Sugarcane (Saccharum sp.), a world-wide known feedstock for producing sugar, bioethanol, and energy, has an extremely complex genome due to its highly polyploid and aneuploid nature. A double digestion restriction site-associated DNA sequencing protocol (ddRADseq) was tested in four commercial sugarcane hybrids and one high-bre biotype for the detection and potential application of single nucleotide polymorphisms (SNPs) in genetic breeding. This genotyping approach compared two Illumina sequencing platforms and different ltering schemes, with and without a reference genome. A greater number of reads were obtained with Illumina platform Novaseq6000 than with Nextseq500, being this consistent with a greater number of SNPs determined with high accuracy. Additionally, more SNPs were found using a denovo pipeline compared to de refmap pipeline of the Stacks software. Longer read size, paired-end reads and 4M reads per individual represent the most e cient combination in recovering SNPs. The optimal combination of lter parameters varied depending on the different matrices created based on the different platforms. In both matrices, the highest number of SNPs localized in the longest chromosome 1, whereas the fewest landed in the shortest chromosomes 5 and 8. Multivariate comparisons of the SNPs matrices showed closer relationships among commercial hybrids than with the high-bre biotype. Functional analysis of the obtained SNPs, performed with the Variant Effect Predictor, demonstrated the appearance of variants throughout the sugarcane genome. The pipeline applied in this study can be exploited to identify useful molecular markers that can be used in sugarcane breeding programs to reduce selection cycles.
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