Thai Oakleaf Lettuce Phenocopies a Phytochrome B Mutant.

Photomorphogenic development in seedlings may be diagnostic of future plant performance. In this report, we characterize the Thai Oakleaf lettuce genotype, as it exhibited abnormalities in photomorphogenic development that were the most conspicuous under red light, including defects in hypocotyl growth inhibition, decreased cotyledon expansion, and constitutive shade avoidance tendencies. These observations are consistent with defects in red light sensing through the phytochrome B (phyB) photoreceptor system. This genotype is sold commercially as a heat-tolerant variety, which aligns with the evidence that phyB acts as a thermosensor.

Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids.

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57-60% GC-content at a rate of 50-100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.