Funded by: PCIEERD
Implementation period: Jan 2013 – Dec 2015 (w/ extension) Project Leaders: Antonio C. Laurena, PHD; Liwayway M. Engle, PHD; Rosalyn T. Luzaran, PHD; RA Ramirez, PHD
This project aims to develop sugarcane varieties with superior agronomic traits exhibiting high sucrose content and resistance to fungal diseases, downy mildew and smut. Three approaches are being used to obtain molecular markers for the targeted traits:
(1) Candidate-gene approach, where primers associated to the targeted genes are synthesized and used to amplify PCR products, the sequences of which are then compared to determine the presence of Single Nucleotide Polymorphisms (SNPs) and Simple Sequence Repeats (SSRs),
(2) Construction of mini-genomic library using genome filtering method (Fellers, 2008), and
(3) whole genome sequencing using Next-Generation-Sequencing (NGS) platform.
Candidate Gene Approach
The presence of SSRs was determined in sequences of genes related to sucrose content in sugarcane. These genes are Sucrose Transporters (SUT1 and SUT4) and Sucrose Phosphate Synthases (SPSA, SPSB and SPSIII). Sequences were screened for SSRs using the online tool SSRPrimerII (http://www.appliedbioinformatics.com.au/projects/ssrPrimer/cgi-bin/index), which also designs primers targeting the SSRs. Thirteen sets of primers were designed from the five genes and are currently being optimized for PCR amplification using 16 sugarcane varieties.
Single nucleotide polymorphisms were identified in the SNLR1 gene (associated to smut resistance) of PHIL 93-1601 sugarcane variety. Six sets of tri/tetra-primers flanking and targeting the SNPs were designed using BatchPrimer3 software. These primers are also being optimized for PCR amplification using different sugarcane varieties.
DNA libraries containing the filtered genome of VMC 86-550 (high sucrose), VMC 87-599 (high resistance to smut and downy mildew, high sucrose), and PHIL 93-1601 (high resistance to smut and downy mildew) varieties are also being developed based on the protocol developed by Fellers (2008). Genomic DNA from the three varieties were subjected to restriction enzyme digestion using methylation-sensitive enzymes, PstI and AatII. Restriction enzyme-specific adaptors were ligated to complement the sticky ends of the DNA and universal primers are used for PCR enrichment of digested and adaptor-ligated DNA. Figure 1 shows the digested DNA for the three varieties as well as the PCR enrichment products. Note the smears found in the PCR enrichment products. These are expected as digestion of genomic DNA can produce differently sized fragments.
For genome sequencing through NGS, three varieties (VMC 86-550, VMC 87-599, and PHIL 93-1601) were sent to the Philippine Genome Center DNA Sequencing Core Facility (PGC-DSCF). PGC-DSCF will perform the sequencing using the MiSeq Technology (Illumina). DNA from the downy mildew pathogen, Peronosclerospora sp., was also sent for whole genome sequencing. However, the samples of the Sugarcane Genomics Project are still on queue for processing at the sequencing facility since June.
In addition to the four samples, genomic DNA from the smut fungal pathogen, Ustilago scitaminea, will also be sequenced through NGS. The smut fungus was already isolated from smut whips collected from smut-infected sugarcane from Batangas and Negros Occidental, and is maintained in pure culture in preparation for DNA extraction.
The Sugarcane Genomics Project is a cross-listed project of PGC with the University of the Philippines Los Baños.