Recombination Events Involving the atp9 Gene Are Associated with Male Sterility of CMS PET2 in Sunflower

Abstract

Cytoplasmic male sterility (CMS) systems represent ideal mutants to study the role of mitochondria in pollen development. In sunflower, CMS PET2 also has the potential to become an alternative CMS source for commercial sunflower hybrid breeding. CMS PET2 originates from an interspecific cross of H. petiolaris and H. annuus as CMS PET1, but results in a different CMS mechanism. Southern analyses revealed differences for atp6, atp9 and cob between CMS PET2, CMS PET1 and the male-fertile line HA89. A second identical copy of atp6 was present on an additional CMS PET2-specific fragment. In addition, the atp9 gene was duplicated. However, this duplication was followed by an insertion of 271 bp of unknown origin in the 5' coding region of the atp9 gene in CMS PET2, which led to the creation of two unique open reading frames orf288 and orf231. The first 53 bp of orf288 are identical to the 5' end of atp9. Orf231 consists apart from the first 3 bp, being part of the 271-bp-insertion, of the last 228 bp of atp9. These CMS PET2-specific orfs are co-transcribed. All 11 editing sites of the atp9 gene present in orf231 are fully edited. The anther-specific reduction of the co-transcript in fertility-restored hybrids supports the involvement in male-sterility based on CMS PET2.

Keywords: CMS PET1; CMS PET2; Helianthus annuus; RNA-editing; atp9; cytoplasmic male sterility; plant mitochondria; recombination; respiration.

Figure 1

Inflorescence in the present of the CMS PET2 in a maintainer and restorer background. (a) CMS PET2 × RHA265, male sterile; (b) CMS PET2 × IH-51, fertility-restored hybrid.

Figure 2

Southern hybridization pattern of mtDNA digested with HindIII using the mitochondrial genes atp6, atp9 and cob as probes. Lanes: 1, CMS PET2 (male sterile); 2, CMS PET1; 3, HA89 (fertile, normal cytoplasm).

Figure 3

Comparison of the organization of the open reading frames on the hybridization fragments obtained by using atp6 as probe in the normal, fertile HA89 and CMS PET2.

Figure 4

Comparison of the organization of the open reading frames on the hybridization fragments obtained by using atp9 as probe in the normal, fertile HA89 and CMS PET2.

Figure 5

Comparison of the organization of the open reading frames on the hybridization fragments obtained by using cob as probe in the normal, fertile HA89 and CMS PET2. (a) 5′ cob fragment (7.3 kb) of fertile cytoplasm and CMS PET2, (b) 3′ cob fragment for the fertile cytoplasm (3.9 kb) and CMS PET2 (5.5 kb).

Figure 6

Model for the creation of the CMS-PET2 specific orf288 and orf231 by an insertion event of 271 bp into the duplicated atp9 gene. (a) Scheme; (b) Amino acid sequences of orf288 and orf231 (edited). Transmembrane domains as predicted by TMHMM are marked by red bars.

Figure 7

RT-PCR analysis of CMS PET2, the fertility-restored hybrid and the male-fertile line HA89 (a) PCR amplification products using primer specific for atp9 (orf300), cob (orf1194), atp6 (orf1056) and 18S rRNA. Expected fragment sizes are given in brackets. Lanes: 1, CMS PET2 (male sterile); 2, PET2 × IH-51 (fertile F1-hybrid); 3, HA89 (fertile, normal cytoplasm); M, 100 bp marker; (b) Quantification of the co-transcript expression level (552 bp) in CMS PET2 and the fertility-restored hybrid in leaves, disk florets and anthers by densitometry. Dark grey: CMS PET2, light grey: fertility-restored hybrid (CMS PET2 × IH-51).

Figure 8

Immuno-blot using complete affinity-purified peptide Anti-ORF288 polyclonal antibody against 10 µg of isolated mitochondria from CMS PET2 and the fertile line HA89, ORF288 recombinant protein and protein plant extracts of CMS PET2 and HA89. Lanes: 1, CMS PET2 mitochondrial extracts; 2, HA89 mitochondrial extracts; 3, recombinant ORF288; 4, CMS PET2 whole plant protein extracts; 5, HA89 whole plant protein extracts; M, prestained protein ladder.

Figure 9

Development of markers specific for CMS PET1, CMS PET2 and the fertile, normal cytoplasm in sunflower. (a) Marker HRO_ATP9-PET2 using primer combination orf300_for/orf300_rev; (b) Marker HRO_PET1 using primers orfH522_for/orfH522_rev; (c) Marker HRO_ATP1-PET1 using primer combination M_atp1_for/M_atp1_rev/orfH522_rev; (d) Marker HRO_ATP1 using primer combination M_atp1_for/M_atp1_rev; Lanes: 1, HA342 (male fertile, normal cytoplasm); 2, CMS PET1 × HA342 (male sterile); 3, CMS PET2 × IH51 (fertility restored F1-hybrid); 4, CMS PET2 × RHA265 (male sterile); 5, RHA265 (restorer line, male fertile, normal cytoplasm); 6, PCR negative control; M, 100 bp marker.