Development of High Density SNP-Based Linkage Map in Pearl Millet.

Pearl millet (Cenchrus americanus (L.) Morrone) is a gluten free grain crop which is additionally gaining importance in the USA due to the increased demand for pearl millet flour by many ethnic groups. As a result, efforts are underway in the Southeast to develop high grain yielding adapted pearl millet cultivars. Genome mapping studies are a pre-requisite for tagging agronomically important traits. As pearl millet lacks a reference genome, development of new markers requires a procedure that does not depend on sequence information. Genotyping-by-sequencing (GBS) is one such approach that results in genome-wide single nucleotide polymorphism (SNP) markers, even in species without a reference genome. We used a recombinant inbred line (RIL) mapping population developed from two parent lines, 99B (Tift 99B) as female parent and 99-17-1 (Tift 454) as male parent. One-hundred eighty seven individuals from this population including two parent lines and a commercial hybrid line were subjected to GBS analysis in a 96-plex ApeKI library sequenced on an Illumina HiSeq2000. The average good reads per individual was 2,089,572 with a filter pass rate of 88% and a coefficient of variation of 50%. Five lines with less than 10% of the mean reads failed to produce good reads.  The sequence reads from 179 RILs were processed with both a reference-free pipeline (UNEAK) and a reference-based pipeline using foxtail millet (Setaria italica (L.) Beauv.) as the reference genome. The data were further filtered to remove probable paralogs and outcrossed lines. The final genetic map contains several thousand SNPs spread across all seven chromosomes. This linkage map will help in the identification and tagging of traits such as blast resistance, pyrucularia resistance and nematode resistance. Future work may include using these markers in marker-assisted selection or genomic selection.