BAC-end sequences analysis provides first insights into coffee (Coffea canephora P.) genome composition and evolution

Coffee is one of the world’s most important agricultural commodities. Coffee belongs to the Rubiaceae family in the euasterid I clade of dicotyledonous plants, to which the Solanaceae family also belongs. Two bacterial artificial chromosome (BAC) libraries of a homozygous doubled haploid plant of Coffea canephora were constructed using two enzymes, HindIII and BstYI. A total of 134,827 high quality BAC-end sequences (BESs) were generated from the 73,728 clones of the two libraries, and 131,412 BESs were conserved for further analysis after elimination of chloroplast and mitochondrial sequences. This corresponded to almost 13 % of the estimated size of the C. canephora genome. 6.7 % of BESs contained simple sequence repeats, the most abundant (47.8 %) being mononucleotide motifs. These sequences allow the development of numerous useful marker sites. Potential transposable elements (TEs) represented 11.9 % of the full length BESs. A difference was observed between the BstYI and HindIII libraries (14.9 vs. 8.8 %). Analysis of BESs against known coding sequences of TEs indicated that 11.9 % of the genome corresponded to known repeat sequences, like for other flowering plants. The number of genes in the coffee genome was estimated at 41,973 which is probably overestimated. Comparative genome mapping revealed that microsynteny was higher between coffee and grapevine than between coffee and tomato or Arabidopsis. BESs constitute valuable resources for the first genome wide survey of coffee and provide new insights into the composition and evolution of the coffee genome.     

Characterization of duplicated Dunaliella viridis SPT1 genes provides insights into early gene divergence after duplication

The sodium-dependent phosphate transporter gene from unicellular green algae Dunaliella viridis, DvSPT1, shares similarity with members of Pi transporter family. Sequencing analysis of D. viridis BAC clone containing the DvSPT1 gene revealed two inverted duplicated copies of this gene (DvSPT1 and DvSPT1-2 respectively). The duplication covered most of both genes except for their 3' downstream region. The duplicated genomic sequences exhibited 97.9% identity with a synonymous divergence of Ks=0.0126 in the coding region. This data indicated very recent gene duplication in D. viridis genome, providing an excellent opportunity to investigate sequence and expression divergence of duplicated genes at an early stage. Scatted point mutations and length polymorphism of simple sequence repeats (SSRs) were predominant among the sequence divergence soon after gene duplication. Due to sequence divergence in the 5' regulatory regions and a swap of the entire 3' downstream regions (3'-UTR), DvSPT1 and DvSPT1-2 showed expression divergence in response to extra-cellular NaCl concentration changes. According to their expression patterns, the two diverged gene copies would provide better adaptation to a broader range of extra-cellular NaCl concentration. Furthermore, Southern blot analysis indicated that there might be a large phosphate transporter gene family in D. viridis.     

Transcriptome analysis provides insights into lignin synthesis and MAPK signaling pathway that strengthen the resistance of bitter gourd (Momordica charantia) to Fusarium wilt

Fusarium wilt is a typical soil-borne disease caused by Fusarium oxysporum f. sp. momordicae (FOM) in bitter gourd. In this study, by comparing sequencing data at multiple time points and considering the difference between resistant (R) and susceptible (S) varieties, differentially expressed genes were screened out. Short time-series expression miner analysis revealed the upregulated expression trend of genes, which were enriched in phenylpropanoid biosynthesis, plant–pathogen interaction, and mitogen-activated protein kinase signaling pathway. Further, observation of the microstructure revealed that the R variety may form tyloses earlier than the S variety to prevent mycelium diffusion from the xylem vessel. After Fusarium wilt infection, the enzymatic activities of superoxide dismutase, peroxidase, phenylalanine ammonia lyase, and catalaseas well as levels of superoxide anion and malondialdehyde were increased in the R variety higher than those in the S variety. This study provides a reference to elucidate the disease resistance mechanism of bitter gourd.     

Identification of 20-hydroxyecdysone late-response genes in the chitin biosynthesis pathway

Background

20-hydroxyecdysone (20E) and its receptor complex ecdysone receptor (EcR) and ultraspiracle (USP) play a crucial role in controlling development, metamorphosis, reproduction and diapause. The ligand-receptor complex 20E-EcR/USP directly activates a small set of early-response genes and a much larger set of late-response genes. However, ecdysone-responsive genes have not been previously characterized in the context of insect chitin biosynthesis.

Principal Findings

Here, we show that injection-based RNA interference (RNAi) directed towards a common region of the two isoforms of SeEcR in a lepidopteron insect Spodoptera exigua was effective, with phenotypes including a high mortality prior to pupation and developmental defects. After gene specific RNAi, chitin contents in the cuticle of an abnormal larva significantly decreased. The expression levels of five genes in the chitin biosynthesis pathway, SeTre-1, SeG6PI, SeUAP, SeCHSA and SeCHSB, were significantly reduced, while there was no difference in the expression of SeTre-2 prior to 72 hr after injection of EcR dsRNA. Meanwhile, injection of 20E in vivo induced the expression of the five genes mentioned above. Moreover, the SeTre-1, SeG6PI, SeUAP and SeCHSB genes showed late responses to the hormone and the induction of SeTre-1, SeG6PI, SeUAP and SeCHSB genes by 20E were able to be inhibited by the protein synthesis inhibitor cycloheximide in vitro indicating these genes are 20E late-response genes.

Conclusions

We conclude that SeTre-1, SeG6PI, SeUAP and SeCHSB in the chitin biosynthesis pathway are 20E late-response genes and 20E and its specific receptors plays a key role in the regulation of chitin biosynthesis via inducing their expression.     

In silico characterization of putative members of the coffee (Coffea arabica) ethylene signaling pathway

The plant hormone ethylene is involved in several developmental and physiological processes in plants, including senescence, fruit ripening and organ abscission, as well as in biotic and abiotic stress responses. Initiation of these processes involves complex regulation of both ethylene biosynthesis and the ability of cells to perceive the hormone and respond in an appropriate manner, a process which is regulated both spatially and temporally. Ethylene is a gaseous hormone whose sensitivity is a key factor to limiting its response in target cells. We made a search of the Coffee Expressed Sequence Tag (CAFEST) database for expressed sequence tags related to known elements of the ethylene signaling pathway. Sequences showing a reliable similarity were clusterized, annotated and analyzed for conserved domains. Multiple alignments comprising the sequences that we found and sequences of ethylene signaling elements from other species were made, and their phylogeny was assessed by phylogenetic trees constructed with the MEGA4 software. The expression profile was assessed by in silico Northern blot analysis performed using the Cluster and TreeView programs. The CAFEST database was found to have a large number of sequences related to previously described ethylene signaling pathway elements, allowing identification of putative members from almost every step of this pathway. The phylogenetic trees demonstrated high similarity between the sequences found in the CAFEST and those from other species, and the electronic Northern blot analysis detected their expression in various tissues, development stages and stress conditions.     

Physiological and molecular analyses of early and late Coffea arabica cultivars at different stages of fruit ripening

Coffee quality is strongly influenced by a great number of factors, among which the fruit ripening stage at harvest time has a major influence on this feature. Studies comprising ethylene production and the regulation of ethylene biosynthesis genes during the ripening process indicate that ethylene plays an important role on coffee fruit ripening. Coffee early cultivars usually show a more uniform ripening process although little is known about the genetic factors that promote the earliness of ripening. Thus, in order to better understand the physiological and genetic factors involved in the regulation of ripening time, and consequently ripening uniformity, this study aimed to analyze ethylene and respiration patterns during coffee ripening, as well as to analyze ACC oxidase, an ethylene biosynthesis enzyme, gene expression, in fruits of early (Catucaí 785-15) and late (Acauã) coffee cultivars. Coffee fruits were harvested monthly from 124 days after flowering (end of February), until complete maturation (end of June). Dry matter, moisture content, color, respiratory rate and ethylene production analysis were performed. In silico analysis identified a coffee ACC oxidase gene (CaACO-like) and its expression was analyzed by real-time PCR. Dry matter and relative water content constantly increased and gradually decreased, respectively, during fruit ripening, and the color analysis enabled the observation of the earliness in the ripening process displayed by Catucaí 785-15 and its higher fruit ripening uniformity. The results obtained from the CaACO-like expression analysis and respiration and ethylene analysis suggest that the differences in ripening behavior between the two coffee cultivars analyzed in this study may be related to the differences in their capacity to produce ethylene, with fruits of Catucaí 785-15 and Acauã showing a typical and an attenuated climacteric phase, respectively, which may have lead to differences in their ripening time and uniformity.     

A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis

Deficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an alpha1,3-mannosyltransferase. The resulting Manalpha1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown alpha1,6-mannosyltransferase.     

Comparative genomic analysis of mammalian NKG2D ligand family genes provides insights into their origin and evolution

NKG2D is a major activating receptor of natural killer cells. Its ligands are major histocompatibility complex (MHC) class I-like molecules whose expression is induced by cellular stresses such as infections and tumorigenesis. Humans have two families of NKG2D ligands (NKG2DL): MHC class I-related chains (MIC) encoded in the MHC and UL16-binding proteins (ULBP) encoded outside the MHC. By contrast, mice have only the latter family of ligands; instead, they have non-MHC-encoded MILL molecules that are closely related to MIC, but do not function as NKG2DL. To gain insights into the origin and evolution of MIC, ULBP, and MILL gene families, we conducted comparative genomic analysis of NKG2DL family genes in five mammalian species. In the opossum MHC, we identified a ULBP-like gene adjacent to a previously described MIC-like gene, suggesting that ULBP genes were originally encoded in the MHC. The opossum genome also contained a transcribed MILL-like gene in a region syntenic to the rodent regions encoding MILL molecules. These observations indicate that MIC-, ULBP-, and MILL-like genes emerged before the divergence of placental and marsupial mammals. Comparison of the human, cattle, rat, mouse, and opossum genomes indicates that after emigration from the MHC, ULBP genes underwent extensive duplications in each species. In mice, some of the ULBP genes appear to have been translocated telomerically on the same chromosome, forming a major cluster of existent NKG2DL genes.     

Transcriptomic analysis provides insights into candidate genes and molecular pathways involved in growth of Manila clam Ruditapes philippinarum

Functional & Integrative Genomics - Growth is one of the most important traits of aquaculture breeding programs. Understanding the mechanisms underlying growth differences between individuals...     

Identification and expression analysis of BMP signaling inhibitors genes of the DAN family in amphioxus

Bone morphogenetic proteins (BMPs) are members of the Transforming Growth Factor-β (TGF-β) family implicated in many developmental processes in metazoans such as embryo axes specification. Their wide variety of actions is in part controlled by inhibitors that impede the interaction of BMPs with their specific receptors. Here, we focused our attention on the Differential screening-selected gene Aberrative in Neuroblastoma (DAN) family of inhibitors. Although they are well-characterized in vertebrates, few data are available for this family in other metazoan species. In order to understand the evolution of potential developmental roles of these inhibitors in chordates, we identified the members of this family in the cephalochordate amphioxus, and characterized their expression patterns during embryonic development. Our data suggest that the function of Cerberus/Dand5 subfamily genes is conserved among chordates, whereas Gremlin1/2 and NBL1 subfamily genes seem to have acquired divergent expression patterns in each chordate lineage. On the other hand, the expression of Gremlin in the amphioxus neural plate border during early neurulation strengthens the hypothesis of a conserved neural plate border gene network in chordates.