Phylogenetic relationships among fertile and petaloid male-sterile accessions of carrot, Daucus carota L.

 Mitochondrial (mt) DNA variation for six petaloid cytoplasmic male-sterile (CMS) and three fertile maintainer lines of carrot was assessed to establish genetic relationships. Total DNA was digested with restriction enzymes and probed with six homologous mtDNA cosmid probes. The six CMS accessions derived from wild carrot, four from Guelph, Ontario, one from Orleans, Massachusetts, and one from Madison, Wisconsin, were more closely related with each other (F=0.91) than with fertile maintainer lines derived from cultivated germplasm (F=0.62). The fertile maintainer lines were likewise found to be more similar to each other (F=0.78) than to the sterile lines. Three sterile lines, originating from wild carrot populations within 1 km of each other in Guelph, Ontario, were most closely related (F=0.96). The high degree of similarity among the six petaloid CMS lines which originated from individual wild carrot plants, some from geographically diverse regions, suggests that the cytoplasm responsible for this trait was imported to, or else evolved, only once in North America. Received: 1 December 1997 / Accepted: 12 December 1997     

Mitochondrial DNA rearrangements in Pennisetum associated with reversion from cytoplasmic male sterility to fertility

Endonuclease restriction fragment patterns of Pennisetum americanum L. mitochondrial DNAs (mtDNAs) from a cytoplasmic male-sterile (CMS-A1), fertile revertants and a normal fertile cytoplasm were variable, while chloroplast DNA from those lines lacked variation. Comparisons between mtDNAs of CMS-A1 (parental) and fertile revertant lines revealed the presence of a unique 4.7 kbp PstI fragment in the sterile line that was not detected in any of the revertant lines. A 9.7 kbp PstI fragment was found in all of the revertants, but not in the CMS-A1. Neither of those fragments was found in the normal cytoplasm mtDNA. Hybridization studies revealed two sets of multiple homologies: 1) the 4.7 kbp fragment had homology with a 10.9 kbp and a 13.6 kbp fragment; and 2) the 9.7 kbp fragment was homologous with the 13.6 kbp fragment. The presence of those two repeated mitochondrial sequences on the altered fragments suggests that they may be involved in the recombinational associated events with reversion from CMS to fertility in P. americanum.Florida Agricultural Experiment Station Journal Series No.7797.     

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).     

Molecular analysis of a new cytoplasmic male sterile genotype in sunflower.

Mitochondrial DNA from 1 fertile and 6 cytoplasmic male sterile (CMS) sunflower genotypes was studied. The CMS genotypes had been obtained either by specific crosses between different Helianthus species or by mutagenesis. CMS-associated restriction fragment length polymorphisms (RFLPs) were found in the vicinity of the atpA locus, generated by various restriction enzymes. The organization of the mitochondrial genes 26S rRNA, 18S + 5S rRNA and coxII was investigated by Southern blot analysis. These genes have similar structures in fertile and all studied sterile sources. Using the atpA probe, 5 from the 6 investigated CMS genotypes showed identical hybridization patterns to the Petiolaris CMS line, which is used in all commercial sunflower hybrids. Only 1 cytoplasm derived from an open pollination of Helianthus annuus ssp. texanus, known as ANT1, contained a unique mitochondrial DNA fragment, which is distinguishable from the fertile and sterile Petiolaris genotypes and from all investigated CMS genotypes. Male fertility restoration and male sterility maintenance of the ANT1 line are different from the Petiolaris CMS system, which is a confirmation that a novel CMS genotype in sunflower has been identified.     

Molecular characterization of a new type of cytoplasmic male sterile sugar beet

A line (named Cl) of cytoplasmic sterility of sugar beet whose cytoplasm derived from Betacicla Turkey was obtained by interspecific hybrid. Its cytoplasm and a spontaneous male sterile cytoplasm from wild beet Beta maritima (named M) were compared with that of Owen's sterile line (S-cms) and a common maintainer of them named N was used as control. RFLP and RAPD methods were mainly used in our experiments. The restriction fragment patterns of mtDNAs were found to be likely but for a few of specific low-lighted electrophoresis bands in Cl. The results of Southern hybridization of six heterogeneous mitochondrial genes as probes to digests of mtDNAs by six restriction enzymes showed to be analogous between S and M lines. But the Cl mtDNA was sorted out by hybridization of atpA probe. Difference of low-molecular-weight mitochondrial DNAs was found among the three sterile lines. Three RNA molecules weighing about 4.2kb stably existed in Cl mitochondria. Our results of RAPD also supported that the Cl cytoplas     

A chimeric <Emphasis Type="Italic">Rfo</Emphasis> gene generated by intergenic recombination cosegregates with the fertility restorer phenotype for cytoplasmic male sterility in radish

In this work, we have identified a chimeric pentatricopeptide repeat (PPR)-encoding gene cosegregating with the fertility restorer phenotype for cytoplasmic male sterility (CMS) in radish. We have constructed a CMS-Rf system consisting of sterile line ‘9802A2’, maintainer line ‘9802B2’ and restorer line ‘2007H’. F₂ segregating population analysis indicated that male fertility is restored by a single dominant gene in the CMS-Rf system described above. A PPR gene named Rfoc was found in the restorer line ‘2007H’. It cosegregated with the fertility restorer in the F₂ segregating population which is composed of 613 fertile plants and 187 sterile plants. The Rfoc gene encodes a predicted protein 687 amino acids in length, comprising 16 PPR domains and with a putative mitochondrial targeting signal. Sequence alignment showed that recombination between the 5′ region of Rfob (EU163282) and the 3′ region of PPR24 (AY285675) resulted in Rfoc, indicating a recent unequal crossing-over event between Rfo and PPR24 loci at a distance of 5.5 kb. The sterile line ‘9802A2’ contains the rfob gene. In the F₂ population, Rfoc and rfob were observed to fit a segregation ratio 1:2:1 showing that Rfoc was allelic to Rfo. Previously we have reported that a fertile line ‘2006H’, which carries the recessive rfob gene, is able to restore the male fertility of CMS line ‘9802A1’ (Wang et al. in Theor Appl Genet 117:313–320, 2008). However, here when conducting a cross between the fertile line ‘2006H’ and CMS line ‘9802A2, the resulting plants were male sterile, which shows that sterile line ‘9802A2’ possesses a different nuclear background compared to ‘9802A1’. Based on these results, the genetic model of fertility restoration for radish CMS is also discussed.     

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.     

优化的反向PCR结合TAIL-PCR法克隆棉花线粒体atpA双拷贝基因及其侧翼序列

双拷贝基因及其侧翼序列的克隆是分子生物学中的一个难点。将优化的反向PCR(Inverse PCR,iPCR)与TAIL-PCR相结合,有效地克隆双拷贝基因及其侧翼序列。先用Southern blotting方法确定一种能获得合适长度片段的限制性内切酶,然后用优化的iPCR方法对该酶切产物进行自连和扩增,将2个拷贝的侧翼序列区分开。根据iPCR结果,进一步用TAIL-PCR扩增更远侧翼的序列。利用这套方法,获得了棉花可育胞质和不育胞质线粒体双拷贝atpA基因的所有EcoR I限制片段(2.2～5.1 kb)和HindⅢ限制片段(8.5～11.7 kb),克隆到2个拷贝各自的侧翼序列。研究结果说明,优化的iPCR与TAIL-PCR相结合是克隆双拷贝基因及其侧翼序列的一种高效方法。     

Linkage analysis of a fertility restoring mutant generated from CMS rice

 DNA polymorphism between a cytoplasmic male-sterile rice line II-32A, the male-fertile maintainer counterpart II-32B, a fertile revertant (T24), as well as two commercial indica restorers, was analyzed with randomly amplified polymorphic DNA (RAPD). A very low degree of polymorphism was found between the revertant T24 and II-32A compared with that of indica rice varieties. This result, together with agronomic and genetic evidence, suggests the revertant to be a product of a nuclear mutation. An analysis of polymorphism between II-32A and the revertant T24 with 510 RAPD decamer primers identified the co-segregating markers OPB07₆₄₀ and OPB18₁₀₀₀ to be linked to a sterile allele of the restoring locus in the revertant T24, at a distance of 5.3 cM. RAPD analysis of a mapping population of Tesanai2/CB with primer OPB07 revealed linkage of OPB07₆₄₀ with RG374 (10.8 cM) and RG394 (8.8 cM) on chromosome 1. Thus the restorer gene, designated Rf ₅, was tentatively localized between RG374 and RG394 on chromosome 1 and appears to be independent of other mapped restorer genes in rice. Received: 11 November 1997 / Accepted: 17 December 1997     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

A comparative study of the atp9 gene between a cytoplasmic male sterile line and its maintainer line and further development of a molecular marker specific for male sterile cytoplasm in kenaf (Hibiscus cannabinus L.)

mtDNA was isolated from cytoplasmic male sterility (CMS) line P3A and its maintainer P3B of kenaf (Hibiscus cannabinus L.). The atp9 gene and its two flanking sequences were obtained using homology cloning and high-efficiency thermal asymmetric interlaced PCR methods. The coding sequences showed only two base pairs difference between the CMS and its maintainer, and shared a homology of over 87 % with atp9 genes from other species in GenBank. However, when comparing the flanking sequences, a 47-bp deletion was characterized at the 3′ flanking sequence of atp9 in the CMS line. Quantitative PCR analysis indicated that the expression level of atp9 in the CMS line was 0.937-fold that of its maintainer. Furthermore, the respiratory rate of anthers in the CMS line was markedly lower than that of its maintainer. The results indicated that the 47-bp deletion at the 3′ flanking sequence of atp9 and/or down-regulated expression of the atp9 gene in the CMS line might be closely related to CMS in kenaf. To confirm whether the 47-bp deletion was specific to cytoplasm of male sterile lines, another 21 varieties were used for further analysis. The results showed that the 47-bp deletion was specific to male sterile cytoplasm (MSC) of kenaf. Based on these, a specific molecular marker was developed to distinguish the MSC from male fertile cytoplasm of kenaf.     

High-rate spontaneous reversion to cytoplasmic male sterility in sugar beet: a characterization of the mitochondrial genomes

Summary Among the fertile sugar beet lines with nuclear sterility maintenance genes, rf, in a homozygous recessive state, sublines capable of reverting spontaneously at a high rate to sterility were identified. Of 24 related fertile sublines studied, 6 were found to spontaneously revert to sterility with a frequency of about 19%. Genetic analysis confirmed the cytoplasmic nature of spontaneously arising sterility. Reversion to sterility in these sublines was accompanied by alterations in the mitochondrial genome structure: loss of the autonomously replicating minicircle c (1.3 kb) and changes in the restriction patterns of high-molecular-weight mitochondrial DNA (mtDNA). Southern hybridixation analysis with cloned minicircle c as a probe revealed no integration of this DNA molecule into the main mitochondrial and nuclear genomes of the revertants. Comparative BamHI and EcoRI restriction analysis of the mtDNA from the sterile revertants and fertile parental subline showed that the spontaneous reversion is accompanied by extensive genomic rearrangement. Southern blot analysis with cloned -subunit of F₁-ATPase (atpA) and cytochrome c oxidase subunit II (COX II) genes as probes indicated that the changes in mtDNA accompanying spontaneous reversion to sterility involved these regions. The mitochondrial genomes of the spontaneous revertants and the sterile analogue were shown to be identical.     

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。     

Unusual Mitochondrial Genome Organization in Cytoplasmic Male Sterile Common Bean and the Nature of Cytoplasmic Reversion to Fertility

Spontaneous reversion to pollen fertility and fertility restoration by the nuclear gene Fr in cytoplasmic male sterile common bean (Phaseolus vulgaris L.) are associated with the loss of a large portion of the mitochondrial genome. To understand better the molecular events responsible for this DNA loss, we have constructed a physical map of the mitochondrial genome of a stable fertile revertant line, WPR-3, and the cytoplasmic male sterile line (CMS-Sprite) from which it was derived. This involved a cosmid clone walking strategy with comparative DNA gel blot hybridizations. Mapping data suggested that the simplest model for the structure of the CMS-Sprite genome consists of three autonomous chromosomes differing only in short, unique regions. The unique region contained on one of these chromosomes is the male sterility-associated 3-kb sequence designated pvs. Based on genomic environments surrounding repeated sequences, we predict that chromosomes can undergo intra- and intermolecular recombination. The mitochondrial genome of the revertant line appeared to contain only two of the three chromosomes; the region containing the pvs sequence was absent. Therefore, the process of spontaneous cytoplasmic reversion to fertility likely involves the disappearance of an entire mitochondrial chromosome. This model is supported by the fact that we detected no evidence of recombination, excision or deletion events within the revertant genome that could account for the loss of a large segment of mitochondrial DNA.     

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated ‘126-1’, was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F₁s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for ‘126-1’ cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of ‘126-1’ cytoplasm for hybrid seed production. The uniqueness of ‘126-1’ has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.     

A variant mitochondrial DNA arrangement specific toPetunia stable sterile somatic hybrids

We have characterized two related regions of twoPetunia mitochondrial genomes in order to understand how plant mt genomes from a cytoplasmic male sterile (cms) line and a fertile line diverge from one another. Restriction maps of these regions indicate that a sequence arrangement shared by the two genomes adjoins sequences which are not shared at the corresponding locations in the two genomes. A point where the mt genomes from the cms line and the fertile lines diverge from each other was identified and mapped. Previously we had observed that somatic hybrids constructed from the cms and the fertile line contained mt genomes carrying new combinations of parental mtDNA restriction fragments (3). Using the restriction maps of the two related mtDNA regions, a mtDNA arrangement unique to the cms parent could be shown to be present in all 17 stable sterile somatic hybrids tested and none of the 24 stable fertile somatic hybrids tested. This data does not exclude the possibility that additional, as yet unidentified, mtDNA arrangements unique to the cms parent might also be found exclusively in sterile somatic hybrids. Whether or not the sterile parental mtDNA arrangement reported here is functionally related to cms, it apparently segregates with cms in somatic hybrids.     

A Moricandia arvensis– based cytoplasmic male sterility and fertility restoration system in Brassica juncea

 A cytoplasmic male-sterility system has been developed in mustard (Brassica juncea) following repeated backcrossings of the somatic hybrid Moricandia arvensis (2n=28, MM)+B. juncea (2n=36, AABB), carrying mitochondria and chloroplasts from M. arvensis, to Brassica juncea. Cytoplasmic male-sterile (CMS) plants are similar to normal B. juncea; however, the leaves exhibit severe chlorosis resulting in delayed flowering. Flowers are normal with slender, non-dehiscent anthers and excellent nectaries. CMS plants show regular meiosis with pollen degeneration occurring during microsporogenesis. Female fertility was normal. Genetic information for fertility restoration was introgressed following the development of a M. arvensis monosomic addition line on CMS B. juncea. The additional chromosome paired allosyndetically with one of the B. juncea bivalents and allowed introgression. The putative restorer plant also exhibited severe chlorosis similar to CMS plants but possessed 89% and 73% pollen and seed fertility, respectively, which subsequently increased to 96% and 87% in the selfed progeny. The progeny of the cross of CMS line with the restorer line MJR-15, segregated into 1 fertile : 1 sterile. The CMS (Moricandia) B. juncea, the restorer (MJR-15), and fertility restored F₁ plants possess similar cytoplasmic organellar genomes as revealed by ‘Southern’ analysis. Received: 17 September 1997 / Accepted: 18 February 1998     

CMS sources in sunflower: different origin but same mechanism?

 The presence of orfH522, orfH708 and orfH873 in the mtDNA, as well as the expression of mitochondrially encoded proteins, were investigated for 28 sources of cytoplasmic male sterility (CMS) and HA89, a fertile line of Helianthus annuus. The whole 5-kb insertion, found in PET1, is also present in all PET1-like CMS sources. However, with regard to the 11-kb inversion ANO1 demonstrated a different organization at the cob locus from the other PET1-like CMS sources. Only orfH873 gave hybridization patterns in all investigated cytoplasms. For the fertile cytoplasm, as well as ANN4, ANN5, ANL1, ANL2, ARG2 and MAX1, hybridizations obtained with orfH708 were highly polymorphic. Hybridization signals with orfH522 were only detectable in the PET1-like CMS sources and MAX1. Comparing the mitochondrially encoded proteins of the CMS sources characteristic patterns could be detected for seven cytoplasms in addition to the PET1-like CMS sources expressing the 16-kDa protein. For ANN1 and ANN3 three CMS-associated proteins of 16.3 kDa, 16.9 kDa and 34.0 kDa could be identified among the in organello translation products. Also ANT1 expressed three additional proteins of 13.4 kDa, 17.8 kDa and 19.7 kDa, respectively. In ARG3 and RIG1 one protein of 17.5 kDa was missing and instead a new protein of 16.9 kDa appeared. In addition, in GIG1 and PET2 a unique protein of 12.4 kDa could be identified. These results indicate that certain types of cytoplasmic male sterility are preferentially present in sunflower. Received: 15 June 1998 / Accepted: 13 July 1998     

The Mitochondrial Genome Organization of a Maize Fertile Cmst Revertant Line Is Generated through Recombination between Two Sets of Repeats

The mitochondrial genome (mtDNA) organization from a fertile revertant line (V3) derived from the maize cytoplasmic male sterile type T (cmsT) callus tissue culture has been determined. We report that the sequence complexity can be mapped on to a circular ``master chromosome' of 705 kb which includes a duplication of 165 kb of DNA when compared to its male sterile progenitor. Associated with this event is also a 0.423-kb deletion, which removed the cmsT-associated urf13 gene. As found for the maize normal type (N) and cmsT mitochondrial genomes, the V3 master chromosome also exists as a multipartite structure generated by recombination through repeated sequences.     

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.