A High-throughput AFLP-based Method for Constructing Integrated Genetic and Physical Maps: Progress Toward a Sorghum Genome Map

Abstract

Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones (∼4× genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified ∼2400 BACs and ordered ∼700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located ∼200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in ∼65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map of the sorghum genome.[The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF218263.]Integrated genetic and physical genome maps are extremely valuable for map-based gene isolation, comparative genome analysis, and as sources of sequenceready clones for genome sequencing projects. Various methods have been developed for assembling physical maps of complex genomes. One of the best characterized approaches uses restriction enzymes to generate large numbers of DNA fragments from genomic subclones (Brenner and Livak 1989;Gregory et al. 1997;Marra et al. 1997). These DNA fingerprints are compared to identify related clones, and to assemble overlapping clones in contigs. The utility of fingerprinting for ordering a complex genome is limited, however, due to variation in DNA migration from gel to gel, the presence of repetitive DNAs, unusual distribution of restriction sites and skewed clone representation. Moreover, fingerprinting, unless combined with other methods, does not link genomic clones directly to gen...

Contigs Built with Fingerprints, Markers, and FPC V4.7

“…The optimal cutoff will vary based on the fingerprint method, data acquisition, image analysis package, number of bands, and the amount of error and must be optimized for a given set of experimental parameters. Some tolerance/ cutoff variations are 7/1e-14 (Klein et al 2000), 5/1e-06 , 7/1e-09 (Zhu et al 1999), and 8/1e-09 (Marra et al 1997), and we use a tolerance 7 cutoff 1e-10 for complete digest BAC data for human chromosomes 9, 10, and 13.…”

“…Zhu et al (1999) built a sequence ready map of chromosome 7 of the rice blast fungus Magnaporthe grisea by using BAC contigs assembled by hybridization and integrated with fingerprinted BAC contigs Hoskins et al (2000) integrated STS content, restriction fingerprinting, and polytene chromosome in situ hybridization to produce a Drosophila melanogaster map for 81% of the genome. Klein et al (2000) used amplified fragment length polymorphism (AFLP)-based markers integrated with fingerprints to map sorghum. To provide confirmation of overlap and information to merge contigs, the Sanger Centre traditionally has used markers with fingerprints (e.g., see Mungall et al 1997;Soderlund et al 1998).…”

Targeted Analysis of Orthologous Phytochrome A Regions of the Sorghum, Maize, and Rice Genomes using Comparative Gene-Island Sequencing

“…These sorghum maps contain DNA markers common to maize and other grass genetic maps, making crossreferencing between these species possible. Construction of an integrated physical and genetic map for sorghum has recently been initiated, using sixdimensional pooling of sorghum bacterial artificial chromosome (BAC) libraries, coupled with amplified fragment-length polymorphism technology (Klein et al, 2000; http://SorghumGenome.tamu.edu). This resource will facilitate map-based cloning of genes and the acquisition of sorghum gene sequences for comparative sequence analysis.…”

Sequence-based physical mapping of complex genomes by whole genome profiling

“…An efficient way to provide these anchor points is by construction of a whole genome physical map from bacterial artificial chromosome (BAC) clones (Shizuya et al 1992;Rounsley et al 2009), in combination with BAC-end sequencing (Nelson and Soderlund 2009). BAC insert clones are relatively easy to generate and store and have proven to be effective for genome-wide physical map construction (Gregory et al 1997;Marra et al 1997;Klein et al 2000;Wu et al 2004). Hence, BAC-based physical maps combined with BAC-end sequencing have formed the basis of several whole genome sequencing projects (Sasaki et al 2005;Wei et al 2007).…”