High-quality Bacterial Artificial Chromosome Research Articles

Large-Scale Preparation and Linearization of Bacterial Artificial Chromosome (BAC) DNA.

A critical step for creating bacterial artificial chromosome (BAC) transgenic organisms is the production of high-quality BAC DNA. The method described here provides a source of DNA for further manipulation and modification, but can also be used for preparing modified BAC DNA, resulting from the one-step or two-step strategy, for use in other applications such as BAC transgenesis. An analysis of precautions to take when working with large DNA molecules is also provided.

Construction of a Genomic Bacterial Artificial Chromosome (BAC) Library for the Prawn Macrobrachium rosenbergii and Initial Analysis of ZW Chromosome-Derived BAC Inserts.

Knowledge on sex determination has proven valuable for commercial production of the prawn Macrobrachium rosenbergii due to sex dimorphism of the male and female individuals. Previous studies indicated that prawn sex is determined by a ZW-ZZ chromosomal system, but no genomic information is available for the sex chromosome. Herein, we constructed a genomic bacterial artificial chromosome (BAC) library and identified the ZW-derived BAC clones for initial analysis of the sex chromosomal DNA sequence. The arrayed BAC library contains 200,448 clones with average insert size of 115.4kb, corresponding to ∼ 4× coverage of the estimated 5.38Gb genome. Based on a short female-specific marker, a Z- and a W-fragment were retrieved with the genomic walking method. Screening the BAC library using a ZW-specific marker as probe resulted in 12 positive clones. From these, a Z-derived (P331M17) and a W-derived (P122G2) BAC clones were randomly selected and sequenced by PacBio method. We report the construction of a large insert, deep-coverage, and high-quality BAC library for M. rosenbergii that provides a useful resource for positional cloning of target genes, genomic organization, and comparative genomics analysis. Our study not only confirmed the ZW/ZZ system but also discovered sex-linked genes on ZW chromosomes for the first time, contributing to a comprehensive understanding of the genomic structure of sex chromosomes in M. rosenbergii.

Construction and characterization of a bacterial artificial chromosome library for Camellia sinensis

Tea is a popular and natural non-alcoholic beverage, and is produced from fresh leaves of Camellia sinensis. Tea leaves contain many bioactive compounds that have significant health benefits. We constructed a high quality bacterial artificial chromosome (BAC) library by using the fresh petals of C. sinensis “Shuchazao” for genome sequencing and improvement of genomic assembly. BAC library is still a significant tool for studies of functional genomes and preservation of precious genetic resources. The BAC library contains 161,280 clones with an average insert size of 113 kb, which represents approximately 6.2-fold coverage of haploid genome equivalents of C. sinensis. We characterized 20 complete BAC clones and 738 BAC end sequences (BESs) ranging from 105 to 917 bp. In addition, we predicted cis-regulatory elements of LAR (leucoanthocyanidin reductase), TCS (caffeine synthase), and TS (theanine synthetase) involved in tea characteristic metabolite synthesis and identified a larger number of light-responsive cis-acting elements in these three genes. Meanwhile, we analyzed alternative splicing of these three genes. Furthermore, 12 pairs of SSR primers were successfully amplified in tea plant DNA. The tea BAC library was a critical resource to accomplish de novo whole-genome sequencing, accelerate gene discovery and enhance molecular breeding of C. sinensis.

Hybrid assembly of the large and highly repetitive genome of Aegilops tauschii, a progenitor of bread wheat, with the MaSuRCA mega-reads algorithm.

Long sequencing reads generated by single-molecule sequencing technology offer the possibility of dramatically improving the contiguity of genome assemblies. The biggest challenge today is that long reads have relatively high error rates, currently around 15%. The high error rates make it difficult to use this data alone, particularly with highly repetitive plant genomes. Errors in the raw data can lead to insertion or deletion errors (indels) in the consensus genome sequence, which in turn create significant problems for downstream analysis; for example, a single indel may shift the reading frame and incorrectly truncate a protein sequence. Here, we describe an algorithm that solves the high error rate problem by combining long, high-error reads with shorter but much more accurate Illumina sequencing reads, whose error rates average <1%. Our hybrid assembly algorithm combines these two types of reads to construct mega-reads, which are both long and accurate, and then assembles the mega-reads using the CABOG assembler, which was designed for long reads. We apply this technique to a large data set of Illumina and PacBio sequences from the species Aegilops tauschii, a large and extremely repetitive plant genome that has resisted previous attempts at assembly. We show that the resulting assembled contigs are far larger than in any previous assembly, with an N50 contig size of 486,807 nucleotides. We compare the contigs to independently produced optical maps to evaluate their large-scale accuracy, and to a set of high-quality bacterial artificial chromosome (BAC)-based assemblies to evaluate base-level accuracy.

Rice blast resistance gene Pikahei-1(t), a member of a resistance gene cluster on chromosome 4, encodes a nucleotide-binding site and leucine-rich repeat protein

Pikahei-1(t) is the strongest quantitative trait locus (QTL) for blast resistance in upland rice cv. Kahei, which has strong field resistance to the rice blast disease. A high-quality bacterial artificial chromosome library was used to fine-map Pikahei-1(t) within ~300 kb on the 31-Mb region on rice chromosome 4. Of the 42 predicted open reading frames, seven resistance gene analogs (RGAs) with the nucleotide-binding site and leucine-rich repeat (NBS-LRR) domain were identified. Among these, RGA1, 2, 3, 5, and 7, but not RGA4 and 6, were found to be expressed in Kahei and monogenic lines containing Pikahei-1(t). Blast inoculation of transgenic rice lines carrying the genomic fragment of each RGA revealed that only RGA3 was associated with blast resistance. On the basis of these results, we concluded that RGA3 is the Pikahei-1(t) and named it Pi63. Pi63 encoded a typical coiled-coil-NBS-LRR protein and showed isolate-specificity. These results suggest that Pi63 behaves like a typical Resistance (R) gene, and the strong and broad-spectrum resistance of Kahei is dependent on natural pyramiding of multiple QTLs. The blast resistance levels of Pi63 were closely correlated with its gene expression levels, indicating a dose-dependent response of Pi63 function in rice resistance. Pi63 is the first cloned R gene in the R gene cluster on rice chromosome 4, and its cloning might facilitate genomic dissection of this cluster region.

Construction of a high-quality genomic BAC library for Chinese peanut cultivar Zhonghua 8 with high oil content.

BackgroundArachis hypogaea L. (2n = 4× = 40, AABB) is one of the most important oil and economic crop plants in the word. This species has the largest genome size of about 2,813 Mb among the oil crop species. Zhonghua 8 is a peanut cultivar planted widely in central China and has several superior traits including high oil content, high yield and disease resistance. A high-quality BAC library of Zhonghua 8 was constructed for future researches on the genomics of Chinese peanut cultivars.ResultsA Hin d III-digested genomic BAC (bacterial artificial chromosome) library was constructed with the genomic DNA from leaves of Zhonghua 8. This BAC library consists of 160,512 clones and the average insert is estimated about 102 kb ranging from 30 to 150 kb. The library represents about 5.55× haploid genome equivalents, and provides a 99.71% probability of finding specific genes. The empty-vector rate is under 5 percent detected from 200 randomly selected clones. Probing of 384 clones with the psbA gene of barley chloroplast and the atp6 gene of rice mitochondrion indicated that the contamination with organellar DNA is insignificant. Successive subculture of three clones showed that the inserts are stable in one hundred generations.ConclusionsThis study presented the construction of a high-quality BAC library for the genome of Chinese cultivated peanut. Many essential experiences were summarized in the present study. This BAC library can serve as a substantial platform for development of molecular marker, isolation of genes and further genome research.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-8) contains supplementary material, which is available to authorized users.

A scalable and flexible approach for investigating the genomic landscapes of phylogenetic incongruence

Analyses of DNA sequence datasets have repeatedly revealed inconsistencies in phylogenetic trees derived with different data. This is termed phylogenetic incongruence, and may arise from a methodological failure of the inference process or from biological processes, such as horizontal gene transfer, incomplete lineage sorting, and introgression. To better understand patterns of incongruence, we developed a method (PartFinder) that uses likelihood ratios applied to sliding windows for visualizing tree-support changes across genome-sequence alignments, allowing the comparative examination of complex phylogenetic scenarios among many species. As a pilot, we used PartFinder to investigate incongruence in the Homo-Pan-Gorilla group as well as Platyrrhini using high-quality bacterial artificial chromosome (BAC)-derived sequences as well as assembled whole-genome shotgun sequences. Our simulations verified the sensitivity of PartFinder, and our results were comparable to other studies of the Homo-Pan-Gorilla group. Analyses of the whole-genome alignments reveal significant associations between support for the accepted species relationship and specific characteristics of the genomic regions, such as GC-content, alignment score, exon content, and conservation. Finally, we analyzed sequence data generated for five platyrrhine species, and found incongruence that suggests a polytomy within Cebidae, in particular. Together, these studies demonstrate the utility of PartFinder for investigating the patterns of phylogenetic incongruence.

Characterizing the walnut genome through analyses of BAC end sequences

Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots of J. regia cv. Chandler using the HindIII and MboI cloning sites. A total of 48,218 high-quality BAC end sequences (BESs) were generated, with an accumulated sequence length of 31.2Mb, representing approximately 5.1% of the walnut genome. Analysis of repeat DNA content in BESs revealed that approximately 15.42% of the genome consists of known repetitive DNA, while walnut-unique repetitive DNA identified in this study constitutes 13.5% of the genome. Among the walnut-unique repetitive DNA, Julia SINE and JrTRIM elements represent the first identified walnut short interspersed element (SINE) and terminal-repeat retrotransposon in miniature (TRIM) element, respectively; both types of elements are abundant in the genome. As in other species, these SINEs and TRIM elements could be exploited for developing repeat DNA-based molecular markers in walnut. Simple sequence repeats (SSR) from BESs were analyzed and found to be more abundant in BESs than in expressed sequence tags. The density of SSR in the walnut genome analyzed was also slightly higher than that in poplar and papaya. Sequence analysis of BESs indicated that approximately 11.5% of the walnut genome represents a coding sequence. This study is an initial characterization of the walnut genome and provides the largest genomic resource currently available; as such, it will be a valuable tool in studies aimed at genetically improving walnut.

Antarctic Notothenioid Fishes: Genomic Resources and Strategies for Analyzing an Adaptive Radiation

The perciform suborder Notothenoidei provides a compelling opportunity to study the adaptive radiation of a marine species-flock in the cold Southern Ocean that surrounds Antarctica. To facilitate genome-level studies of the diversification of these fishes, we present estimates of the genome sizes of 11 Antarctic species and describe the production of high-quality bacterial artificial chromosome (BAC) libraries for two, the red-blooded notothen Notothenia coriiceps and the white-blooded icefish Chaenocephalus aceratus. Our results indicate that evolution of phylogenetically derived notothenioid families (e.g., the crown group Channichthyidae [icefishes]), was accompanied by genome expansion. Six species from the basal family Nototheniidae had C-values between 0.98 and 1.20 pg, a range that is consistent with the genome sizes of proposed outgroups (e.g., percids) of the notothenioid suborder. In contrast, four icefishes had C-values in the range 1.66-1.83 pg. The BAC libraries VMRC-19 (N. coriiceps) and VMRC-21 (C. aceratus) comprise 12× and 10× coverage of the respective genomes and have average insert sizes of 138 and 168 kb. Paired BAC-end reads representing ∼0.1% of each genome showed that the repetitive element landscapes of the two genomes (13.4% of the N. coriiceps genome and 14.5% for C. aceratus) were similar. The availability of these high-quality and well-characterized BAC libraries sets the stage for targeted genomic analyses of the unusual anatomical and physiological adaptations of the notothenioids, some of which mimic human diseases. Here we consider the evolution of secondary pelagicism by various taxa of the group and illustrate the utility of Antarctic icefishes as an evolutionary-mutant model of human osteopenia (low-mineral density of bones).

An overview of the apple genome through BAC end sequence analysis

The apple, Malus x domestica Borkh., is one of the most important fruit trees grown worldwide. A bacterial artificial chromosome (BAC)-based physical map of the apple genome has been recently constructed. Based on this physical map, a total of approximately 2,100 clones from different contigs (overlapping BAC clones) have been selected and sequenced at both ends, generating 3,744 high-quality BAC end sequences (BESs) including 1,717 BAC end pairs. Approximately 8.5% of BESs contain simple sequence repeats (SSRs), most of which are AT/TA dimer repeats. Potential transposable elements are identified in approximately 21% of BESs, and most of these elements are retrotransposons. About 11% of BESs have homology to the Arabidopsis protein database. The matched proteins cover a broad range of categories. The average GC content of the predicted coding regions of BESs is 42.4%; while, that of the whole BESs is 39%. A small number of BES pairs were mapped to neighboring chromosome regions of A. thaliana and Populus trichocarpa; whereas, no pairs are mapped to the Oryza sativa genome. The apple has a higher degree of synteny with the closely related Populus than with the distantly related Arabidopsis. BAC end sequencing can be used to anchor a small proportion of the apple genome to the Populus and possibly to the Arabidopsis genomes.

Characterization of a BAC Library from Channel Catfish Ictalurus punctatus: Indications of High Levels of Chromosomal Reshuffling Among Teleost Genomes

The CHORI-212 bacterial artificial chromosome (BAC) library was constructed by cloning EcoRI/EcoRI partially digested DNA into the pTARBAC2.1 vector. The library has an average insert size of 161 kb, and provides 10.6-fold coverage of the channel catfish haploid genome. Screening of 32 genes using overgo or cDNA probes indicated that this library had a good representation of the genome as all tested genes existed in the library. We previously reported sequencing of approximately 25,000 BAC ends that generated 20,366 high-quality BAC end sequences (BES) and identified a large number of sequences similar to known genes using BLASTX searches. In this work, particular attention was given to identification of BAC mate pairs with known genes from both ends. When identified, comparative genome analysis was conducted to determine syntenic regions of the catfish genome with the genomes of zebrafish and Tetraodon. Of the 141 mate pairs with known genes from channel catfish, conserved syntenies were identified in 34 (24.1%), with 30 conserved in the zebrafish genome and 14 conserved in the Tetraodon genome. Additional analysis of three of the 34 conserved syntenic groups by direct sequencing indicated conserved gene contents in all three species. This indicates that comparative genome analysis may provide shortcuts to genome analysis in catfish, especially for short genomic regions once the conserved syntenies are identified.

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (http://biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 x coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.

Construction and Identification of Bacterial Artificial Chromosome Library for 0‐613‐2R in Upland Cotton

AbstractA bacterial artificial chromosome (BAC) library containing a large genomic DNA insert is an important tool for genome physical mapping, map‐based cloning, and genome sequencing. To isolate genes via a map‐based cloning strategy and to perform physical mapping of the cotton genome, a high‐quality BAC library containing large cotton DNA inserts is needed. We have developed a BAC library of the restoring line 0‐613‐2R for isolating the fertility restorer (Rf1) gene and genomic research in cotton (Gossypium hirsutum L.). The BAC library contains 97 825 clones stored in 255 pieces of a 384‐well microtiter plate. Random samples of BACs digested with the NotI enzyme indicated that the average insert size is approximately 130 kb, with a range of 80–275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with NotI and HindIII enzymes. Thus, the stability of a single BAC clone can be sustained at least for 100 generations. Eight simple sequence repeat (SSR) markers flanking the Rf1 gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.(Managing editor: Li‐Hui Zhao)

The genome sequence of Drosophila melanogaster.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.