Adventures in the Enormous: A 1.8 Million Clone BAC Library for the 21.7 Gb Genome of Loblolly Pine

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu)

Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

<p>Abstract</p> <p>Background</p> <p><it>Eucalyptus </it>species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing.</p> <p>Results</p> <p>We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of <it>E. grandis </it>(clone BRASUZ1) digested with <it>Hind</it>III and <it>BstY</it>I, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest <it>via </it>hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the <it>E. grandis </it>chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes.</p> <p>Conclusions</p> <p>The two <it>E. grandis </it>BAC libraries described in this study represent an important milestone for the advancement of <it>Eucalyptus </it>genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in <it>Eucalyptus </it>and possibly in related species of <it>Myrtaceae</it>, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (<it>E. grandis </it>BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming <it>Eucalyptus </it>reference genome sequence.</p

How buildings become landmarks and their contribution to imageability of urban street scapes

The experiencing of strong images within an urban environment help to generate clues of identification within our mind. The intensity of these clues help to recognize that location accurately. The imageability of a location is strongly influenced by various kinds of landmarks found in that particular environment. These landmarks could be doorknob or a stone mark, or a sophisticated building. The dissertation is a study of landmarks and their characteristics in order to understand their contribution to imageability and legibility in an urban street. Here the main focus was to identify the object buildings in an urban streetscape through an extensive survey, and then determine to what extent they possess the attributes of successful landmarks, which were identified through a literature study. The buildings that were determined to be landmarks were then discussed in terms of their contribution to the overall imageability of the selected street segments considered in the case studies. Imeagibility of streets play a major role which directly effects the day to day life of the community. Streets contribute to the social and urban life of the society which generates feelings of "safety, "sense of place", "sense of belongingness" etc in an urban context. In this context a person should be able to feel and understand his relative position in the street environment to ensure his sense of place and orientation within the street, which facilitates free movement without any confusion or fear of being lost. This results through developing an image of that particular environment, and this will vary from person to person. A clear sense of orientation helps to perceive a good environmental image, which often influences behavior pattern and psychology. Therefore when designing a building to a street facade, it is vital to identify whether the proposed building will act as a landmark or not. The analysis was focused to identify the basic characteristics of a landmark as physical and associational attributes. Physical attributes mainly contribute to the visibility of the object and to dive a symbolical meaning attached to it. Therefore in every context these two characteristics involved to build up "mental pictures" in the minds of its inhabitants. Through this study one can easily understand the role landmarks play within the streetscape and the factors that directly contribute to making more meaningful landmarks within a context. It would also be a valuable reference for architectural students. Through this dissertation it was intended to introduce some basic guidelines for architectural students, town planners, architects etc to understand and stimulate further exploration on the role landmarks play within the streetscape and how these factors contribute to the an overall meaningful Imageability in a particular urban context

Energy Balance Assessment in Agricultural Systems; An Approach to Diversification

The energy in agricultural systems is two-fold: transformation and utilization. The assessment and proper use of energy in agricultural systems is important to achieve economic benefits and overall sustainability. Therefore, this study was conducted to evaluate the energy balance of crop and livestock production, net energy ratio (NER), and water use efficiency (WUE) of crops of a selected farm in Sri Lanka using the life cycle assessment (LCA) approach. In order to assess the diversification, 18 crops and 5 livestock types were used. The data were obtained from farm records, personal contacts, and previously published literature. Accordingly, the energy balance in crop production and livestock production was −316.87 GJ ha−1 Year−1 and 758.73 GJ Year−1, respectively. The energy related WUE of crop production was 31.35 MJ m−3. The total energy balance of the farm was 736.2 GJ Year−1. The results show a negative energy balance in crop production indicating an efficient production system, while a comparatively higher energy loss was shown from the livestock sector. The procedure followed in this study can be used to assess the energy balance of diversified agricultural systems, which is important for agricultural sustainability. This can be further developed to assess the carbon footprint in agricultural systems

Phylogenetic relationships of the sweetpotato in Ipomoea series Batatas (Convolvulaceae) based on nuclear beta-amylase gene sequences.

Phylogenetic relationships of 13 accessions and a cultivar representing the sweetpotato, Ipomoea batatas (L.) Lam., and its wild progenitors, were investigated using the nucleotide sequence variation of a nuclear-encoded beta-amylase gene. A 1.1-1.3 kb fragment of the gene spanning two exons separated by a long intron was PCR-amplified, cloned, and sequenced. Exon sequences proved highly conservative, while intron sequences yielded large differences. Intron analyses grouped species in a phylogenetic context according to the presence of two genome types: A and B. These groups are consistent with results of previous analyses, save for the novel placement of I. tiliacea, among the A-genome species. Sequences specific to both A and B genome species have been identified. Exon sequences indicate that I. ramosissima and I. umbraticola are quite different from other A-genome species. Placement of I. littoralis is questionable; its intron is similar to other B-genome species, but its exons are quite different. Exon evolution indicates that the B-genome has evolved faster than the A-genome. Interspecific intron and exon variation indicates I. trifida, I. tabascana, and I. batatas form a monophyletic group

Energy balance assessment in agricultural systems:an approach to diversification

Abstract The energy in agricultural systems is two-fold: transformation and utilization. The assessment and proper use of energy in agricultural systems is important to achieve economic benefits and overall sustainability. Therefore, this study was conducted to evaluate the energy balance of crop and livestock production, net energy ratio (NER), and water use efficiency (WUE) of crops of a selected farm in Sri Lanka using the life cycle assessment (LCA) approach. In order to assess the diversification, 18 crops and 5 livestock types were used. The data were obtained from farm records, personal contacts, and previously published literature. Accordingly, the energy balance in crop production and livestock production was −316.87 GJ ha⁻¹ Year⁻¹ and 758.73 GJ Year⁻¹, respectively. The energy related WUE of crop production was 31.35 MJ m⁻³. The total energy balance of the farm was 736.2 GJ Year⁻¹. The results show a negative energy balance in crop production indicating an efficient production system, while a comparatively higher energy loss was shown from the livestock sector. The procedure followed in this study can be used to assess the energy balance of diversified agricultural systems, which is important for agricultural sustainability. This can be further developed to assess the carbon footprint in agricultural systems

Lr34 multi-pathogen resistance ABC transporter: molecular analysis of homoeologous and orthologous genes in hexaploid wheat and other grass species

The Triticum aestivum (bread wheat) disease resistance gene Lr34 confers durable, race non-specific protection against three fungal pathogens, and has been a highly relevant gene for wheat breeding since the green revolution. Lr34, located on chromosome 7D, encodes an ATP-binding cassette (ABC) transporter. Both wheat cultivars with and without Lr34-based resistance encode a putatively functional protein that differ by only two amino acid polymorphisms. In this study, we focused on the identification and characterization of homoeologous and orthologous Lr34 genes in hexaploid wheat and other grasses. In hexaploid wheat we found an expressed and putatively functional Lr34 homoeolog located on chromosome 4A, designated Lr34-B. Another homoeologous Lr34 copy, located on chromosome 7A, was disrupted by the insertion of repetitive elements. Protein sequences of LR34-B and LR34 were 97% identical. Orthologous Lr34 genes were detected in the genomes of Oryza sativa (rice) and Sorghum bicolor (sorghum). Zea mays (maize), Brachypodium distachyon and Hordeum vulgare (barley) lacked Lr34 orthologs, indicating independent deletion of this particular ABC transporter. Lr34 was part of a gene-rich island on the wheat D genome. We found gene colinearity on the homoeologous A and B genomes of hexaploid wheat, but little microcolinearity in other grasses. The homoeologous LR34-B protein and the orthologs from rice and sorghum have the susceptible haplotype for the two critical polymorphisms distinguishing the LR34 proteins from susceptible and resistant wheat cultivars. We conclude that the particular Lr34-haplotype found in resistant wheat cultivars is unique. It probably resulted from functional gene diversification that occurred after the polyploidization event that was at the origin of cultivated bread wheat

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning