Genotyping-by-Sequencing Using Double Digest Restriction Associated DNA (ddRAD) Approach for Fine Mapping LG2 Drought Tolerance QTL in Pearl Millet

Genotyping-by-sequencing is a powerful and inexpensive technique which captures RADTags distributed across the genome. ddRAD is a variant of RADtag sequencing and is a reduced representation sequencing method useful for genotyping and genetic mapping in organisms with or without reference sequences. We evaluated different RADTag approaches for application in pearl millet using both single and double enzyme protocols for RADTag generation on Illumina HiSeq2500. We used RAD and ddRAD technique to genotype NILs for LG2 drought tolerance QTL. Single enzyme protocol (ApeKI) compared poorly with that of two enzyme protocol (SphI and MluCI). We generated ~50Mb data using both RAD and ddRAD techniques for NILs, and analyzed using custom scripts for polymorphic markers in de-novo approach using uclust and samtools. More than 91% reads aligned to the reference in ddRAD and 78-86% aligned in RAD sequence data. A total of 14,294 SNPs and 188 Indels were identified with read depth of 10 in ddRAD data, and 3,465 SNPs and 26 Indels from RAD data, demonstrating effectiveness of ddRAD technique over RAD technique. Further analysis of homozygous polymorphic markers between parental lines revealed 84 markers for ddRAD and 33 markers for RAD technique in the NILs. Based on this evaluation, ddRAD technique is being further employed for genotyping LG2 drought tolerance QTL fine mapping population.