Next Generation Sequencing, Assembly and Annotation of Chloroplasts for Phylogenetic Analysis of Amaranths

Amaranthus species (amaranths) are an emerging nutritious traditional leafy vegetable food source in Africa, and have high protein content, vitamins and minerals. Uninformative morphological characters and poorly resolved marker dendograms have led to interest in alternative forms of molecular identifications. Chloroplast DNA has sufficient informative gene and intergenic regions to discriminate efficiently between closely related amaranth species. Species discriminations based solely on taxonomic and morphological descriptors are inefficient due to influences from the environment. Progress to date on whole genome sequencing includes a draft genome assembly of Amaranthus tricolor with approximately one TB of sequence data generated on the Illumina high throughput platforms. The assembly incorporated 1.5 billion reads, spanning 382 Mb of genomic region, thus representing approximately 80% of the genome.  During the present study, it was further aimed to identify and classify 38 unknown amaranth accessions collected, through groupings with 45 previously positively identified amaranth species. The accessions were first phylogenetically characterized using inter- and intra-species informative chloroplast barcoding genes (MatK and rbcL).  These two genes revealed poor resolution within and between amaranth species. Using the sequence data from the draft genome assembly, it was possible to extract, assemble and annotate full-length chloroplast sequences for each of the 83 amaranth accessions in the germplasm set. Maximum parsimony algorithms were used for the identification and classification of unknown Amaranthus species by grouping accessions based on their genetic similarity. A high number of informative sites across the plastid genome revealed more efficient clade representation of the different amaranth species groups