Development and validation of candidate gene-specific markers for the major fertility restorer genes, <Emphasis Type="Italic">Rf4</Emphasis> and <Emphasis Type="Italic">Rf3</Emphasis> in rice

Two major nuclear genes, Rf3 and Rf4, are known to be associated with fertility restoration of wild-abortive cytoplasmic male sterility (WA-CMS) in rice. In the present study, through a comparative sequence analysis of the reported putative candidate genes, viz. PPR9-782-(M,I) and PPR762 (for Rf4) and SF21 (for Rf3), among restorer and maintainer lines of rice, we identified significant polymorphism between the two lines and developed a set of PCR-based codominant markers, which could distinguish maintainers from restorers. Among the five markers developed targeting the polymorphisms in PPR9-782-(M,I), the marker RMS-PPR9-1 was observed to show clear polymorphism between the restorer (n = 120) and maintainer lines (n = 44) analyzed. Another codominant marker, named RMS-PPR762 targeting PPR762, displayed a lower efficiency in identification of restorers and maintainers, indicating that PPR9-782-(M,I) is indeed the candidate gene for Rf4. With respect to Rf3, a codominant marker, named RMS-SF21-5 developed targeting SF21, displayed significantly lower efficiency in identification of restorers and non-restorers as compared to the Rf4-specific markers. Validation of these markers in a F₂ mapping population segregating for fertility restoration indicated that Rf4 has a major influence on fertility restoration and Rf3 is a minor gene. Further, the functional marker RMS-PPR9-1 was observed to be very useful in identification of impurities in a seed lot of the popular hybrid, DRRH3. Interestingly, when RMS-PPR9-1 and RMS-SF21-5 were considered in conjunction with analysis, near-complete, marker–trait co-segregation was observed, indicating that deployment of the candidate gene-specific markers both Rf4 and Rf3, together, can be helpful in accurate identification of fertility restorer lines and can facilitate targeted transfer of the two restorer genes into elite varieties through marker-assisted breeding.     

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.     

Mitotype-specific sequences related to cytoplasmic male sterility in Oryza species

Plant mitochondrial genomes contain a large number of mitotype-specific sequences (MSS) which establish a mitochondrial genome structure distinct from other mitotypes. In rice, nine mitochondrial genomes have been sequenced, which provides us with the possibility of characterizing the MSS of rice and probing their relationship to cytoplasmic male sterility (CMS) in rice. We therefore analyzed the mitochondrial genomes of CW-CMS, LD-CMS, WA-CMS, N and Nipponbare lines, and found 57 MSS with sizes ranging from 102 to 5,745 bp, and with an aggregate length of 92.4 kb. The MSS account for more than 14.5 % of the rice mitochondrial genome and are a significant contributing factor in the variation of mitochondrial genome sizes. Of the MSS tested, 34 MSS exhibited polymorphism among rice lines, and 14 MSS were further confirmed as being specific to CMS. This includes nine MSS specific to sporophytic CMS, three specific to gametophytic CMS, and two shared by all types of CMS. Interestingly, except for CMS genes orf(H)79 and orf352 which are partly or fully overlapping with some MSS fragments, there are ten more open reading frames of unknown function that were detected in CMS-specific MSS, hinting at their possible roles in plant CMS. These novel findings provide us with potential new molecular tools to direct the breeding of CMS lines in hybrid rice breeding programs.     

Genic markers for wild abortive (WA) cytoplasm based male sterility and its fertility restoration in rice

Commercial exploitation of heterosis is essential for enhancing productivity of rice. The use of cytoplasmic male sterility (CMS) and fertility restoration system greatly facilitates large scale production of hybrid seed. The wild abortive (WA) cytoplasm is most widely used for hybrid seed production in rice. The present study was undertaken to develop molecular markers for both WA cytoplasm based male sterility and its fertility restoration for use in efficient hybrid breeding. High degree of genetic differentiation of WA-cytoplasm from its normal fertile counterpart was observed due to DNA rearrangements involving five (coxI, coxIII, cob, atp6 and rps3) mitochondrial genes. Cleaved amplified polymorphic sequence (CAPS) markers based on five mitochondrial genes namely, coxIII, cob, atp9, rps3 and 18SrRNA polymorphic between CMS and maintainer line were developed. The utility of these informative markers was demonstrated in purity testing of the CMS line Pusa6A being used in commercial hybrid seed production. Fertility restoration was found to be controlled by a major locus in the Basmati restorer line PRR78, which was mapped to a short marker interval of 0.8 cM and a physical interval of 163.6 kb on rice chromosome 10. A total of 13 pentatricopeptide repeat (PPR) motif containing genes were predicted in a 1.66 Mb region on the long-arm of this chromosome of which, four were present in the marker interval containing the fertility restorer gene. High degree of conservation of gene order was observed between japonica and indica for the predicted PPR genes. A sequence tagged site (STS) and a genic non-coding microsatellite (GNMS) marker were designed based on one of the candidate PPR motif containing genes present in the marker interval, which were validated using F₂ population and other known restorer lines. The candidate gene based marker identified in the present study would be useful in marker assisted selection (MAS) for fertility restorer gene in hybrid breeding programme based on WA-CMS of rice.     

Small mitochondrial DNA molecules of wild abortive cytoplasm in rice are not necessarily associated with CMS

Summary Mitochondrial DNA was isolated from leaf tissue of both the cytoplasmic male sterile line of Indica rice variety V41, which carries wild abortive (WA) cytoplasm, and from the corresponding maintainer line. In addition to the main mitochondrial DNA, four small plasmid-like DNA molecules were detected in both the male sterile and fertile lines. Restriction analysis of total mitochondrial DNA from the male sterile and fertile lines showed DNA fragments unique to each. Our findings suggest that the four small mitochondrial DNA (mtDNA) molecules are conserved when WA cytoplasm is transferred into different nuclear backgrounds. However, there is no simple correlation between the presence/ absence of small mitochondrial DNA molecules and the expression of WA cytoplasmic male sterility (CMS).     

Localization and organization of tRNA genes on the mitochondrial genomes of fertile and male sterile lines of maize

Summary Maize mitochondrial (mt) tRNA genes were localized on the mt master circles of two fertile lines (WF9-N and B37-N) and of one cytoplasmic male sterile line (B37-cmsT) of maize. The three genomes contain 16 tRNA genes with 14 different anticodons which correspond to 13 amino acids. Out of these 16 tRNA genes, 6 show a high degree of homology with the corresponding chloroplast (cp) tRNA genes and were shown to originate from cp DNA insertions and to be expressed in the mitochondria. The organization of the mt tRNA genes in both fertile lines is similar. The same genes are found, in the same environment, as judged from the restriction maps, in fertile and male sterile lines that have the same nuclear background, but the relative organization of the mt tRNA genes on the master circle is completely different.     

Wheat beta-expansin (EXPB11) genes: Identification of the expressed gene on chromosome 3BS carrying a pollen allergen domain

Background

Cytoplasmic male sterility (CMS) has often been associated with abnormal mitochondrial open reading frames. The mitochondrial gene orfH79 is a candidate gene for causing the CMS trait in CMS-Honglian (CMS-HL) rice. However, whether the orfH79 expression can actually induce CMS in rice remains unclear.

Results

Western blot analysis revealed that the ORFH79 protein is mainly present in mitochondria of CMS-HL rice and is absent in the fertile line. To investigate the function of ORFH79 protein in mitochondria, this gene was fused to a mitochondrial transit peptide sequence and used to transform wild type rice, where its expression induced the gametophytic male sterile phenotype. In addition, excessive accumulation of reactive oxygen species (ROS) in the microspore, a reduced ATP/ADP ratio, decreased mitochondrial membrane potential and a lower respiration rate in the transgenic plants were found to be similar to those in CMS-HL rice. Moreover, retarded growth of primary and lateral roots accompanied by abnormal accumulation of ROS in the root tip was observed in both transgenic rice and CMS-HL rice (YTA).

Conclusion

These results suggest that the expression of orfH79 in mitochondria impairs mitochondrial function, which affects the development of both male gametophytes and the roots of CMS-HL rice.     

Characterization of fertility alteration and marker validation for male sterility genes in novel PTGMS lines hybrid rice

Photoperiod and thermosensitive genetic male sterile (PTGMS) lines have become one of the main sources of global rice production increasing. This study was conducted to evaluate the fertility alteration and validate the male sterility genes using validation markers in novel Egyptian Indica and Japonica PTGMS lines under natural conditions. The study revealed that the new genetic male sterile lines belong to the type of photo–thermosensitive genetic male sterility (PTGMS). The fertility alteration of these lines has influenced by photoperiod and temperature interaction. The new PTGMS lines have three sensitive periods of fertility alteration; transformation, sterility, and fertility period. Furthermore, the sensitive stage of fertility transformation might be from secondary branch primordial to pollen mother cells (PMC) meiosis. Under the natural Sakha condition, the new PTGMS lines were stable sterile under the condition of day length upper 13,75 h and temperature over 25 °C, while its convert to fertile under day length under 13 h, and temperature lower than 24 °C. The co-dominant markers identified the pms3 and tms5 genes in the new PTGMS lines, indicated that the fertility alteration in these lines controlled by photoperiod and thermosensitive stages.     

Development and validation of co-dominant gene based markers for <Emphasis Type="Italic">Pi9</Emphasis>, a gene governing broad-spectrum resistance against blast disease in rice

Rice production and grain quality are severely affected by blast disease caused by the ascomycetous fungus Magnaporthe oryzae. Incorporation of genes that confer broad-spectrum resistance to blast has been a priority area in rice breeding programs. The blast resistance gene Pi9 sourced from Oryza minuta has shown broad spectrum and durable resistance to blast world-wide. In the present study co-dominant gene-based markers were developed for the precise marker-assisted tracking of Pi9 in breeding programs. The developed markers were validated across a diverse set of cultivars including basmati, indica and japonica varieties. Two markers, Pi9STS-1 and Pi9STS-2, effectively differentiated Pi9 donors from all the indicas and commercial basmati varieties tested. However, these markers were monomorphic between Pi-9 donors (IRBL9-W and Pusa 1637) and japonica type varieties. An additional gene-derived CAPS marker Pi91F_ 2R was developed to differentiate Pi9 donors from japonicas and traditional basmati lines. The co-dominant markers developed in the present study will be of immense utility to rice breeders for precise and speedy incorporation of Pi-9 into susceptible rice varieties through marker-assisted selection.     

Distribution of fertility-restorer genes for wild-abortive and Honglian CMS lines of rice in the AA genome species of genus Oryza

BACKGROUND AND AIMS: Rice (Oryza sativa) is one of the most important cereal plants in the world. Wild-abortive (WA) and Honglian (HL) cytoplasmic male sterility (CMS) have been used extensively in the production of hybrid seeds. Although a variable number of fertility-restorer genes (Rf) for WA and HL-CMS have been identified in various cultivars, information on Rf in Oryza species with the AA-genome is sparse. Therefore the distribution and heredity of Rf for WA and HL-CMS in wild rice species of Oryza with the AA-genome were investigated. METHODS: Fertility-restorer genes for WA and HL-CMS in wild rice species with the AA-genome were investigated by following the fertility of microspores identified by I2-KI staining and by following the seed-setting rate of spikelets. A genetic model of Rf in some selected restorer accessions was analysed based on the fertility segregation of BC1F1 populations. KEY RESULTS: Fertility analysis showed that 21 out of 35 HL-type F1s, and 13 out of 31 WA-type F1s were scored as fertile. The frequency of Rf in wild rice was 60% for HL-CMS and 41.9% for WA-CMS, respectively. The fertility-restorer accessions, especially those with complete restoring ability, aggregated mainly in two species of O. rufipogon and O. nivara. The wild rice accessions with Rf for HL-CMS were distributed in Asia, Oceania, Latin American and Africa, but were centered mainly in Asia, whilst the wild restorer accessions for WA-CMS were limited only to Asia and Africa. Apart from one restorer accession that possessed two pairs of Rf for WA-CMS, all of the other nine tested wild restorer accessions each contained only a single Rf for WA-CMS or HL-CMS. Allele analysis indicated that there existed at least three Rf loci for the WA and HL-CMS systems. CONCLUSIONS: These data support the hypothesis that fertility-restorer genes exist widely in Oryza species with the AA-genome, and that Rf in Oryza sativa originated from the Oryza rufipogon/Oryza nivara complex, the ancestor of cultivated rice in Asia. The origin and evolution of Rf is tightly linked to that of CMS in wild rice, and fertility of a given CMS type is controlled by several Rf alleles in various wild restorer accessions.     

Organization of the mitochondrial Cob 2 pseudogene in different lines of rice

In the fertile rice line IR 36 there are two copies of the apocytochrome b (cob) gene: a functional copy, cob 1, and a pseudogene, cob 2 (Kaleikau et al. 1992). In a survey of diverse rice lines, we found that cob 2 was absent in the wild abortive(WA)-type cytoplasmic male-sterile cytoplasm, but was present in the fertile lines. While cob 1 was conserved among all the lines, fertile and sterile, the cob 2 region was different in the fertile lines tested. The 5′ regions of most cob 2 loci were similar to cob 1 (about 4 kb of the flanking region and most of the coding region), but the 3′ region varied among different fertile lines. The point of divergence, the break-point, from the cob 1 sequence was conserved in all the cob 2 regions tested. In all the cob 2 regions, this break-point seems to be linked to the variable region of cob 2 through a conserved 192-bp segment, which is not a part of cob 1. It is proposed that the cob 2 regions could have been produced by recombination or insertion events involving cob 1 and the 192-bp segment which is present at different locations in the mitochondrial genomes of the various rice lines.     

Organizational differences between cytoplasmic male sterile and male fertile Brassica mitochondrial genomes are confined to a single transposed locus.

Comparison of the physical maps of male fertile (cam) and male sterile (pol) mitochondrial genomes of Brassica napus indicates that structural differences between the two mtDNAs are confined to a region immediately upstream of the atp6 gene. Relative to cam mtDNA, pol mtDNA possesses a 4.5 kb segment at this locus that includes a chimeric gene that is cotranscribed with atp6 and lacks an approximately 1kb region located upstream of the cam atp6 gene. The 4.5 kb pol segment is present and similarly organized in the mitochondrial genome of the common nap B.napus cytoplasm; however, the nap and pol DNA regions flanking this segment are different and the nap sequences are not expressed. The 4.5 kb CMS-associated pol segment has thus apparently undergone transposition during the evolution of the nap and pol cytoplasms and has been lost in the cam genome subsequent to the pol-cam divergence. This 4.5 kb segment comprises the single DNA region that is expressed differently in fertile, pol CMS and fertility restored pol cytoplasm plants. The finding that this locus is part of the single mtDNA region organized differently in the fertile and male sterile mitochondrial genomes provides strong support for the view that it specifies the pol CMS trait.     

Identification of a novel cytoplasmic male sterile line M2BS induced by partial-length <Emphasis Type="Italic">HcPDIL5-2a</Emphasis> transformation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A cytoplasmic male sterile line (designated as M2BS) was obtained from an indica rice maintainer M2B induced by partial-length HcPDIL5-2a (Hibiscus cannabinus protein disulfide isomerase-like) transformation. The anther of M2BS was short, slender, hygrophanous, and fissured. I2-KI staining method showed that there was typical and spherical abortion in pollen grains. M2BS was found abortive at middle and late stage of monocyte by the modified carbol fuchsin stained observation and paraffin section observation. The tapetum was observed pre-degenerated in M2BS. Hereditary analysis indicated that the male sterility of M2BS was a maternally inherited inability after six backcross generations with M2B and the combinations of M2BS hybridized with other two male fertile materials. The M2BS could be affirmed a cytoplasmic male sterile (CMS) type. Moreover, it was a transgenic plant confirmed by PCR, Southern blot and RT-PCR detection. M2BS could be distinguished from M2B and its CMS line M2A by RFLP analysis. The overall mitochondrial genome sequencing results showed, that in M2BS, the main differences of mitochondrial gene sequence were located in nad4, nad5, nad7, orf194 and intergenic region, relatively to those of M2A. The obtained results indicate that M2BS is a novel cytoplasmic male sterile line.     

Construction of rice cybrid plants

Summary The mitochondrial genomes of rice cells were transferred to a fertile rice variety (N8) from a cytoplasmic male sterile variety (CMS) by asymmetric protoplast fusion based on metabolic complementation. Protoplasts derived from CMS were X-irradiated (125 krad) and electrofused with protoplasts which had been treated with iodoacetamide. Metabolic complementation, presumably between nuclear and cytoplasmic compartments, enabled fused protoplasts to form colonies at high efficiency. Restriction digest analysis of mitochondrial DNA (mtDNA) indicated that hybrid cells carried mtDNA derived from both parents. Of the plants regenerated from hybrid calli, 68% carried a diploid chromosome set (2n=24) and the rest of them carried 48 chromosomes. All of them expressed the aryl acylamidase I deficient phenotype encoded by the recessive allele of the fertile N8 parent. These results indicate that the novel somatic hybrid plants regenerated were cybrids, deriving their nucleus from the iodoacetamide treated parent and their mitochondria from both parents.     

Molecular basis of cytoplasmic male sterility and fertility restoration in rice

Cytoplasmic male sterility (CMS) is a maternally inherited trait that causes dysfunctions in pollen and anther development. CMS is caused by the interaction between nuclear and mitochondrial genomes. A product of a CMS-causing gene encoded by the mitochondrial genome affects mitochondrial function and the regulation of nuclear genes, leading to male sterility. In contrast, the RESTORER OF FERTILITY gene (Rf gene) in the nuclear genome suppresses the expression of the CMS-causing gene and restores male fertility. An alloplasmic CMS line is often bred as a result of nuclear substitution, which causes the removal of functional Rf genes and allows the expression of a CMS-causing gene in mitochondria. The CMS/Rf system is an excellent model for understanding the genetic interactions and cooperative functions of mitochondrial and nuclear genomes in plants, and is also an agronomically important trait for hybrid seed production. In this review article, pollen and anther phenotypes of CMS, CMS-associated mitochondrial genes, Rf genes, and the mechanism that causes pollen abortion and its agronomical application for rice are described.     

Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice

The so-called ＂wild abortive＂ （WA） type of cytoplasmic male sterility （CMS） derived from a wild rice species Oryza rufipogon has been extensively used for hybrid rice breeding. However, extensive analysis of the structure of the related mitochondrial genome has not been reported, and the CMS-associated gene（s） remain unknown. In this study, we exploited a mitochondrial genome-wide strategy to examine the structural and expressional variations in the mitochondrial genome conferring the CMS. The entire mitochondriai genomes of a CMS-WA line and two normal fertile rice lines were amplified by Long-polymerase chain reaction into tilling fragments of up to 15.2 kb. Restriction and DNA blotting analyses of these fragments revealed that structural variations occurred in several regions in the WA mitochondrial genome, as compared to those of the fertile lines. All of the amplified fragments covering the entire mitochondrial genome were used as RNA blot probes to examine the mitochondriai expression profile among the CMS-WA and fertile lines. As a result, only two mRNAs were found to be differentially expressed between the CMS-WA and the fertile lines, which were detected by a probe containing the nad5 and orf153 genes and the other having the ribosomal protein gene rpl5, respectively. These mRNAs are proposed to be the candidates for further identification and functional studies of the CMS gene.