The presence of specific nuclear genes mediates the restoration of male fertility to CMS plants. In T cytoplasm, this is accomplished by the combined action of Rf1 and Rf2a (Wise et al., 1999a). Rf1 mediates a series of mitochondrial transcript processing events, resulting in the accumulation of additional 1.6- and 0.6-kbp T-urf13 transcripts (Dewey et al., 1987; Wise et al., 1996) whereas Rf2a is an aldehyde dehydrogenase and is not involved in transcript processing (Cui et al., 1996; Liu et al., 2001). Two additional genes, Rf8 and Rf*, restore partial fertility when they, like Rf1, are combined with Rf2a (Dill et al., 1997). However, Rf8 and Rf* mediate the accumulation of additional 1.42- and 0.42-kpb transcripts as well as 1.4- and 0.4-kbp T-urf13 transcripts, respectively (Dill et al., 1997).
Of the five species with cloned restorer of fertility (Rf) genes, four encode pentatricopeptide repeat (PPR) proteins (Akagi et al., 2004; Bentolila et al., 2002; Klein et al., 2005; Koizuka et al., 2003). Pentatricopeptide repeat proteins are RNA binding proteins that are predominantly localized to mitochondria and chloroplasts with functions in editing, stabilization, and cleavage (Lurin et al., 2004; Small and Peeters, 2000). The cloned Rf genes of radish (Raphanus sativus L.), rice (Oryza sativa L.), and petunia [Petunia ×atkinsiana (Sweet) D. Don ex W. H. Baxter [= P. axillaris × P. integrifolia] (syn. Petunia hybrida L.)] are present in clusters of PPR-encoding genes with one functional gene and multiple pseudogenes suggesting recent gene duplication (Akagi et al., 2004; Bentolila et al., 2002; Brown et al., 2003; Desloire et al., 2003; Komori et al., 2003). Three of the restorer genes in sorghum [Sorghum bicolor (L.) Moench] reside in PPR clusters that share similarity to rice OsRf1 (Klein et al., 2005; Jordan et al., 2011). Because Rf1, Rf8, and Rf* are associated with the additional accumulation of T-urf13 transcripts, the possibility exists that they encode PPR proteins as well.
Analogous to T-urf13, the mitochondrial gene region encompassing orf355 and orf77 is responsible for male sterility in S-cytoplasm maize (cms-S) (Zabala et al., 1997). Likewise, cms-S utilizes the nuclear gene Rf3 to restore fertility to male-sterile plants. Rf3 cosegregates with a novel orf355 and orf77 transcript accumulation, suggesting an RNA editing function (Wen and Chase, 1999). The rf3 locus was mapped to maize chromosome 2L by Laughnan and Gabay (1978) by translocation and inversion heterozygotes. Kamps and Chase (1997) placed rf3 4.3 cM distal to restriction fragment length polymorphism (RFLP) whp1 and 6.4 cM proximal to RFLP bnl7.14. Shi et al. (1997) mapped rf3 4.8 cM distal to RFLP umc49 and 2.7 cM proximal to a random amplified polymorphic DNA (RAPD) marker, E08-1.2. Zhang et al. (2006) placed rf3 2.4 cM distal to a cleaved amplified polymorphic sequence (CAPS) marker and 1.8 cM proximal to a sequence characterized amplified region (SCAR) marker. Ultimately, Xu et al. (2009) observed cosegregation of Rf3-mediated fertility with three PPR-encoding genes on chromosome 2L in 900 segregating individuals.
While Rf1 and Rf2a have been genetically mapped to chromosomes 3 and 9, respectively (Schnable and Wise, 1994), the position of Rf8 has not yet been reported. To understand T cytoplasm in greater detail, the sequence and function of the rf8 locus needs to be elucidated. These experiments describe the high-resolution mapping of the rf8 locus to a 4.55-Mbp region on chromosome 2L, positioned within bacterial artificial chromosome contig 108 of MaizeSequence release 5b.60 (Ware et al., 2011). This region harbors 10 PPR-encoding genes, including candidates for the cms-S restorer, Rf3. Plants restored to partial fertility segregated independently of the Rf8-associated 1.42- and 0.42-kbp transcripts, suggesting the possibility of additional factors affecting pollen exsertion in the genome.
MATERIALS AND METHODS
Loci and recessive alleles are designated by lowercase symbols, and dominant alleles are designated by uppercase symbols; for example, the rf8 allele of the rf8 locus is recessive to the Rf8 allele. Lines that carry T cytoplasm (sterile or fertile) are referred to as T-cytoplasm lines. Male-sterile lines that carry T cytoplasm are designated cms-T. Restored T cytoplasm designates lines restored to fertility via the presence of nuclear restorer genes. Except in rare circumstances, normal (N) -cytoplasm lines are male fertile.
General Mapping Strategy
Rf8 and Rf1 have similar molecular phenotypes. Both genes are associated with additional accumulation of T-urf13 transcripts, and both are reported to restore at least some fertility to T-cytoplasm plants (Dill et al., 1997; Wise et al., 1996). Both genes are also associated with decreased accumulation of URF13 (Dewey et al., 1987; Dill et al., 1997). The Rf8-mediated URF13 reduction is less pronounced in ears than tassels whereas the Rf1-mediated reduction occurs equally in ears and tassels (Dill et al., 1997; Wise et al., 1987a). Because Rf8-associated anther exsertion is environmentally sensitive, the most reliable way to assay the plants for Rf8-associated T-urf13 accumulation is to determine if the 1.42- and 0.42-kbp transcripts are present via RNA gel blot analysis.
Development of Plant Material
Normal (N) W64A (rf1/rf1, Rf2/Rf2, rf8/rf8) and (N) wx1-m8 (rf1/rf1, Rf2/Rf2, Rf8/Rf8) were the two primary inbred lines used in this study. The original Rf8 allele, Rf8-8703, originated from plant number 90 8703 (rf1/rf1, Rf2/rf2, Rf8/rf8) (Schnable and Wise, 1994). It was identified by its sterile phenotype in a 1990 screen for rf2 mutants and was fertilized with pollen from our wx1-m8 stock (rf1/rf1, Rf2/Rf2, Rf8/rf8). Partially fertile plants (90 g 1138-5 and -6; Table 4 in Schnable and Wise, 1994) derived from this cross that were homozygous for Rf2 (rf1/rf1, Rf2/Rf2, Rf8/rf8) were self pollinated; subsequent progeny derived from these plants via self pollinations or outcrosses are designated as being derived from the 8703 pedigree (Dill et al., 1997). The Rf* allele originated from the rf1-m7212 pedigree in a screen for rf1 mutants wherein a novel 1.4-kbp T-urf13 transcript was identified as being mediated by Rf* (Fig. 5 in Wise et al., 1996; Fig. 3 and Table 6 in Dill et al., 1997).
As illustrated in Fig. 1, our initial population consisted of progeny derived from a single cross, (T) Rf8-8703/rf8-W64A × (N) rf8-W64A/rf8-W64A BC2, grown in the 1997 summer nursery at the Iowa State University Curtiss Research Farm in Ames, IA. One hundred seventeen segregating individuals were crossed by (N) rf8-W64A/rf8-W64A and second (unfertilized) ears were collected from each for DNA and RNA extractions. Ten plants from this 1997 population that possessed the T-urf13-derived 1.42- and 0.42-kbp transcripts were interpreted as harboring the Rf8 allele (genotype Rf8-8703/rf8-W64A), and thus crosses derived from them were selected to create the 2008 high-resolution BC3 population. This population was also grown at the Curtiss Research Farm in 2008. Young leaf tissue was collected from 1731 individuals for DNA extractions and fertility phenotypes were recorded from adult plants. Tissue for total RNA extractions was collected from immature second ears from 952 individuals and 1584 plants were crossed by (N) rf8-W64A/rf8-W64A.
Observation of Fertility Phenotypes (Anther Exsertion)
Male fertility was scored based on the four category system—S, sterile; “S,” partially fertile; “F,” mostly fertile; F, fertile—as described in Schnable and Wise (1994). Sterile indicates no anther exsertion, partially fertile indicates >0 but <50% of the anthers on the tassel exserted, mostly fertile indicates >50 but <100% of the anthers exserted, and fertile indicates 100% of the anthers exserted. The 2008 field was observed for fertility every day for 17 consecutive days, starting at the beginning of flowering (4 Aug. 2008) and ending 3 d after anther exsertion from the last plant (20 Aug. 2008).
Plants containing an Rf8 allele appear to lose fertility with increasing numbers of backcrosses to (N) W64A. To account for this observation, plants with successive amounts of backcrossing to (N) W64A and test crossing to (N) wx1-m8 were grown in 2009 at Curtiss Research Farm and are illustrated in Fig. 1. The origin of the wx1-m8 stock is described in detail in Wise and Schnable (1994). Sixty progeny each from five crosses, BC5TC1, BC4 (2008 cross only), BC6TC1, BC5TC2, and BC6TC2, were grown. Fertility observations and leaf tissue for DNA analysis were collected from all plants. A subset of plants was tested for fertility by crossing as males onto (T) W64A or (N) wx1-m8. Pollen was tested from each genotype that flowered and all plants tested produced kernels.
DNA Isolation and Analysis
For the 1997 mapping population, DNA was extracted using a 1 g hexadecyltri-methylammonium bromide (CTAB) extraction (Wise et al., 1996). One hundred seventeen individuals were subjected to bulk segregant analysis amplified fragment length polymorphism (BSA-AFLP) analysis as described by Wei et al. (1999).
For the 2008 mapping population, isolation of DNA was performed using a modified 96-well CTAB extraction (Dietrich et al., 2002). Polymerase chain reaction (PCR) primers were designed from the filtered gene set at MaizeSequence (Ware et al., 2011; Schnable et al., 2009) to amplify introns or 3′ untranslated regions (UTRs) from linked genes (see Table 1). Primers were designed to be codominant markers, CAPS markers, or size polymorphic markers. Polymerase chain reaction conditions were 3 min at 95°C, 30 sec at 95°C, 30 sec at melting temperature, 1.5 min at 72°C, 40 cycles, 10 min for 72°C, and then hold at 4°C.
|Marker||Located in gene||Marker type||Primer sequence (5′-3′)||Restriction enzyme||Tm|
|76755p2||GRMZM2G527387||Codominant||TGAAGAAATGGTGATGCGAGC / ACAGATGGCACTCCTGATGTGTC||–||56|
|135087p1||GRMZM2G135087||Size polymorphism||TGGAATACTTGCTTCTTGCTTGG / CAATGGTTATGCGTGAACGGG||–||56|
|144635p4||GRMZM2G144635||Codominant||TTGTGCTTGGGCTTTTCACG / CCTGACTTCCTGCTTTTGTATCGC||–||54|
|66902p1||GRMZM2G066902||Codominant||CGCTAACGCTTTCCTCTTGGAC / CTGTTCCCCATCCTTTCTACATC||–||56|
|csu811_p9||AC217293.3_FGT007||Codominant||CGAGGTCGAATCAAATTCTTCC / GTACGGGCGGTTAAAGAAAC||–||56|
|J04p6||GRMZM2G108171||Size polymorphism||CAAAGTCTCTGTCACTGTCACCTGG / TCTTCTTCCTCCTCCCTTGGAC||–||60|
|J04p8||GRMZM2G108171||Size polymorphism||TTAGTTGATTAGAGGAGGTTGCGG / GTCATTTAGCGTTTAGCGTCCAAG||–||60|
|18p4||GRMZM2G092284||CAPS marker||AAGATCATTCGGCGCGAGAA / CGGAGCCAAAACATGTGAAA||MseI||56|
|87p2||GRMZM2G035807||CAPS marker||ACATTGGTCTTTGTGGAGAC / TTCACACCCAACAGGTTGAC||DraI||56|
|10p2||GRMZM2G149935||CAPS marker||CGTAATGAAATGCGACGACG / CGTAGCCAGGTCCATTAGCA||MseI||56|
|26p3||GRMZM2G147819||CAPS marker||AGTTAAGGCTATCAGAATGA / ACTGACGATCAAATCTGATC||ApoI||56|
|umc36_p10||GRMZM2G059033||Codominant||CCTGGTGCACCATGTGATAGTTT / TTACCATGCCAATGGAATTG||–||56|
To efficiently screen the large 2008 mapping population, PCR primers were derived from the RFLP markers used in the 1997 mapping study. Overgo sequences were located at MaizeGDB (Lawrence et al., 2008) for the csu811 and umc36 RFLPs. These sequences were blasted against the maize genome using MaizeSequence release 5b.60 (Ware et al., 2011; Schnable et al., 2009). These overgos aligned to two genes on 2L (see Table 1). From these genes, PCR primers were designed to amplify interior portions of these genes.
RNA Isolation and Analysis
Total RNA was isolated from one gram of frozen second immature ear tissue via a Trizol-like reagent: 38% saturated phenol pH 4.3, 1 M guanidine thiocyanate, 1 M ammonium thiocyanate, 0.1 M sodium acetate pH 5.0, and 5% glycerol (Caldo et al., 2004). Eight micrograms of RNA were denatured with glyoxal (Ambion, Austin, TX) and size fractionated on a 1.8% SeaKem GTG agarose gel (FMC, Rockland, ME) with 0.01 M iodoacetic acid (Sigma, St. Louis, MO) for 14 h at 4°C. The gel and the circulating running buffer was 10 mmol Na2HPO4 pH 7.0. Ribonucleic acid was transferred to Hybond XL membranes (GE Healthcare/Amersham Biosciences, Piscataway, NJ) for 4 h using 20x saline-sodium citrate (SSC) (3 M NaCl and 0.3 M sodium citrate, pH 7.0) as a transfer buffer and crosslinked with 220 MJ of ultraviolet light emitted by 312 nm bulbs in a Stratalinker 2400 (Stratagene, La Jolla, CA) followed by baking at 80°C for 1 h. The fixed RNA was de-glyoxylated by treating the membrane in 20 mmol Tris-Cl pH 8.0 at 65°C for 30 min. The T-urf13 derived T-st308 DNA probe (Wise et al., 1996) was used for hybridization. Probe DNA was random primed with α-32P deoxycytidine triphosphate (dCTP) (PerkinElmer, Waltham, MA) (Feinberg and Vogelstein, 1983). Hybridization was performed for 18 h at 65°C in 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin, 1 mmol Na2 ethylenediaminetetraacetic acid (EDTA), and 0.5 M NaHPO4, pH 7.2 (Church and Gilbert, 1984). Membranes were incubated at 65°C in 1x saline sodium phosphate EDTA (SSPE), 0.1% SDS (20x SSPE contains 0.2 M monobasic sodium phosphate, 3.6 M NaCl, and 20 mmol EDTA, pH 7.4) for two 30 min washes followed by a 1 h wash. A more stringent wash in 0.1x SSPE, and 0.1% SDS was done for 15 to 20 min and membranes were exposed to CL-XPosure film (Thermo Scientific, Rockford, IL) for 1 to 10 d at −80°C using two Dupont Cronex Lightning Plus intensifying screens (Sigma, St. Louis, MO).
The rf8 Locus Maps to Maize Chromosome 2L
To position the gene encoding Rf8-associated T-urf13 transcript accumulation on the maize genetic map, DNA from 117 individuals from the 1997 mapping population were tested for Rf8-associated transcripts via RNA gel blot analyses. This information was utilized in the design of a BSA-AFLP strategy (see Methods). Two hundred fifty-six pairwise combinations of EcoRI and MseI primers were tested on the mapping parents, (T) Rf8-8703/rf8-W64A and (N) rf8-W64A/rf8-W64A, and two contrasting DNA pools, one representing 16 progeny displaying the T-urf13-derived 1.42- and 0.42-kbp transcripts and the other representing 16 progeny without the 1.42- and 0.42-kbp transcripts. Three hundred twenty-five polymorphisms were found with 20 conserved between the mapping parents. Of these, three polymorphic amplified fragment length polymorphisms (AFLPs) confirmed linkage to the locus mediating Rf8-associated T-urf13 transcript accumulation. Sequence tagged site markers were designed from these cloned AFLP fragments (Yu and Wise, 2000) and one, designated ias21, displayed a polymorphism between the T232 and CM37 parents of the Brookhaven mapping population (Burr et al., 1988). The ias21 forward and reverse primers, 5′-TGCCACACTTTATCTAAGGTT-3′ and 5′-TTGCTTTTGCGACAACGACGA-3′, respectively, corresponding to Arf8.3 (E-AGA and M-CTA) AFLP, were used to amplify a DNA fragment that cosegregated in the Brookhaven low-resolution mapping population with whp1 (white pollen 1) on 2L. Restriction fragment length polymorphism markers linked to whp1 were also tested and csu811 and umc36 cosegregated closely with Rf8.
Positioning the rf8 locus on the Maize Genome Sequence
Results derived from the 1997 BC2 mapping population indicated that the gene mediating the accumulation of the additional 1.42- and 0.42-kbp T-urf13 transcripts is closely linked to the csu811 and umc36 RFLPs near whp1 on 2L (Pei, 2000). To further characterize the rf8 locus and take advantage of the newly sequenced maize genome (Schnable et al., 2009), progeny from 10 1997 BC3 crosses were used to create a large 2008 BC3 mapping population. Restriction fragment length polymorphism markers csu811 and umc36 were converted into the PCR markers csu811_p9 and umc36_p10 by amplifying the gene associated with the RFLP (see Table 1).
Two hundred fifty-three primer pairs were designed to amplify 3′ UTRs and introns of genes in the region from whp1 to umc36. Amplicons derived from these primers were screened against (N) wx1-m8 and (N) W64A for size polymorphisms. Cleaved amplified polymorphic sequence markers were developed from the sequence of monomorphic amplicons of the parents and a small subset of segregants. This facilitated the identification of informative single nucleotide polymorphisms that differentiate the wx1-m8 and W64A parents present in the mapping population (Table 1). These CAPS and size-polymorphic markers were screened against the 2008 BC3 population, and RNA gel blot analysis was used to determine the accumulation of the 1.42- and 0.42-kbp T-urf13 transcripts for recombinant individuals in the region from whp1 to umc36 recombinant individuals (see Fig. 2 and Table 2). Based on these analyses, the rf8 locus (i.e., specifying the accumulation of the 1.42- and 0.42-kbp T-urf13 transcripts) resides between polymorphic markers 76755p2 and 135087p1, which corresponds to a 4.55-Mbp region within B73 contig 108 (Fig. 2 and 3; MaizeSequence release 5b.60 [Ware et al., 2011]).
|Interval||Number of plants tested||Confirmed informative recombinants||Maximum no. recombinants||Genetic distance||B73 physical size||kbp:cM ratio|
|76755p2 to 135087p1||616||31||71||8.25 ± 3.25||4.55 Mbp||552|
|76755p2 to Rf8||607||28||66||7.14 ± 3.13||–||–|
|Rf8 to 135087p1||702||2||50||3.70 ± 3.42||–||–|
|135087p1 to 144635p4||701||1||54||3.92 ± 3.78||0.81 Mbp||207|
|144635p4 to 66902p1||700||22||75||6.93 ± 3.79||1.99 Mbp||287|
|66902p1 to csu811_p9||1141||22||30||2.28 ± 0.35||0.85 Mbp||371|
|csu811_p9 to umc36_p10||1106||40||43||3.75 ± 0.14||1.00 Mbp||268|
Candidate Gene Analysis
As shown in Table 3, the rf8 flanking region between the 76755p2 and 135087p1 contains 4.55 Mbp and 146 genes from the B73 filtered gene set. This region also contains PPR-encoding genes that cosegregated perfectly with 900 segregating individuals in an Rf3 S-cytoplasm mapping population (Xu et al., 2009). There are a total of sixteen genes of interest in the 4.55 Mbp Rf8-flanking region (Table 3). Ten of these are PPR-encoding genes, one is a tetratricopeptide repeat (TPR)-encoding gene, four genes have only one 35 amino acid PPR repeat, and one is a pre-messenger RNA (mRNA) processing gene. The PPR-encoding genes fall into four subclusters on 2L (Fig. 2). Subcluster 1 spans 24 kbp and contains two genes: one PPR encoding gene and one TPR encoding gene. Subcluster 2 spans 232 kbp and harbors two PPR-encoding genes and one gene containing a single PPR. Subcluster 3 spans 123 kbp and contains five PPR-encoding genes and two genes containing a single PPR. Subcluster 4 spans 82 kbp and contains two PPR-encoding genes and one gene containing a single PPR. It is important to note that the architecture of the region is likely not the same for all inbred lines and genetic backgrounds. Thus, the possibility exists that the wx1-m8 background has a different structure for this PPR cluster.
|Gene||Predicted function||Sequence coordinates|
|GRMZM2G527387||Unknown and/or sodium symporter||223,510,475–223,522,044|
|GRMZM2G130379||Unknown and/or GPCR||223,587,853–223,589,462|
|GRMZM2G410567||GH3 auxin-responsive promoter||223,632,037–223,638,458|
|GRMZM2G322844||Natural resistance-associated macrophage protein||223,721,037–223,727,516|
|GRMZM2G151227||whp1 Thiolase and/or chalcone synthase||223,888,706–223,892,691|
|GRMZM2G166776||Unknown and/or antifreeze||224,064,456–224,065,350|
|GRMZM2G166718||Unknown and/or antifreeze||224,106,079–224,107,632|
|GRMZM2G414114||TCP transcription factor||224,523,636–224,527,847|
|GRMZM2G114948||Unknown and/or DUF247||224,536,161–224,537,608|
|GRMZM2G097896||Patatin and/or storage protein||224,734,195–224,741,296|
|GRMZM2G408768||14-3-3 protein binding domain||224,904,656–224,907,978|
|GRMZM2G057056||MAF and/or putative inhibitor of septum formation||225,019,032–225,020,646|
|GRMZM2G355940||Peptide chain release factors||225,023,880–225,033,407|
|GRMZM5G838671||Unknown and/or zinc finger domain||225,094,961–225,096,904|
|GRMZM2G090559||Unknown and/or ankyrin repeat||225,097,180–225,128,205|
|GRMZM2G099862||Unknown and/or DNA binding||225,159,420–225,162,831|
|GRMZM2G074496||Unknown and/or defense||225,227,431–225,230,275|
|GRMZM2G448692||Unknown and/or DUF724||225,318,983–225,321,945|
|GRMZM2G024641||AT hook-like and/or Tudor-like||225,364,989–225,366,192|
|GRMZM2G372058||Unknown and/or leucine rich repeat||225,781,716–225,784,625|
|GRMZM2G007028||Unknown and/or AT hook-like||225,825,290–225,833,496|
|GRMZM2G023652||Unknown and/or Xklp2-like||226,201,724–226,206,198|
|GRMZM2G106604||Unknown and/or DUF593||226,373,905–226,378,075|
|GRMZM2G150813||Unknown and/or zinc finger CCHC domain||226,422,679–226,426,851|
|GRMZM2G083095||Chaperone and/or tailless complex polypeptide||226,447,069–226,451,914|
|GRMZM2G053384||Pentatricopeptide repeat protein||226,676,462–226,678,841|
|GRMZM2G000936||Tetratricopeptide repeat and/or protein binding||226,699,977–226,705,433|
|GRMZM2G053713||Protein phosphatase 2C-like||226,963,057–227,123,377|
|GRMZM2G318412||Unknown and/or homeodomain-like||227,029,696–227,031,991|
|GRMZM2G450166||Pentatricopeptide repeat protein||227,045,894–227,046,871|
|AC196106.2_FG001||Unknown and/or C terminal domain of Paramyxovirinae RNA polymerase||227,173,374–227,175,299|
|GRMZM2G165173||Pentatricopeptide repeat protein||227,209,438–227,212,064|
|GRMZM2G165216||Unknown and/or kinase-like||227,213,495–227,214,494|
|GRMZM2G303463||Unknown and/or HLH DNA binding domain||227,264,500–227,268,560|
|GRMZM2G004521||Kelch motif and/or pentatricopeptide repeat||227,277,639–227,282,413|
|GRMZM2G145930||Unknown and/or kinase-like||227,516,080–227,537,665|
|GRMZM2G158308||Pentatricopeptide repeat protein||227,540,645–227,542,487|
|GRMZM2G158288||Nucleic acid-binding proteins and/or OB fold||227,546,551–227,551,614|
|GRMZM2G439814||Pentatricopeptide repeat protein||227,599,623–227,600,372|
|AC215723.3_FG001||Pentatricopeptide repeat protein||227,600,700–227,601,599|
|GRMZM2G453956||Pentatricopeptide repeat protein||227,604,054–227,608,803|
|GRMZM2G408216||Unknown and/or peptidase||227,610,560–227,618,077|
|GRMZM2G104286||Pentatricopeptide repeat protein||227,663,963–227,665,778|
|GRMZM2G416498||Pentatricopeptide repeat protein||227,876,866–227,880,456|
|GRMZM2G124602||Pentatricopeptide repeat protein||227,958,844–227,962,673|
|GRMZM2G124616||Unknown and/or peptidase-like||227,964,374–227,971,891|
|GRMZM2G135087||Unknown and/or DUF295 domain||228,064,519–228,066,636|
Six PPRs in this region belong to a clade of PPRs that encompass known Rf genes in plants (Akagi et al., 2004; Bentolila et al., 2002; Brown et al., 2003; Desloire et al., 2003; Klein et al., 2005). These genes are GRMZM2G450166, GRMZM2G158308, GRMZM2G439814, GRMZM2G453956, GRMZM2G416498, and GRMZM2G124602. However, GRMZM2G053384 is a PPR-encoding gene in this region that does not belong to the Rf clade (Fujii et al., 2011; I. Small, personal communication, 2010). In addition, GRMZM2G070831 is a pre-mRNA processing factor positioned 910 kbp proximal to the PPRs. Previously cloned Rf genes encode PPR proteins that alter RNA transcript accumulation (with the exception of maize Rf2a). Because Rf8 mediates T-urf13 transcript accumulation and is located in a PPR cluster, it is reasonable to speculate that Rf8 may also be a PPR-encoding gene.
Additional Restorer of Fertility Loci in the whp1 to umc36 Interval
As described above, T-cytoplasm plants segregating for Rf8 accumulate additional 1.42- and 0.42-kbp T-urf13 transcripts. Likewise, plants segregating for Rf1 accumulate additional 1.6- and 0.6-kbp transcripts (Wise et al., 1996) and those segregating for an additional Rf locus, Rf*, accumulate additional 1.40- and 0.40-kbp T-urf13 transcripts (Dill et al., 1997; Wise et al., 1999b). All three of these T-cytoplasm restorers share a small, conserved target sequence in the T-urf13 open reading frame yet independently control the modification of T-urf13 CMS-associated transcripts (Dill et al., 1997). Interestingly, this same target sequence is also highly conserved among sites for Rf*-mediated T-urf13 processing and the CMS-associated orf107 transcript accumulation regulated by sorghum Rf3 (Tang et al., 1996; Wise et al., 1999a).
Since we already identified Rf8-linked DNA markers, we further tested the relationship between Rf8 and the additional partial restorer, Rf*, by testing Rf8-linked markers on segregating progeny of an Rf* mapping population. A population of 88 progeny segregating for Rf* was established using the same procedure used to generate the Rf8 mapping population (see Methods) and the Rf8-linked 144635p4 PCR-based marker was found to be linked to the Rf* locus (Fig. 4).
Having established that Rf* was in the whp1 to umc36 interval on maize chromosome 2L, we performed an additional test to see whether we could directly identify recombinants between Rf8 and Rf*. The experiment was based on RNA blot analysis using two different probes that can differentiate the Rf8 and Rf* specific T-urf13 transcripts. Probe T-st308 hybridizes to the Rf8 specific transcripts (1.42- and 0.42 kbp) as well as the Rf*-specific transcripts (1.40- and 0.40 kbp). The oligo probe CD1721 only hybridizes to the Rf8-specific transcripts (Dill et al., 1997).
An individual heterozygous for Rf* (Rf*-7212/rf*-W64A) was used to pollinate a plant heterozygous for Rf8 (Rf8-8703/rf8-W64A) and a BC1 population was generated by pollination with (N) W64A (rf8/rf8, rf*/rf*). Two BC1 families derived from individuals carrying both Rf8 and Rf* (98 1233 and 98 1236) were identified from 24 planted families via RNA blot analysis in the 1998 summer nursery. A total of 53 individual plants from the two families (12 from 98 1233 and 41 from 98 1236) were analyzed via RNA blot analysis using probes T-st308 and CD1721 (Table 4).
|Number of progeny accumulating Rf8- and/or Rf*-mediated T-urf13 transcripts
||Number of progeny accumulating Rf8- or Rf*-mediated T-urf13 transcripts
|Assumptions||Rf8 or Rf*||rf8 or rf*||χ2||Rf8||Rf*||χ2|
|Independent||Expected (3:1)||39.7||13.3||17.6||Expected (1:1)||26.5||26.5||0.17|
|Allelic||Expected (1:0)||53||0||0||Expected (1:1)||26.5||26.5||0.17|
If Rf8 and Rf* are encoded by separate but linked open reading frames, then the BC1 families would be derived from an individual with the genotype Rf8-8703, Rf*-7212/rf8-W64A, rf*-W64A and any of four genotypes could arise (Rf8-8703, Rf*-7212/rf8-W64A, rf*-W64A; rf8-W64A, rf*-W64A/rf8-W64A, rf*-W64A; Rf8-8703, rf*-W64A/rf8-W64A, rf*-W64A; and rf8-W64A, Rf*-7212/rf8-W64A, rf*-W64A). If Rf8 and Rf* are alleles of one locus, the two BC1 families would be derived from an individual with the genotype Rf8-8703/Rf*-7212 and the expected segregation pattern would consist of two genotypes (Rf8-8703/rf-W64A and Rf*-7212/rf-W64A). As described above, plants carrying Rf8 and Rf* can be distinguished by hybridization of RNA gel blots with the T-st308 and CD1721 probes. When hybridized with T-st308, all 53 individuals displayed either the 1.42-kbp or the 1.40-kbp transcripts (χ21:0 = 0 < χ21, 0.05 = 3.84, p value > 0.05). Twenty-five of these individuals hybridized to the 1.42-kbp transcript when hybridized with the probe CD1721, confirming that these individuals contained Rf8. The remaining 28 individuals did not hybridize to the 1.42-kbp transcript when probed with CD1721; this result implies that these progeny do not harbor Rf8 and thus must contain Rf*.
Figure 5 illustrates the accumulation of T-urf13 transcripts in segregating progeny of BC1 families. Panel A shows the expected transcript accumulation detected by the T-st308 and CD1721 probes in different cms-T genotypes. Panel B illustrates the position of these probes on the T-urf13 sequence and the respective splice sites within T-urf13 mediated by each of the different Rf genes (from Dill et al., 1997). Panel C shows that the T-st308 probe hybridizes to all segregating BC1 progeny; thus, they all must carry one or both of the Rf8 or Rf* genes. Panel D illustrates the CD1721 probe hybridizes only to progeny containing the 1.42- and 0.42-kbp transcripts; implying they contain the Rf8 gene. Those individuals not displaying hybridizing transcripts to CD1721 are interpreted as containing Rf*. As shown in Table 4, no double recessive individuals (rf8-W64A/rf8-W64A, rf*-W64A/rf*-W64A, or rf8-W64A/rf*-W64A) were identified among 53 progeny, indicating that Rf8 and Rf* are either alleles of the same locus or tightly linked. To calculate the upper limit of recombination based on 53 progeny, we used the formula (1 − r)53 = α, where r represents the recombination ratio and α represents the type I error level. Based on this formula, the 95% confidence interval for recombination is (0, 0.055).
Transcript Accumulation and Fertility Phenotypes Do Not Cosegregate
Table 5 displays the fertility segregation of the 1997 and 2008 populations. The 2008 population exhibited 151 partially male-fertile plants and 1575 sterile plants while the 1997 population exhibited 44 partially male-fertile and 118 male sterile plants. Previously, Dill et al. (1997) observed that every partially male-fertile individual tested displayed the Rf8-associated transcripts while the sterile plants segregated for the presence of the transcripts. It was also observed that sterile plants exhibiting presence of the transcripts could produce fertile progeny in subsequent generations. Based on these observations, it was concluded that Rf8 is incompletely penetrant. Further complicating these conclusions was the empirical observation that fertility was environmentally sensitive; if the temperature was cool (24–28°C) during tassel development before anthesis, there was a high probability that plants with the potential for fertility would be fertile whereas if the temperature was hot (29–34°C) during the same period, plants with the identical genotype would be sterile (Dill et al., 1997). To further characterize these phenomena in the 2008 population, 126 partially male-fertile plants were genotyped using the tightly linked markers csu811_p9 and umc36_p10. Since it was thought that Rf8 was incompletely penetrant, it was expected that most or all partially male-fertile plants in the 2008 population would carry the dominant Rf8-8703 allele and would be heterozygous (Rf8-8703/rf8-W64A) for the tightly linked genotypic markers. However, the plants displayed a 1:1 segregation (χ21:1 = 0.008; p = 0.93) for these two markers—five were recombinant, 61 were heterozygous (Rf8-8703/rf8-W64A), and 60 were homozygous recessive (rf8-W64A/rf8-W64A).
|Progenitor fertility phenotype||Number of plants with the indicated phenotypes
|Progenitor||Progeny rows||S||“S”||Total no. of plants|
|95 3233-2||“S”||97 2219–97 2226||118||44||166|
|97 2220-11||“S”||08 7243–08 7247||167||14||183|
|97 2220-12||“S”||08 7248–08 7250 and 08 7301–08 7302||179||14||194|
|97 2220-17||S||08 7303–08 7307||117||7||125|
|97 2220-22||“S”||08 7308–08 7312||147||14||161|
|97 2221-3||S||08 7313–08 7316||129||32||163|
|97 2222-15||“S”||08 7327–08 7331||163||25||188|
|97 2222-3||S||08 7322–08 7326||162||12||174|
|97 2223-15||“S”||08 7332–08 7336||175||4||179|
|97 2224-3||“S”||08 7337–08 7341||164||12||176|
|97 2225-1||S||08 7342–08 7346||172||17||189|
Because a 1:1 genotypic segregation was not expected in the partially fertile plants, we designed an experiment to test the partially fertile and sterile plants for the Rf8-associated transcripts. One hundred eighty plants that were selected based on prior knowledge of their genotype score and fertility phenotype were assayed for presence of Rf8-associated 1.42- and 0.42-kbp T-urf13 transcripts. Table 6 and Fig. 6 show a lack of cosegregation of partially male-fertile and male-sterile plants with and without the Rf8-associated transcripts. Of the 44 partially male-fertile plants, 17 individuals did not contain the 1.42- and 0.42-kbp transcripts while 27 individuals displayed the 1.42- and 0.42-kbp transcripts. Of the 136 male-sterile plants, 48 did not contain the 1.42- and 0.42-kbp transcripts while 88 displayed the transcripts. The findings that partially male fertile individuals are segregating 1:1 for tightly linked markers and at least 17 of these partially male-fertile plants do not display the 1.42- and 0.42-kbp transcripts suggest that fertility restoration could be under the control of an additional unlinked locus. This opens the possibility that fertility and transcript accumulation could be uncoupled and therefore Rf8 would not be incompletely penetrant as postulated previously (Dill et al., 1997). These observations further imply that accumulation of Rf8-associated T-urf13 transcripts is not necessary or sufficient for fertility restoration.
|T-urf13 transcript accumulation||Number of partially male fertile plants||Number of male sterile plants||Total|
|1.42 and 0.42 kbp present||27||88||115|
|1.42 and 0.42 kbp absent||17||48||65|
Molecular Marker Genotypes in the rf8 Region Segregate as Expected but Fertility Phenotypes Do Not
Individual segregating progeny were assessed for anther exsertion and/or accumulation of the 1.42- and 0.42-kbp T-urf13 transcripts to determine the number of factors responsible for these phenotypes. To test the hypothesis that rf8 is a single locus, tightly linked genotypic markers should segregate 1:1 for heterozygous and homozygous recessive individuals in the Rf8 backcross population, respectively. Table 7 demonstrates that the p value for segregation of the tightly linked PCR marker 66902p1 was greater than 0.05 and therefore not significantly different from a 1:1. All 1997 progenitor plants of the 2008 population displayed the Rf8- associated 1.42- and 0.42-kbp T-urf13 transcripts and were heterozygous for RFLP markers csu811 and umc36. Progeny from one progenitor, 97 2220-22, did not segregate for 66902p1. This could be explained by a crossover occurring at meiosis in the 97 2220-22 plant between 144635p4 and 66902p1. The adjusted population total listed in Table 7 removes these progenies.
|Number of plants with the indicated 66902p1 score|
|Progenitor||Progeny rows||Heterozygous||Recessive||χ21:1||p value|
|97 2220-11||08 7243–08 7247||60||67||0.386||0.535|
|97 2220-12||08 7248–08 7250 and 08 7301–08 7302||33||41||0.865||0.352|
|97 2220-17||08 7303–08 7307||37||42||0.316||0.574|
|97 2220-22||08 7308–08 7312||0||121||121.000||0.000|
|97 2221-3||08 7313–08 7316||34||47||2.086||0.149|
|97 2222-15||08 7327–08 7331||54||71||2.312||0.128|
|97 2222-3||08 7322–08 7326||82||67||1.510||0.219|
|97 2223-15||08 7332–08 7336||61||67||0.281||0.596|
|97 2224-3||08 7337–08 7341||57||76||2.714||0.099|
|97 2225-1||08 7342–08 7346||43||51||0.681||0.409|
Dill et al. (1997) reported environmental sensitivity in Rf8 plants. Greater anther exsertion is observed at lower temperatures while less is observed at higher temperatures. Segregation for fertility categories in the 2008 mapping population is reported in Table 5. This population displayed a ratio of 1:10 (χ2 1:10 = 0.244; p = 0.62) partially fertile to sterile plants. This ratio does not align with the hypothesis of one, two, three, or four completely dominant independently assorting genes; however, flowering is a complex process and other phenomena are likely. A 1:10 ratio lies between a three-gene test cross (1:7) and a four-gene test cross (1:15). Linkage, additivity, epistatis, or incomplete dominance could be involved in skewing a standard ratio to 1:10.
Effect of Genetic Background on Rf8-Mediated Fertility
The observation was made from 1994 to 2000 that fertility of plants carrying Rf8 decreased with each successive generation backcrossed to (N) rf8-W64A/rf8-W64A. Starting in 2000, plants were crossed by (N) wx1-m8 in addition to (N) W64A (see Fig. 1) to test if the wx1-m8 background would increase fertility compared to (N) W64A. As shown in Table 8, five generations were observed for fertility in our 2009 summer Ames, IA, nursery. When backcrossed to (N) W64A, fertility decreased whereas crossing to (N) wx1-m8 increased fertility. A subset of the plants that displayed fertility, including the one partially male-fertile plant from the BC4 cross, were used as pollen donors and seed was obtained, demonstrating their fertility. The generations tested generally displayed the expected trend of increased fertility with greater amounts of (N) wx1-m8 in the pedigree as opposed to (N) W64A. This is suggestive of other factors involved in fertility present in the background of (N) wx1-m8 yet absent in the background of (N) W64A.
|Parental genotype||Pollen donor||Parental fertility phenotype||Parental transcript accumulation||2009 progeny rows||Number of plants with the indicated phenotypes
||Total no. of plants|
|08 7330-1 × 7319||BC4||(N) W64A||S||1.42 and 0.42 present||8132, 8136, 8147, and 8148||53||1||0||54|
|01 4136-4 × 4018||BC6TC1||(N) W64A||“S”||1.42 and 0.42 present||8123, 8126, 8130, and 8145||23||20||11||54|
|02 5230-21 × 5229-3||BC6TC2||(N) wx1-m8||“S”||1.42 and 0.42 present||8131, 8141, 8146, and 8150||8||18||22||48|
|00 3437-1 × 3446||BC5TC1||(N) wx1-m8||NA||1.42 and 0.42 present||8124, 8133, 8140, and 8142||7||23||17||47|
|01 4136-3 × 4149||BC5TC2||(N) wx1-m8||“S”||1.42 and 0.42 present||8135, 8137, 8139, and 8144||13||20||2||35|
Delay of Fertility in Partially Male-Fertile Plants
In addition to the genetic background affecting the amount of fertility in Rf8 plants, the timing of anther exsertion influences the degree of fertility observed. To test if mostly male-fertile plants flower earlier than partially male-fertile plants, five generations were grown and observed in 2009. All plants were observed daily for exsertion of anthers. Four generations, BC5TC1, BC5TC2, BC6TC1, and BC6TC2, segregated for three of the fertility categories: sterile, partially male fertile, and mostly male fertile. As shown in Table 9, the average days after planting to the first flowering (DAPFF) was calculated for all cross types, with DAPFF defined as the first day an exserted plump yellow anther was observed. A two-tailed paired t test was used to calculate significance between flowering time of partially fertile and mostly fertile plants. Within a given genotype, all mostly male-fertile plants showed a significantly earlier DAPFF of flowering than the partially male-fertile plants. Flowering in 2009 was delayed by 3.7 to 2.4 d depending on the genotype. These results suggest that there may be other factors responsible for flowering time segregating in the partially fertile and mostly fertile plants.
|Genotype||Phenotype||Average DAPFF||p value|
rf8 is Positioned in a 4.55-Mbp Region on Maize 2L
Cytoplasmic male sterility systems have been established as models for studying nuclear-cytoplasmic interactions. This is because restoration of fertility depends on nuclear-encoded gene products to overcome mitochondrial dysfunction. Genetic and physical mapping of Rf loci with easily assayable molecular markers is a step toward the physiological understanding of fertility restoration because individual factors can then be tracked in the progeny of various crosses. An important factor to the success of this genetic mapping was the use of the T-urf13 transcript phenotype as opposed to the fertility phenotype. This allowed us to observe the uncoupling of the fertility from the transcript accumulation pattern. These experiments demonstrate that gene mediating the Rf8-associated T-urf13 accumulation pattern is located in the 4.55-Mbp region on 2L between PCR markers 76755p2 and 135087p1, corresponding to contig 108 in MaizeSequence release 5b.60 (Ware et al., 2011).
Dill et al. (1997) reported that Rf8 was incompletely penetrant based on fertility data and transcript accumulation of 79 individuals. All partially male-fertile plants in that particular study accumulated the 1.42- and 0.42-kbp T-urf13 transcripts. However, the results reported here demonstrate that partially male-fertile individuals in the 2008 mapping population segregate 1:1 for tightly linked genotypic markers and 17 of these did not display the Rf8-associated transcripts. Thus, the current results are not congruent with the interpretation of incomplete penetrance reported by Dill et al. (1997). The locus designated Rf8 on maize chromosome 2L appears to control transcript accumulation, while fertility appears to be at least partially controlled by additional factors. Given the existence of partially male–fertile individuals without the 1.42- and 0.42-kbp transcripts and male-sterile individuals with the 1.42- and 0.42-kbp transcripts, transcript presence does not appear to be necessary or sufficient for fertility restoration. This could indicate the presence of at least one other factor in the genome responsible for partial fertility restoration. One could postulate that other RF2-like aldehyde dehydrogenase (ALDH) proteins affect fertility; however, the Rf2a allele was fixed and homozygous in these populations. Nevertheless, there are other segregating Rf2 family members, that is, Rf2b, Rf2c, and Rf2d, encoding functional ALDH proteins (Skibbe et al., 2002), which may be contributing to anther exsertion.
To test the hypothesis that genetic background affects fertility, five generations of Rf8 plants were grown in 2009. The fertility observations suggest a difference in the backgrounds of W64A and wx1-m8 because plants reintroduced with wx1-m8 displayed greater fertility. This could be interpreted as wx1-m8 harboring other unlinked genes favorable to fertility that W64A does not possess. To test for differences between partially fertile and mostly fertile plants, the DAPFF was recorded. This demonstrated that partially fertile plants flower significantly later than mostly fertile plants. This suggests the presence of other factors segregating for the timing of flowering.
Clusters of Linked Restorer of Fertility Genes are Conserved across Plant Taxa
Fine mapping of ZmRf8 (T cytoplasm) as well as ZmRf3 (S cytoplasm) suggest that both genes map to the same cluster of PPR genes on 2L. As illustrated in Table 10, the phenomenon of linked restorer genes is not unique to maize. Rice contains a PPR cluster spanning 450 kbp on chromosome 10L that contains six Rf genes that restore four different cytoplasms (Tan et al., 2011). OsRf4 and OsqRf-10-2 restore wild abortive (WA) and dwarf abortive (DA) cytoplasm, respectively, which are characterized by sporophytic restoration (Xie et al., 2002; Yao et al., 1997). OsRf1a and OsRf1b restore Boro II (BT) cytoplasm, whereas OsRf5 and OsRf6 restore Honglian (HL) cytoplasm (Akagi et al., 2004; Komori et al., 2004; Liu et al., 2004; Wang et al., 2006). Both BT and HL cytoplasms are characterized by gametophytic restoration. In this way, the rice 10L locus is analogous to the maize 2L locus. Both species contain linked Rf genes capable of restoring cytoplasms with different modes of restoration. Similar to rice and maize, cotton (Gossypium hirsutum L.) has two linked Rf genes, GhRf1 and GhRf2. These genes restore two different cytoplasms that also utilize different modes of restoration: D2 cytoplasm uses sporophytic restoration by GhRf1 whereas D8 cytoplasm uses gametophytic restoration via GhRf2 (Meyer, 1975; Zhang and Stewart, 2001). Two studies have mapped these genes within 1 cM of each other (Wang et al., 2007, 2009). Likewise, common bean (Phaseolus vulgaris L.) contains two linked fertility restorer genes, PvFr and PvFr2, on linkage group K (He et al., 1995; Jia et al., 1997). Sorghum, a close relative of maize, contains PPR clusters around its unlinked Rf genes. SbRf1, SbRf2, and SbRf5 reside in a cluster of five, four, and seven PPR-encoding genes, respectively (Jordan et al., 2011). The PPR-encoding genes within these clusters display high similarity, and the clusters around SbRf2 and SbRf5 show high similarity to rice OsRf1 (Jordan et al., 2011). Thus, precedent exists for linked Rf genes to exist in one PPR cluster capable of restoring multiple cytoplasms characterized by various modes of restoration.
|Cytoplasm||Mode of restoration||Restorer genes||Gene location||Reference|
|Oryza sativa L. (rice)||WA||Sporophytic||OsRf4||10L||Yao et al., 1997|
|DA||Sporophytic||Osq-Rf-10-2||10L||Xie et al., 2002|
|BT||Gametophytic||OsRf1a, OsRf1b||10L||Akagi et al., 2004; Komori et al., 2004; Wang et al., 2006|
|HL||Gametophytic||OsRf5, OsRf6||10L||Liu et al., 2004|
|Zea mays L. (maize)||T||Sporophytic||ZmRf1||3||Schnable and Wise, 1994|
|ZmRf2||9S||Schnable and Wise, 1994|
|ZmRf8, ZmRf*||2L||Pei, 2000|
|S||Gametophytic||ZmRf3||2L||Kamps and Chase, 1997; Xu et al., 2009|
|Gossypium hirsutum L. (cotton)||D2||Sporophytic||GhRf1||D5||Meyer, 1975; Wang et al., 2007; Wang et al., 2009;|
|D8||Gametophytic||GhRf2||D5||Zhang and Stewart, 2001|
|Sorghum bicolor (L.) Moench (sorghum)||A1||Sporophytic||SbRf1||SBI-08L||Klein et al., 2005|
|SbRf2||SBI-02||Jordan et al., 2010|
|SbRf5||SBI-05||Jordan et al., 2011|
|Brassica napus L. (canola)||Ogura||Sporophytic||BnRfo||CN19||Brown et al., 2003; Feng et al., 2009|
|Petunia ×atkinsiana (Sweet) D. Don ex W. H. Baxter (petunia)||RM||Gametophytic||Phrf-PPR592||4||Bentolila et al., 2002; Bentolila et al., 1998|
|Phaseolus vulgaris L. (bean)||Sprite||NA||PvFr, PvFr2||K||He et al., 1995; Jia et al., 1997|
|Helianthus annuus L. (sunflower)||PET1||Sporophytic||HaRf1||13||Yue et al., 2010|
Canola (Brassica napus L.) and petunia contain Rf genes present in PPR clusters containing only one known Rf gene and multiple pseudogenes. BnRfo of canola is located in a cluster with two other PPR-encoding genes (Brown et al., 2003; Feng et al., 2009). Phrf-PPR592 of petunia is adjacent to another PPR gene, PhPPR591 (Bentolila et al., 2002; Bentolila et al., 1998). The nonrestoring allele of Phrf-PPR592 contains a promoter deletion and most likely a recombination event involving similar PPR genes (Bentolila et al., 2002). Currently, it is unknown if sunflower (Helianthus annuus L.) contains linked Rf genes residing in a PPR cluster. HaRf1 restores PET1 cytoplasm in sunflower; however, the landscape of this fertility locus needs to be elucidated (Yue et al., 2010).
The mapping of ZmRf8 in this study places it in or near a PPR cluster on 2L, tightly linked to PCR marker 135087p1 (Fig. 2 and Table 3). Mapping of ZmRf3 for cms-S positions ZmRf3 4.3 cM distal to whp1 (Kamps and Chase, 1997). Further investigations of the whp1 region revealed a cluster of rice OsRf1-orthologus PPR genes in B73 (Xu et al., 2009). This is the same PPR cluster to which ZmRf8 maps. It is therefore reasonable to postulate that ZmRf3 and ZmRf8 reside in the same cluster of PPR-encoding genes.
The molecular phenotype of ZmRf8 is the accumulation of the additional 1.42- and 0.42-kbp T-urf13 transcripts. Pentatricopeptide repeat proteins mediate organelle promoter recognition and RNA editing and translation, processes that could alter patterns of transcript accumulation. For example, maize PPR10 defines both 5′ and 3′ transcript termini simply by site-specific RNA binding and thus does not mediate RNA processing directly (Pfalz et al., 2009; Prikryl et al., 2011). Thus, PPR-encoding genes are the most promising candidates for Rf8, based on previous cloned Rf genes and the pattern of T-urf13 transcript accumulation.
Fujii et al. (2011) identified Rf-like PPR genes in many species. There are five Rf-like-identified genes in the maize B73 genome. Finer mapping in the region containing the PPR-encoding genes could elucidate whether ZmRf3 and ZmRf8 map to the same Rf-like PPR encoding gene. If ZmRf3 and ZmRf8 are alleles, this would be one of the first Rf genes with alleles capable of restoring two different types of cytoplasm with different modes of restoration. Even if they are not alleles, these loci will provide insight into the evolution of CMS and Rf systems. Clearly, this complex locus is a hotspot for fertility restoration.