In species devoid of complete genomic sequence information, libraries of bacterial artificial chromosome (BAC) clones [1
] are an indispensable genomic tool. The utility of BAC libraries has been further enhanced by the development of high-information-content-fingerprinting (HICF) techniques [2
] and the FPC program for the assembly of fingerprinted BAC clones into contigs [5
], which opened the door to the construction of BAC-based physical maps of plant and animal genomes (for review see [8
]). An operation common to virtually all applications of BAC libraries is screening the libraries for clones harboring specific nucleotide sequences. Current screening techniques utilize either DNA-DNA hybridization or polymerase chain reaction (PCR). In some applications, such as the construction of a physical map, a BAC library must be screened for the presence of hundreds or thousands of different molecular markers. To maximize the efficiency of such screening, multidimensional pooling of clones or probes is employed [9
]; probes are pooled if a library is screened by DNA-DNA hybridization [10
] and clones are pooled if it is screened by PCR [11
All current screening strategies are laborious, slow, error prone, and often result in ambiguous assignments of BAC clones to loci on a genetic map. The identification of BAC clones harboring specific nucleotide sequences by hybridization of multidimensional pools of overgo probes with BAC library screening membranes [10
], in addition to some of the above problems, also requires handling large amounts of radioactive material. The greatest impediment to screening of BAC libraries with pools of cDNA clones or overgo probes is that a single probe often hybridizes with clones in multiple contigs, either due to gene duplication, the presence of repeated sequences, or other reasons. Unequivocal assignment of BAC clones to loci on the genetic map requires additional work.
We describe here a BAC library screening strategy that is largely devoid of these limitations and can be performed in a high-throughput mode. The strategy employs Illumina GoldenGate™ oligonucleotide assays, also referred to as Oligonucleotide Pool Assays (OPAs), that are currently used for the highly parallel SNP genotyping of genomic DNAs [12
]. Each assay targets a specific SNP locus and utilizes two allele-specific oligonucleotides to discriminate between SNP alleles. The allele discriminating nucleotide of an allele-specific oligonucleotide is at its 3' end. Another primer, the locus-specific oligonucleotide, which contains an address sequence for the SNP locus, anneals downstream of the SNP. After annealing one of the two allele-specific oligonucleotides to the genomic DNA template, the oligonucleotide is extended by DNA polymerase and ligated to the locus-specific oligonucleotide downstream forming a contiguous PCR template. Primer extension and ligation can be performed at up to 1536 loci simultaneously. The templates are PCR amplified using three PCR primers complementary to specific sequences inserted into all oligonucleotides. Two primers anneal to the allele-specific oligonucleotides; one for each SNP is labeled with the Cy3 fluorochrome and the other with the Cy5 fluorochrome. The third anneals to the locus specific oligonucleotide. The ratio of the Cy3 and Cy5 fluorescence is used to determine the genotype at a SNP locus. If the ratio is near 0 or 1 (near pure Cy3 or near pure Cy5 fluorescence), the locus is homozygous. If the ratio is about 1: 1 the locus is heterozygous.
It is shown here that annealing of allele- and locus-specific oligonucleotides to a pool of BAC DNAs and the subsequent primer extension and ligation reaction can be used to determine whether or not a BAC pool DNA harbors a specific locus. This allows genotyping the BAC pool for the presence or absence of the locus. It is also shown that BAC genotyping with Illumina oligonucleotide assays results in a high percentage of unequivocal assignments of BAC clones and BAC contigs to loci on a genetic map.
A six-dimensional BAC pooling strategy has previously been successfully used to genotype 24576 sorghum BAC clones (about 4× sorghum genome equivalents) for the presence or absence of specific amplified fragment length polymorphism (AFLP) amplicons during sorghum physical map construction [11
]. The pooling strategy utilized 184 pools. Although the six-dimensional strategy worked well for the sorghum genome, it results in too many pools for large genomes, such as those of wheat and its diploid ancestors. One genome equivalent of Aegilops tauschii
(1C = 4,020 Mb [13
]), one of the three diploid ancestors of polyploid wheat, amounts to 30000 to 40000 BAC clones, depending on the average size of DNA inserts. To facilitate screening multiple genome equivalents of large genomes, such as that of Ae. tauschii
, but keeping the numbers of pools manageably low, a simple, five-dimensional pooling strategy was designed and evaluated here.
Contigs built from 199190 Ae. tauschii
BAC clones [14
] fingerprinted with the SNaPshot HICF technology [4
] were simultaneously screened with 1384 multiplexed Illumina GoldenGate assays. Assignments of BAC clones and contigs to gene loci on the Ae. tauschii
chromosome 2D genetic map were analyzed to assess the proportion of BAC clones and contigs unequivocally assigned to individual loci on the genetic map.