New genomic resources for the honey bee(Apis mellifera L): development of a deep-coverage BAC library and a preliminary STC database

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2 empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99 probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/)

New genomic resources for the honey bee(Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database.

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2% empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99% probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/).

A marker-dense physical map of the Bradyrhizobium japonicum genome.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes the development of a physical framework for the genome of Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybean. A BAC library for B. japonicum was constructed that provides a 77-fold genome coverage based on an estimated genome size of 8.7 Mb. The library contains 4608 clones with an average insert size of 146 kb. To generate a physical map, the entire library was fingerprinted with HindIII, and the fingerprinted clones were assembled into contigs using the software (; Sanger Centre, UK). The analysis placed 3410 clones in six large contigs. The ends of 1152 BAC inserts were sequenced to generate a sequence-tagged connector (STC) framework. To join and orient the contigs, high-density BAC colony filters were probed with 41 known gene probes and 17 end sequences from contig boundaries. STC sequences were searched against the public databases using and algorithms. Query results allowed the identification of 113 high probability matches with putative functional identities that were placed on the physical map. Combined with the hybridization data, a high-resolution physical map with 194 positioned markers represented in two large contigs was developed, providing a marker every 45 kb. Of these markers, 177 are known or putative B. japonicum genes. Additionally, 1338 significant results (E < 10(-4)) were manually sorted by function to produce a functionally categorized database of relevant B. japonicum STC sequences that can also be traced to specific locations in the physical map.

Physical map and gene survey of the Ochrobactrum anthropi genome using bacterial artificial chromosome contigs.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.

A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance.

We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.

A bacterial artificial chromosome library for sugarcane.

Modern cultivated sugarcane is a complex aneuploid polyploid with an estimated genome size of 3000 Mb. Although most traits in sugarcane show complex inheritance, a rust locus showing monogenic inheritance has been documented. In order to facilitate cloning of the rust locus, we have constructed a bacterial artificial chromosome (BAC) library for the cultivar R570. The library contains 103,296 clones providing 4.5 sugarcane genome equivalents. A random sampling of 240 clones indicated an average insert size of 130 kb allowing a 98% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4 × 4 double-spotted array on 22.5-cm(2) filters. Each set of five filters provides a genome coverage of 4x with 18,432 clones represented per filter. Screening of the library with three different barley chloroplast gene probes indicated an exceptionally low chloroplast DNA content of less than 1%. To demonstrate the library's potential for map-based cloning, single-copy RFLP sugarcane mapping probes anchored to nine different linkage groups and three different gene probes were used to screen the library. The number of positive hybridization signals resulting from each probe ranged from 8 to 60. After determining addresses of the signals, clones were evaluated for insert size and HindIII-fingerprinted. The fingerprints were then used to determine clone relationships and assemble contigs. For comparison with other monocot genomes, sugarcane RFLP probes were also used to screen a Sorghum bicolor BAC library and two rice BAC libraries. The rice and sorghum BAC clones were characterized for insert size and fingerprinted, and the results compared to sugarcane. The library was screened with a rust resistance RFLP marker and candidate BAC clones were subjected to RFLP fragment matching to identify those corresponding to the same genomic region as the rust gene.