Chloroplast Comparative Genomics: Implications For Phylogeny, Evolution, and Biotechnology

Lack of complete chloroplast genome sequences is still a limiting factor determining phylogenetic relationships, discerning evolutionary forces, and extending chloroplast genetic engineering to useful crops. Therefore, the chloroplast genomes from six economically important crops were isolated and sequenced. The results will have an impact on chloroplast biology and biotechnology. The complete soybean chloroplast genome was compared to the other completely sequenced legumes, Lotus and Medicago. The rpl22 gene was found to be missing from all three legumes, a very informative phylogenetic marker. There is a single, large inversion changing the gene order in the legumes from the typical order found in Arabidopsis. Detailed analysis of repeat elements within the chloroplast genomes analyzed indicate they may play some functional role in evolution, and that the psbA and rbcL repeats indicate that the loss of an inverted repeat has only occurred once during the evolutionary history of the legumes. Ideal sites for integration of transgenes were also determined. Next, the chloroplast genomes of the agriculturally important solanacaeae crops Solanum lycopersicum and Solanum bulbocastanum were isolated and sequenced. Analysis of the complete chloroplast genome sequences revealed significant insertions and deletions (indels) within certain coding regions. Photosynthesis, RNA, and atp synthase genes are the least divergent and the most divergent genes are clpP, cemA, ccsA, and matK. The identified repeats characterized across the solanaceae are similar to the legumes, located in the same genes or intergenic regions indicating a possible functional role. A comprehensive genome-wide analysis of all coding sequences and intergenic spacer regions was done for the first time in chloroplast genomes. Analysis of RNA editing sites demonstrated they were less common than what was previously observed in tobacco and Atropa, suggesting a loss of editing sites and a possible increase in variation at the RNA level. Finally, the complete chloroplast genome sequences of barley, sorghum, and creeping bentgrass, were identified and compared to six published grass chloroplast genomes to reveal that gene content and order are similar, but two microstructural changes have occurred. First, the expansion of the inverted repeat at the small single copy/inverted repeat boundary that duplicates a portion of the 5\u27 end of ndhH is restricted to three genera of the subfamily Pooideae (Agrostis, Hordeum, and Triticum). Second, a 6bp deletion in ndhK is shared by creeping bentgrass, barley, rice, and wheat, and this event supports the sister relationship between the subfamilies Erhartoideae and Pooideae. Repeat analysis revealed many dispersed repeats shared among the grasses, as well as repeats that flank a major genome rearrangement common only to the grasses suggesting this repeat had a functional role in the genome rearrangement. Examination of simple sequence repeat markers identified 16-21 potential SSRs. Distances based on intergenic spacer regions were analyzed as well as RNA editing sites. Phylogenetic trees based on DNA sequences of 61 protein-coding genes of 38 taxa using both maximum parsimony and likelihood methods provide moderate support for a sister relationship between the subfamilies Erhartoideae and Pooideae

Gene discovery and differential expression analysis of humoral immune response elements in female Culicoides sonorensis (Diptera: Ceratopogonidae)

Female Culicoides sonorensis midges (Diptera: Ceratopogonidae) are vectors of pathogens that impact livestock and wildlife in the United States. Little is known about their biology on a molecular-genetic level, including components of their immune system. Because the insect immune response is involved with important processes such as gut microbial homeostasis and vector competence, our aims were to identify components of the midge innate immune system and examine their expression profiles in response to diet across time

Meeting Minutes

Meeting regarding international education, salaries, independent contractors, affirmative action and freshman seminar

Using next-generation sequencing technology, RNA-Seq, to understand the Chinese Hamster Ovary (CHO) cell transcriptome under industrially relevant conditions

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Choline Supplementation and DNA Methylation in the Hippocampus and Prefrontal Cortex of Rats Exposed to Alcohol During Development

BACKGROUND: Some of the most frequent deficits seen in children with fetal alcohol spectrum disorders (FASD) and in animal models of FASD are spatial memory impairments and impaired executive functioning, which are likely related to alcohol-induced alterations of the hippocampus and prefrontal cortex (PFC), respectively. Choline, a nutrient supplement, has been shown in a rat model to ameliorate some of alcohol\u27s teratogenic effects, and this effect may be mediated through choline\u27s effects on DNA methylation. METHODS: Alcohol was given by intragastric intubation to rat pups during the neonatal period (postnatal days 2 to 10) (ET group), which is equivalent to the third trimester in humans and a period of heightened vulnerability of the brain to alcohol exposure. Control groups included an intubated control group given the intubation procedure without alcohol (IC) and a nontreated control group (NC). Choline or saline was administered subcutaneously to each subject from postnatal days 2 to 20. On postnatal day 21, the brains of the subjects were removed and assayed for global DNA methylation patterning as measured by chemiluminescence using the cpGlobal assay in both the hippocampal region and PFC. RESULTS: Alcohol exposure caused hypermethylation in the hippocampus and PFC, which was significantly reduced after choline supplementation. In contrast, control animals showed increases in DNA methylation in both regions after choline supplementation, suggesting that choline supplementation has different effects depending upon the initial state of the brain. CONCLUSIONS: This study is the first to show changes in global DNA methylation of the hippocampal region and PFC after neonatal alcohol exposure. Choline supplementation impacts global DNA methylation in these 2 brain regions in alcohol-exposed and control animals in a differential manner. The current findings suggest that both alcohol and choline have substantial impact on the epigenome in the PFC and hippocampus, and future studies will be needed to describe which gene families are impacted in such a way that function of the nervous system is changed

Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (\u3cem\u3eRubus\u3c/em\u3e L.)

Background Due to a relatively high level of codominant inheritance and transferability within and among taxonomic groups, simple sequence repeat (SSR) markers are important elements in comparative mapping and delineation of genomic regions associated with traits of economic importance. Expressed sequence tags (ESTs) are a source of SSRs that can be used to develop markers to facilitate plant breeding and for more basic research across genera and higher plant orders. Methods Leaf and meristem tissue from ‘Heritage’ red raspberry (Rubus idaeus) and ‘Bristol’ black raspberry (R. occidentalis) were utilized for RNA extraction. After conversion to cDNA and library construction, ESTs were sequenced, quality verified, assembled and scanned for SSRs. Primers flanking the SSRs were designed and a subset tested for amplification, polymorphism and transferability across species. ESTs containing SSRs were functionally annotated using the GenBank non-redundant (nr) database and further classified using the gene ontology database. Results To accelerate development of EST-SSRs in the genus Rubus (Rosaceae), 1149 and 2358 cDNA sequences were generated from red raspberry and black raspberry, respectively. The cDNA sequences were screened using rigorous filtering criteria which resulted in the identification of 121 and 257 SSR loci for red and black raspberry, respectively. Primers were designed from the surrounding sequences resulting in 131 and 288 primer pairs, respectively, as some sequences contained more than one SSR locus. Sequence analysis revealed that the SSR-containing genes span a diversity of functions and share more sequence identity with strawberry genes than with other Rosaceous species. Conclusion This resource of Rubus-specific, gene-derived markers will facilitate the construction of linkage maps composed of transferable markers for studying and manipulating important traits in this economically important genus

Enhancing Genome Investigations in the Mosquito Culex quinquefasciatus via BAC Library Construction and Characterization

Background Culex quinquefasciatus (Say) is a major species in the Culex pipiens complex and an important vector for several human pathogens including West Nile virus and parasitic filarial nematodes causing lymphatic filariasis. It is common throughout tropical and subtropical regions and is among the most geographically widespread mosquito species. Although the complete genome sequence is now available, additional genomic tools are needed to improve the sequence assembly. Findings We constructed a bacterial artificial chromosome (BAC) library using the pIndigoBAC536 vector and HindIII partially digested DNA isolated from Cx. quinquefasciatus pupae, Johannesburg strain (NDJ). Insert size was estimated by NotI digestion and pulsed-field gel electrophoresis of 82 randomly selected clones. To estimate genome coverage, each 384-well plate was pooled for screening with 29 simple sequence repeat (SSR) and five gene markers. The NDJ library consists of 55,296 clones arrayed in 144 384-well microplates. Fragment insert size ranged from 50 to 190 kb in length (mean = 106 kb). Based on a mean insert size of 106 kb and a genome size of 579 Mbp, the BAC library provides ~10.1-fold coverage of the Cx. quinquefasciatus genome. PCR screening of BAC DNA plate pools for SSR loci from the genetic linkage map and for four genes associated with reproductive diapause in Culex pipiens resulted in a mean of 9.0 positive plate pools per locus. Conclusion The NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes

Construction of Papaya Male and Female BAC Libraries and Application in Physical Mapping of the Sex Chromosomes

Papaya is a major fruit crop in the tropics and has recently evolved sex chromosomes. Towards sequencing the papaya sex chromosomes, two bacterial artificial chromosome (BAC) libraries were constructed from papaya male and female genomic DNA. The female BAC library was constructed using restriction enzyme BstY I and consists of 36,864 clones with an average insert size of 104 kb, providing 10.3x genome equivalents. The male BAC library was constructed using restriction enzyme EcoR I and consists of 55,296 clones with an average insert size of 101 kb, providing 15.0x genome equivalents. The male BAC library was used in constructing the physical map of the male-specific region of the male Y chromosome (MSY) and in filling gaps and extending the physical map of the hermaphrodite-specific region of the Yh chromosome (HSY) and the X chromosome physical map. The female BAC library was used to extend the X physical map gap. The MSY, HSY, and X physical maps offer a unique opportunity to study chromosomal rearrangements, Y chromosome degeneration, and dosage compensation of the papaya nascent sex chromosomes

Phylogenetic Analyses of Vitis (Vitaceae) Based on Complete Chloroplast Genome Sequences: Effects of Taxon Sampling and Phylogenetic Methods on Resolving Relationships Among Rosids

Background The Vitaceae (grape) is an economically important family of angiosperms whose phylogenetic placement is currently unresolved. Recent phylogenetic analyses based on one to several genes have suggested several alternative placements of this family, including sister to Caryophyllales, asterids, Saxifragales, Dilleniaceae or to rest of rosids, though support for these different results has been weak. There has been a recent interest in using complete chloroplast genome sequences for resolving phylogenetic relationships among angiosperms. These studies have clarified relationships among several major lineages but they have also emphasized the importance of taxon sampling and the effects of different phylogenetic methods for obtaining accurate phylogenies. We sequenced the complete chloroplast genome of Vitis vinifera and used these data to assess relationships among 27 angiosperms, including nine taxa of rosids. Results The Vitis vinifera chloroplast genome is 160,928 bp in length, including a pair of inverted repeats of 26,358 bp that are separated by small and large single copy regions of 19,065 bp and 89,147 bp, respectively. The gene content and order of Vitis is identical to many other unrearranged angiosperm chloroplast genomes, including tobacco. Phylogenetic analyses using maximum parsimony and maximum likelihood were performed on DNA sequences of 61 protein-coding genes for two datasets with 28 or 29 taxa, including eight or nine taxa from four of the seven currently recognized major clades of rosids. Parsimony and likelihood phylogenies of both data sets provide strong support for the placement of Vitaceae as sister to the remaining rosids. However, the position of the Myrtales and support for the monophyly of the eurosid I clade differs between the two data sets and the two methods of analysis. In parsimony analyses, the inclusion of Gossypium is necessary to obtain trees that support the monophyly of the eurosid I clade. However, maximum likelihood analyses place Cucumis as sister to the Myrtales and therefore do not support the monophyly of the eurosid I clade. Conclusion Phylogenies based on DNA sequences from complete chloroplast genome sequences provide strong support for the position of the Vitaceae as the earliest diverging lineage of rosids. Our phylogenetic analyses support recent assertions that inadequate taxon sampling and incorrect model specification for concatenated multi-gene data sets can mislead phylogenetic inferences when using whole chloroplast genomes for phylogeny reconstruction

Characterization of extrachromosomal circular DNA (eccDNA) structure, function, and dynamics in a CHO K-1 clone

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