiently characterized thus far. In this study, we performed nuclear RNA sequencing (RNA-seq) to identify snoRNAs globally and investigate their roles in latex regeneration. We identified a total of 3,626 snoRNAs by computational prediction with nuclear RNA-seq data. Among these snoRNAs, 50 were highly expressed in latex; furthermore, the results of reverse transcription polymerase chain reaction (RT-PCR) showed the abundant expression of 31 of these snoRNAs in latex. The correlation between snoRNA expression and adjusted total solid content (TSC/C) identified 13 positively yield-correlated snoRNAs. To improve the understanding of latex regeneration in rubber trees, we developed a novel insulated tapping system (ITS), which only measures the latex regenerated in specific laticifers. Using this system, a laticifer-abundant snoRNA, HbsnoR28, was found to be highly correlated with latex regeneration. To the best of our knowledge, this is the first report to globally identify snoRNAs that might be involved in latex regeneration regulation and provide new clues for unraveling the mechanisms underlying the regulation of latex regeneration.
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BACKGROUND: The processabilities and mechanical properties of natural rubber depend greatly on its molecular weight (MW) and molecular weight distribution (MWD). However, the mechanisms underlying the regulation of molecular weight during rubber biosynthesis remain unclear. RESULTS: In the present study, we determined the MW and particle size of latex from 1-year-old virgin trees and 30-year-old regularly tapped trees of the Hevea clones Reyan7-33-97 and RRIM600. The results showed that both the MW and the particle size of latex varied between these two clones and increased with tree age. Latex from RRIM600 trees had a smaller average particle size than that from Reyan7-33-97 trees of the same age. In 1-year-old trees, the Reyan7-33-97 latex displayed a slightly higher MW than that of RRIM600, whereas in 30-year-old trees, the RRIM600 latex had a significantly higher MW than the Reyan7-33-97 latex. Comparative analysis of the transcriptome profiles indicated that the average rubber particle size is negatively correlated with the expression levels of rubber particle associated proteins, and that the high-MW traits of latex are closely correlated with the enhanced expression of isopentenyl pyrophosphate (IPP) monomer-generating pathway genes and downstream allylic diphosphate (APP) initiator-consuming non-rubber pathways. By bioinformatics analysis, we further identified a group of transcription factors that potentially regulate the biosynthesis of IPP. CONCLUSIONS: Altogether, our results revealed the potential regulatory mechanisms involving gene expression variations in IPP-generating pathways and the non-rubber isoprenoid pathways, which affect the ratios and contents of IPP and APP initiators, resulting in significant rubber MW variations among same-aged trees of the Hevea clones Reyan7-33-97 and RRIM600. Our findings provide a better understanding of rubber biosynthesis and lay the foundation for genetic improvement of rubber quality in H. brasiliensis.
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Hevea/genética , Látex/metabolismo , Transcriptoma , Hevea/metabolismo , Peso MolecularRESUMO
Five TERMINAL FLOWER 1 (TFL1)/CENTRORADIALIS (CEN)-like genes were isolated and characterized from rubber tree (Hevea brasiliensis). All genes, except HbCEN1, were found to have conserved genomic organization, characteristic of the phosphatidyl ethanolamine-binding protein (PEBP) family. Overexpression of all of them delayed flowering and altered flower architecture compared with the wild-type (wt) counterpart. In addition, as premature-flowering of the terminal bud was successfully overcome in the tfl1-1 mutant of Arabidopsis, all these genes have a potential function similar to TFL1. Quantitative reverse transcriptase-polymerase chain reaction analysis showed higher expressions of HbCEN1 and HbCEN2 in the shoot apices and stems of both immature and mature rubber trees than in reproductive organs. HbTFL1-1 and HbTFL1-2 expression was confined to roots of 3-month-old seedlings and HbTFL1-3 was significantly higher in the shoot apices of these seedlings. These results suggested that HbCEN1 and HbCEN2 could be associated with the development of vegetative growth, whereas HbTFL1-1, HbTFL1-2 and HbTFL1-3 seem to be mainly related with maintenance of juvenility. In addition, four of the five genes displayed variable diurnal expression, HbTFL1-1 and HbTFL1-3 being mainly expressed during the night whereas HbCEN1 and HbCEN2 showed irregular diurnal rhythms.
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Flores/metabolismo , Flores/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Hevea/metabolismo , Hevea/fisiologia , Proteínas de Plantas/metabolismo , Flores/genética , Regulação da Expressão Gênica de Plantas/genética , Hevea/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Plântula/genética , Plântula/metabolismo , Plântula/fisiologiaRESUMO
The rubber tree (Hevea brasiliensis Muell. Arg) is a tropical, perennial, woody plant that is susceptible to cold stress. In China, cold stress has been found to severely damage rubber plants in plantations in past decades. Although several Hevea clones that are resistant to cold have been developed, their cold hardiness mechanism has yet to be elucidated. For the study reported herein, we subjected the cold-resistant clone CATAS93-114 and the cold-sensitive clone Reken501 to chilling stress, and characterized their transcriptomes at 0, 2, 8 and 24 h after the start of chilling. We found that 7870 genes were differentially expressed in the transcriptomes of the two clones. In CATAS93-114, a greater number of genes were found to be up- or downregulated between 2 h and 8 h than in Reken501, which indicated a more rapid and intensive response by CATAS93-114 than by Reken501. The differentially expressed genes were grouped into seven major clusters, according to their Gene Ontology terms. The expression profiles for genes involved in abscisic acid metabolism and signaling, in an abscisic acid-independent pathway, and in early signal perception were found to have distinct expression patterns for the transcriptomes of the two clones. The differential expression of 22 genes that appeared to have central roles in response to chilling was confirmed by quantitative real-time PCR.
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Resposta ao Choque Frio/genética , Hevea/genética , Transcriptoma , Ácido Abscísico/genética , Ácido Abscísico/metabolismo , Resposta ao Choque Frio/fisiologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Hevea/fisiologia , Proteínas de Plantas/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/genéticaRESUMO
Rubber trees are economically important tropical tree species and the major source of natural rubber, which is an essential industrial material. This tropical perennial tree is susceptible to cold stress and other abiotic stresses, especially in the marginal northern tropics. Recent years, the genome sequencing and RNA-seq projects produced huge amount of sequence data, which greatly facilitated the functional genomics study. However, the characterization of individual functional gene is in urgent demands, especially for those involved in stress resistance. Here we identified and characterized the rubber tree gene ErbB-3 binding protein 1, which undergoes changes in expression in response to cold, drought stress and ABA treatment. HbEBP1 overexpression (OE) in Arabidopsis increased organ size, facilitated root growth and increased adult leaf number by delaying the vegetative-to-reproductive transition. In addition, HbEBP1 OE enhanced the resistance of the Arabidopsis plants to freezing and drought stress, demonstrating that this gene participates in the regulation of abiotic stress resistance. RD29a, RD22 and CYCD3;1 expression was also greatly enhanced by HbEBP1 OE, which explains its regulatory roles in organ size and stress resistance. The regulation of drought stress resistance is a novel function identified in plant EBP1 genes, which expands our understanding of the roles of EBP1 gene in response to the environment. Our results provide information that may lead to the use of HbEBP1 in genetically engineered crops to increase both biomass and abiotic stress resistance.
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BACKGROUND: Forward genetics approaches are not popularly applied in non-model plants due to their complex genomes, long life cycles, backward genetic studies etc. Researchers have to adopt reverse genetic methods to characterize gene functions in non-model plants individually, the efficiency of which is usually low. RESULTS: In this study, we report a gain-of-function in Arabidopsis (GAINA) strategy which can be used for batch identification of functional genes in a plant species. This strategy aims to obtain the gain-of-function of rubber tree genes through overexpressing transformation ready full-length cDNA libraries in Arabidopsis. An initial transformation test produced about two thousand independent transgenic Arabidopsis lines, in which multiple obvious aberrant phenotypes were observed, suggesting the gain-of-function of rubber tree genes. The transferred genes were further isolated and identified. One gene identified to be metallothionein-like protein type 3 gene was further transferred into Arabidopsis and reproduced a similar aberrant phenotype. CONCLUSION: The GAINA system proves to be an efficient tool for batch identification of functional genes in Hevea brasiliensis, and also applicable in other non-model plants.
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A homolog of MOTHER OF FT AND TFL1 (MFT) was isolated from Hevea brasiliensis and its biological function was investigated. Protein multiple sequence alignment and phylogenetic analysis revealed that HbMFT1 conserved critical amino acid residues to distinguish MFT, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1)-like proteins and showed a closer genetic relationship to the MFT-like group. The accumulation of HbMFT1 was generally detected in various tissues except pericarps, with the highest expression in embryos and relatively higher expression in roots and stems of seedlings, flowering inflorescences, and male and female flowers. HbMFT1 putative promoter analysis showed that tissue-specific, environmental change responsive and hormone-signaling responsive elements were generally present. HbMFT1 was strongly induced under a short-day condition at 28 °C, with the highest expression after the onset of a day. Overexpression of HbMFT1 inhibited seed germination, seedling growth, and flowering in transgenic Arabidopsis. The qRT-PCR further confirmed that APETALA1 (AP1) and FRUITFULL (FUL) were drastically down-regulated in 35S::HbMFT1 plants. A histochemical ß-glucuronidase (GUS) assay showed that HbMFT1::GUS activity was mainly detected in stamens and mature seeds coinciding with its original expression and notably induced in rosette leaves and seedlings of transgenic Arabidopsis by exogenous abscisic acid (ABA) due to the presence of ABA cis-elements in HbMFT1 promoter. These results suggested that HbMFT1 was mainly involved in maintenance of seed maturation and stamen development, but negatively controlled germination, growth and development of seedlings and flowering. In addition, the HbMFT1 promoter can be utilized in controlling transgene expression in stamens and seeds of rubber tree or other plant species.
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Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Transporte/genética , Hevea/crescimento & desenvolvimento , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Flores/química , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Hevea/química , Hevea/genética , Hevea/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteína de Ligação a Fosfatidiletanolamina/análise , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Filogenia , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Plântula/química , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Alinhamento de SequênciaRESUMO
Rubber trees (Hevea brasiliensis) are susceptible to low temperature and therefore are only planted in the tropical regions. In the past few decades, although rubber trees have been successfully planted in the northern margin of tropical area in China, they suffered from cold injury during the winter. To understand the physiological response under cold stress, we isolated a C-repeat binding factor 1 (CBF1) gene from the rubber tree. This gene (HbCBF1) was found to respond to cold stress but not drought or ABA stress. The corresponding HbCBF1 protein showed CRT/DRE binding activity in gel shift experiment. To further characterize its molecular function, the HbCBF1 gene was overexpressed in Arabidopsis. The HbCBF1 over expression (OE) line showed enhanced cold resistance and relatively slow dehydration, and the expression of Arabidopsis CBF pathway downstream target genes, e.g. AtCOR15a and AtRD29a, were significantly activated under non-acclimation condition. These data suggest HbCBF1 gene is a functional member of the CBF gene family, and may play important regulation function in rubber tree.
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Hevea/metabolismo , Proteínas de Plantas/metabolismo , Transativadores/metabolismo , Aclimatação , Sequência de Aminoácidos , Sequência de Bases , Resposta ao Choque Frio , Hevea/genética , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Transativadores/química , Transativadores/genéticaRESUMO
In order to enrich gene encoding region of Hevea brasiliensis, a methylation filtration library was constructed using Escherichia coli McrBC restriction-modification system. The titers of the non-amplified library and the amplified library were 2.6×106 pfu/ml and 9.0×109, respectively. The rate of positive clones was 86.4%. The lengths of inserted DNA sequence ranged from 1 kb to 2.5 kb and the average size of inserts was 1.2 kb. One hundred clones were selected randomly for sequencing, resulting in splicing out of 81 non-redundant sequences, including 6 contigs and 75 singlets. The redundancy was 17.35%. Blast analysis showed that 39.5% of non-redundant sequences were homologous with the Nr database, 14.81% with the EST database, and 32.1% were unknown sequences. Some sequences were related genes for flowering, insect and disease resistance. Therefore, the rubber tree methylation library is helpful for discovery and cloning of functional genes.