Barley chloroplast and mitochondrial genomes diversity evaluation by ngs of the organelle DNA mixtures

In this study, complete chloroplast and mitochondrial genomes of wild (Hordeum vulgare subsp. spontaneum) and cultivated (Hordeum vulgare subsp. vulgare) barley forms were compared to evaluate the diversity of these genomes within the species.

NGS (next generation sequencing) was conducted for mixtures of chloroplast and mitochondrial DNA of 17 barley samples. The chloroplast DNA was obtained from the fraction of these organelles, isolated by differential centrifugation. Such fraction contained an admixture of mitochondrial DNA, which made it possible to conduct sequencing of both genomes simultaneously. The NGS data processing algorithm included some features to prevent mistakes during the alignment of reads from the homology regions within and between the genomes. Five sequences of barley chloroplast genome and two sequences of mitochondrial genome accessible in NCBI GenBank were also involved in the full-genome comparative analysis.

Comparison of 22 complete barley chloroplast genome sequences revealed a high level of diversity of these genomes within Hordeum vulgare. There were 107 polymorphic loci: 9 INDELs, 79 SNPs and 19 polymorphisms of SSR-regions (simple sequence repeats). Twenty from 79 found SNPs were located in coding sequences.

Analysis of 19 complete sequences of mitochondrial genomes (8 H. vulgare subsp. vulgare and 11 H. vulgare subsp. spontaneum) showed a lower level of variability of these organelles: 1 INDEL and 22 SNP (4 in coding sequences). Two SNPs were found in one of the genes of the small subunit of ribosomes and two were located in the exon of a pseudogene.

The results of the study proved the applicability of NGS of chloroplast and mitochondrial DNA mixtures to the obtaining of complete sequences of these genomes. Revealed polymorphic loci could be used for barley intraspecific identification and phylogeny.

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