A high-throughput assay for rapid and simultaneous analysis of perfect markers for important quality and agronomic traits in rice using multiplexed MALDI-TOF mass spectrometry.
The application of single nucleotide polymorphisms (SNPs) in plant breeding involves the analysis of a large number of samples, and therefore requires rapid, inexpensive and highly automated multiplex methods to genotype the sequence variants. We have optimized a high-throughput multiplexed SNP assay for eight polymorphisms which explain two agronomic and three grain quality traits in rice. Gene fragments coding for the agronomic traits plant height (semi-dwarf, sd-1) and blast disease resistance (Pi-ta) and the quality traits amylose content (waxy), gelatinization temperature (alk) and fragrance (fgr) were amplified in a multiplex polymerase chain reaction. A single base extension reaction carried out at the polymorphism responsible for each of these phenotypes within these genes generated extension products which were quantified by a matrix-assisted laser desorption ionization-time of flight system. The assay detects both SNPs and indels and is co-dominant, simultaneously detecting both homozygous and heterozygous samples in a multiplex system. This assay analyses eight functional polymorphisms in one 5 microL reaction, demonstrating the high-throughput and cost-effective capability of this system. At this conservative level of multiplexing, 3072 assays can be performed in a single 384-well microtitre plate, allowing the rapid production of valuable information for selection in rice breeding.
Masouleh
Ardashir K
AK
Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia.
Waters
Daniel L E
DL
Reinke
Russel F
RF
Henry
Robert J
RJ
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D017343:Genes, Plant; D005819:Genetic Markers; D005838:Genotype; D054643:INDEL Mutation; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D017931:DNA Primers; D018744:DNA, Plant; D005819:Genetic Markers