Direct isolation of differentially expressed genes from a specific chromosome region of common wheat: application of the amplified fragment length polymorphism-based mRNA fingerprinting (AMF) method in combination with a deletion line of wheat.
The amplified restriction fragment length polymorphism (AFLP)-based mRNA fingerprinting (AMF) method makes it possible systematically and conveniently to identify differentially expressed cDNAs with high reproducibility. We have applied the AMF method to the cloning of the Q gene of common wheat, which is located on the long arm of chromosome 5A and pleiotropically controls the spike morphology and the threshing character of seeds. Using the AMF method, we compared the fingerprints of mRNA samples extracted from the young spikes of Triticum aestivum cv. Chinese Spring (CS) carrying the Q gene to those of a chromosome deletion line of CS, namely, q5, which lacks 15% of 5AL including the Q gene. Approximately 12,200 fragments were produced after PCR with 256 primer combinations. Of these, 92 fragments were differentially expressed between CS and q5. Northern and Southern analyses showed that 16 fragments gave specific or relatively stronger transcript signals in CS, and these clones were present in single copy or in low copy numbers in the wheat genome. Four clones were genetically mapped to the region deleted in q5. Subsequently, one clone, pTaQ22, was mapped at the same locus as the Q gene, indicating that pTaQ22 corresponds to the Q gene or is tightly linked to it. DNA sequence data showed that pTaQ22 had no homology to any known genes, thus suggesting a novel function for this gene in flower morphogenesis. This AMF method might provide a straightforward method for isolating genes in the hexaploid background of common wheat.
Kojima
T
T
Kihara Institute for Biological Research and Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan.
Habu
Y
Y
Iida
S
S
Ogihara
Y
Y
D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D017353:Gene Deletion; D018506:Gene Expression Regulation, Plant; D005819:Genetic Markers; D005821:Genetic Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D015203:Reproducibility of Results; D014908:Triticum / Q000235:genetics*
D003062:Codon; D005819:Genetic Markers; D012333:RNA, Messenger