Mitochondrial aldehyde dehydrogenase activity is required for male fertility in maize

Some plant cytoplasms express novel mitochondrial genes that cause male sterility. Nuclear genes that disrupt the accumulation of the corresponding mitochondrial gene products can restore fertility to such plants. The Texas (T) cytoplasm mitochondrial genome of maize expresses a novel protein, URF13, which is necessary for T cytoplasm-induced male sterility. Working in concert, functional alleles of two nuclear genes, rf1 and rf2, can restore fertility to T cytoplasm plants. Rf1 alleles, but not Rf2 alleles, reduce the accumulation of URF13. Hence, Rf2 differs from typical nuclear restorers in that it does not alter the accumulation of the mitochondrial protein necessary for T cytoplasm-induced male sterility. This study established that the rf2 gene encodes a soluble protein that accumulates in the mitochondrial matrix. Three independent lines of evidence establish that the RF2 protein is an aldehyde dehydrogenase (ALDH). The finding that T cytoplasm plants that are homozygous for the rf2-R213 allele are male sterile but accumulate normal amounts of RF2 protein that lacks normal mitochondrial (mt) ALDH activity provides strong evidence that rf2-encoded mtALDH activity is required to restore male fertility to T cytoplasm maize. Detailed genetic analyses have established that the rf2 gene also is required for anther development in normal cytoplasm maize. Hence, it appears that the rf2 gene was recruited recently to function as a nuclear restorer. ALDHs typically have very broad substrate specificities. Indeed, the RF2 protein is capable of oxidizing at least three aldehydes. Hence, the specific metabolic pathway(s) within which the rf2-encoded mtALDH acts remains to be discovered.     

Isolation and Analysis of Rice Rf1-Orthologus PPR Genes Co-segregating with <Emphasis Type="Italic">Rf3</Emphasis> in Maize

Using an in silico cloning approach, five putative maize pentatricopeptide repeat (PPR)-containing protein genes (PPR-814a, PPR-814b, PPR-814c, PPR-816, PPR-817) with complete open reading frames were identified in the inbred line S-Mo17^Rf3Rf3. The amino acid sequence indicated that these genes encoded mitochondrially targeted proteins containing repeats of a 35-aa PPR motif. The genes were mapped into the interval umc1525–bnlg1520 on chromosome 2. In a non-restoring genotype, we identified three homologous genes that contained deletions or nucleotide substitutions in the coding region. Sequence analysis revealed that one of the three genes (PPR-814a, PPR-814b, PPR-814c) could be considered a candidate restorer gene for S male sterility cytoplasm, and linkage analysis demonstrated that the genes co-segregated with the fertility restorer gene Rf3.     

Recovery of Heritable, Transposon-Induced, Mutant Alleles of the Rf2 Nuclear Restorer of T-Cytoplasm Maize

T (Texas) cytoplasm is associated with a mitochondrial disruption that is phenotypically expressed during microsporogenesis resulting in male sterility. Restoration of pollen fertility in T-cytoplasm maize is controlled by dominant alleles at two unlinked, complementary, nuclear-encoded genes, rf1 and rf2. As a first step in the molecular isolation of the rf2 gene, 178,300 gametes derived from plants that carried the Mutator, Cy or Spm transposon families were screened for rf2 mutant alleles (rf2-m) via their inability to restore pollen fertility to T-cytoplasm male-sterile maize. Seven heritable rf2-m alleles were recovered from these transposon populations. Pedigrees and restriction fragment length polymorphism (RFLP)-based analyses indicated that all seven rf2-m alleles were derived independently. The ability to obtain rf2-m derivatives from Rf2 suggests that Rf2 alleles produce a functional product necessary to restore pollen fertility to cmsT. Molecular markers flanking the rf1 and rf2 loci were used to decipher segregation patterns in progenies segregating for the rf2-m alleles. These analyses provided preliminary evidence of a weak, third restorer gene of cmsT that can substitute for Rf1.     

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.     

Molecular markers linked to the Rf2 fertility restorer gene in cotton.

Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants do not produce viable pollen. Fertility in plants with CMS can be recovered by nuclear restorer genes. Most restorer genes cloned so far are members of the pentatricopeptide repeat (PPR) protein family. The objective of our study was to use the CMS-D8 and restoration (Rf2) system of cotton (Gossypium hirsutum L.) to develop more DNA markers for the Rf2 gene. In a backcross population with 112 plants, segregation of male fertility was 1 fertile : 1 sterile. Three new RAPD markers were identified for Rf2, one of which was converted to a CAPS marker. In addition, 2 AFLP markers and 1 SSR marker were identified to be linked to the fertility restorer gene (Rf2). PPR motif primers were designed based on the conserved PPR motifs and used in combination with AFLP primers to test the mapping population, and 1 PPR-AFLP marker was identified. A linkage map with 9 flanking markers including 1 from a previous study was constructed.     

The petunia restorer of fertility protein is part of a large mitochondrial complex that interacts with transcripts of the CMS-associated locus

A class of nuclear genes termed "restorers of fertility" (Rf) acts to suppress the expression of abnormal mitochondrial genes associated with cytoplasmic male sterility (CMS). In petunia, both the nuclear Rf gene and mitochondrial CMS-associated gene have previously been identified. The CMS-associated gene is an aberrant chimera in which portions of several mitochondrially encoded genes are fused to an unknown reading frame. The dominant Rf allele reduces the CMS-associated protein to nearly undetectable levels and alters the RNA population derived from the CMS locus, but its mechanism of action has not been determined. The petuniaRf gene is a member of the pentatricopeptide repeat gene family (PPR), an unusually large gene family in Arabidopsis (approximately 450 genes) compared with yeast (five genes) and mammalian genomes (six genes). The PPR gene family has been implicated in the control of organelle gene expression. To gain insight into the mode of action of PPR genes, we generated transgenic petunia plants expressing a functional tagged version of Rf. Analysis of the restorer protein revealed that it is part of a soluble mitochondrial inner-membrane-associated, RNase-sensitive high-molecular-weight protein complex. The complex is associated with mRNA derived from the CMS locus.     

Genetics of fertility restoration in the C-group of cytoplasmic male sterility in maize

The genetics of fertility restoration of cms-C group cytoplasm of maize was studied using crosses involving stable maintainer lines and lines that restored full pollen fertility. Pollen fertility in the sources of cms-C sterile cytoplasms studied was restored by a single dominant restorer (Rf4) gene. The fertility restoration was sporophytic. Allelism tests among five restorer lines showed that they all apparently carried the same alleles (Rf4 Rf4). Similar tests also demonstrated that seven nonrestoring maintainer lines had apparently the same genotype (rf4 rf4), although a partial "late break" of fertility was observed at low levels in some maintainer crosses. Comparative studies among different cms-C sources (C, Bb, ES, PR and RB) indicated that similar inheritance of fertility restoration was involved. The data indicated that a single, dominant Rf gene is involved in the restoration of several C-group cytoplasms, at least in the lines studied here. This is the first single-gene, sporophytic restorer system described in maize to date.     

Molecular mapping and candidate gene identification of the Rf2 gene for pollen fertility restoration in sorghum [Sorghum bicolor (L.) Moench]

The A1 cytoplasmic–nuclear male sterility system in sorghum is used almost exclusively for the production of commercial hybrid seed and thus, the dominant genes that restore male fertility in F₁ hybrids are of critical importance to commercial seed production. The genetics of fertility restoration in sorghum can appear complex, being controlled by at least two major genes with additional modifiers and additional gene–environment interaction. To elucidate the molecular processes controlling fertility restoration and to develop a marker screening system for this important trait, two sorghum recombinant inbred line populations were created by crossing a restorer and a non-restoring inbred line, with fertility phenotypes evaluated in hybrid combination with three unique cytoplasmic male sterile lines. In both populations, a single major gene segregated for restoration which was localized to chromosome SBI-02 at approximately 0.5 cM from microsatellite marker, Xtxp304. In the two populations we observed that approximately 85 and 87% of the phenotypic variation in seed set was associated with the major Rf gene on SBI-02. Some evidence for modifier genes was also observed since a continuum of partial restored fertility was exhibited by lines in both RIL populations. With the prior report (Klein et al. in Theor Appl Genet 111:994–1012, 2005) of the cloning of the major fertility restoration gene Rf1 in sorghum, the major fertility restorer locus identified in this study was designated Rf2. A fine-mapping population was used to resolve the Rf2 locus to a 236,219-bp region of chromosome SBI-02, which spanned ~31 predicted open reading frames including a pentatricopeptide repeat (PPR) gene family member. The PPR gene displayed high homology with rice Rf1. Progress towards the development of a marker-assisted screen for fertility restoration is discussed.     

Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments

Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4. Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but will also be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of the gene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F₁s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F₁s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363 and PRR 396 possessing both Rf4 and Rf3 genes and good fertility restoration have been identified which could be used further in hybrid rice breeding.     

用微卫星标记定位小麦T型CMS的恢复基因

以T型细胞质雄性不育系 75 336 9A×恢复系 72 6 9 10的F2 群体作为育性调查和基因定位群体。通过育性分析 ,确定该恢复系含有 2个主效恢复基因 ;结合群分法 ,对恢复基因进行了SSR分子标记定位 ,在 2 30对微卫星引物中 ,微卫星标记Xgwm136和Xgwm5 5 0分别与 2个主效恢复基因连锁。这两个标记与Rf基因之间的遗传距离分别为 6 7cM和 5 1cM ,从而将该恢复基因定位在 1AS、1BS染色体上。