A Pale Shade of Green: Abundance and Structural Integrity of the Chloroplast Genome in Barley Microspores and Albino Plants

Androgenesis is a highly valuable technology rapidly yielding homozygous lines from a heterozygous donor in a single generation. By reducing the time to genetic fixation, it is of benefit in plant breeding and genetic research. One of the drawbacks of the technology is the occurrence of albinism, a condition that results in plants that are incapable of performing photosynthesis and are thus unviable. Past studies have revealed that albinism is associated with morphological anomalies of the chloroplast and the loss of certain portions of the chloroplast genome (cpDNA). In this work, our goal was to examine the abundance and structural integrity of the cpDNA in microspores and albino plants of barley (Hordeum vulgare) using next-generation sequencing. Total DNA was extracted from green leaves of the donor plant (control), microspores undergoing androgenesis, and albino leaves. These DNAs were sequenced and chloroplast reads were aligned against the chloroplast reference genome. We observed an eight-fold decrease in the abundance of cpDNA reads in microspores relative to the green leaf control; no evidence of any structural rearrangement was found. In albino leaves, contrary to the situation in green leaves and microspores, depth of coverage of the cpDNA varied widely, with inverted repeats (IRs) enjoying a mean abundance 16 fold higher than the single copy region. Even across the single copy regions, coverage was highly variable and seemed to follow a gradient sloping downward with increasing distance from the IRs. Such a profile of coverage in albino plants is inconsistent with a circular chloroplast genome.