Long range PCR, deep parallel sequencing and multiplexed Maldi-TOF mass spectrometry reveals new SNP variants associated with starch functional properties
Waters, DLE, Masouleh, AK, Reinke, RF & Henry, RJ, 2011, 'Long range PCR, deep parallel sequencing and multiplexed Maldi-TOF mass spectrometry reveals new SNP variants associated with starch functional properties', paper presented to the 15th Australian Barley Technical Symposium: Barley for business, Adelaide, SA, 18-21 September.
The barley genome sequence will be available soon, allowing barley to follow the rice model of genetic analysis, accelerating the rate of knowledge acquisition surrounding the genetic control of important traits. We have used the rice genome sequence to understand the genetic control of rice starch quality within the Australian rice breeding program. Illumina GAIIx sequencing of 17 rice starch synthesis genes from 233 genotypes within the Australian rice breeding program detected 501 SNPs and 113 Indels in both coding and non-coding regions. Five genes encoding the enzymes AGPL2a, Isoamylase1, SPHOL, SSIIb and SSIVb showed no polymorphism. Associations between 52 of the 110 functional SNP and the pasting and cooking quality of the rice were detected. GBSSI and SSIIa had a major influence on starch properties and the genes encoding other enzymes had minor associations. The ´G/T´ SNP at the boundary site of exon1/intron1 of GBSSI showed the strongest association with retrogradation and amylose content. The GC/TT SNP at exon 8 of SSIIa showed a very significant association with pasting temperature, gelatinisation temperature and peak time. A new ´C/T´ SNP was found at position 1188 in the GPT encoding gene which alters Leu24 to Phe. This SNP may be associated with retrogradation and amylose content. These associations provide new tools for deliberate selection of rice genotypes for specific functional and nutritional outcomes.
Acknowledgements: This project was funded by NSW DPI and the Australian Research Council. Thanks to Rachelle Ward NSW DPI for provision of rice starch phenotypic data and Stirling Bowen and Mark Edwards for technical assistance with Illumina GAIIx sequencing and MALDI-TOF MS (Sequenom MassARRAY) analysis.