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Understanding the Effects of Selection on Genetic Diversity

Overview | Selection and Sequencing | Analysis | People | Publications

Selection and Sequencing

Selection and sequencing of homologous gene pairs.

The first 50 gene pairs (stage 1) to sequence will be chosen according to several criteria. First, we will constrain our selection to genes for which we have confidently inferred both homoeologous sequences from present EST assemblies. This is important in that it will serve to exclude aberrant sequences from cloning artifacts or PCR recombination. From this initial set of several thousand homoeologs, we will constrain the selection to include only those genes that align to a close Arabidopsis homolog such that primers may be designed that are in exons but which target ~750-1000 bp amplicons that yield sequences that are at least 50% intron. This list will be further filtered and automated using primer design software using criteria of primer optimization for Tm and possible multiplexing. The set of 50 genes for stage 2 will be selected after phenotypic and microarray analysis, in addition to the aforementioned criteria. For both stage 1 and stage 2 sequencing, we will initially target 50% more genes than will be used in the nucleotide diversity surveys, using as a final filter the simple criterion of what works best.

Genes will be amplified, cloned, and sequenced using updates of procedures we have employed in past studies. The ~6000 PCRs (2000 from stage 1 + 2000 from stage 2, multiplied by 1.5 for primer effectiveness) will require ~60, 96-well plates. Following clean-up and gel evaluation of PCR success, amplicons will be pooled in lots of 10 genes (5 pools) per accession, in approximately equimolar ratios, for cloning. Following plating, approximately 100 colonies will be sequenced per cloning reaction by colony PCR using the appropriate primer mixture; this design will ensure that for nearly all genes studied, both homoeologs are recovered following bioinformatic sorting of the randomly sequenced clones (the average will be 10 copies per gene pair). PCR recombinants will readily be identified (see above) and discarded. With 400 cloning reactions X 100 sequences, we will generate 40,000 sequences.

We welcome your comments and suggestions.