ARS | Publication request: Whole Chloroplast Genome Sequencing in Fragaria Using Deep Sequencing: A Comparison of Three Methods

Submitted to: Plant and Animal Genome Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: November 4, 2009
Publication Date: N/A

Interpretive Summary: Scientists have been wondering about different types of strawberries. Some of them may belong to different species but look very similar to each other by their physical traits. We decided to look at three different molecular DNA preparation techniques – looking at the DNA sequence or code of the strawberries – to determine differences that allow species groupings. We also are very interested in determining the evolutionary history of the strawberry species, how they came to be this way since the last ice age. We would like to identify which species may most closely resemble the ancestors of other more modern species. Strawberries and other plants have DNA in several locations. In the strawberry leaves there are tiny organelles called “chloroplasts.” The chloroplast contains green pigment and makes the leaf green. In addition, a small section of DNA is found there. Usually, this small DNA loop is inherited directly from one of the plant’s parent: the mother or the father. We used new sequencing technology called “ultra high throughput sequencing” and were able to look at the sequence of DNA codes for the chloroplast from 22 strawberry species. In strawberry, the chloroplast DNA was found to be inherited directly from the mother plant. DNA preparation using an approach called “low coverage genomic sequencing” was the most successful of the three DNA preparation techniques in determining up to 99% of the chloroplast DNA sequence.

Technical Abstract: Chloroplast sequences previously investigated in Fragaria revealed low amounts of variation. Deep sequencing technologies enable economical sequencing of complete chloroplast genomes. These sequences can potentially provide robust phylogenetic resolution, even at low taxonomic levels within plant groups. We compared three different approaches for sequencing chloroplast genomes on the Illumina Genome Analyzer: PCR amplification, physical chloroplast isolation, and plastome assembly from low coverage genomic sequencing. Illumina libraries were tagged with sample specific barcodes, and three to six libraries were multiplexed in each lane. Sample preparation was time consuming in the case of PCR amplification and plastid genome completion ranged from 36-82%. Incomplete assembly likely resulted from errors arising from PCR product preparation and/or PCR failures due to lack of plastome-specific primers. Physical isolation required large amounts of leaf tissue and was very inefficient, resulting in only 0.01-0.61% of reads aligning to the chloroplast genome. Preliminary analyses reveal that six of nine genomic DNA preparations have plastome assemblies ranging from 85-99% complete, with 1-10% of the reads aligning to the chloroplast genome. Based on these results, we recommend low coverage genomic sequencing as the most efficient approach for obtaining complete chloroplast genome sequences.