|Nipper, Rick -|
|Brown, Patrick -|
Submitted to: Sugar Journal
Publication Type: Abstract Only
Publication Acceptance Date: June 16, 2010
Publication Date: June 17, 2010
Citation: Glynn, N.C., Nipper, R.W., Brown, P.J. 2010. Second-generation Sequencing for Marker Development in Sugarcane. Sugar Journal. 136. Technical Abstract: Second generation sequencing (also known as next-generation or massively parallel sequencing) involves the simultaneous generation of millions of short DNA sequences. The impact and applications of this technology are still emerging; however, strategies that reduce the complexity of the DNA sample prior to sequencing and the utilization of reference genome sequences are two important tools which greatly enhance the application of the technology. Here, we examined the utility of Restriction site Associated DNA (RAD) LongRead sequencing for the identification of Single Nucleotide Polymorphism (SNP) markers in sugarcane. RAD libraries were prepared for sugarcane clone CP 92-1167 and wild, Saccharum spontaneum accession IND 81-146 through a combination of enzyme digestion and physical shearing of genomic DNA. Sequencing consisted of paired end 54 bp sequence reads (2 x 54 bp) using the Illumina GAII platform. A total of 0.628 Gb of raw sequence was generated and assembled into approximately 6,500 contigs per genotype. Since no reference genome is currently available for sugarcane, the fully sequenced Sorghum genome was used as a framework for alignment of assembled sequences. Approximately 25% of the total number of contigs aligned with the sorghum genome, 500 of which were common to both genotypes and Sorghum at sequence identities of >90%. Mining the alignments revealed a total of 1,239 SNPs and 153 short insertion/ deletions with an average SNP frequency of 1 SNP per 127 bp. This study has shown the utility of SNP marker identification in sugarcane using complexity reduction sequencing and utilization of the fully sequenced Sorghum genome. Further exploration of sugarcane-sorghum synteny will be presented.