Do potatoes and tomatoes have a single evolutionary history, and what proportion of the genome supports this history?

BACKGROUND: Phylogenies reconstructed with only one or a few independently inherited loci may be unresolved or incongruent due to taxon and gene sampling, horizontal gene transfer, or differential selection and lineage sorting at individual loci. In an effort to remedy this situation, we examined the utility of conserved orthologous set (COSII) nuclear loci to elucidate the phylogenetic relationships among 29 diploid Solanum species in the sister clades that include tomato and potato, and in Datura inoxia as a far outgroup. We screened 40 COSII markers with intron content over 60% that are mapped in different chromosomes; selected a subset of 19 by the presence of single band amplification of size mostly between 600 and 1200 bp; sequenced these 19 COSII markers, and performed phylogenetic analyses with individual and concatenated datasets. The present study attempts to provide a fully resolved phylogeny among the main clades in potato and tomato that can help to identify the appropriate markers for future studies using additional species. RESULTS: Among potatoes, when total evidence is invoked, one single predominant history is highlighted with complete resolution within and among the three main clades. It also supports the hypothesis of the North and Central American B-genome origin of the tuber-bearing members of Solanum sect. Petota and shows a clear division between A genomes in clades 3 and 4, and B genomes in clade 1+2. On the other hand, when a prior agreement approach is invoked other potato evolutionary histories are revealed but with less support. These alternative histories could be explained by past hybridization, or fast rates of speciation. In the case of tomato, the analyses with all sequence data completely resolved 19 of 21 clades, for the first time revealed the monophyly of five clades, and gave further support for the recent segregation of new species from the former Solanum peruvianum. Concordance analyses revealed and summarized the extensive discordance among COSII markers. Some potential reasons for discordance could be methodological, to include systematic errors due to using a wrong model of sequence evolution, coupled with long branches, or mixtures of branch lengths within COSII, or undetected paralogy or alignment bias. Other reasons could be biological processes such as hybridization or lineage sorting. CONCLUSION: This study confirms and quantifies the utility of using DNA sequences from different parts of the genome in phylogenetic studies to avoid possible bias in the sampling. It shows that 11-18 loci are enough to get the dominant history in this group of Solanum, but more loci would be needed to discern the distribution of gene genealogies in more depth, and thus detect which mechanism most likely shaped the discordance.

Differential regulation of grain sucrose accumulation and metabolism in Coffea arabica (Arabica) and Coffea canephora (Robusta) revealed through gene expression and enzyme activity analysis.

* Coffea arabica (Arabica) and Coffea canephora (Robusta) are the two main cultivated species used for coffee bean production. Arabica genotypes generally produce a higher coffee quality than Robusta genotypes. Understanding the genetic basis for sucrose accumulation during coffee grain maturation is an important goal because sucrose is an important coffee flavor precursor. * Nine new Coffea genes encoding sucrose metabolism enzymes have been identified: sucrose phosphate synthase (CcSPS1, CcSPS2), sucrose phosphate phosphatase (CcSP1), cytoplasmic (CaInv3) and cell wall (CcInv4) invertases and four invertase inhibitors (CcInvI1, 2, 3, 4). * Activities and mRNA abundance of the sucrose metabolism enzymes were compared at different developmental stages in Arabica and Robusta grains, characterized by different sucrose contents in mature grain. * It is concluded that Robusta accumulates less sucrose than Arabica for two reasons: Robusta has higher sucrose synthase and acid invertase activities early in grain development - the expression of CcSS1 and CcInv2 appears to be crucial at this stage and Robusta has a lower SPS activity and low CcSPS1 expression at the final stages of grain development and hence has less capacity for sucrose re-synthesis. Regulation of vacuolar invertase CcInv2 activity by invertase inhibitors CcInvI2 and/or CcInvI3 during Arabica grain development is considered.

Euchromatin and pericentromeric heterochromatin: comparative composition in the tomato genome.

Eleven sequenced BACs were annotated and localized via FISH to tomato pachytene chromosomes providing the first global insights into the compositional differences of euchromatin and pericentromeric heterochromatin in this model dicot species. The results indicate that tomato euchromatin has a gene density (6.7 kb/gene) similar to that of Arabidopsis and rice. Thus, while the euchromatin comprises only 25% of the tomato nuclear DNA, it is sufficient to account for approximately 90% of the estimated 38,000 nontransposon genes that compose the tomato genome. Moreover, euchromatic BACs were largely devoid of transposons or other repetitive elements. In contrast, BACs assigned to the pericentromeric heterochromatin had a gene density 10-100 times lower than that of the euchromatin and are heavily populated by retrotransposons preferential to the heterochromatin-the most abundant transposons belonging to the Jinling Ty3/gypsy-like retrotransposon family. Jinling elements are highly methylated and rarely transcribed. Nonetheless, they have spread throughout the pericentromeric heterochromatin in tomato and wild tomato species fairly recently-well after tomato diverged from potato and other related solanaceous species. The implications of these findings on evolution and on sequencing the genomes of tomato and other solanaceous species are discussed.

Cloning, expression, crystallization and preliminary X-ray analysis of the XMT and DXMT N-methyltransferases from Coffea canephora (robusta).

Caffeine is a secondary metabolite produced by a variety of plants including Coffea canephora (robusta) and there is growing evidence that caffeine is part of a chemical defence strategy protecting young leaves and seeds from potential predators. The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-L-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). The crystals are orthorhombic, with space group P2(1)2(1)2(1) for XMT and C222(1) for DXMT. X-ray diffraction to 2.8 A for XMT and to 2.5 A for DXMT have been collected on beamline ID23-1 at the ESRF.

Oleosin gene family of Coffea canephora: quantitative expression analysis of five oleosin genes in developing and germinating coffee grain.

Coffee grains have an oil content between 10% and 16%, with these values associated with Coffea canephora (robusta) and C. arabica (arabica), respectively. As the majority of the oil stored in oil seeds is contained in specific structures called oil bodies, we were interested in determining whether there are any differences in the expression of the main oil body proteins, the oleosins, between the robusta and arabica varieties. Here, we present the isolation, characterization and quantitative expression analysis of six cDNAs representing five genes of the coffee oleosin family (CcOLE-1 to CcOLE-5) and one gene of the steroleosin family (CcSTO-1). Each coffee oleosin cDNA encodes for the signature structure for oleosins, a long hydrophobic central sequence containing a proline KNOT motif. Sequence analysis also indicates that the C-terminal domain of CcOLE-1, CcOLE-3 and CcOLE-5 contain an 18-residue sequence typical of H-form oleosins. Quantitative RT-PCR showed that the transcripts of all five oleosins were predominantly expressed during grain maturation in robusta and arabica grain, with CcOLE-1 and CcOLE-2 being more highly expressed. While the relative expression levels of the five oleosins were similar for robusta and arabica, significant differences in the absolute levels of expression were found between the two species. Quantitative analysis of oleosin transcripts in germinating arabica grain generally showed that the levels of these transcripts were lower in the grain after drying, and then further decreased during germination, except for a small spike of expression for CcOLE-2 early in germination. In contrast, the levels of CcSTO-1 transcripts remained relatively constant during germination, in agreement with suggestions that this protein is actively involved in the process of oil body turnover. Finally, we discuss the implications of the coffee oleosin expression data presented relative to the predicted roles for the different coffee oleosins during development and germination.

Traumatic Visual Loss and a Limitation of Point-of-Care Ocular Ultrasound: A Case Report.

Incorporation of point-of-care ultrasound into the skill set of Special Operations medical providers should come with an appreciation of the potential limitations of the technology. We present a case of a U.S. Army Special Forces Soldier who suffered traumatic monocular vision loss after being struck in the eye during a combatives tournament. Evaluation in the emergency department (ED) included an unremarkable ocular ultrasound, despite a high clinical suspicion of intraocular pathology. Ophthalmologic consultation was obtained emergently. Optical coherence topography and a dilated fundoscopic examination were performed, which revealed a small subretinal hemorrhage. We will review the history of ocular ultrasound and its sensitivity to detect intraocular pathology. We will also emphasize the need to obtain specialty consultation when the clinical suspicion for intraocular pathology is high despite a negative ocular ultrasound.

An investigation of carotenoid biosynthesis in Coffea canephora and Coffea arabica.

Carotenoids are essential components of the photosynthetic apparatus in a wide range of organisms. They participate in the adaptation of plastids to changing environmental light conditions and prevent photo-oxidative damage of the photosynthetic apparatus by detoxifying reactive oxygen species. We identified eight cDNAs from the carotenoid biosynthetic pathway (PSY, PDS, ZDS, PTOX, LCY-E, CRTR-B, ZEP and VDE) and two cDNA encoding carotenoid cleavage dioxygenase family members (NCED3 and CCD1) in Coffea canephora. We also obtained cDNA encoding several different fibrillin proteins involved in carotenoid sequestration (FIB). Expression of the coffee carotenoid genes was determined in leaf, branch and flower tissues using quantitative RT-PCR. Expression analysis of these genes in leaf tissue from osmotically stressed plants was also carried out. These experiments showed that the transcript levels of PTOX, CRTR-B, NCED3, CCD1 and FIB1 increased under these stress conditions, while LCY-E decreased, indicating that the metabolic flux towards the xanthophyll cycle branch of the carotenoid biosynthetic pathway may be favoured in leaves under drought conditions. Functional analysis of CcCRTR-B using an in vivo method employing Escherichia coli strains engineered to make carotenoids confirmed that the beta-carotene hydroxylase activity of CcCRTR-B generates beta-cryptoxanthin and zeaxanthin from beta-carotene. A similar approach was also used to show that CcCCD1 encoded a functional 9,10(9'10') carotenoid cleavage dioxygenase, and thus that this enzyme is capable of forming one or more apocarotenoids in vivo. Finally, high-performance liquid chromatography analysis of coffee leaves revealed the presence of alpha-carotene and suggests that Coffea arabica may have higher levels of alpha-carotene than C. canephora.

Isolation and characterization of cDNA encoding three dehydrins expressed during Coffea canephora (Robusta) grain development.

BACKGROUND AND AIMS: Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized. METHODS: The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT-PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2). KEY RESULTS: The CcDH1 and CcDH2 genes encode Y(3)SK(2) dehydrins and the CcDH3 gene encodes an SK(3) dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized. CONCLUSIONS: cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.

Comparative analyses of potato expressed sequence tag libraries.

The cultivated potato (Solanum tuberosum) shares similar biology with other members of the Solanaceae, yet has features unique within the family, such as modified stems (stolons) that develop into edible tubers. To better understand potato biology, we have undertaken a survey of the potato transcriptome using expressed sequence tags (ESTs) from diverse tissues. A total of 61,940 ESTs were generated from aerial tissues, below-ground tissues, and tissues challenged with the late-blight pathogen (Phytophthora infestans). Clustering and assembly of these ESTs resulted in a total of 19,892 unique sequences with 8,741 tentative consensus sequences and 11,151 singleton ESTs. We were able to identify a putative function for 43.7% of these sequences. A number of sequences (48) were expressed throughout the libraries sampled, representing constitutively expressed sequences. Other sequences (13,068, 21%) were uniquely expressed and were detected only in a single library. Using hierarchal and k means clustering of the EST sequences, we were able to correlate changes in gene expression with major physiological events in potato biology. Using pair-wise comparisons of tuber-related tissues, we were able to associate genes with tuber initiation, dormancy, and sprouting. We also were able to identify a number of characterized as well as novel sequences that were unique to the incompatible interaction of late-blight pathogen, thereby providing a foundation for further understanding the mechanism of resistance.