Recent work has used a transgenic apple tree which produces no de

Recent work has used a transgenic apple tree which produces no detect able ethylene to examine gene expression changes and production of volatile compounds associated with apple aroma. Fruit from this tree mature, but do not ripen or soften, unless treated with exogenous ethylene. Schaf fer et al. used the apple oligonucleotide array described here to identify 944 apple cortex and skin Crenolanib Sigma genes that respond to ethylene. Because the ripe fruit samples in the fruit development experiment consisted of cortex tis sue only we identified only those genes that change by at least 2 fold in cortex, giving a list of 456 genes that respond strongly to ethylene in fruit cortex. Of these 456 ethylene responsive genes, 106 also changed significantly during the ripening phase of normal fruit development.

These ethylene responsive fruit cortex ripening genes are shown in Table 7 and are grouped by ripening sub cluster. The distribution Inhibitors,Modulators,Libraries of the genes was uneven between the three clusters with a greater percentage of the R2 cluster also identified as ethylene responsive in R1, 48 of 195 genes in R2 and 48 of 408 genes in R3. Included amongst these genes was one gene identified as a putative ethylene receptor, most sim ilar to the ETR2 EIN4 receptors from Arabidopsis. The apple microar ray also contains oligonucleotide probes for four addi tional putative ethylene receptor genes. Expression of three of these Inhibitors,Modulators,Libraries genes was not significantly changed during the ethylene microarray experiment or during normal fruit ripening was selected as induced by ethylene, and although it was not selected as significantly changing during fruit develop ment, it does show some induction in normal Inhibitors,Modulators,Libraries fruit ripen ing.

Inhibitors,Modulators,Libraries Discussion Confirmation of microarray expression patterns by qRT PCR At each of the steps used to produce microarray data, var iability can be introduced leading to potential errors. We used qRTPCR of cDNA from the same samples of RNA used in the microarray experiment itself to estimate the overall accuracy of our data. Overall we found good corre lation between qRT PCR and microarray results with 75% of microarray expression patterns reproducible by qRT PCR. However, 25% of expression patterns for which the qRT PCR results did not match the microarray result. In some cases, this difference seems to be associated with genes where the genomic DNA reference sample gave very high intensity binding.

Inhibitors,Modulators,Libraries It is possible that this high level of gDNA binding distorted the ratios observed or the gDNA binding may have interfered with cDNA binding for those genes. Another possible explanation for qRT PCR results disagreeing with microarray results is that the oligo on the microarray was able to hybridise to more than one allele of a gene in the sample, and qRT PCR primer binding was more specific. Alternatively the micro array oligo may be hybridizing to more than one member of a gene family.

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