Targeted sequencing of the Panicum miliaceum gene space and genotyping of variant sites from population genetics studies, combined in a single assay, as a tool for broomcorn millet assisted breeding

Broomcorn millet ( Panicum miliaceum L.) is a prehistorical cereal, today cultivated as a minor crop with low yields but with a renewed interest for its high water use efficiency and gluten-free grains. To reverse the downward trend in broomcorn millet cultivation, the crop needs genetic improvement and creation of novel genetic variation to increase productivity. In order to facilitate genomics-assisted breeding, we designed a reduced representation genome-sequencing assay that investigates 1.8% of the nuclear DNA in a targeted and reproducible way, with an intensity of genomic sampling that is a direct function of local recombination rate. We used this tool and set up bioinformatics analyses tailored to the polyploid genome of P. miliaceum for maternity and paternity testing, quantification and genomic distribution of homozygous regions and estimation of parental genome contribution for individual seedlings in advanced inbred lines from a breeding program and compared their genomic composition with registered varieties. We found several clues that suggest that the genetic purification process to ensure genetic uniformity is incomplete in varieties of this species. Residual heterozygosity was detected in the genome of three registered varieties ranging from 4.4 to 6.25% of their haploid genome length. Other registered varieties show genome-wide homozygosity. We found, however, evidence of intravarietal genetic variation in three cases that suggest that the breeder seed or commercial seed production had fixed by self-pollination multiple inbred lines with very similar, though not identical, genotypes within each variety.