Transcriptomic and mitochondrial analysis of cytoplasmic male sterility and fertility restoration in cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants, resulting in failure to produce functional pollen. The CMS system is widely used in crop breeding to produce F1 hybrid crops to utilize heterosis and is also a valuable avenue to study cytoplasmic and nuclear gene interactions. CMS is caused by the dysfunction of mitochondrial DNA genes encoding for ATP synthase subunits (atp) or cytochrome oxidases (cox) in many plants and RNA editing is also implicated in some CMS systems. CMS-associated genes are located in the mitochondrial (mt) genome and present adjacent to functional mitochondrial genes as novel chimeric genes generated due to mt genome rearrangements. Male fertility in CMS plants can be recovered by nucleus-encoded fertility restorer gene(s) and the communication between mitochondrial and nucleus genomes, termed retrograde/anterograde signaling, plays an important role in pollen fertility. The CMS-D8 and restoration ( Rf2) system was developed by introducing the exotic cytoplasm and the restorer gene into the cultivated tetraploid Upland cotton (AD1), G. hirsutum from the wild diploid cotton species (D8), G. trilobum. However, the molecular mechanism of CMS in cotton is currently unknown. The objectives of the present study were to identify differentially expressed (DE) genes associated with fertility restoration, to study the relationship between RNA editing in mtDNA genes and CMS, and to develop mtDNA gene-based markers for the CMS-D8 system. Part 1: Through a comparative microarray analysis followed by quantitative RT-PCR between a fertile restorer line and a sterile CMS line in the same genetic background, 458 DE genes (of 24,132) with >2-fold changes in flower buds were identified, 127 of which were up-regulated and 331 of which were down-regulated in the CMS line. The most frequent DE genes encoded putative proteins involved in cell wall expansion and cytoskeleton, such as pectinesterase, pectate lyase, pectin methylesterase, glyoxal oxidase, polygalacturonase, indole-3-acetic acid-amino synthetase, xyloglucan endo-transglycosylase and actin. The results provide evidence that cell wall expansion is involved in male fertility of CMS-D8. Part 2: Through a comparative sequencing analysis of PCR and RT-PCR products in a CMS-D8, its maintainer and restorer line, a total of 67 RNA editing sites in coding sequences and 1 in 3' untranslated region (UTR) were detected for eight mtDNA genes atp1, 4, 6, 8 and 9, and cox1, 2 and 3. The full length of atp6 and cox2 genes was cloned and sequenced in cotton for the first time. Twelve editing efficiency differences between CMS-D8 and its restorer line in all sequenced mitochondrial genes except for atp1 were found, suggesting that the fertility restorer gene may modulate the RNA editing efficiency. However, the correlation between RNA editing and CMS is weak even though cytoplasm may affect RNA editing efficiency. Part 3: A comparative sequencing analysis resulted in the identification of single nucleotide polymorphisms (SNPs) between AD1 and CMS-D8 cytoplasm in the above eight mtDNA genes except for atp9. An allelic specific PCR (AS-PCR) was then utilized for SNP typing by incorporating artificial mismatched nucleotides into the third or fourth bases from the 3' terminus in both the specific and nonspecific primers. The method modifying allele specific primers obtained seven polymorphic SNP markers using seven primer pairs designed from the seven mtDNA genes to discriminate two alleles between AD1 and CMS-D8 cytoplasm. Additionally, a PCR-based marker for a nine nucleotide insertion-deletion (InDel) sequence (AATTGTTTT) at the 59 to 67 bp positions from the start codon of atp6, which is present in the CMS and restorer lines with the D8 cytoplasm but absent in the maintainer line with the AD1 cytoplasm, was also developed. These PCR-based SNP markers will be useful in discriminating CMS-D8 and AD1 cytoplasms as a rapid, simple, inexpensive, and reliable genotyping tool to assist hybrid cotton breeding.