Molecular characterization of cytoplasmic male sterility conditioned by Gossypium harknessii cytoplasm (CMS-D2) in upland cotton

Abstract

Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and to develop CMSspecific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed that the three genes were differentially expressed between the two lines. These results indicated that there existed structural and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from ATG codon were also changed between the two cytoplasms (i.e., CG?AA), and this mutation also exists in RNA sequences. Interestingly, nine nucleotides (ATGCAACTA) were also inserted at the location of 899 bp upstream of ATG codon in the CMS line. The results suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either normal-or CMS-D2cytoplasm. This will allow quick and reliable Jianyong Wu and Yangcang Gong contributed equally to this work.Electronic supplementary material The online version of this article (

A genome-wide identification and analysis of the DYW-deaminase genes in the pentatricopeptide repeat gene family in cotton (Gossypium spp.)

“…However, little is known about the RNA editing events of mitochondrial genes in CMS-D2. Wu et al (2011) found a C-U and a C-G editing site at position 1178 and 1214 bp downstream of the ATG codon in atpA and a A-U and a C-U editing site at position 18 and 35 bp upstream of ATG codon in nad6 between maintainer and CMS-D2 lines, respectively [16]. Thus, we speculate that RNA editing events in D2 mitochondrial genes may regulate ATP synthase activity, and the impaired ATP synthesis results in CMS occurrence.…”

“…Cytoplasm male sterility (CMS-D2) A S(rf1rf1) with cytoplasm from Gossypium harknessii and its isogenic restorer line R S(Rf1rf1) were planted under normal conditions in the farm. Floral buds about 3 mm in length (nearly represent the stage of male meiosis) were collected from the near isogenic line with three biological replicates as previous studies, respectively [16, 17]. All collected samples were immediately frozen in liquid nitrogen and stored at -76°C before use.…”

Mitochondrial SCAR and SSR Markers for distinguishing cytoplasmic male sterile lines from their isogenic maintainer lines in cotton

“…Meanwhile, the analysis on CMS‐D2 and CMS‐D8 did confirm the polymorphism for atpA (Wang et al. 2010, Wu et al. 2011).…”

“…2011). Wu et al. (2011) found that two nucleotides at the −4 and −5 position upstream ATG codon of the atpA gene were different.…”

“…1999, Wang 2000), AFLP (Zhu et al. 2001, Wang and Li 2002) and SCAR (Wu et al. 2011) markers were used to distinguish N‐cytoplasm from sterile CMS‐D2 cytoplasm in cotton.…”