Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display

CMS-D8 and its restorer were developed by introducing the cytoplasm and nuclear gene Rf ₂ from the wild diploid Gossypium trilobum (D8) into the cultivated tetraploid Upland cotton (Gossypium hirsutum). No information is available on how the Rf ₂ gene interacts with CMS-associated genes and how CMS-D8 cytoplasm affects nuclear gene expression. The objective of this study was to identify differentially expressed genes in anther tissues between the non-restoring fertile maintainer ARK8518 (rf ₂ rf ₂) and its isogenic heterozygous D8 restorer line, ARK8518R (Rf ₂ rf ₂) with D8 cytoplasm, by mRNA differential display (DD). Out of more than 3,000 DDRT-PCR bands amplified by 31 primer combinations from 12 anchor primers and 8 arbitrary decamer primers, approximately 100 bands were identified as being qualitatively differentially displayed. A total of 38 cDNA fragments including 12 preferentially expressed cDNA bands in anther were isolated, cloned and sequenced. Reverse northern blot analysis showed that only 4 genes, including genes encoding a Cys-3-His zinc finger protein and aminopeptidase, were up-regulated, while 22 genes, including genes for phosphoribosylanthranilate transferase (PAT), starch synthase (SS), 4-coumarate-CoA ligase, electron transporter, calnexin, arginine decarboxylase, and polyubiquitin, were down-regulated in the heterozygous restorer ARK8518R. The down-regulation of SS explains the lack of starch accumulation in sterile rf ₂ pollen grains in the heterozygous restored plants. The molecular mechanism of CMS and its restoration, specifically the possible roles of SS and PAT genes in relation to restoration of Rf ₂ to CMS-D8, are discussed. This investigation represents the first account of such an analysis in cotton.