HIGH THROUGHPUT GENOME SPECIFIC MOLECULAR MARKERS FOR ERUCIC ACID CONTENT GENES IN BRASSICA NAPUS (L.)

摘要

A single base change in the Bn-FAE1.1 gene in the A genome and atwo-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearlyzerocontent of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1from aB. rapa BAC library and a BAC clone anchoring Bn-FAE'1.2 from a B. oleraceaBAClibrary were used in this research. After sequencing the gene flankingregions, it wasfound that the dissimilarity of the flanking sequences of these two FAE1homologsfacilitated the design of genome specific primers that could amplify thecorrespondinggenome in allotetraploid B. napus. The two-base deletion in the C genome genewasdetected as a sequence characterized sequence region (SCAR) marker. Toincreasethe throughput, one genome specific primer was labeled with four fluorescencedyesand combined with 20 different primers to produce PCR products with differentfragment sizes. Eventually, a super pool of 80 samples was detectedsimultaneously,making it possible to analyze over half a million of samples per day using amediumcapacity ABI 3100 Genetic Analyzer. This dramatically reduces the cost ofmarkerdetection. The single base change in the Bn-FAE1.1 gene was detected as singlenucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexingprimerset was designed by adding a polyT to the 5' primer end to increase SNPdetectionthroughput through sample pooling. These multiplexed high throughput molecularmarkers have been successfully implemented in our canola/rapeseed breedingprograms.