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.     

籼稻二元不育系9730A及三交种的初步研究

通过对所选育的籼稻二元不育系9730A的花粉育性、自交结实率、雌性育性、对"920"的敏感性、柱头外露情况、花时、雄性不育的可恢复性及三交种的抽穗整齐度、有效穗、株高、单株产量、结实率、主茎穗长、主茎叶长、穗粒等变异度的分析,对杂交水稻三交法育种的应用价值进行初步探讨.结果认为:通过引进具有不同优良品种特性的第二保持系,可以在保持所选育杂种品种主要农艺性状整齐一致的前提下,较大幅度地拓宽品种的遗传基础,改进雄性不育系的异交性能,从而为提高杂种种子生产水平、及时利用优良常规稻育种成果材料和综合实现杂交稻品种选育过程中的多抗、广适、高产、优质育种目标提供新的途径.     

Functional markers developed from multiple loci in <Emphasis Type="Italic">GS3</Emphasis> for fine marker-assisted selection of grain length in rice

The gene GS3 has major effect on grain size and plays an important role in rice breeding. The C to A mutation in the second exon of GS3 was reported to be functionally associated with enhanced grain length in rice. In the present study, besides the C-A mutation at locus SF28, three novel polymorphic loci, SR17, RGS1, and RGS2, were discovered in the second intron, the last intron and the final exon of GS3, respectively. A number of alleles at these four polymorphic loci were observed in a total of 287 accessions including Chinese rice varieties (Oryza sativa), African cultivated rice (O. glaberrima) and AA-genome wild relatives. The haplotype analysis revealed that the simple sequence repeats (AT)_n at RGS1 and (TCC)_n at RGS2 had differentiated in the wild rice whilst the C-A mutation occurred in the cultivated rice recently during domestication. It also indicated that A allele at SF28 was highly associated with long rice grain whilst various motifs of (AT)_n at RGS1 and (TCC)_n at RGS2 were mainly associated with medium to short grain in Chinese rice. The C-A mutation at SF28 explained 33.4% of the grain length variation in the whole rice population tested in this study, whereas (AT)_n at RGS1 and (TCC)_n at RGS2 explained 26.4 and 26.2% of the variation, respectively. These results would be helpful for better understanding domestication of GS3 and its manipulation for grain size in rice. The genic marker RGS1 based on the motifs (AT)_n was further validated as a functional marker using two sets of backcross recombinant inbred lines. These results suggested that the functional markers developed from four different loci within GS3 could be used for fine marker-assisted selection of grain length in rice breeding.     

Overexpression of TIFY genes promotes plant growth in rice through jasmonate signaling

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.     

Planthopper‐rice interactions: unequal stresses on pure‐line and hybrid rice under similar experimental conditions

Hybrid and pure‐line (inbred) rice [Oryza sativa L. (Poaceae)] varieties have distinct physiologies, particularly as related to their nutrient requirements. These differences could confound the results and interpretation of experiments that compare rice varieties grown in pots for their resistance and responses to herbivores. In this study, a series of experiments was conducted to identify potentially confounding interactions between pot size (soil volume), fertilizer regime, and the use of acetate insect cages with rice line type (hybrid, fertile parental inbred, and male sterile inbred) during bioassays with the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). The growth of hybrid rice was often limited (relatively low biomass and low grain production) compared to fertile inbred lines even in large pots (7 200 ml) and when grown without added fertilizer. Several interactions between the effects of growth conditions and line type were detected. Acetate cages caused a significant reduction in grain yield in hybrids, but not in inbreds, mainly resulting from a cage‐induced decrease in grain weight (smaller grains). Hybrids and male sterile lines often had higher root or above‐ground biomass in the largest caged pots compared with fertile inbred lines, but biomass was similar in smaller pots, indicating that the large pots allowed longer unimpeded growth of fertile inbreds, but not other line types. There were no interactions between the presence or absence of planthoppers with line type or experimental conditions. All line types were equally susceptible to planthoppers. Often the effects of planthopper feeding on plant fitness (i.e., tolerance) were apparent when plants were grown in large pots but not in small pots, particularly for hybrid lines and under high nitrogen regimes. On the basis of these results we recommend that researchers ensure that plants are not unequally stressed by inadequate growth conditions during comparative studies with herbivores on physiologically distinct rice varieties or rice species. We recommend the use of large pots (soil volume) and lower fertilizer levels with young, non‐reproductive plants during comparative bioassays with planthoppers. Field cages are recommended for hybrid‐inbred comparisons during older plant stages but these are subject to a range of other confounding variables.     

Male Sex Interspecies Divergence and Down Regulation of Expression of Spermatogenesis Genes in Drosophila Sterile Hybrids

Male sex genes have shown a pattern of rapid interspecies divergence at both the coding and gene expression level. A common outcome from crosses between closely-related species is hybrid male sterility. Phenotypic and genetic studies in Drosophila sterile hybrid males have shown that spermatogenesis arrest is postmeiotic with few exceptions, and that most misregulated genes are involved in late stages of spermatogenesis. Comparative studies of gene regulation in sterile hybrids and parental species have mainly used microarrays providing a whole genome representation of regulatory problems in sterile hybrids. Real-time PCR studies can reject or reveal differences not observed in microarray assays. Moreover, differences in gene expression between samples can be dependant on the source of RNA (e.g., whole body vs. tissue). Here we survey expression in D. simulans, D. mauritiana and both intra and interspecies hybrids using a real-time PCR approach for eight genes expressed at the four main stages of sperm development. We find that all genes show a trend toward under expression in the testes of sterile hybrids relative to parental species with only the two proliferation genes (bam and bgcn) and the two meiotic class genes (can and sa) showing significant down regulation. The observed pattern of down regulation for the genes tested can not fully explain hybrid male sterility. We discuss the down regulation of spermatogenesis genes in hybrids between closely-related species within the contest of rapid divergence experienced by the male genome, hybrid sterility and possible allometric changes due to subtle testes-specific developmental abnormalities.     

几个优良籼稻亲本品质性状的配合力和杂种优势分析

以3个不育系和10个恢复系为材料,采用NCII交配设计研究10个米质性状的配合力和杂种优势。结果表明：①大多数品质性状的量值介于双亲之间,除粒重表现一定的超亲优势、垩白度和粒宽表现一定的正向平均优势外,其他品质性状优势不明显。②杂种稻米的品质性状主要受不育系或恢复系的影响,其中粒长、粒宽和直链淀粉含量3个性状,不育系的影响要高于恢复系;而对于整精米率、粒重、垩白率、垩白度和糊化温度,则恢复系的影响要高于不育系。③就优质育种的利用价值而言,不育系以广占63-4S为好,恢复系以扬稻6号为好,R527、镇恢084次之,用上述亲本选配的杂交组合米质较好;恢复系特青、盐恢559表现为一般配合力效应低,特殊配合力方差小,优质育种利用价值不大。     

Catalytic assays in blue native gels revealed normal ATPase but deficient NADH dehydrogenase activity in ZidaoA CMS line of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

Cytoplasmic male sterility (CMS) is an important trait in rice (Oryza sativa L.) breeding because it provides a source for producing hybrid seeds. In rice CMS lines, ATPases involved in the oxidative phosphorylation complexes are believed to be dysfunctional due to the expression of rice CMS-related gene orf79. In the present study, a new type of CMS line named CMS-ZA (ZidaoA) was used. We found an orf79 homologous gene (named orfZ79) in three different rice lines (a CMS line, a maintainer line, and a hybrid). However, no detectable expression products of orfZ79 were found in the three lines. We evaluated the ATPase and NADH dehydrogenase activities of the three lines using in-gel catalytic assays. Our results show that the sterile line has intact ATPase activity, while NADH DHase activity is clearly decreased. To investigate NADH dehydrogenase deficiency, we measured NADH DHase activity in etiolated seedlings and green seedlings from the ZidaoA CMS sterile line and its corresponding maintainer line. We note that the NADH DHase activity of the sterile line was more deficient in green seedlings than that in etiolated seedlings. Our results show a possible role of NADH DHase deficiency to cause rice CMS.     

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.     

Improving panicle exsertion of rice cytoplasmic male sterile line by combination of artificial microRNA and artificial target mimic

The adoption of hybrid rice caused the second leap in rice yield after the ‘green revolution’ and contributes substantially to food security of China and the world. However, almost all cytoplasmic male sterile lines (A lines) as females of hybrid rice have a natural deficiency of ‘panicle enclosure’, which blocks pollination between the A line and the fertility restorer line as the male (R line) of hybrid rice and decreases seed yield. In hybrid rice seed production, exogenous ‘920’ (the active ingredient is gibberellin A₃) must be applied to eliminate or alleviate panicle enclosure of the A line; however, this not only increases production cost and pollutes the environment, it also decreases seed quality. In this study, we designed a transgenic approach to improve plant height and panicle exsertion of the A line to facilitate hybrid rice production and maintain the semi‐dwarf plant type of the hybrid. This approach comprising two components—artificial microRNA (amiRNA) and artificial target mimicry—can manipulate the differential expression of the endogenous Eui1 gene that is associated with rice internode elongation in the A line and the hybrid. amiRNA is a recently developed gene silencing method with high specificity, while target mimicry is a natural mechanism inhibiting the miRNA function that was also recently characterized. This approach provides a paradigm to tune the expression of endogenous genes to achieve the desired phenotype by combining amiRNA and artificial target mimicry technologies.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Natural variation of MS2 confers male fertility and drives hybrid breeding in soybean

A complete and genetically stable male sterile line with high outcrossing rate is a prerequisite for the development of commercial hybrid soybean. It was reported in the last century that the soybean male sterile ms2 mutant has the highest record with seed set. Here we report the cloning and characterization of the MS2 gene in soybean, which encodes a protein that is specifically expressed in the anther. MS2 functions in the tapetum and microspore by directly regulating genes involved in the biosynthesis of secondary metabolites and the lipid metabolism, which is essential for the formation of microspore cell wall. Through comparison of the field performance with the widely used male sterile mutants in the same genetic background, we demonstrated that the ms2 mutant conducts the best in outcrossing rate and makes it an ideal tool in building a cost-effective hybrid system for soybean.     

Generation of a novel germline stem cell line expressing a germline‐specific reporter in the mouse

Germline stem (GS) cells are stem cell lines derived from postnatal male germline cells. Remarkably, GS cells can form functional spermatozoa when transplanted into infertile host mouse testes, indicating that GS cells have spermatogonial stem cell (SSC) activity. As GS cells are the only type with SSC activity, they are most suitable for in vitro studies on male germ cell differentiation. However, GS cells can deviate from the germ cell state to become other types of cells, depending on culture conditions. Therefore, it is desirable to have a monitor system to ensure that GS cells are kept at the germ cell state in culture. Here, we established GS cell lines from neonatal testes of transgenic mice that express the fluorescent protein, Venus, whose gene expression is driven by the promoter of Mvh (mouse Vasa homolog), a gene highly specific to mammalian germ cells. This novel cell line has genuine GS cell properties equivalent to existing GS lines, including the ability to generate viable offspring. This Mvh–Venus GS cell line, to our knowledge, is the first one expressing a germ cell‐specific reporter. This valuable resource should provide new opportunities for studies on male germ cell differentiation. genesis 51:498–505. © 2013 Wiley Periodicals, Inc.     

Validation of gene based marker-QTL association for grain dimension traits in rice

Validation of marker-QTL association for genes grain size 3 (GS3), grain weight 2 (GW2), seed width 5 (qSW5) and a QTL qgrl7.1 for grain length was undertaken in a set of 242 diverse rice germplasm. Further, the study was extended to an F₂ mapping population derived from cross of Sonasal, a short grain aromatic rice landrace with Pusa Basmati 1121, a variety with extra long slender grains. Seven gene specific markers, namely, SF28, SR17, RGS1and RGS2 based on GS3, W004 for GW2, MS40671 for qSW5 and RM505 for qgrl7.1, were used for validation. Single marker analysis revealed significant association of these markers to grain size and shape. The marker SF28 explained highest phenotypic variance (37 %) while the marker W004 explained lowest variance (2.6 %) for grain length in the germplasm set at the significance level P?<?0.05. Three markers namely, SF28, MS40671 and RM505 were polymorphic between the parents Sonasal and Pusa Basmati 1121. In the F₂ population, the marker SF28 linked to gene GS3 explained highest phenotypic variance (32.5 %), while RM505 linked to qgrl7.1 explained 5.4 % of phenotypic variance for grain length. The marker SF28 was found to be most robust in the validation studies both in germplasm and F₂ population. The validated gene specific markers can be utilised in marker assisted selection for improving grain size and shape as these traits have significant contribution towards grain quality and grain yield. This is the first study on validation of gene based markers for grain dimension traits in Indian rice germplasm.