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 ( approximately 4x 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 approximately 2400 BACs and ordered approximately 700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located approximately 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 approximately 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.

Figure 1

Autoradiograph of a typical ³³P-labeled DNA fingerprint gel. Each lane represents the fingerprint analysis from an individual sorghum BAC clone digested with HindIII and HaeIII followed by labeling at the HindIII terminus. Labeled λ/Sau3A standard was loaded in the first and every ninth lane (λ). The asterisk marks the position of the common cloning vector band appearing in all BAC clone lanes. Arrows at the top and bottom of the image delineate the region of the gel that was used for band calling in Image 3.5.

Figure 2

Sixfold BAC DNA pooling strategy. Two hundred-fifty six 96-well microtiter plates containing 24,576 individual BAC clones were arranged in a three-dimensional stack consisting of 32 layers (or plates) × 24 columns × 32 rows. The stack was pooled on the six unique coordinate axes as shown to generate a total of 184 DNA pools.

Figure 3

PCR-based screening of BAC DNA pools for SSR markers. (A) BAC DNA plate pools (PP) and side pools (SP) were analyzed for the presence of two SSRs, Xtxp84 and Xtxp211. BAC DNA pools positive for a given SSR are identified above the respective lane. Plant genomic DNA (BTx623) was used as PCR template in control lanes labeled BTx. Fluorescent-labeled molecular weight standards (LI-COR) were loaded in the first and last lanes and their sizes (bp) are indicated to the right of the gels. (B) FPC V4.5 fingerprint analysis window displaying the representative fingerprint patterns of individual BAC clones from ctg190. Four BAC clones in ctg190 were identified as positive for Xtxp84, and three BACs were identified as positive for Xtxp211 (·) following analysis of the BAC DNA pools from A. (C) FPC V4.5 contig window displaying a horizontal representation of ctg190 that contains BAC clones positive for Xtxp84 and Xtxp211. The characters (*, = and ∼) to the right of individual clone names represent canonical, equal, and approximate clones, respectively (Soderlund et al. 1998). The shaded bar below the contig display indicates the length of the contig, measured in number of bands.

Figure 4

PCR-based screening of BAC DNA pools for SAS-DNA markers using AFLP technology. AFLP templates were prepared from BAC DNA PPs (1–32) and SPs (1–16) and selectively amplified with fluorescent-labeled EcoRI + TGA and MseI + CGG. Labeled products were analyzed on a LI-COR DNA sequencer. AFLP template from genomic DNAs (IS3620C and BTx623) were run as controls and are indicated above the respective lanes. Arrows to the right of the gel show selected SAS DNA markers that were analyzed in the DNA pools. Asterisks to the right of a subset of the markers indicate those SAS DNAs that revealed polymorphisms between BTx623 and IS3620C and could be mapped as AFLPs on the sorghum genetic map. Fluorescent-labeled molecular weight markers (LI-COR) were run in lanes marked M and their sizes (bp) are shown to the left of the gel.

Figure 5

Analysis of individual BAC clones for SAS-DNA/AFLP markers. BAC clones identified as positive for SAS-DNA/AFLP markers following analysis of the BAC DNA pools were individually tested for the presence of the marker. AFLP templates were prepared from individual BAC clones as well as from genomic DNA (IS3620C, lanes marked P₁; BTx623, lanes marked P₂). AFLP templates were selectively amplified with fluorescent-labeled EcoRI + TGA/MseI + CGG (A); EcoRI + TGA/MseI + CTG (B); EcoRI + CAA/MseI + CAA (C); EcoRI + CAA/MseI + CCT (D); and EcoRI + CAA/MseI + CGT (E). Fluorescent-labeled products were run on a LI-COR DNA sequencer. Numbers to the left of panel A indicate the sizes (bp) of molecular weight standards. SAS-DNA/AFLP markers identified in the individual BAC clones and BTx623 genomic DNA are indicated to the right of each panel.

Figure 6

Integrated genetic and physical map of LG B from S. bicolor BTx623 x IS3620C recombinant inbred population. LG B is shown in three parts from the upper end to the bottom end. All SSR and RFLP markers shown were previously mapped to LG B (Peng et al. 1999; Kong et al. 2000; G. Hart, pers. comm.). AFLP markers (labeled Xtxa along LG B) were mapped in the present study. BACs were linked to SSRs (Xtxp markers), STSs (Xtxs markers) or AFLPs (bold-type Xtxa markers) by PCR-based screening of the BAC DNA pools. The individual BAC clones positive for each genetic marker are indicated by symbols next to the clone name. BAC clones that were placed into the same contig by DNA fingerprint analysis are grouped in boxes. Genetic markers linked to two different contigs or one contig and a BAC singleton clone indicate those BAC clones positive for a common marker but not placed into the same contig by DNA fingerprint analysis.

Figure 6

Integrated genetic and physical map of LG B from S. bicolor BTx623 x IS3620C recombinant inbred population. LG B is shown in three parts from the upper end to the bottom end. All SSR and RFLP markers shown were previously mapped to LG B (Peng et al. 1999; Kong et al. 2000; G. Hart, pers. comm.). AFLP markers (labeled Xtxa along LG B) were mapped in the present study. BACs were linked to SSRs (Xtxp markers), STSs (Xtxs markers) or AFLPs (bold-type Xtxa markers) by PCR-based screening of the BAC DNA pools. The individual BAC clones positive for each genetic marker are indicated by symbols next to the clone name. BAC clones that were placed into the same contig by DNA fingerprint analysis are grouped in boxes. Genetic markers linked to two different contigs or one contig and a BAC singleton clone indicate those BAC clones positive for a common marker but not placed into the same contig by DNA fingerprint analysis.

Figure 7

Integrated genetic and physical map of the region of sorghum LG B containing markers Xtxp50, Xtxa532, Xtxp211, and Xtxp84. The three SSR markers, Xtxp50, Xtxp211, and Xtxp84, were previously mapped to this region of LG B (G. Hart, pers. comm.), whereas the AFLP marker, Xtxa532, was mapped between Xtxp50 and Xtxp211 in the present study. BACs linked to these four genetic markers by PCR-based screening of the BAC DNA pools are shown below the genetic map with the dashed lines extending from each marker through the respective, positive BAC clones. Four BAC clones in ctg806 were positive for Xtxa532 as well as the singleton BAC clone, sbb15971. Three BAC clones in ctg190 were positive for Xtxp211 as well as the singleton clone, sbb15971. Therefore, sbb15971 was used as a bridging clone to merge ctg190 and ctg806.