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.