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 |
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Authors: | Ardashir K Masouleh Daniel L E Waters Russel F Reinke Robert J Henry |
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Institution: | Centre for Plant Conservation Genetics, Southern Cross University, Lismore, NSW 2480, Australia; Yanco Agricultural Institute, Yanco, NSW 2703, Australia |
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Abstract: | 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 µL 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. |
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Keywords: | matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) multiplex perfect markers rice single nucleotide polymorphism |
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