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Project Progress

We have carried out the bulked segregant analysis described in Specific Aim 1 for the evolved multicellular Chlamydomonas reinhardtii from the centrifugation experiment (Herron). This analysis has revealed two chromosomal regions that are substantially enriched for alleles from the multicellular strain in the multicellular pool (insert Fig. 1). Among these are several genes involved in the production of extracellular matrix and one known to function in cell cycle control. We have developed primer sets to amplify length polymorphism markers in these regions, and we are currently PCR amplifying these markers for a large set of F2 progeny to “ground truth” the results of the bulked segregant analysis. If multicellular F2s are found to always carry markers from the multicellular parent, this will provide strong evidence that the genes causing the multicellular phenotype are indeed within these regions.

In addition, we have carried out the RNA-Seq experiment described in Specific Aim 1, comparing global patterns of gene expression between the multicellular strain from the centrifugation experiment and a unicellular ancestor (Herron). Across several functional categories, the largest number of genes are overexpressed in the multicellular strain, and the smallest number underexpressed, during the release of unicellular propagules and the beginning of multicellular development (insert Fig. 2).

We have isolated evolved clones from two experimental populations and one control population from the Paramecium predation experiment, and we are carrying out detailed analyses of the evolved life cycles (Walker, under the supervision of Ratcliff). By culturing the isolates in the absence of predators over several transfers, we have confirmed that the multicellular phenotypes are genetically fixed. We have quantified the motility of both multicellular and unicellular isolates, showing that all multicellular isolates and some unicellular isolates are impaired for motility (Boyd). We have also carried out experiments to show that the multicellular phenotype provides a viability advantage under predation (Knox).

We have isolated DNA from all 24 isolates from the Paramecium predation experiment (8 from each of 3 populations) and carried out paired-end Illumina sequencing of whole genomic DNA libraries. Analysis of these data are underway (Chen). We have also carried out crossing experiments to identify the mating type of each clone in preparation for the evolution experiments described in Specific Aim 3.