Nuclear-mitochondrial interactions in cytoplasmic male-sterile sorghum

Summary Variation in mitochondrial genome organization and expression between male fertile and sterile nuclear-cytoplasmic combinations of sorghum has been examined. Cytoplasmic genotypes were classified into eleven groups on the basis of restriction endonuclease digestion of mitochondrial DNA (mtDNA) and five groups on the basis of mitochondrial translation products. These cytoplasms were further characterized by hybridization of specific gene probes to Southern blots of EcoRI digested mtDNA, and identification of the fragment location of four mitochondrial genes. Variation was observed in the genomic location and copy number of the F₁ ATPase -subunit gene, as well as the genomic location and gene product of the cytochrome c oxidase subunit I gene. The effect of nuclear genotype on mitochondrial genome organization, expression and the presence of two linear plasmid-like mtDNA molecules was examined. Our results indicate that nuclear-mitochondrial interactions are required for regulation of mitochondrial gene expression. When a cytoplasm is transferred from its natural to a foreign nuclear background some changes in the products of in organello mitochondrial protein synthesis occur. In a number of cytoplasmic genotypes these changes correlate with the expression of cytoplasmic male sterile phenotype, suggesting a possible molecular basis for this mutation.     

Phosphatase and ATPase activities in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Summary Soluble and membrane-bound fractions of plant leaves, cell suspension cultures and seedlings of petunia were examined for phosphohydrolase activity on p-nitrophenyl phosphate (pNPPase) and adenosine triphosphate (ATPase). One cytoplasmic male-sterile (CMS) and one fertile (F) line was examined for each tissue. Both pNPPase and ATPase exhibited a broad optimal activity between pH 5.5–7.0 for the membrane-bound fraction and between 4.5–7.0 for the soluble fractions. The activity of both were inhibited by divalent ions including Mg²⁺. At pH 7.2, the activities on various triphosphonucleotides were similar and they were hydrolyzed by a rate of 20–50% of that of ATP. Significant differences between CMS and F extracts were: (a) higher activities in CMS membranes; (b) lower Ea (energy of activation) values for activities in CMS membrane functions; (c) seedling and cell-culture CMS extracts exhibited a higher sensitivity to high temperature denaturation; (d) the hydrolase activity on monoand triphospho-cytosine compounds was significantly higher in CMS than in F membranes.Contribution from the Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel No. 355-E, 1992 series     

Differences in amino acid transport in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Summary Two pairs of isonuclear lines of cytoplasmic male-sterile (CMS) and fertile (F) petunia cells grown in suspension culture in the presence or absence of amino acid sources were examined for uptake of 11 amino acids and adenosine. Cells from CMS lines exhibited a significant lower rate of uptake than F cells. These differences, for various amino acids, are a result of lower affinity (high Km) values and of lower maximal velocities. Although the uptake of most of the amino acids examined was affected by the availability of energy in the cell, the differences in uptake seem to be less dependent on the energy status of the cell.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 3351-E, 1991 series     

高粱七种胞质恢保体系的创新与利用

通过6年10代南北育种,对征集的1000份国内外种质资源、育种试材进行大量测交育性鉴定,建立了A1至A7七种胞质恢保体系,进一步拓展和丰富了高粱杂种优势利用的基因型范围,培育出A1至A7七种胞质雄性不育系30多个系列和新胞质杂交种.通过对A1、A2、A3、A5、A6型五种胞质不育的育性恢复基因的遗传机制的研究,找到了A1、A2、A5、A6胞质不育恢复基因的差异和有许多共同恢复系和保持系的原因.研究并分析了A3胞质不育杂交种F2分离比率和A3不育系育性恢复基因的遗传方式,发现了A3胞质不育系小花不败育,培育出矮秆、穗大粒多、配合力强、育性又非常稳定的一批A3型不育系.找到了优点多、实用价值大、长期科学实践中难恢复的不育系--A3不育系的恢复系,进一步配制出A3杂交种并转育了一大批A3恢复系后代材料.     

Sequence analysis of a chloroplast intergenic spacer for phylogenetic estimates in Allium section Cepa and a PCR-based polymorphism detecting mixtures of male-fertile and male-sterile cytoplasmic onion

Restriction-enzyme analysis of the chloroplast (cp) DNA yielded maternal phylogenies supporting a close phylogenetic relationship among normal (N) male-fertile and male-sterile (S) cytoplasmic bulb onion (Allium cepa), Allium altaicum, Allium fistulosum, Allium galanthum, Allium roylei, and Allium vavilovii. The S cytoplasm of onion is most likely an alien cytoplasm introduced in antiquity into onion populations. We previously showed that size differences in an intergenic spacer in the cp DNA distinguish N and S cytoplasms of onion. We cloned and sequenced this intergenic spacer from the N and S cytoplasms of onion, A. altaicum, A. fistulosum, A. galanthum, Allium pskemense, Allium oschaninii, A. roylei, and Allium ampeloprasm (outgroup) to identify the nature of previously described RFLPs and to develop a PCR-based marker revealing N-cytoplasmic contamination of S-cytoplasmic hybrid seed lots. Phylogenies based on restriction-enzyme analysis of the entire cp DNA were similar, but not identical, to those based on sequence divergence in this intergenic region. Received: 29 November 1999 / Accepted: 28 April 2000     

Rapid hybridization-based assays for identification by DNA probes of male-sterile and male-fertile cytoplasms of the sugar beet Beta vulgaris L.

Summary Methods are described whereby hybridization of mitochondrial (mt) DNA with different DNA probes can definitely distinguish male-fertile and and male-sterile (cms) cytoplasms of sugar beet Beta vulgaris L. We have developed two types of miniassays. (1) Comparative methods requiring the isolation and restriction of total cellular DNA, hybridization with cloned mtDNA fragments from either fertile or male-sterile cytoplasms, and comparison of the hybridization patterns to the fertile-and sterile-specific patterns of mtDNA of sugar beet for the given mtDNA probe. For these analyses, we routinely used 1 g of plant material to determine the type of cytoplasm. (2) Noncomparative (plus-minus) methods requiring neither the isolation of pure DNA nor restriction, electrophoresis, or Southern blotting. Instead, alkaline-SDS plant extracts from as little as 50 mg of plant material were dot-blotted and hybridized with fertile-specific (mitochondrial minicircular DNA) and/or cms-specific probes (consisting of a 2.3-kb mtDNA sequence exclusively occurring in the cms cytoplasm). The assays are simple to perform, give definitive results, are nonde-structive to the plants, and may be used in mass screening of sugar beet populations for hybrid production or in in vitro culture processes.     

萝卜细胞质雄性不育恢复基因的RAPD标记

以萝卜恢复系9802和不育系9802A配制杂交组合,并以174株个体组成的F2分离群体作为恢复基因的标记群体.以分离群体的不育株和可育株分别建立不育池和恢复池,利用100个RAPD引物对两池间的多态性进行研究.分析表明引物OPC6在两池间扩增出稳定的多态性差异.经连锁分析,证明标记OPC61900与萝卜细胞质雄性不育恢复基因连锁,遗传距离为11.6cM(Centimorgan).这个标记可应用于对育性恢复基因的标记辅助选择.     

Development and mapping of AFLP markers linked to the sorghum fertility restorer gene <Emphasis Type="Italic">rf4</Emphasis>

The restoration of male fertility in the sorghum IS1112 C (A3) male-sterile cytoplasm is through a two-gene gametophytic system involving complementary action of the restoring alleles Rf3 and Rf4. To develop markers suitable for mapping rf4, AFLP technology was applied to bulks of sterile and fertile individuals from a segregating BC₃F₁ population. Three AFLP markers linked to rf4 were identified and subsequently converted to STS/CAPS markers, two of which are co-dominant. Based on a population of 378 BC₁F₁ individuals, two STS/CAPS markers, LW7 and LW8, mapped to within 5.31 and 3.18 cM, respectively, of rf4, while an STS marker, LW9, was positioned 0.79 cM on the flanking side of rf4. Markers LW8 and LW9 were used to screen sorghum BAC libraries to identify the genomic region encoding rf4. A series of BAC clones shown to represent a genomic region of linkage group E were identified by the rf4-linked markers. A contig of BAC clones flanking the LW9 marker represent seed clones on linkage group E, from which fine mapping of the rf4 locus and chromosome walking can be initiated. Received: 20 June 2001 / Accepted: 3 August 2001     

Diversity among male-sterility-inducing and male-fertile cytoplasms of onion

Hybrid-onion (Allium cepa) seed is produced using systems of cytoplasmic-genic male sterility (CMS). Two different sources of CMS (S and T cytoplasms) have been genetically characterized. Testcrosses of N-cytoplasmic maintaining and restoring genotypes to S and T cytoplasmic lines demonstrated that different alleles, or loci, restore male fertility for these two male-sterile cytoplasms. Other sources of CMS have been used or reported in Europe, Japan and India, and their relationships to S and T cytoplasms are not clear. Restriction fragment length polymorphisms were identified in the organellar genomes among commercially used male-sterile cytoplasms from Holland, Japan and India, and were compared to S and T cytoplasms. Mitochondrial DNA diversity among 58 non-S-cytoplasmic open-pollinated onion populations was also assessed. All five putative CMS lines selected from the Indian population Nasik White Globe were identical to S cytoplasm for all polymorphisms in the chloroplast genome, and always possessed the same-sized mitochondrial fragments as S cytoplasm. T cytoplasm, the male-sterile cytoplasm used to produce the Dutch hybrid Hygro F₁, and two sources of CMS from Japan, were similar and showed numbers of mitochondrial polymorphisms similar to those observed among the 58 non-S-cytoplasmic open-pollinated populations. This research demonstrates that the same, or very similar, male-sterile cytoplasms have been independently isolated and exploited for hybrid-seed production in onion. Received: 27 October 1999 / Accepted: 12 February 2000     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.     

Comparative analysis of gene expression between CMS-D8 restored plants and normal non-restoring fertile plants in cotton by differential display

CMS-D8 and its restorer were developed by introducing the cytoplasm and nuclear gene Rf ₂ from the wild diploid Gossypium trilobum (D8) into the cultivated tetraploid Upland cotton (Gossypium hirsutum). No information is available on how the Rf ₂ gene interacts with CMS-associated genes and how CMS-D8 cytoplasm affects nuclear gene expression. The objective of this study was to identify differentially expressed genes in anther tissues between the non-restoring fertile maintainer ARK8518 (rf ₂ rf ₂) and its isogenic heterozygous D8 restorer line, ARK8518R (Rf ₂ rf ₂) with D8 cytoplasm, by mRNA differential display (DD). Out of more than 3,000 DDRT-PCR bands amplified by 31 primer combinations from 12 anchor primers and 8 arbitrary decamer primers, approximately 100 bands were identified as being qualitatively differentially displayed. A total of 38 cDNA fragments including 12 preferentially expressed cDNA bands in anther were isolated, cloned and sequenced. Reverse northern blot analysis showed that only 4 genes, including genes encoding a Cys-3-His zinc finger protein and aminopeptidase, were up-regulated, while 22 genes, including genes for phosphoribosylanthranilate transferase (PAT), starch synthase (SS), 4-coumarate-CoA ligase, electron transporter, calnexin, arginine decarboxylase, and polyubiquitin, were down-regulated in the heterozygous restorer ARK8518R. The down-regulation of SS explains the lack of starch accumulation in sterile rf ₂ pollen grains in the heterozygous restored plants. The molecular mechanism of CMS and its restoration, specifically the possible roles of SS and PAT genes in relation to restoration of Rf ₂ to CMS-D8, are discussed. This investigation represents the first account of such an analysis in cotton.     

植物雄性不育基因的研究进展

本文概述了植物雄性核不育基因的分子标记及其定位,综述了植物细胞质雄性不育中不育系与保持系在叶绿体和线粒体基因组的结构、转录和翻译产物方面的差异以及和雄性不育之间的可能关系,以及恢复系中的恢复基因分子水平的研究现状;讨论了环境条件光周期和温度对雄性不育的影响在分子水平上的研究现状,指出了植物雄性不育基因研究方面存在的问题和解决贩思路。