Construction and characterization of a soybean bacterial artificial chromosome library and use of multiple complementary libraries for genome physical mapping

Abstract

Two plant-transformation-competent large-insert binary clone bacterial artificial chromosome (hereafter BIBAC) libraries were previously constructed for soybean cv. Forrest, using BamHI or HindIII. However, they are not well suited for clone-based genomic sequencing due to their larger ratio of vector to insert size (27.6 kbp:125 kbp). Therefore, we developed a larger-insert bacterial artificial chromosome (BAC) library for the genotype in a smaller vector (pECBAC1), using EcoRI. The BAC library contains 38,400 clones; about 99.1% of the clones have inserts; the average insert size is 157 kbp; and the ratio of vector to insert size is much smaller (7.5 kbp:157 kbp). Colony hybridization with probes derived from several chloroplast and mitochondrial genes showed that 0.89% and 0.45% of the clones were derived from the chloroplast and mitochondrial genomes, respectively. Considering these data, the library represents 5.4 haploid genomes of soybean. The library was hybridized with six RFLP marker probes, 5S rDNA and 18S-5.8S-25S rDNA, respectively. Each RFLP marker hybridized to about six clones, and the 5S and 18S-5.8S-25S rDNA probes collectively hybridized to 402 BACs--about 1.05% of the clones in the library. The BAC library complements the existing soybean Forrest BIBAC libraries by using different restriction enzymes and vector systems. Together, the BAC and BIBAC libraries encompass 13.2 haploid genomes, providing the most comprehensive clone resource for a single soybean genotype for public genome research. We show that the BAC library has enhanced the development of the soybean whole-genome physical map and use of three complementary BAC libraries improves genome physical mapping by fingerprint analysis of most of the clones of the library. The rDNA-containing clones were also fingerprinted to evaluate the feasibility of constructing contig maps of the rDNA regions. It was found that physical maps for the rDNA regions could not be readily constructed by fingerprint analysis, using one or two restriction enzymes. Additional data to fingerprints and/or different fingerprinting methods are needed to build contig maps for such highly tandem repetitive regions and thus, the physical map of the entire soybean genome.

The construction and characteristics of a BAC library for Cucumis sativus L. ‘B10’

“…6) range from < 50 kb to 260 kb. Despite the double selection, there is quite a large number of clones shorter than 50 kb, which correlates with the results obtained by other authors [21,[25][26]. The average length of clones in the library is 135 kb.…”

Impact of Genomic Technologies on Chickpea Breeding Strategies

“…In the case of chickpea, a BIBAC library consisting of 23,780 clones, with an average insert size of 100 kb and covering about 3.8X genomes of chickpea was developed [93]. However, multi-enzyme BAC libraries with higher genome coverage are required for comprehensive genome research [94][95][96]. Subsequently, Lichtenzveig et al, [54] developed a BAC and BIBAC library in chickpea from the Hadas genotype; digested with HindIII and BamHI enzymes, respectively.…”

Additional Polymorphisms Linked to Soybean Cyst Nematode Resistance At The Rhg4 Locus

“…The construction of BAC libraries and the physical map helped put the fragmented genome sequence of Forrest together (Shultz et al, 2006). Although the Forrest cultivar library and the BAC libraries for the Asgrow 3244 and the Williams 82 susceptible cultivars were available, complete BAC libraries did not exist for either cultivar (Wu et al, 2004). Wu et al (2004) reported a complete physical map from three BAC libraries derived from the Forrest cultivar.…”

“…Although the Forrest cultivar library and the BAC libraries for the Asgrow 3244 and the Williams 82 susceptible cultivars were available, complete BAC libraries did not exist for either cultivar (Wu et al, 2004). Wu et al (2004) reported a complete physical map from three BAC libraries derived from the Forrest cultivar. As the BAC libraries created complement each other, they expanded the number of clones used soybean genome analysis.…”