High-throughput sequencing of three Lemnoideae (duckweeds) chloroplast genomes from total DNA

Abstract

Background: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth.

Methods: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform.

Conclusions: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

Figure 1. Pipeline of chloroplast genome assembly.

Details are described under Methods.

Figure 2. Coverage of Lemnoideae chloroplast genome by SOLiD system reads.

Depth of coverage was plotted along the genome coordinates. Blue peaks show the coverage.

Figure 3. Alignment of Lemnoideae chloroplast genomes.

The sequence of L. minor chloroplast genome was compared to those of S. polyrhiza (top), W. lingulata (middle), W. australiana (bottom). Sequences were aligned in mVISTA and the annotation shown above the alignment corresponds to the L. minor genome. Grey arrows above the alignment indicate genes and their orientation. Thick black lines show the position of the IRs. The grey peaks determine the percent identity between two sequences of L. minor as the reference and our sequenced genomes.

Figure 4. Complete chloroplast genome phylogeny of Lemnoideae.

The phylogram was drawn by Maximum Parsimony with 1000 replicates of bootstrap test. The tree was rooted by Phoenix dactylifera as an outgroup. Support from bootstrap value was shown at the nodes. The GenBank accessions used for the analyses are JN160603 (S. polyrhiza), DQ400350 (L. minor), JN160604 (W. lingulata), JN160605 (W. australiana) and GU811709 (P. dactylifera). The whole genome sequences were aligned by Multi-LAGAN and MEGA 5 was used to draw the tree.