An extreme cytoplasmic bottleneck in the modern European cultivated potato (Solanum tuberosum) is not reflected in decreased levels of nuclear diversity

We have used the polymorphic chloroplast (cp) and nuclear simple sequence repeats (SSRs) to analyse levels of cytoplasmic and nuclear diversity in the gene pool of the European cultivated potato (Solanum tuberosum ssp. tuberosum). Primers designed from the complete chloroplast sequence of tobacco (Nicotiana tabacum) were used to amplify polymorphic products in a range of potato cultivars. Combining the data from seven polymorphic cpSSR loci gave 26 haplotypes, one of which (haplotype A) accounted for 151 out of the 178 individuals studied and corresponded to the T-type cytoplasm previously identified in cultivated potatoes using chloroplast restriction fragment length polymorphism analysis. Phylogenetic and diversity analyses of the relationships between cpSSR haplotypes confirmed much higher levels of cytoplasmic diversity outwith the T-type group. Diversity levels at eight nuclear SSR loci, however, were not significantly different between cytoplasmic groups, suggesting a severe maternal bottleneck in the evolution of the modern cultivated potato. These results highlight the importance in quantifying levels of cytoplasmic as well as nuclear diversity and confirm the need for a change in breeding practices to increase levels of non-T-type cytoplasm in the cultivated gene pool, thus helping reduce problems associated with pollen sterility. This may be facilitated by germplasm analysis using cpSSRs, which will allow efficient selection of diverse cytoplasm donors.     

细胞质雄性不育高粱叶绿体 ndh D 基因的序列变异

片段SAAU－02 700特异地扩增自7种具可育细胞质的高粱材料的总DNA,含有叶绿体psa C(88bp)和ndh D(192bp)基因的部分序列。该片段与Eco Ri HindⅢ酶切的总DNA,线粒体DNA和叶绿体DNA杂交,在总DNA中获得了0.74kb的杂交带,而在叶绿体中获得0.74kb和0．45kb两条杂交带。与线粒体DNA无杂交;与经Hae Ⅲ酶切的总DNA杂交,在不育系中获得4．9kb的杂交带,而保持系的杂交带为4．45kb。参考GenBank中高粱的近缘物种玉米叶绿体基因组的序列,构建了ndh D基因区的酶切位点图谱,借此分析得出高粱不育系的叶绿体ndh D基因序列已发生改变。这种变异与高粱细胞质雄性不育反生的关系正在探讨中。     

The structure of sterile cytoplasm types within a maize genebank collection

Maize Research Institute (MRI) gene bank maintains a collection of 6000 maize accessions. Within this collection over 100 sources of cytoplasmic male sterility (CMS) were found in field trials, i.e. more than 2% of the total accession numbers. These sources are distributed among Yugoslav open-pollinated varieties (4.56% of them contain CMS), as well as introduced heterozygous genotypes and inbred lines. In order to identify cytoplasm types the gene-bank sources of CMS were screened using a PCR assay with specific primers for C, T and S cytoplasms. Predominant cytoplasmic male sterility type among the analyzed accessions was CMS-S. Results were inconclusive for three accessions, i.e. different results for the progenies of two ears per accession were obtained. For another two accessions a new PCR product profile was identified, consisting of one band characteristic for CMS-S and one unspecific for any of the three CMS types. The PCR approach enabled a simple, fast and reliable large scale screening of maize cytoplasm among MRI gene bank accessions, significantly reducing time for cytoplasm characterizations compared to classical method of testing with restorers for each known type of CMS.     

Structural and Expressional Variation Analyses of Mitochondrial Genomes Reveal Candidate Transcripts for the SV Cytoplasmic Male Sterility in Wheat (Triticum aestivum L.)

Common wheat (Triticum aestivum L.) is one of the most important crops,and intra-specific wheat hybrids have obvious heterosis in yield and protein quality.Therefore,utilization of hybrid wheat varieties offers an effective way to increase yield and nutrition.Cytoplasmic male sterility (CMS) systems are a useful genetic tool for hybrid crop breeding,and are ideal models for studying the genetic interaction and cooperative function of mitochondrial and nuclear genomes in plants (Schnable and Wise,1998;Hanson and Bentolila,2004).The breeding of hybrid wheat using male sterility caused by the cytoplasm of T.timopheevii has been studied since the early 1960's.But it is unsuccessful because there are some deficiencies in the practical application of this cytoplasm,including limited restoration resources,thin seeds,pre-harvest sprouting and lower germination rate (Wilson and Ross,1962).The Sv type of cytoplasmic male sterility (CMS-Sv) in wheat is general accessions for four types of CMS lines that were derived from four Aegilops species:Ae.kotschyi,Ae.variabilis,Ae.ventricosa,and Ae.bicornis.Based on the observation of alloplasmic lines produced in all possible combinations between 12 wheat nuclear genotypes and 47 cytoplasms of two related genera,Triticum (wheat) and Aegilops,Ogihara and Tsunewaki (1988) concluded that the D2-cytoplasm of Ae.crassa and its relatives,N-cytoplasm of Ae.uniaristata,and SV-cytoplasm of Ae.kotschyi and its relatives might be used as the alternative male sterile cytoplasm to replace the T.timopheevii cytoplasm for hybrid wheat breeding.Ikeguchi et al.(1999) proposed that spring-type hybrid wheat may be bred by combination of the 1BL-1RS chromosome and Ae.kotschyi cytoplasm with a new fertility-restorer gene discovered in a wheat variety Kitamiharu 48.Zhang et al.(1996) also proposed the use of CMS-Sv lines as the most effective male sterile cytoplasm.     

Paternal inheritance of mitochondria in rapeseed (Brassica napus)

Summary Transfer of a mitochondrially associated plasmid following sexual crosses in Brassica napus rapeseed suggested that paternal mitochondria were being transferred to the cytoplasm of the egg. To examine this possibility further, plants carrying the chloroplast (cp) marker of triazine resistance, but which had lost the plasmid associated with the mitochondria of this cytoplasm, were crossed as females to males carrying the polima cytoplasm. The males carried a nuclear fertility restorer gene on an extra chromosome to overcome the male sterility marker conferred by the mitochondria of this cytoplasm. Approximately 10% of the F₁ progeny displayed the male sterility and flower morphology of the male parent. Mitochondrial (mt) DNA from the progeny showed the combined restriction patterns of both parents, but this rut heterogeneity did not continue into subsequent generations. All progeny retained the cp DNA restriction patterns of the maternal plant as well as resistance to the herbicide atrazine. To date, sexually mediated cybrid plants have shown no morphological abnormalities and have maintained their unique combination of cp and mt traits through several sexual generations.     

DNA polymorphism in Allium cepa cytoplasms and its implications concerning the origin of onions

Summary Mitochondrial and chloroplast DNA was isolated from fertile and cytoplasmic male sterile cultivars of cultivated onions. Restriction fragment length polymorphism led to the distinction between cytoplasms S and M. Mitochondrial DNA patterns from S cytoplasms appeared dentical and characterized mostly male sterile lines. An open-pollinated variety was found to bear this cytoplasm and thought to be the origin of S types. Mitochondrial DNA patterns from M cytoplasms were subdivided into four types, M₁ and M₂ corresponding to normal N cytoplasm, M₃ and M₄ probably corresponding to T cytoplasms. S and M cytoplasms were also distinguished by chloroplast DNA restriction patterns. Our results confirm previous genetic distinction between S, N and T cytoplasms.     

二角型非1B/1R 小麦CMS不育恢复体系的建立

通过对具有二角山羊草细胞质的1B/1R型小麦雄性不育系m s(A e.bicorn is)-5-1回交置换,获得了新型二角山羊草细胞质小麦雄性不育系m s(A e.bicor)-V 9125和m s(A e.bicor)-M 853.利用染色体制片、分子标记、原位杂交和酸性聚丙烯酰胺凝胶电泳对其保持系V 9125和M 853进行分子细胞遗传学检测,并对其育性恢复性机理进行了初步研究.结果表明:(1)V 9125和M 853均为非1B/1R易位系.V 9125为小麦-簇毛麦易位系,m s(A e.bicor)-V 9125是二角山羊草细胞质与普通小麦核背景中簇毛麦外源染色体互作产生的一类新的核质互作不育系.(2)M 853为小麦-滨麦易位系,m s(A e.bicor)-M 853是二角山羊草细胞质与普通小麦核背景中滨麦外源染色体互作产生的一类新的核质互作不育系.(3)利用一些普通小麦品种(系)与这两个不育系杂交,m s(A e.bicor)-V 9125和m s(A e.bicor)-M 853仅与T 6-3杂交育性得到恢复,且恢复度较高,变异小,而与其它大部分普通小麦杂交F1表现雄性不育,且扬花期花药不外露.而m s(A e.bicor)-5-1与包括T 6-3在内的普通小麦品种(系)杂交F1育性得到不同程度的恢复.从而得出结论,二角山羊草细胞质与小麦细胞核的互作存在两个不同的不育-恢复系统.     

RAPD Comparison Study on Mitochondrial DNA of the D2 Cytoplasmic Male Sterile (CMS) Line with K-, V-and T-type CMS Lines in Wheat

A new line of cytoplasmic male sterile (CMS) wheat ( Triticum aestivum L. ) named msD2-CA8057 (briefly as D:) was compared with K-, V- and T-type CMS lines by mitochondrial DNA (mtDNA) RAPD analysis. It was revealed that the mtDNA of this D2 line was different from that of the other three types although differences were also found between the K-, V- and T-type lines themselves. This result provided some evidences at the molecular level for the identification of the cytoplasm of this D2 line as a new type of CMS line. In the experiment the authors also found polymorphism between two CMS lines, which had the same source of T-type cytoplasm but had different nuclear background. This was the first time to provide direct evidence about the mtDNA mutation in T-type CMS wheat lines during the routine backcross process. Such changes are most likely resulted from the influence of different nuclear background.     

细胞融合与植物细胞质雄性不育性状改良

原生质体融合技术不仅可以实现核基因组的重组,而且可以实现胞质基因组的重组,为转移由线粒体DNA或叶绿体DNA控制的性状提供了一条捷径。本文综述了获得胞质杂种的方法及其原理,细胞融合在胞质转移及创造新的细胞质雄性不育(CMS)资源上的研究进展及其应用情况,以及CMS在有性后代中的遗传规律,探讨了此育种途径的优势及其在木本果树无籽育种上的应用。     

Cybrids and tetrad sterility for developing true potato seed hybrids

Potato cybrids result from the fusion between cytoplasm and nuclear gene donors. Such genetic materials are an alternative means to broaden the breeding pool by non‐sexual gene transfer. Tetrad pollen sterility provides also another source of male sterility with some potential for true potato seed breeding. The objective of this research was to investigate cybrid‐derived offspring for both agronomic and reproductive characteristics in two contrasting Peruvian locations, and to examine new exotic germplasm for tetrad sterility, with the aim of broadening the breeding pool available at the Centro Internacional de la Papa (CIP). The cybrids were derived from fusions between Y‐245.7, a clone with tetrad sterility, and Atzimba. These cybrids were crossed with selected male parents from the CIP breeding population, and their hybrid offspring were tested in La Molina (coastal desert) and Huancayo (cool highlands). In addition, other clones with tetrad sterility were also crossed with selected testers to determine their breeding value. There were significant differences for tuber yield, style length, and berry number among the hybrid offspring, and the genotype by environment interaction was significant for tuber yield and berry number. The top 25% highest yielding cybrid‐derived offspring across both locations showed the same tuber yield although they were significantly different for some of the reproductive characteristics. With the exception of one cybrid, the others did not exhibit segregation for tetrad sterility in their hybrid offspring, which were male fertile. However, the offspring derived from crosses between other sources of tetrad sterility and the same testers all showed tetrad sterility, and some of them had outstanding tuber yield at La Molina. The lack of segregation for tetrad sterility in these new crosses suggests that the non‐cybrid, male sterile, female parents are triplex or quadriplex for the Tr nuclear locus, which interacts with a sensitive cytoplasm (e.g. Trs from S. verrucosum or S. stoloniferum) to produce tetrad sterility in potato.     

Determination of cytoplasmic male sterile factors in onion plants (Allium cepa L.) using PCR-RFLP and SNP markers

We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Cultivated potato chloroplast DNA differs from the wild type by one deletion — evidence and implications

Summary The chloroplast DNA (ctDNA) of Solanum tuberosum ssp. tuberosum (T type) and S. chacoense (W type) yield five different restriction fragment patterns with five different restriction endonucleases. DNA-DNA hybridization tests revealed that these differences were all caused by one physical deletion (about 400 bp in size) in the ctDNA of ssp. tuberosum. This suggests that T type ctDNA of the common potato and of Chilean tuberosum originated from W type ctDNA. The deleted region of the T type ctDNA is probably not concerned with gene-cytoplasmic male sterility.Reference to a specific brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned     

Molecular genetic analysis of maize mitochondrial regions associated with CMS

The Mmolecular-genetic polymorphism of 86 maize lines of world and Ukrainian breeding with S-, C- and T-types of cytoplasmic male sterility (CMS) and with wild-type mitochondra was studied by PCR analysis of mitochondrion regions. A molecular marker system was able to detect and identify a certain type of CMS in the maize lines and differentiate maize lines with a certain type of CMS from both lines with a different type of CMS and those with the wild-type cytoplasm.     

水稻籼型光温敏核雄性不育性遗传研究

1　引　　言水稻光温敏核雄性不育性是一种典型的生态遗传现象 ,其遗传行为既受内部基因控制 ,又受外部光、温等生态因子的调节 ,还与所处的遗传背景密切相关 .前人已对农垦 5 8Ｓ及其衍生系等粳型光温敏核不育性有过较系统地研究 ,并提出一对、二对、三对和重复基因突变等多种假说[3 ,5,7～ 9] ;但对籼型及非农垦 5 8Ｓ基因源的光温敏核不育性研究较少 .本文采用极大似然法 ,对不同来源的籼型光温敏核不育性进行系统研究 ,旨在揭示其遗传本质 ,为解决两系法杂交水稻推广过程中出现的不育起点温度“漂移”等问题提供理论依据 .2　材料与方法…     

Species of small DNA molecules found in mitochondria from sugarbeet with normal and male sterile cytoplasms

Summary Mitochondrial DNA and RNA were isolated from a range of normal and cytoplasmically male sterile sugarbeet varieties and breeding lines. When these nucleic acids were analysed by electrophoresis on agarose gels it was found that mitochondria from normal sugarbeet contain DNA species of sizes 1.3 kilobase pairs (kbp), 1.4 kbp and one of two other species of sizes 1.45 kbp and 1.5 kbp, in addition to their DNA of relatively much higher molecular weight. In contrast mitochondria from cytoplasmically male sterile sugarbeet contained only one of these DNA species, that of 1.5 kbp. Treatment with DNaseI, RNase and nuclease S1 showed that these species consisted of supercoiled circular DNA. It is not known whether the lack of the two smaller DNA species causes cytoplasmic male sterility, or whether the two traits are associated by chance.In addition it was found that mitochondria from some individual sugarbeets contained one or more types of high molecular weight RNA molecules which were probably double stranded. Also mitochondria from some sugarbeet lines and varieties contained a series of DNA molecules with molecular weights in the range 2 to 10 kbp. Neither these DNA molecules nor the RNA molecules were apparently correlated with cytoplasmic male sterility.     

Organelle DNA variation in parental<Emphasis Type="Italic"> Solanum</Emphasis> spp. genotypes and nuclear-cytoplasmic interactions in<Emphasis Type="Italic"> Solanum tuberosum</Emphasis> (+)<Emphasis Type="Italic"> S. commersonii</Emphasis> somatic hybrid-backcross progeny

Nuclear-cytoplasmic interactions can influence fertility and agronomic performance of interspecific hybrids in potato as well as other species. With the aim of assessing the potential value of a novel recombinant cytoplasm derived by interspecific somatic hybridization, backcross progeny were produced by crossing a somatic hybrid between Solanum tuberosum (tbr) and the wild incongruous species S. commersonii (cmm) with various potato clones. BC₁ clones were evaluated for male fertility and other agronomic traits. Male fertility clearly depended on the cross direction and the cytoplasm source. Genotypes with cytoplasms sensitive to nuclear genes derived from Solanum commersonii and inducing male sterility showed identical mtDNA composition, as based on mtDNA analyses with various PCR-based and RFLP markers. On the other hand, genotypes with cytoplasms not inducing male sterility in the presence of the cmm nuclear genes showed a different mtDNA organisation. Analysis of cpDNA confirmed similarity of cytoplasmic composition in CMS-inducing genotypes and clear differences with the others. Genotypes with recombinant cytoplasm induced by somatic hybridization generally showed similar agronomic performances in reciprocal hybrids with tbr cytoplasm, suggesting that the novel cytoplasm can be used in potato breeding.Contribution no. 24 from the Institute of Plant Genetics, Research Division of Portici     

Alloplasmic male sterility in AD allotetraploid Gossypium hirsutum upon replacement of its resident A cytoplasm with that of D species G. harknessii

Summary Alloplasmic male sterile (cms) and restoration-of-fertility (Rf) lines of the AD allotetraploid Gossypium hirsutum were earlier derived from the presumed introgression of the cytoplasm of the D species G. harknessii. To confirm that this happened and address its significance, cytoplasms of the maternal progenitor, backcross intermediates, derived breeding lines, related A, D, and F species, and a synthetic AD tetraploid were examined by agarose and polyacrylamide gel electrophoresis of 140 restriction enzyme fragments of chloroplast DNA. Length mutations of 10–50 nucleotides predominate over site loss/gain mutations. Chloroplast DNA is maternally inherited and that of G. harknessii has been maintained in the cms lines for at least 13 successive generations without detectable alteration. Chloroplast DNA divergence is consistent with current nuclear genome classification and shows that the A progenitor was the maternal parent of the AD tetraploids. As predicted from incompatability models of cms, the degree of male sterility in alloplasmic Gossypium tetraploids is correlated with the extent of evolutionary divergence of their cytoplasms. It is suggested that the A genome in the AD tetraploids dominates those nuclear-cytoplasm interactions reflected by male fertility.     

A light sensitive recipient for the effective transfer of chloroplast and mitochondrial traits by protoplast fusion in Nicotiana

Summary A light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wildtype donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with mesophyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits. On leave from: Department of Genetics, Section of Biosciences, Martin Luther University, Domplatz 1, DDR-4020 Halle/ S., German Democratic Republic     

Development of PCR assays diagnostic for RFLP marker alleles closely linked to allelesGro1 andH1, conferring resistance to the root cyst nematodeGlobodera rostochiensis in potato

The use of RFLPs for marker-assisted selection schemes in potato breeding is hampered by the fact that RFLP technology requires good laboratory facilities, technical skills and high financial input. Marker technology based on the polymerase chain reaction (PCR) would facilitate the application of marker-assisted selection. PCR assays have been developed that are diagnostic for RFLP alleles at two marker loci,CP56 andCP113, which are closely linked in coupling to the nematode resistance allelesGro1 on chromosome VII andH1 on chromosome V of potato. By comparing DNA sequences among different marker alleles, point mutations were identified based on which allele-specific oligonucleotides were designed. Using allele-specific oligonucleotides as primers in PCR reactions, single-marker alleles were amplified by which the inheritance ofGro1 andH1 could be followed in crosses of diploid potato genotypes containing the genetically characterizedGro1 orH1 resistance allele. When tested in 136 unrelated tetraploid potato varieties, the marker allele indicative ofGro1 was not correlated with the presence of nematode resistance. The marker allele indicative for theH1 resistance allele was correlated with nematode resistance. It was, however, found in four varieties only of the 136 tested.