Bacterial Artificial Chromosome End Research Articles

Improved protocols for BAC insert DNA isolation, BAC end sequencing and FISH for construction of BAC based physical map of genes on the chromosomes.

Bacterial artificial chromosome (BAC) library is an important genomic resource useful in targeted marker development, positional cloning, physical mapping and a substrate for genome sequencing for better understanding the genome organization of a species. The present manuscript elucidates the improvement in protocols for economical and efficient BAC insert DNA isolation, BAC end sequencing and FISH for physical localization on the metaphase chromosome complements. BAC clones of Clarias magur, maintained in 384-well plate format in our laboratory, were used in this study. The protocols gave consistent and efficient results. We use routinely these protocols for BAC insert DNA extraction, generating end sequence data of the clone and constructing DNA probes to hybridize on the metaphase spreads of C. magur using FISH for physical their localization.

A high-throughput BAC end analysis protocol (BAC-anchor) for profiling genome assembly and physical mapping.

SummaryTraditional approaches for sequencing insertion ends of bacterial artificial chromosome (BAC) libraries are laborious and expensive, which are currently some of the bottlenecks limiting a better understanding of the genomic features of auto‐ or allopolyploid species. Here, we developed a highly efficient and low‐cost BAC end analysis protocol, named BAC‐anchor, to identify paired‐end reads containing large internal gaps. Our approach mainly focused on the identification of high‐throughput sequencing reads carrying restriction enzyme cutting sites and searching for large internal gaps based on the mapping locations of both ends of the reads. We sequenced and analysed eight libraries containing over 3 200 000 BAC end clones derived from the BAC library of the tetraploid potato cultivar C88 digested with two restriction enzymes, Cla I and Mlu I. About 25% of the BAC end reads carrying cutting sites generated a 60–100 kb internal gap in the potato DM reference genome, which was consistent with the mapping results of Sanger sequencing of the BAC end clones and indicated large differences between autotetraploid and haploid genotypes in potato. A total of 5341 Cla I‐ and 165 Mlu I‐derived unique reads were distributed on different chromosomes of the DM reference genome and could be used to establish a physical map of target regions and assemble the C88 genome. The reads that matched different chromosomes are especially significant for the further assembly of complex polyploid genomes. Our study provides an example of analysing high‐coverage BAC end libraries with low sequencing cost and is a resource for further genome sequencing studies.

Molecular marker development and primary physical map construction for the tuber shape Ro gene locus in diploid potato (Solanum tuberosum L.)

Potato (Solanum tuberosum L.) is an important crop worldwide that is consumed fresh and processed in many different ways. The tuber shape is a critical trait in potato breeding, because it directly affects processing quality. Previous studies have demonstrated that the Ro locus on chromosome X is the major locus controlling tuber shape, and the round shape allele, Ro, is dominant over the long shape allele, ro. However, no reports on the cloning or physical mapping of this locus have been published. In this study, whole genome sequencing and bacterial artificial chromosome (BAC) library construction were applied to develop molecular markers and construct a physical map of Ro. Eight round-tuber genotypes were used to construct the BAC library, which contains about 40,000 clones with an average insert size of 85 kb and represents approximately four haploid potato genomes. A total of 66 individuals with extreme round or long tubers were selected to construct a round-tuber shape DNA bulk and a long-tuber shape DNA bulk for WGS. Single-nucleotide polymorphisms (SNPs) identified from WGS data were used to develop nine polymorphic markers linked to Ro. These markers were used to screen the BAC library, and eight positive BAC clones were ultimately identified and then analyzed by restriction enzyme digestion and BAC end sequencing and used to develop markers and identify new positive BAC clones. Finally, a primary physical map of Ro was constructed using a chromosome walking strategy. This map provides an important reference for cloning the potato tuber shape gene and studying tuber shape variation.

Genome sequence comparison between Chinese hamster ovary (CHO) DG44 cells and mouse using end sequences of CHO BAC clones based on BAC-FISH results.

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology as a mammalian host cell platform for expressing genes of interest. Previously, we constructed a detailed physical chromosomal map of the CHO DG44 cell line by fluorescence in situ hybridization (FISH) imaging using 303 bacterial artificial chromosome (BAC) clones as hybridization probes (BAC-FISH). BAC-FISH results revealed that the two longest chromosomes were completely paired. However, other chromosomes featured partial deletions or rearrangements. In this study, we determined the end sequences of 303 BAC clones (BAC end sequences), which were used for BAC-FISH probes. Among 606 BAC-end sequences (BESs) (forward and reverse ends), 558 could be determined. We performed a comparison between all determined BESs and mouse genome sequences using NCBI BLAST. Among these 558 BESs, 465 showed high homology to mouse chromosomal sequences. We analyzed the locations of these BACs in chromosomes of the CHO DG44 cell line using a physical chromosomal map. From the obtained results, we investigated the regional similarities among CHO chromosomes (A-T) and mouse chromosomes (1-19 and sex) about 217 BESs (46.7% of 465 high homologous BESs). Twenty-three specific narrow regions in 13 chromosomes of the CHO DG44 cell line showed high homology to mouse chromosomes, but most of other regions did not show significant correlations with the mouse genome. These results contribute to accurate alignments of chromosomes of Chinese hamster and its genome sequence, analysis of chromosomal instability in CHO cells, and the development of target locations for gene and/or genome editing techniques.

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.

PBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping.

Applications that use Bacterial Artificial Chromosome (BAC) libraries often require paired-end sequences and knowledge of the physical location of each clone in plates. To facilitate obtaining this information in high-throughput, we generated pBACode vectors: a pool of BAC cloning vectors, each with a pair of random barcodes flanking its cloning site. In a pBACode BAC library, the BAC ends and their linked barcodes can be sequenced in bulk. Barcode pairs are determined by sequencing the empty pBACode vectors, which allows BAC ends to be paired according to their barcodes. For physical clone mapping, the barcodes are used as unique markers for their linked genomic sequence. After multi-dimensional pooling of BAC clones, the barcodes are sequenced and deconvoluted to locate each clone. We generated a pBACode library of 94,464 clones for the flounder Paralichthys olivaceus and obtained paired-end sequence from 95.4% of the clones. Incorporating BAC paired-ends into the genome preassembly improved its continuity by over 10-fold. Furthermore, we were able to use the barcodes to map the physical locations of each clone in just 50 pools, with up to 11 808 clones per pool. Our physical clone mapping located 90.2% of BAC clones, enabling targeted characterization of chromosomal rearrangements.

Novel 307 polymorphic SSR markers from BAC-end sequences in walnut (Juglans regia L.): Effects of motif types and repeat lengths on polymorphism and genetic diversity

Walnut, Juglans regia L., has been cultivated for its edible nut and timber since ancient times. It is one of the most valuable and widely cultivated horticultural commodities in the world. Simple sequence repeats (SSRs) have a wide range of applications in plants, and there is still a necessity to develop novel SSR markers for each plant species. Bacterial artificial chromosome-end sequences (BES) are good sources to generate novel SSR markers. In pursuit of this objective, a total of 558 BES-SSR primer pairs were designed for J. regia. Of them, 507 (91%) had amplifications, and 307 were polymorphic when tested in eight walnut cultivars. A total of 1097 alleles were generated from 307 polymorphic SSR loci, ranging from two to eleven, with an average of 3.6 per locus. Polymorphism information contents (PIC) varied from 0.11 to 0.88 with an average of 0.46. Di-nucleotide motifs had higher polymorphism and genetic diversity values than the other motif types. The rate of polymorphism increased with the repeat length, therefore, class I SSRs are useful source of polymorphic DNA markers in walnut. Novel SSR markers developed in this study may possess potential applications such as fingerprinting, marker-assisted breeding, germplasm characterization and genetic linkage mapping in walnut and other species of Juglans.

Physical Mapping of Bread Wheat Chromosome 5A: An Integrated Approach.

The huge size, redundancy, and highly repetitive nature of the bread wheat [Triticum aestivum (L.)] genome, makes it among the most difficult species to be sequenced. To overcome these limitations, a strategy based on the separation of individual chromosomes or chromosome arms and the subsequent production of physical maps was established within the frame of the International Wheat Genome Sequence Consortium (IWGSC). A total of 95,812 bacterial artificial chromosome (BAC) clones of short-arm chromosome 5A (5AS) and long-arm chromosome 5A (5AL) arm-specific BAC libraries were fingerprinted and assembled into contigs by complementary analytical approaches based on the FingerPrinted Contig (FPC) and Linear Topological Contig (LTC) tools. Combined anchoring approaches based on polymerase chain reaction (PCR) marker screening, microarray, and sequence homology searches applied to several genomic tools (i.e., genetic maps, deletion bin map, neighbor maps, BAC end sequences (BESs), genome zipper, and chromosome survey sequences) allowed the development of a high-quality physical map with an anchored physical coverage of 75% for 5AS and 53% for 5AL with high portions (64 and 48%, respectively) of contigs ordered along the chromosome. In the genome of grasses, Brachypodium [Brachypodium distachyon (L.) Beauv.], rice (Oryza sativa L.), and sorghum [Sorghum bicolor (L.) Moench] homologs of genes on wheat chromosome 5A were separated into syntenic blocks on different chromosomes as a result of translocations and inversions during evolution. The physical map presented represents an essential resource for fine genetic mapping and map-based cloning of agronomically relevant traits and a reference for the 5A sequencing projects.

Substantial genome synteny preservation among woody angiosperm species: comparative genomics of Chinese chestnut (Castanea mollissima) and plant reference genomes.

BackgroundChinese chestnut (Castanea mollissima) has emerged as a model species for the Fagaceae family with extensive genomic resources including a physical map, a dense genetic map and quantitative trait loci (QTLs) for chestnut blight resistance. These resources enable comparative genomics analyses relative to model plants. We assessed the degree of conservation between the chestnut genome and other well annotated and assembled plant genomic sequences, focusing on the QTL regions of most interest to the chestnut breeding community.ResultsThe integrated physical and genetic map of Chinese chestnut has been improved to now include 858 shared sequence-based markers. The utility of the integrated map has also been improved through the addition of 42,970 BAC (bacterial artificial chromosome) end sequences spanning over 26 million bases of the estimated 800 Mb chestnut genome. Synteny between chestnut and ten model plant species was conducted on a macro-syntenic scale using sequences from both individual probes and BAC end sequences across the chestnut physical map. Blocks of synteny with chestnut were found in all ten reference species, with the percent of the chestnut physical map that could be aligned ranging from 10 to 39 %.The integrated genetic and physical map was utilized to identify BACs that spanned the three previously identified QTL regions conferring blight resistance. The clones were pooled and sequenced, yielding 396 sequence scaffolds covering 13.9 Mbp. Comparative genomic analysis on a microsytenic scale, using the QTL-associated genomic sequence, identified synteny from chestnut to other plant genomes ranging from 5.4 to 12.9 % of the genome sequences aligning.ConclusionsOn both the macro- and micro-synteny levels, the peach, grape and poplar genomes were found to be the most structurally conserved with chestnut. Interestingly, these results did not strictly follow the expectation that decreased phylogenetic distance would correspond to increased levels of genome preservation, but rather suggest the additional influence of life-history traits on preservation of synteny. The regions of synteny that were detected provide an important tool for defining and cataloging genes in the QTL regions for advancing chestnut blight resistance research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1942-1) contains supplementary material, which is available to authorized users.

Construction of a Spinach Bacterial Artificial Chromosome (BAC) Library as a Resource for Gene Identification and Marker Development

Spinach (Spinacae oleracae) is a nutritious vegetable and an economically important crop worldwide. Spinach is threatened by a number of diseases, and the most significant of which is downy mildew, caused by Peronospora farinosa f. sp. spinaciae (Pfs). The spinach locus RPF1 confers resistance to 11 of the 15 known races of Pfs. To study the RPF1 locus in depth, a BAC library was constructed from DNA of a single spinach plant that was homozygous at the RPF1 locus. The BAC library contained 73,728 clones, with an average insert size of 183 kb, which represented approximately 13 spinach genome equivalents (989 Mb). The ends of 1920 BAC clones were sequenced from both directions, and 3405 BAC end sequences (BESs) ranging from 100 to 935 bp were obtained. About 38.59 % of the BESs contained repetitive sequences, including 363 copies of retrotransposable elements and 32 copies of transposable elements. Less than 3 % of the clones originated from chloroplasts or mitochondria. Various categories of protein-encoding genes, including transcription factors, stress responsive genes, and reproduction-associated genes, were identified. Putative disease resistance genes containing nucleotide binding site-leucine-rich repeat (NBS-LRR) domains were also identified, one of which was closely linked to the RPF1 locus. Simple sequence repeat (SSR) motifs were identified from 154 BESs, including various di-, tri-, tetra-, and penta-nucleotide repeats. One hundred pairs of SSR primers and 24 retrotransposon primers were designed from BESs, all of which successfully amplified spinach DNA. This spinach BAC library will provide a useful resource for spinach genetic studies and breeding efforts.

Draft sequences of the radish (Raphanus sativus L.) genome.

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.

A BAC based physical map and genome survey of the rice false smut fungus Villosiclava virens

BackgroundRice false smut caused by Villosiclava virens is a devastating fungal disease that spreads in major rice-growing regions throughout the world. However, the genomic information for this fungal pathogen is limited and the pathogenic mechanism of this disease is still not clear. To facilitate genetic, molecular and genomic studies of this fungal pathogen, we constructed the first BAC-based physical map and performed the first genome survey for this species.ResultsHigh molecular weight genomic DNA was isolated from young mycelia of the Villosiclava virens strain UV-8b and a high-quality, large-insert and deep-coverage Bacterial Artificial Chromosome (BAC) library was constructed with the restriction enzyme HindIII. The BAC library consisted of 5,760 clones, which covers 22.7-fold of the UV-8b genome, with an average insert size of 140 kb and an empty clone rate of lower than 1%. BAC fingerprinting generated successful fingerprints for 2,290 BAC clones. Using the fingerprints, a whole genome-wide BAC physical map was constructed that contained 194 contigs (2,035 clones) spanning 51.2 Mb in physical length. Bidirectional-end sequencing of 4,512 BAC clones generated 6,560 high quality BAC end sequences (BESs), with a total length of 3,030,658 bp, representing 8.54% of the genome sequence. Analysis of the BESs revealed general genome information, including 51.52% GC content, 22.51% repetitive sequences, 376.12/Mb simple sequence repeat (SSR) density and approximately 36.01% coding regions. Sequence comparisons to other available fungal genome sequences through BESs showed high similarities to Metarhizium anisopliae, Trichoderma reesei, Nectria haematococca and Cordyceps militaris, which were generally in agreement with the 18S rRNA gene analysis results.ConclusionThis study provides the first BAC-based physical map and genome information for the important rice fungal pathogen Villosiclava virens. The BAC clones, physical map and genome information will serve as fundamental resources to accelerate the genetic, molecular and genomic studies of this pathogen, including positional cloning, comparative genomic analysis and whole genome sequencing. The BAC library and physical map have been opened to researchers as public genomic resources (http://gresource.hzau.edu.cn/resource/resource.html).

Exploring the genome of the salt-marsh Spartina maritima (Poaceae, Chloridoideae) through BAC end sequence analysis

Spartina species play an important ecological role on salt marshes. Spartina maritima is an Old-World species distributed along the European and North-African Atlantic coasts. This hexaploid species (2n = 6x = 60, 2C = 3,700 Mb) hybridized with different Spartina species introduced from the American coasts, which resulted in the formation of new invasive hybrids and allopolyploids. Thus, S. maritima raises evolutionary and ecological interests. However, genomic information is dramatically lacking in this genus. In an effort to develop genomic resources, we analysed 40,641 high-quality bacterial artificial chromosome-end sequences (BESs), representing 26.7 Mb of the S. maritima genome. BESs were searched for sequence homology against known databases. A fraction of 16.91% of the BESs represents known repeats including a majority of long terminal repeat (LTR) retrotransposons (13.67%). Non-LTR retrotransposons represent 0.75%, DNA transposons 0.99%, whereas small RNA, simple repeats and low-complexity sequences account for 1.38% of the analysed BESs. In addition, 4,285 simple sequence repeats were detected. Using the coding sequence database of Sorghum bicolor, 6,809 BESs found homology accounting for 17.1% of all BESs. Comparative genomics with related genera reveals that the microsynteny is better conserved with S. bicolor compared to other sequenced Poaceae, where 37.6% of the paired matching BESs are correctly orientated on the chromosomes. We did not observe large macrosyntenic rearrangements using the mapping strategy employed. However, some regions appeared to have experienced rearrangements when comparing Spartina to Sorghum and to Oryza. This work represents the first overview of S. maritima genome regarding the respective coding and repetitive components. The syntenic relationships with other grass genomes examined here help clarifying evolution in Poaceae, S. maritima being a part of the poorly-known Chloridoideae sub-family.

Identification of Citrus sinensis BAC clones containing genes relevant to fruit quality by two-dimensional overgo hybridization

As a complement to whole-genome sequencing efforts, we are comparing targeted genomic regions among sweet orange cultivars to identify coding and conserved noncoding regions, including regulatory elements, responsible for biological features unique to this species. Here, we report the identification of 1,018 bacterial artificial chromosome (BAC) clones containing genes relevant to fruit quality from a Citrus sinensis cv. “Vaniglia” 19.3X BAC library by two-dimensional 9 × 9 overgo hybridization. To design the overgo probes, we used the “C38” expressed sequence tag assembly ( http://harvest.ucr.edu/ ) and OligoSpawn software ( http://138.23.178.42 ). For BAC library screening, we selected 81 overgo probes associated with unigenes that putatively code for enzymes relevant to fruit quality (flavonol, anthocyanin, carotenoid, cellulose, starch, ascorbic acid, aromatic amino acid, and lignin biosynthesis; sucrose catabolism; glycolysis; oxidative/nonoxidative pentose phosphate pathway; fatty acid biosynthesis and oxidation; Krebs cycle). Hybridization probes were pooled and hybridized in groups of intersecting rows and columns to high-density BAC filters, followed by a deconvolution process that established BAC-probe addresses. BAC addresses were obtained for 75 of the 81 overgo probes initially selected, for a total of 1,018 BAC clones, a number consistent with the depth of coverage of the BAC library. BAC end sequencing was carried out, and end-sequence pairs were mapped to their best location in the Citrus clementina genome sequence assembly using the comparative genomic database Phytozome ( http://www.phytozome.net/ ). The BAC clones corresponding to each probe were mapped within the same scaffold as the target gene, demonstrating that the approach we used was successful in isolating the targeted genomic regions.

Development of Genomic Resources in the Species of Trifolium L. and Its Application in Forage Legume Breeding

Clovers (genus Trifolium) are a large and widespread genus of legumes. A number of clovers are of agricultural importance as forage crops in grassland agriculture, particularly temperate areas. White clover (Trifolium repens L.) is used in grazed pasture and red clover (T. pratense L.) is widely cut and conserved as a winter feed. For the diploid red clover, genetic and genomic tools and resources have developed rapidly over the last five years including genetic and physical maps, BAC (bacterial artificial chromosome) end sequence and transcriptome sequence information. This has paved the way for the use of genome wide selection and high throughput phenotyping in germplasm development. For the allotetraploid white clover progress has been slower although marker assisted selection is in use and relatively robust genetic maps and QTL (quantitative trait locus) information now exist. For both species the sequencing of the model legume Medicago truncatula gene space is an important development to aid genomic, biological and evolutionary studies. The first genetic maps of another species, subterranean clover (Trifolium subterraneum L.) have also been published and its comparative genomics with red clover and M. truncatula conducted. Next generation sequencing brings the potential to revolutionize clover genomics, but international consortia and effective use of germplasm, novel population structures and phenomics will be required to carry out effective translation into breeding. Another avenue for clover genomic and genetic improvement is interspecific hybridization. This approach has considerable potential with regard to crop improvement but also opens windows of opportunity for studies of biological and evolutionary processes.

A BAC library of the SP80-3280 sugarcane variety (saccharum sp.) and its inferred microsynteny with the sorghum genome.

BackgroundSugarcane breeding has significantly progressed in the last 30 years, but achieving additional yield gains has been difficult because of the constraints imposed by the complex ploidy of this crop. Sugarcane cultivars are interspecific hybrids between Saccharum officinarum and Saccharum spontaneum. S. officinarum is an octoploid with 2n = 80 chromosomes while S. spontaneum has 2n = 40 to 128 chromosomes and ploidy varying from 5 to 16. The hybrid genome is composed of 70-80% S. officinaram and 5-20% S. spontaneum chromosomes and a small proportion of recombinants. Sequencing the genome of this complex crop may help identify useful genes, either per se or through comparative genomics using closely related grasses. The construction and sequencing of a bacterial artificial chromosome (BAC) library of an elite commercial variety of sugarcane could help assembly the sugarcane genome.ResultsA BAC library designated SS_SBa was constructed with DNA isolated from the commercial sugarcane variety SP80-3280. The library contains 36,864 clones with an average insert size of 125 Kb, 88% of which has inserts larger than 90 Kb. Based on the estimated genome size of 760–930 Mb, the library exhibits 5–6 times coverage the monoploid sugarcane genome. Bidirectional BAC end sequencing (BESs) from a random sample of 192 BAC clones sampled genes and repetitive elements of the sugarcane genome. Forty-five per cent of the total BES nucleotides represents repetitive elements, 83% of which belonging to LTR retrotransposons. Alignment of BESs corresponding to 42 BACs to the genome sequence of the 10 sorghum chromosomes revealed regions of microsynteny, with expansions and contractions of sorghum genome regions relative to the sugarcane BAC clones. In general, the sampled sorghum genome regions presented an average 29% expansion in relation to the sugarcane syntenic BACs.ConclusionThe SS_SBa BAC library represents a new resource for sugarcane genome sequencing. An analysis of insert size, genome coverage and orthologous alignment with the sorghum genome revealed that the library presents whole genome coverage. The comparison of syntenic regions of the sorghum genome to 42 SS_SBa BES pairs revealed that the sorghum genome is expanded in relation to the sugarcane genome.

A Second Generation Integrated Map of the Rainbow Trout (Oncorhynchus mykiss) Genome: Analysis of Conserved Synteny with Model Fish Genomes

DNA fingerprints and end sequences from bacterial artificial chromosomes (BACs) from two new libraries were generated to improve the first generation integrated physical and genetic map of the rainbow trout (Oncorhynchus mykiss) genome. The current version of the physical map is composed of 167,989 clones of which 158,670 are assembled into contigs and 9,319 are singletons. The number of contigs was reduced from 4,173 to 3,220. End sequencing of clones from the new libraries generated a total of 11,958 high quality sequence reads. The end sequences were used to develop 238 new microsatellites of which 42 were added to the genetic map. Conserved synteny between the rainbow trout genome and model fish genomes was analyzed using 188,443 BAC end sequence (BES) reads. The fractions of BES reads with significant BLASTN hits against the zebrafish, medaka, and stickleback genomes were 8.8%, 9.7%, and 10.5%, respectively, while the fractions of significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 6.2%, 5.8%, and 5.5%, respectively. The overall number of unique regions of conserved synteny identified through grouping of the rainbow trout BES into fingerprinting contigs was 2,259, 2,229, and 2,203 for stickleback, medaka, and zebrafish, respectively. These numbers are approximately three to five times greater than those we have previously identified using BAC paired ends. Clustering of the conserved synteny analysis results by linkage groups as derived from the integrated physical and genetic map revealed that despite the low sequence homology, large blocks of macrosynteny are conserved between chromosome arms of rainbow trout and the model fish species.

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.

BAC end sequencing of Pacific white shrimp Litopenaeus vannamei: a glimpse into the genome of Penaeid shrimp

Little is known about the genome of Pacific white shrimp (Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 pairedends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.

A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo) and chicken (Gallus gallus) genomes

BackgroundA robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species.ResultsThe turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes.ConclusionThe BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.