Using Mass Spectrometry (Sequenom MALDI-TOF) to genotype sugarcane

DNA markers of various kinds (AFLPs, SSRs, SNPs etc.) are being used for many crop species as a means to improve genetic gains in breeding programs by marker assisted selection (MAS – selecting parent plants based on the presence of a marker rather than measuring the trait of interest each generation). For the purposes of MAS, DNA markers should ideally be reliable and cheap to assay. Point mutations or single nucleotide polymorphisms (SNPs) as they are now more commonly known are an excellent source of markers because of their occurrence throughout the genome. Various methods have been developed to assay SNPs for genotyping in both plants and animals. The Sequenom MASS Array system provides a high throughput method of assaying SNPs (genotyping) that is both reliable and cheap. The Sequenom MASS Array system has been used at Southern Cross to assay SNPs in sugarcane genes. A particular difficulty when working at the DNA level with sugarcane is its highly polyploid nature (multiple copies of each chromosome – compared with only two in diploids), which has meant that it has been difficult to detect low dose and in particular single dose SNP markers due to the low signal from the single dose allele. However with the Sequenom system the proportion of the homologous chromosomes which carry a particular SNP allele can be estimated. This method has been used in two projects within the CRC-SIIB marker program. About 300 genes are being targeted based on independent evidence from other CRC projects that these genes may influence important sugarcane traits. In one project a large number of sugarcane clones have been genotyped using a large number of SNPs. The results will be used to determine if there are associations between SNP alleles and the traits of interest that have been scored across this broad range of sugarcane clones. In a second project SNPs have been genotyped across the parents of a genetic linkage mapping population. SNPs that appeared to be single dose were tested across a subset of the progeny of the parents of the mapping population and if segregation was observed in this subset, the entire mapping population of 220 individuals was genotyped. SNPs genotyped in this way have been added to the linkage map being constructed by CRC-SIIB at CSIRO which can be used for the detection of trait associations within the mapping population. The number of SNPs has to date been limited due to a lack of knowledge about SNP sites in the parents of the sugarcane mapping population. However a much larger number of SNPs are expected to be found in this cross using new methods of DNA sequencing.