Obtaining an Ogura-type CMS line from asymmetrical protoplast fusion between cabbage (fertile) and radish (fertile)

Cytoplasmic male sterile (CMS) plants were obtained by asymmetrical protoplast fusion between red cabbage (fertile) and normal radish (fertile). The CMS plants showed restriction fragment length polymorphism (RFLP) patterns of mitochondrial (mt) DNA that were different from those of both parental lines. PCR analysis of mtDNA of the CMS plants and though Southern blot analysis showed that the mtDNA of the CMS line had the characteristics of the ogura CMS type. The results suggested that the orf138 gene and the ogura type atp6 gene are present in normal fertile cabbage and radish at a low copy level. This revised version was published online in July 2006 with corrections to the Cover Date.     

小麦T、 K、 V型胞质不育系和杂种mtDNA的RAPD分析及育性相关片段的克隆

用RAPD技术对细胞质分别来源于粘果山羊草（Ae.kotschyi）、偏凸山羊草（Ae.ventricosa）、提莫菲维小麦（T.timopheevii）的三种普通小麦雄性不育系--K型、V型、T型及相应的保持系、恢复系以及杂种F1代的线粒体DNA（mtDNA）进行了比较分析。结果如下：（1）发现在mtDNA组织结构上K型、V型、T型不育系之间以及与保持系之间均     

Changes in sugar beet mitochondrial DNA induced during callus stage

The organization of mitochondrial DNA was studied in seed‐derived plants of sugar beet, Beta vulgaris L., and compared with the structure of DNA isolated from calli propagated in vitro. Tissues representing three genotypes (diploid male sterile, diploid maintainer and diploid fertile) were maintained under in vitro conditions for different periods before the DNA was analysed. Restriction fragment length polymorphism with BamHI and XhoI enzymes, and Southern hybridization with atpA and atp6 homologous probes were used. Hybridization experiments showed conspicuous differences in the organization of both genes in long‐term callus cultures (2 years or older). The novel organization of mitochondrial DNA in the calli of the male sterile genotype showed an additional 3.9 kb fragment after hybridization with the atp6, and 7.9 kb and 3.5 kb bands after hybridization with atpA. In the fertile genotype, changes in mitochondrial DNA structure were manifested in the disappearance of a 2.1 kb fragment after probing with atpA. Alterations induced in the mitochondrial genome of the male sterile line showed unique features in mtDNA rearrangements.     

RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton

Cytoplasmic male sterility (CMS) in higher plants is a maternally inherited trait and CMS-associated genes are known to be located in the mitochondrial genome. However, CMS-inducing genes in CMS-D2 and CMS-D8 of Upland cotton (Gossypium hirsutum L., AD1) are currently unknown. The objective of this study was to identify potential candidate DNA or gene sequences for CMS-D2 and CMS-D8 through restriction fragment length polymorphism (RFLP) analysis. Seven mtDNA gene probes and five restriction enzymes were first used to compare D2 (from G. harknessii Brandegee) and AD1 cytoplasms. With cox1, cox2, and atp1 as probes, RFLP polymorphisms were detected with one or more restriction enzyme digestions. The most notable difference was an additional fragment in the normal AD1 cytoplasm detected by cox2 in digests of three enzymes, and by cox1 and atp1 in digests with PstI. The RFLP analysis was then conducted among CMS-D2, CMS-D8 (from G. trilobum (DC.) Skovst.), and AD1 cytoplasms. Two probes from maize, atp1 and atp6, detected polymorphism among the different cytoplasmic lines. However, no difference in RFLP patterns was noted between male sterile (A) and restorer (R) lines with the D2 or D8 cytoplasm, indicating that the presence of the D2 or D8 restorer gene does not affect mtDNA organization in Upland cotton. The results demonstrate that RFLP using atp1 and atp6 as probes can distinguish the three cytoplasms. The atp1 and atp6 in CMS-D8 and these two genes together with cox1 and cox2 in CMS-D2 could be the candidates of CMS-associated genes in the mitochondrial genome, providing information for further molecular studies and developing PCR-based markers for the CMS cytoplasms in breeding. This research represents the first work using RFLP to analyze the genetic basis of CMS in cotton.     

RFLP analysis of cytoplasmic male-sterile lines of pigeonpea [Cajanus cajan (L.) Millsp.] developed by interspecific crosses

Total DNA from three putative cytoplasmic male sterile (CMS) progenies derived from crosses between the wild species Cajanus sericeus and the cultivated species Cajanus cajan, five C. cajan, one accession of C. sericeus and two genetic male sterile lines of pigeonpea were compared for their RFLP patterns using maize mitochondrial DNA (mtDNA) specific probes. Three putative cytoplasmic male sterile (CMS) progenies from the multiple cross genome transfer of pigeonpea lines (CMS 7–1, CMS 12–3, and CMS 33–1) showed hybridization patterns identical to that of C. sericeus when DNA was digested with EcoRI and HindIII and probed with maize mtDNA clones. The results suggested that these putative CMS progenies have the mitochondria of the female wild species parent. The hybridization patterns of the three male parental lines used in the development of the CMS progenies were similar in all the restriction enzyme-probe combinations except HindIII-atp6. The genetic male sterile lines, MS Prabhat and QMS 1 differed from each other in their hybridization pattern. The genomic DNA hybridization pattern of HindIII digested DNA from ICPL 87 differed from the other pigeonpea lines when probed with the maize mtDNA clones. The cluster analysis of the hybridization data suggested the occurrence of variation in the mitochondrial genome even among the cultivated species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of the Putative Cytoplasmic Donor of a CMS System in Brassica juncea

Chloroplast (cp) and mitochondrial (mt) DNA restriction profiles of a cytoplasmic male sterile (CMS) line of Brassica juncea and its maintainer line were compared and found to be markedly different. Comparison of cpDNA restriction profiles of fifty different species of genus Brassica and some allied genera showed that the cpDNA profiles of CMS lines were similar to that of B. tournefortii for twenty different restriction endonucleases. This CMS system is thus not of spontaneous origin as reported earlier, but is alloplasmic in nature. Comparison of restriction profiles of mtDNA of B. tournefortii and CMS lines revealed some differences which might either be due to changes in DNA pattern during the transfer, or, due to the cytoplasm coming from a B. tournefortii line different from the one used in this study.     

水稻野败不育系与保持系线粒体DNA限制酶酶切图谱分析

采用限制酶酶切片段长度多态性（ＲＦＬＰ）分析广泛比较了水稻野败不育系及其籼型保持系线粒体ＤＮＡ酶切图谱。野败不育系与籼型保持系线粒体ＤＮＡ在组成上存在十分明显的差异。根据具有相同迁移位置与大小的片段比例判断，这２种不同细胞质线粒体ＤＮＡ顺序同源性约为３０％。分子杂交结果显示，编码功能蛋白的基因及其邻近顺序存在在相当程度的保守性。本文并对线粒体ＤＮＡ的组成变异与细胞质雄性不育的关系进行了分析与讨论。     

水稻质核互作雄性不育系选育的反向杂交法研究

不同的部分保持系可能存在不同的微效恢复基因,通过有性杂交,产生基因重组,能够获得完全保持系类型,但只有在不育细胞质中才能观察得到微效恢复基因是否存在以及它们的作用大小.反向杂交法以不育细胞质为选择背景,在杂交后代植株中直接观察到微效恢复基因的表达,获得的完全不育株,在一定程度上排除微效恢复基因,不育株再通过高温处理转换为可育后自交,来自不育株的微效恢复基因可以进一步排除掉,从而产生出没有(或很少)微效恢复基因的"亲本",利用该"亲本"高温处理后仍可转换为可育的特性,作为父本进行杂交或回交育种,在其后代中获得没有微效恢复基因的完全保持系.该研究为Cp 26不育细胞质源创造出了完全保持系.如果在田间鉴定出优异的完全不育株,对其进行单倍体育种(诱导孤雌生殖或花培),选择到没有(或很少)微效恢复基因的纯合不育株.再对其进行花培,筛选可育的突变体;或者利用纯合不育株的原生质体与一个已破坏细胞核的可育系原生质体融合,都可能得到具有纯合不育株细胞核和可育细胞质的保持系,而能够完善和改进反向杂交法.反向杂交法不但能够为所谓不能保持的不育细胞质源创造出保持系,而且有利于加强新不育系选育的目标性和预见性,提高不育系配合力和培育不同类型优异不育系.     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. IV. Ogura-type cytoplasm found in the wild radish, Raphanus raphanistrum

The orf138 gene, which is specific to Ogura male-sterile cytoplasm, was analysed in mitochondrial DNA (mtDNA) of the wild radish, Raphanus raphanistrum, by polymerase chain reaction (PCR), Southern hybridization and sequencing. The effect of R. raphanistrum cytoplasm on the expression of male sterility was also examined in progeny with R. sativus. A PCR-aided assay and Southern hybridization revealed that three out of six strains analysed included plants with orf138. The sequence of wild type orf138 was same as that of Ogura, except for one or two nucleotide substitutions. Southern hybridization showed a novel mtDNA configuration in R. raphanistrum, in addition to the normal and Ogura types identical to those in R. sativus. Among interspecific hybrids, all the F₁ had normal pollen fertility. In the F₂ progeny between female wild plants having orf138 and the maintainer of Ogura male sterility, male-sterile plants were segregated, fitting the ratio of 3 fertile: 1 sterile plant. R. raphanistrum has cytoplasm that induces male sterility in radishes, and contains a dominant fertility restorer gene.     

Molecular analysis of the fourth progeny of plants derived from a cytoplasmic male sterile chicory cybrid

Cytoplasmic male sterile (CMS) chicory cybrids have previously been obtained by fusion between chicory and CMS sunflower protoplasts. Preliminary restriction fragment length polymorphism analysis has shown mitochondrial recombination events in CMS plants and their progeny. The aim of this study was to investigate the fate of the mitochondrial genome of the fourth progeny derived from one CMS cybrid. Southern hybridization using several specific mitochondrial sequences as probes have revealed polymorphic patterns between the plants analysed and, consequently, the genetic instability of this genome. Presence of the sequence responsible for cytoplasmic male sterility in sunflower (orf522) was detected by polymerase chain reaction and confirmed by molecular hybridization in the CMS chicory plants; however, its part in CMS expression in chicory did not appear obvious. Moreover, different flowering phenotypes, including completely fertile plants occurred and no correlation could be established between the different mitochondrial profiles and the flowering types. These results suggested some hypotheses and questions about the molecular determinant of CMS in chicory, its regulation and expression and questions about the mitochondrial-nuclear interactions.     

Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.)

The main objective of the present study was to identify mitochondrial DNA based marker, which can distinguish male sterile and fertile counterparts of the cytoplasmic male sterile (CMS) lines used in production of rice hybrids. Amplified fragment length polymorphism (AFLP) analysis in CMS lines: IR58025A & IR62829A and their respective maintainers: IR58025B & IR62829B identified a polymorphic DNA fragment of about 510 bp size that was present in both CMS (A) and absent in their maintainer (B) lines. Sequencing followed by database analysis of the polymorphic fragment indicated about 97% similarity with mitochondrial NADH gene subunits of rice, maize and wheat. Based on the variable sequence regions, a site specific primer pair (BF-STS-401) was designed. PCR analysis showed that BF-STS-401 could amplify a strong band of 464 bp size in CMS and a faint band of the same size in maintainer line. To act as a positive control and avoid possible errors in PCR, BF-STS-401 was multiplexed with a new primer pair (BF-STS-402), derived from mitochondrial atp9 subunit of rice, producing monomorphic amplification indiscriminately in both CMS and maintainer lines. Both the primer pairs in combination clearly differentiated CMS lines from their corresponding maintainer lines. This primer combination was validated in a set of diverse genotypes consisting of different sources of CMS lines, restorer lines, hybrids, varieties and mixed samples from private seed companies. Our results suggested that the multiplex primer pairs developed in this study can be effectively utilized to assess the genetic purity in commercial seed lots of CMS lines and hybrids of rice.     

Molecular characterization of cytoplasmic male sterility conditioned by <Emphasis Type="Italic">Gossypium harknessii</Emphasis> cytoplasm (CMS-D2) in upland cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and to develop CMS-specific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed that the three genes were differentially expressed between the two lines. These results indicated that there existed structural and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from ATG codon were also changed between the two cytoplasms (i.e., CG→AA), and this mutation also exists in RNA sequences. Interestingly, nine nucleotides (ATGCAACTA) were also inserted at the location of 899 bp upstream of ATG codon in the CMS line. The results suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either normal- or CMS-D2cytoplasm. This will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F₁ seed lots.     

A short review of research on male sterility and prospects for F1 hybrid varieties in field beans (Vicia faba L.)

Summary It has not yet been possible to utilise cytoplasmically inherited male sterility (CMS) in Vicia faba, in the commerical production of F₁ hybrids. The main problem has been a rapid increase in the proportion of fertile revertants during multiplication. Investigations, mainly in France, have clarified environmental causes of phenotypic instability, CMS and maintainer lines with improved stability have been selected, and two new cytoplasms have been produced by mutation of the first two. CMS is known to be associated with cytoplasmic spherical particles, and recognisable types of double stranded RNA, and there may also be an association with mitochondrial DNA, and polypeptides of mitochondrial origin.The better understanding of CMS may eventually lead to more easily managed forms, either through genetic manipulation or biochemical assays that breeders can use for screening for stability.     

线粒体DNA由来的RAPD标记能鉴别水稻雄性不育和可育细胞质

以野败型细胞质的水稻雄性不育系珍汕97A及其保持系珍汕97B的总DNA为模板,从100个引物中筛选到OPA12对珍汕97A能扩增出一条1600 bp的特异带.用OPA12扩增野败型细胞质的龙特浦A及其保持系龙特浦B、矮败型细胞质的协青早A及其保持系协青早B、恢复系明恢63、珍汕97A/明恢63的F1和F2个体的总DNA,不育系、F1和F2所有调查个体都有16     

Random amplified polymorphic DNA analyses of cytoplasmic male sterile and male fertile Pigeonpea (Cajanus cajan (L.) Millsp.)

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of two pigeonpea cytoplasmic genic male sterile (CMS) lines derived from crosses between the wild (Cajanus scarabaeoides & C. sericeus) and the cultivated species of Cajanus cajan. The male sterile (A) line and its maintainer (B) line could be easily differentiated with certain random primers. The two male sterile (288 A and 67 A)systems are based on C. scarabaeoidesand the other is based on C. sericeus could also be differentiated. Amplification product of 600bp amplified by primer OPC-11 was observed in both the cytoplasmic male sterile lines (288 A and 67A), which was absent in the maintainer lines (288 B and 67 B) and the putative R-line (TRR 5 and TRR 6). CMS lines, putative R lines, other cultivars and wild species under the study could be easily distinguished with the help of different primers. Dendrogram constructed based on the similarity index showed that considerable genetic variation exist sbetween CMS lines, two putative R line and wild species studied. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of male-sterile cytoplasm in resistance to barley yellow mosaic virus and Fusarium head blight in barley

Two types of male‐sterile cytoplasm, designated msm1 and msm2, in barley were investigated to determine whether these cytoplasms confer resistance to barley yellow mosaic virus (Ba YMV) and Fusarium head blight (FHB). Alloplasmic lines and isogenic lines of two cultivars showed the same reaction to each Ba YMV as that of their euplasmic lines. This demonstrates that the barley male‐sterile cytoplasms msm1 and msm2 have no effect on resistance to BaYMV. No significant difference in reactions to FHB was recognized among fertile alloplasmic lines of ‘Adorra’, but the difference in reactions to FHB between fertile and sterile isogenic lines of ‘Adorra’ was significant. The damage caused by FHB in the male‐sterile lines that produced sterile pollen was significantly greater than the damage in a sterile line that did not produce pollen. These results suggest that pollen or anthers are important factors in infection with or spread of FHB. For production of hybrid seeds, male‐sterile lines with no pollen production, such as those with msm1 male‐sterile cytoplasm, would reduce FHB infestation.     

Isolation and genetic characterization of a fertility-restoring revertant induced from cytoplasmic male sterile rice

Summary A male fertile revertant was isolated from M₁ of a cytoplasmic male sterile indica rice line II-32A, the dry seeds of which were treated with ⁶⁰Co- rays at a dose of 290 Gy. The acquired revertant T24 was morphologically and agronomically similar to II-32B, the maintainer of II-32A. Testcrosses of the revertant with II-32A and Zhenshan 97A showed that the revertant was able to restore the fertility of CMS lines. Genetic analysis of the progenies between T24 and II-32A, Zhenshan 97A XieqingzaoA and DZhenshan 97A, which have different cytoplasms, showed that the fertility restoration of four CMS lines by T24 involved one nuclear gene, indicating that T24 was a result of the mutation of one nuclear gene. The mechanism of the restoration of CMS line by T24 was obviously different from other restorers such as Minghui 63 and 20964, which were shown to behave in two gene mode in fertility restoration. The discovery of the revertant T24 contributes to the understanding of CMS and fertility restoration of CMS in rice. The T24 and its parent II-32A may constitute a pair of near isogenic lines for the restoring gene, which should be valuable materials for molecular genetic analysis of CMS.     

Chromosomal location of fertility restoring genes for ‘wild abortive’ cytoplasmic male sterility using primary trisomics in rice

Summary Identification and location of fertility restoring genes facilitates their deployment in a hybrid breeding program involving cytoplasmic male sterility (CMS) system. The study aimed to locate fertility restorer genes of CMSWA system on specific chromosomes of rice using primary trisomics of IR36 (restorer), CMS (IR58025A) and maintainer (IR58025B) lines. Primary trisomic series (Triplo 1 to 12) was crossed as maternal parent with the maintainer line IR58025B. The selected trisomic and disomic F₁ plants were testcrossed as male parents with the CMS line IR58025A. Plants in testcross families derived from disomic F₁ plants (Group I crosses) were all diploid; however, in the testcross families derived from trisomic F₁ plants (Group II crosses), some trisomic plants were observed. Diploid plants in all testcross families were analyzed for pollen fertility using 1% IKI stain. All testeross families from Group I crosses segregated in the ratio of 2 fertile: 1 partially fertile+partially sterile: 1 sterile plants indicating that fertility restoration was controlled by two independent dominant genes: one of the genes was stronger than the other. Testcross families from Group II crosses segregated in 2 fertile: 1 partially fertile+ partially sterile: 1 sterile plants in crosses involving Triplo 1, 4, 5, 6, 8, 9, 11 and 12, but families involving triplo 7 and triplo 10 showed significantly higher X² values, indicating that the two fertility restorer genes were located on chromosome 7 and 10. Stronger restorer gene (Rf-WA-1) was located on chromosome 7 and weaker restorer gene (Rf-WA-2) was located on chromosome 10. These findings should facilitate tagging of these genes with molecular markers with the ultimate aim to practice marker-aided selection for fertility restoration ability.     

Characterization for agronomic use of cytoplasmic male-sterility in sunflower (Helianthus annuus L.) introduced from H. resinosus Small

A backcrossing programme was carried out both to assess the stability of a cytoplasmic male‐sterility (CMS) source from Helianthus resinosus, designated RES1, and to incorporate it into inbred sunflower lines (HA89, RHA271, RHA801). All the progenies, grown in different environments, were completely male‐sterile. This suggests that the expression of this cytoplasm is stable. Female‐fertility of lines HA89, RHA271 and RHA801 carrying CMS RES1 were compared with those of the corresponding fertile inbred lines. There were no differences in the number of seeds per head. This indicates that female‐fertility is not affected by RES1 cytoplasm. Cytological studies showed that meiosis proceeds normally until the tetrad stage; consequently, the absence of pollen is caused by alterations that take place during postmeiotic stages. With the aim of identifying male‐fertility restorer genotypes, crosses were made between HA89 (CMS RES1) plants and different annual diploid and perennial hexaploid Helianthus species. All the diploid germplasm evaluated behaved as a CMS RES1 maintainer. However, the hexaploid species, H. resinosus, H. x laetiflorus, H. pauciflorus and H. tuberosus, restored pollen fertility in CMS RES1 plants.     

Introgression of a gene restoring fertility to CMS (Trachystoma) Brassica juncea and the genetics of restoration

A dominant gene restoring fertility to a cytoplasmic male sterile (CMS) line of Brassica juncea was derived from the somatic hybrid Trachystoma ballii+B. juncea. Its introgression resulted from forced pairing between chromosomes of the cultivar ‘Pusa Bold’ and chromosomes of the fusion hybrid. Segregation ratios of this fertility restorer gene followed a monogenic pattern. The introgression of the fertility restorer gene did not cause any abnormalities, such as reduced fertility; pollen and seed fertilities of the restored plants were over 90%. Restored fertile and CMS plants exhibited similar Southern hybridization patterns when probed with the mitochondrial probe atp6.