Two large-insert soybean genomic libraries constructed in a binary vector: applications in chromosome walking and genome wide physical mapping

Abstract

Large DNA insert libraries in binary T-DNA vectors can assist in the isolation of the gene(s) underlying a quantitative trait locus (QTL). Binary vectors facilitate the transfer of large-insert DNA fragments containing a QTL from E. coli to Agrobacterium sp. and then to plants. We constructed two soybean large-insert libraries from cv. Forrest in the pCLD04541 (V41) binary vector after partial digestion of genomic high-molecular-weight DNA with BamHI or HindIII. The libraries contain 76,800 clones with an average insert size of 125 kb, and therefore represent 9.5-fold haploid genome equivalents. Colony hybridization using a chloroplast-specific probe infers that the libraries contain less than 0.5% clones of chloroplast DNA origin. These two libraries have provided clones for physical mapping of the soybean genome and for isolation of a number of disease resistance genes. One microsatellite marker was identified from the clone that hybridized to the Bng122 RFLP probe. The sequence-tagged site was used for genetic mapping and marker-assisted selection for genes underlying resistance to the soybean cyst nematode and sudden death syndrome.

Construction of BAC libraries from two apomictic grasses to study the microcolinearity of their apospory-specific genomic regions

“…BAC clones were assigned to the ASGR in the polyhaploid library (25 or 5.0 per probe from an approximately 4× genome coverage after excluding the A14M clones that had no counterpart from buffelgrass) and in the buffelgrass library (28 or 5.6 per probe from an approximately 5× genome coverage). The representation of a probe in our libraries did not differ substantially from the two to eight clones per probe recovered from two soybean libraries, each with 4.7 haploid genome equivalents (Meksem et al 2000). The larger total number of clones from buffelgrass (83 vs. 58 from polyhaploid) could be due to the slightly greater genome coverage of the library, but also to the larger number of unmapped, multiplex RFLP fragments detected in genomic DNA that could represent allelic, linked, or unlinked sequences (Ozias-Akins et al 1998;Roche et al 1999).…”

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

“…Forrest cultivar sequence derived from two BAC libraries using BamHI and HindIII enzymes (Meksem et al, 2000). The construction of BAC libraries and the physical map helped put the fragmented genome sequence of Forrest together (Shultz et al, 2006).…”

Recombination suppression at the dominant Rhg1/Rfs2 locus underlying soybean resistance to the cyst nematode

“…Forrest provided a unique set of tools for genomics (Meksem et al 2000;Wu et al 2004;Shultz et al 2006;Lightfoot 2008). Forrest had introgressed only resistance to Hg type 0 (Hartwig and Epps 1973) but Peking also resisted two other Hg types.…”