Figure 1.
Figure 1.

Pedigree of Rf8. Each box represents a cross. Plants were either backcrossed to (normal [N]) W64A (rf1/rf1, Rf2/Rf2, rf8/rf8) or test crossed to (N) wx1-m8 (rf1/rf1, Rf2/Rf2, Rf8/Rf8). Transcripts associated with Rf8 were identified from the 1994 generation. The 1997 mapping population is a BC3 generated from one cross. The 2008 mapping population is a BC4 generation and is generated from 10 1997 individuals possessing the Rf8-associated 1.42-kbp transcripts. Plants grown for fertility observation in 2009 are progeny from crosses BC6, BC5TC1, BC4 (2008 cross only), BC6TC1, BC5TC2, BC7TC1, and BC6TC2. T, Texas.

 


Figure 2.
Figure 2.

wx1-m8 × W64A genetic map, B73 physical map, and candidate genes in the flanking region. Rf8 is located between polymerase chain reaction markers 76755p2 and 135087p1 on chromosome 2L corresponding to contig 108. Interval distances in the genetic map are in centimorgans and interval distances in the physical maps are in megabase pairs. Positions of primers and names of genes are taken from MaizeSequence release 5b.60 (Ware et al., 2011; Schnable et al., 2009). The flanking region contains 10 pentatricopeptide repeat genes (designated ‡) and one pre-messenger RNA (mRNA) processing gene (designated †).

 


Figure 3.
Figure 3.

Polymerase chain reaction (PCR) markers illustrating the Rf8 flanking region. Panel A contains marker 76755p2, panel B contains 135087p1, and panel C contains 144635p4. Lanes 1 and 2 display the polymorphism between the mapping parents of the 2008 mapping population. Lanes 3 through 14 represent a subset of the segregating 2008 progeny in order of recombination breakpoint. Lanes 3, 4, and 9 through 12 demonstrate the absence of the Rf8-associated 1.42-kbp transcript while lanes 5 through 8, 13, and 14 display the presence of the 1.42-kbp transcript. Lanes 4 and 13 display a recombination breakpoint on the distal side of the 1.42-kbp transcript while lanes 5 through 12 display a recombination breakpoint on the proximal side of the 1.42-kbp transcript. N, normal; T, Texas.

 


Figure 4.
Figure 4.

Polymerase chain reaction (PCR) marker 144635p2 illustrating linkage to the Rf* locus. Inbred lines (normal [N]) wx1-m8 and (N) W64A and 17 progeny from the Rf* mapping population were tested for PCR marker 144635p4. The presence or absence of the 1.42- and 0.42-kbp Rf*-associated T-urf13 transcript, as detected by probe T-st308 is given. T, Texas.

 


Figure 5.
Figure 5.

Analysis of progeny from BC1 families segregating for Rf8 and Rf*. Panel A shows the expected transcript accumulation detected by the T-st308 probe, and the CD1721 26nt oligo probe for all Texas (T)-cytoplasm maize (cms-T), Rf1, Rf8, and Rf*, respectively. Panel B shows the position of the probes on the T-urf13 sequence and the respective splice sites mediated by each of the different restorer of fertility (Rf) genes (vertical arrowheads designated as nucleotides from the T-urf13 AUG; from Dill et al., 1997). Panel C illustrates that the T-st308 probe hybridizes to both the 1.42- and 1.40-kbp transcripts in backcross progeny that carry Rf8 or Rf*, respectively, whereas panel D shows that the CD1721 probe only hybridizes to the 1.42-kbp transcript in progeny that carry Rf8 and not the 1.40-kbp transcript mediated by Rf*, thus differentiating the two types of progeny.

 


Figure 6.
Figure 6.

Ribonucleic acid (RNA) gel blot of partially male-fertile and -sterile plants showing segregation between fertility and the Rf8-associated 1.42-kbp transcript. The probe is st308, a portion of the T-urf13 gene (Wise et al., 1996). No hybridizing bands are expected on the RNA blot in lanes 1 and 2 because the probe is specific to Texas (T) cytoplasm while the samples are normal (N) cytoplasm. Lane 3 contains the Rf1 control illustrating the 1.6-kbp transcript. Lanes 5 through 14 are partially male fertile with lanes 5 through 9 displaying the 1.42-kbp transcript. Lanes 10 through 14 illustrate the absence of the transcript. Lanes 15 through 23 are male sterile with lanes 15 through 19 demonstrating the 1.42-kbp transcript. Lanes 20 through 23 display the absence of the transcript.