Transgenic rice hybrids that carry the Rf-1 gene at multiple loci show improved fertility at low temperature

By using a genomic fragment that carries the rice (Oryza sativa L.) fertility restorer gene, Rf-1, rice restorer lines harbouring multiple Rf-1 genes on different chromosomes were developed by genetic engineering and crossing. Hybrid lines that were obtained by crossing the restorer lines having two and three Rf-1 genes with a cytoplasmic male sterile (CMS) line had nearly 75 and 87.5% pollen fertility rates under a normal condition, respectively, whereas a conventional hybrid line showed a 50% pollen fertility rate. Furthermore, the seed set percentage under low temperature conditions was much higher in the hybrid lines with multiple Rf-1 genes than the conventional hybrid line. These results indicate that multiplication of the Rf-1 gene conferred cold tolerance at the booting stage to hybrid rice through increasing the potentially fertile pollen grains. This strategy to improve fertility at low temperature of hybrids could be applied to any grain crops that are developed based on CMS and its gametophytic restorer gene, let alone rice.     

A pentatricopeptide repeat-containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic male-sterile rice

A fertility restorer gene (Rf-1) of [ms-bo] cytoplasmic male sterility (CMS) in rice has been reported to be responsible for the processing of RNA of aberrant atp6 of mitochondria. We have carried out map-based cloning of the Rf-1 gene and found that a 4.7-kb genomic fragment of a restorer line promoted the processing of aberrant atp6 RNA when introduced into a CMS line. The genomic fragment contained a single open reading frame encoding 18 repeats of the 35 amino acid pentatricopeptide repeat (PPR) motif. The cloned PPR gene is a possible candidate of Rf-1. A non-restoring genotype was identified to have deletions within the coding region.     

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.     

Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers

The cytoplasmic male sterility (CMS) of wild-abortive (WA) cytoplasm has been widely used for breeding hybrid rice. Two restorer genes for the CMS have been found by traditional genetic analysis. To tag the restorer genes we used a set of near-isogenic lines (NILs) of Zhenshan 97 carrying different genotypes for fertility restoration from IR24, to perform RAPD analysis. From the survey of 720 random primers, six RAPD markers were identified to be associated with Rf-3. Three of these OPK05-800, OPU10-1100 and OPW01-350, were mapped on chromosome 1. Two populations from the crosses between Zhenshan 97 A and a near-isogenic restorer line ZSR21 and between Zhenshan 97 A and IR24 were used for mapping Rf-3. The three RAPD markers and three RFLP markers, RG532, RG140 and RG458, were found to be closely linked to Rf-3 in the two populations. The same location of Rf-3 was also found in a population from the cross of IR58025 A//IR36/IR58025 B. At the RG532 locus, different alleles were found between two CMS lines, Zhenshan 97 A and IR58025 A, and between two restorer lines, IR24 and IR36. The use of these molecular markers closely linked to Rf-3 in facilitating the development of hybrid rice is discussed. Received: 3 January 1996 / Accepted: 17 May 1996     

Structural diversity and evolution of the Rf-1 locus in the genus Oryza

The Rf-1 locus in rice is agriculturally important as it restores fertility in plants with BT-type cytoplasmic male sterility (CMS). The Rf-1 locus contains several duplicated copies of the gene responsible for restoration of fertility. We analyzed the genomic structure of the Rf-1 locus in the genus Oryza to clarify the structural diversity and evolution of the locus. We identified six genes (Rf-1A to Rf-1F) with homology to Rf-1 at this locus in Oryza species with an AA genome. The Rf-1 locus structures in the rice accessions examined were very complex and fell into at least six classification types. The nucleotide sequences of the duplicated genes and their flanking regions were highly conserved suggesting that the complex Rf-1 locus structures were produced by homologous recombination between the duplicated genes. The fact that complex Rf-1 locus structures were common to Oryza species that have evolved independently indicates that a duplication of the ancestral Rf-1 gene occurred early in rice evolution and that homologous recombination resulted in the diversification of Rf-1 locus structures. Additionally, the amino acid sequences of each duplicated gene were conserved between species. This suggests that the duplicated genes in the Rf-1 locus may have divergent functions and may act by controlling mitochondrial gene expression in rice as occurs in the restoration of CMS.     

几种农作物细胞质雄性不育恢复基因的定位和分子标记研究进展

利用杂种优势提高作物产量时, 生产杂交种的主要授粉控制系统是细胞质雄性不育及其恢复系统。在杂交品种的选育过程中, 优良恢复系选育至关重要。为了高效并准确地鉴定选择恢复材料, 同时更深入地研究恢复基因的作用机理, 近年来植物细胞质雄性不育恢复基因分子标记研究受到了广泛重视。本文综述了主要农作物水稻、油菜、小麦、棉花和玉米等细胞质雄性不育类型恢复基因的定位和分子标记研究进展, 并讨论了恢复基因的精确定位和分子标记鉴定在基因克隆和分子标记辅助选择育种中的意义和应用前景。     

几种农作物细胞质雄性不育恢复基因的定位和分子标记研究进展

利用杂种优势提高作物产量时,生产杂交种的主要授粉控制系统是细胞质雄性不育及其恢复系统。在杂交品种的选育过程中,优良恢复系选育至关重要。为了高效并准确地鉴定选择恢复材料,同时更深入地研究恢复基因的作用机理,近年来植物细胞质雄性不育恢复基因分子标记研究受到了广泛重视。本文综述了主要农作物水稻、油菜、小麦、棉花和玉米等细胞质雄性不育类型恢复基因的定位和分子标记研究进展,并讨论了恢复基因的精确定位和分子标记鉴定在基因克隆和分子标记辅助选择育种中的意义和应用前景。     

A rapid PCR-aided selection of a rice line containing the Rf-1 gene which is involved in restoration of the cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is widely known in higher plants, the mechanism of which is believed to involve incompatibility between nuclei and cytoplasms. In rice lines with the CMS trait, fertility is restored by the aid of a nuclear-encoded gene, Rf-1, whose locus has been determined in chromosome 10. We found a particular PCR-amplified fragment, designated fL601, that specifically amplified using the DNAs from Rf-1 lines tested as templates. RFLP mapping of the fL601 locus revealed that there are two loci for the fL601, and that both are tightly linked to the Rf-1 locus. Progeny analysis also showed high frequency of their co-segregation. Southern analysis of the genomic DNA demonstrated that the Rf-1 lines shared a unique sequence in the fL601 region. These results enabled us to construct a system for specific detection of the corresponding regions. Utilizing this detection system, we established a simple PCR-mediated selection method for the Rf-1 lines, which may facilitate the breeding for hybrid rice.     

苎麻atp6和atp9基因的克隆表达及与细胞质雄性不育的相关性

摘要: 根据GenBank报道的双子叶植物线粒体atp6和atp9基因编码区保守序列设计简并引物, 通过PCR技术从苎麻细胞质雄性不育系、保持系和恢复系(简称“三系”) mtDNA中扩增目的基因片段, 发现所得序列开放阅读框虽不完整, 但与GenBank报道的其他植物线粒体atp6和atp9基因同源性分别高于94%和85%。采用DNA Walking步移法分别从3′端和5′端扩增两个基因片段的未知侧翼序列, 分离出完整的苎麻线粒体atp6和atp9基因, 包含了完整的开放阅读框。其中“三系”的atp6基因在mtDNA水平、转录和翻译调控水平、蛋白质水平上均无差异。不育系atp9基因在编码区3′端与保持系和恢复系相比存在若干个碱基的差异和缺失; RT-PCR分析还表明, 不育系atp9基因在现蕾期和盛花期的表达量很高。推测不育系atp9基因的结构变异和/或异常表达与苎麻细胞质雄性不育(CMS)的关系密切。     

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.     

Towards molecular breeding of reproductive traits in cereal crops

The transition from vegetative to reproductive phase, flowering per se , floral organ development, panicle structure and morphology, meiosis, pollination and fertilization, cytoplasmic male sterility (CMS) and fertility restoration, and grain development are the main reproductive traits. Unlocking their genetic insights will enable plant breeders to manipulate these traits in cereal germplasm enhancement. Multiple genes or quantitative trait loci (QTLs) affecting flowering (phase transition, photoperiod and vernalization, flowering per se ), panicle morphology and grain development have been cloned, and gene expression research has provided new information about the nature of complex genetic networks involved in the expression of these traits. Molecular biology is also facilitating the identification of diverse CMS sources in hybrid breeding. Few Rf (fertility restorer) genes have been cloned in maize, rice and sorghum. DNA markers are now used to assess the genetic purity of hybrids and their parental lines, and to pyramid Rf or tms (thermosensitive male sterility) genes in rice. Transgene(s) can be used to create de novo CMS trait in cereals. The understanding of reproductive biology facilitated by functional genomics will allow a better manipulation of genes by crop breeders and their potential use across species through genetic transformation.     

棉花细胞核雄性不育两用系差异表达基因分析

应用cDNA-AFLP对棉花ms5ms6双隐性核雄性不育两用系的不育株和可育株花粉发育的3个时期—造孢细胞时期、花粉母细胞时期和花粉粒时期进行对比分析,共得到17个差异表达片段,它们分别属于11种表达模式,其中14个片段可以在NCBI数据库中找到同源序列,功能分析表明这些片段所编码的基因可能参与了信号转导、转录、能量代谢、细胞壁发育等相关过程。Northern杂交结果证明检测片段的表达模式与cDNA-AFLP结果吻合。同时还在可育花药中发现了与玉米T型细胞质雄性不育恢复因子RF2基因高度同源的育性恢复因子类基因。     

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.     

Unveiling the molecular arms race between two conflicting genomes in cytoplasmic male sterility?

Cytoplasmic male sterility can be thought of as the product of a genetic conflict between two genomes that have different modes of inheritance. Male sterilizing factors, generally encoded by chimeric mitochondrial genes, can be down-regulated by specific nuclear restorer genes. The recent cloning of a restorer gene in rice and its comparison with restorer genes cloned in petunia and radish could be regarded as the beginning of a general molecular scenario in this peculiar arms race.     

稻米营养品质种子效应和母体效应的遗传分析

采用谷类作物种子数量性状的遗传模型,以珍汕９７Ａ等６个籼型不育系与测早２－２等,３个早籼恢复系进行不完全双列杂交,对籼型杂交稻稻米营养品质性状进行了遗传分析。结果表明：稻米蛋白质含量和蛋白质指数主要受制于母体遗传效应,但亦受到种子基因效应的影响;赖氨酸含量和赖氨酸指数则主要与种子基因效应有关,其中赖氨酸含量还受到母体加性效应的影响。除赖氨酸指数外,其它营养品质性状的种子直接遗传率和母体遗传率均已达到极显著水平。遗传效应预测值表明,选用浙南１号Ａ和２６７１５较易获得稻米营养品质较为理想的籼型杂交稻组合。     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.     

水稻偏粳型广谱性强恢复系种质GR38的创新和评价

用无育性恢复力的意大利粳稻巴利拉,与具有广亲和性的我国改良籼稻广中接杂交,创新出偏粳型广谱性强恢复系种质GR38。与亲本度现有4类恢复基因型仅能恢复部分不育细胞质的情况不同,GR38对几乎所有主要不育细胞质均具强恢复力。SSR标记分析发现,GR38的一个恢复基因位于第1染色体长臂中部,与已报道的恢复基因位点均有所不同,推测GR38至少携带一个新恢复基因。GR38具有很强的产量配合力,与许多不育系配制的籼粳杂交稻组合具有很强的杂种优势和产量潜力,可以直接用于生产。GR38的育成,将地理远缘的粳稻基因导入到恢复系中,创造了地理远缘粳稻／广亲和籼稻的杂交选育途径,打破了常用的籼恢／籼恢单一途径,以度恢复基因源局限于IR24度其衍生系的状况,拓宽了恢复基因源和遗传基础．有助于杂交稻育种的新突破。