Genome-wide scan of the soybean genome using degenerate oligonucleotide primed PCR: an example for studying large complex genome structure
The availability of next generation sequencing instruments has made large-scale and genome-wide sequence analysis more feasible in a wild variety of species with large complex genomes, especially crops. This report suggested an approach for characterizing large complex genomes of less-studied/orphan crops. Degenerate oligonucleotide primed PCR (DOP-PCR) is a useful tool for the survey of genomes in less-studied/orphan crops, as prior sequence information is not necessary. Here, four different degenerate primers were redesigned from previously described DOP-PCR primers. The degeneracy of these primers was increased with the addition of two more ‘Ns’. The amplified DOP-PCR products from Sinpaldalkong 2, a soybean genotype, were applied to GS-FLX and the reads from Sinpaldalkong 2 were mapped against Williams 82 as a reference (http://www.phytozome.net/soybean.php), using the Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/). These results suggest the identification of 4 single nucleotide polymorphisms between Sinpaldalkong 2 and Williams 82 and recent duplication of the soybean genome. The sequenced reads were subsequently assembled into contigs by Newbler under default conditions. A total of 29 Sinpaldalkong 2 contigs exhibited 95% similarity and < E-100 when mega-blasted with Williams 82 reference sequences. These contigs were mapped to the soybean chromosomes and positioned as clusters within each chromosome. Most of the contigs also showed similarity with the Arabidopsis RNase H domain-containing protein, suggesting a potential way to study retrotransposons in less-studied/orphan crops. Using these modified DOP-PCR primers and GS-FLX, it is possible to obtain insight into the large complex genomes of less-studied/orphan crops.
KeywordsDOP-PCR Genome duplication GS-FLX Less-studied/orphan crops Retrotransposons Soybean
Unable to display preview. Download preview PDF.
- Barabaschi D, Guerra D, Lacrima K, Laino P, Michelotti V, Urso S, Vale G, and Cattivelli L (2011) Emerging knowledge from genome sequencing of crop species. Mol. Biotechnol. DOI 10.1007/s12033-011-9443-1.Google Scholar
- Choulet F, Wicker T, Rustenholz C, Paux E, Salse J, Leroy P, Schlub S, Le Paslier M-C, Magdelenat G, Gonthier C, et al. (2010) Megabase level sequencing reveals contrasted organization and evolution patterns of the wheat gene and transposable element spaces. Plant Cell 22: 1686–1701.PubMedCrossRefGoogle Scholar
- Iqbal SM and Bashir R (2011) Nanopores: sensing and fundamental biological interactions. Springer, New YorkGoogle Scholar
- Kim SD, Hong EH, Song YG, Kim YH, Lee YH, Hwang YH, Kim HS, Lee SH, Kim WH, Ryu YH, Park RK (1994) New soybean variety resistant to disease and lodging, with adapted high yielding “Sinpaldalkong 2”. RDA J. Agric. Sci. 36: 153–157.Google Scholar
- Van K, Kim D, Cai C.M, Kim MY, Shin JH, Graham MA, Shoemaker RC, Choi B-S, Yang T-J, Lee S-H (2008) Sequence level analysis of recently duplicated regions in soybean [Glycine max (L.) Merr.] genome. DNA Res. 15: 93–102.Google Scholar