Molecular characterization of fertile and sterile cytoplasms in Beta spp.

Mitochondrial DNA (mtDNA) from sugar beet carrying fertile (F) and male sterile (CMS) cytoplasms, and from male sterile accession of Beta maritima collected in Brittany (France) were characterized and compared by restriction fragment length polymorphism (RFLP) and Southern hybridization with coxII. The F and CMS cytoplasms could be clearly distinguished from each other by RFLP when XhoI, EcoRI and BamHI endonucleases were used. Southern hybridization with the coxII gene provided further evidence that mitochondrial genome organization differs between fertile and sterile plants. All cytoplasmic male sterile lines from different breeding stations showed the same restriction and hybridization patterns, which confirms the uniformity of mitochondrial genomes within the materials used for hybrid seed production in several European countries. No visible differences were found between the maintainer lines studied. However, comparisons of XhoI restriction profiles of mtDNA from maintainer lines and from fertile monogerm populations revealed slight differences, which were reflected by the appearance of a unique 0.9 kb fragment in the latter. Analysis of mtDNA from male sterile plants of the wild beet B. maritima showed different restriction and hybridization patterns in comparison with normal and sterile sugar beet cytoplasms. This shows the unique nature of cytoplasmic male sterility in this species.     

甘蓝细胞质雄性不育相关基因orf138的分子特性分析

为了研究甘蓝雄性不育机制,根据萝卜CMS相关基因orf138的序列信息,设计特异引物,并在甘蓝不同类型不育系和保持系中鉴定PCR产物的稳定性。随后利用Tail-PCR技术,扩增获得此基因的侧翼序列并进行了生物信息学分析。结果表明在甘蓝不育型材料中,能够稳定扩增出300 bp左右的单一条带,而在其他细胞质不育类型和可育材料中均未扩出条带,经多次验证结果稳定可靠。甘蓝中orf138的上下游侧翼序列有效碱基1789 bp,通过生物信息学分析,获得包括起始密码和终止密码的orf138的完整序列共417 bp。同源性比对结果显示：与甘蓝型油菜﹑白菜和萝卜的orf138片段具有高度保守性。分析侧翼序列表明甘蓝orf138的3’端是由ORF83、trnfM、ORF125等基因片段构成的一个复杂序列。获得了甘蓝OguCMS特异的分子鉴定标记,明确了orf138在甘蓝线粒体中的位置,以上结果为甘蓝雄性不育的进一步研究奠定良好的基础。     

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.     

萝卜胞质紫菜薹雄性不育系花药发育的细胞形态学研究

通过2种细胞质紫菜薹雄性不育系小孢子发生的细胞形态学观察表明,供试不育系的雄蕊都深度退化,花药内无花粉。改良不育系小孢子在四分体前发育正常,在单核小孢子期,绒毡层异常膨大挤压小孢子,造成小孢子发育营养不良,引起小孢子败育。Ogura不育系的小孢子发育有所不同,它在孢原细胞分化期之前就已有53%败育,不形成花粉囊;形成花粉囊的孢原细胞大多只形成2个体积很小的的花粉囊,其中的小孢子发育与改良不育系的小孢子发育相似:单核早期绒毡层膨大挤压小孢子,使小孢子败育。     

Characterization of different cytoplasmic male sterility systems in Indian mustard (Brassica juncea L. Czern & Coss)

Six cytoplasmic male sterility (CMS) systems, viz. moricandia, ogura, oxyrrhina, siifolia, tournefortii and trachystoma of Indian mustard (Brassica juncea L.) were characterized for agronomic and floral characteristics. Introgression of alien cytoplasm caused alterations in different floral traits in ogura, siifolia, tournefortii and trachystoma CMS systems. Varied response to different genetic backgrounds of CMS lines indicated the presence of cytoplasmic–nuclear interaction in alteration of floral traits. On the basis of floral characteristics, CMS systems could be grouped into distinct classes. Siifolia, tournefortii and trachystoma CMS lines had narrow petals, while moricandia, ogura and oxyrrhina had wider petals, which were distinguishable on the basis of visual observations. The ratio between length and width of petals were >2.0 in wide petal group but <2.0 in narrow petal group. Further, the relative position of anther and stigma, which was estimated as the ratio between stamen and style length could differentiate the CMS systems. Stamens were longer than styles in oxyrrhina, equal in moricandia and shorter in ogura, siifolia, tournefortii and trachystoma male sterile lines. Non‐viable pollen grains were present in moricandia and oxyrrhina systems, but absent in other systems. In tournefortii and trachystoma, few flowers showed petaloid corolla. All male sterile lines, except trachystoma, which showed crooked siliqua formation were at par with their respective maintainers for flower initiation, plant height, primary branches, seeds per siliqua, seed yield, harvest index, oil and protein content. In general, flower senescence and maturity occurred earlier in male sterile lines than in their respective maintainer lines.     

大白菜CMS96细胞质雄性不育分子特性研究

大白菜CMS96细胞质不育系是由大白菜株系与甘蓝型油菜细胞质不育源杂交、多代回交后获得的。该不育系生长势旺、不育性稳定,不育度和不育株率均为100%,蜜腺正常,在大白菜杂种一代生产中具有广阔的应用前景。为了定位大白菜CMS96细胞质不育所属的类型和获得其不育有关的特异序列,依据atp9、coxI、orf138和orf224保守序列设计4对引物,对3组11份大白菜材料线粒体DNA进行PCR扩增,每组材料内包含同核异质PolCMS、OguCMS、CMS96三种大白菜不育系和一种共用保持系。结果表明,atp9和coxI引物在所有材料中均有扩增产物,供试材料间没有差异;而orf138和orf224引物扩增产物存在差异。orf138引物仅在全部OguCMS和CMS96不育系中扩增出309bp特异带,而保持系和PolCMS不育系没有扩增产物;orf224引物仅在所有PolCMS中扩增出689bp特异带,而保持系、OguCMS和CMS96不育系没有扩增产物。同时,对保持系和不育系花组织mRNA进行RT-PCR分析,进一步验证了orf138和orf224引物扩增产物的特异性和一致性。同源性分析结果表明,利用orf138引物所获得的309bp大白菜mtDNA特异片段均与萝卜OguCMS、甘蓝型油菜OguCMS萝卜体细胞杂种所具有的Oguorf138高度同源,二者有172个核苷酸完全相同,有58个氨基酸完全相同;orf224引物所获得的689bp大白菜mtDNA特异片段与甘蓝型油菜的Polorf224高度同源,二者有677个核苷酸完全相同,有225个氨基酸完全相同,同源性均达到100%。初步认为大白菜OguCMS和CMS96不育系具有相似性,OguCMS萝卜所具有的orf138是导致二者不育的原因;Pol甘蓝型油菜所具有的Polorf224是导致大白菜PolCMS不育的原因。     

Induction of cytoplasmic male sterility in the seed progeny derived from artificially-synthesized interspecific chimera in Brassica

We produced artificial interspecific chimeras by in vitro grafting, and obtained cytoplasmic male sterile (CMS) variants in the seed progenies derived from backcrossing the chimera with one of the mother plants, B. campestris cv. Komatsuna. The induced CMS has been stably inherited by crossing it with `Komatsuna', not with `Ruby Ball' cabbage. The nuclear component of CMS is complete `Komatsuna' type in morphology, chromosome number (2n = 20) and Southern blot using ribosomal 17S RNA gene as a probe. PCR analysis by using mitochondrial atpA primer showed the complete `Ruby Ball' type, suggesting nuclear-cytoplasmic exchange. However, Southern blot patterns were different among those of the CMS and both parents by using atpA. Recombination or some unknown change is supposed in the mitochondrial genome via the processes of synthesis and propagation of the chimeras.     

PCR primers for the estimation of contamination by seeds with normal cytoplasm in rapeseed lots bearing male-sterility-inducing Ogu-INRA cytoplasm

A polymerase chain reaction (PCR)-based test was developed to detect seeds bearing the ‘so-called’ normal rapeseed cytoplasm in seed lots with an OGU-INRA type cytoplasm. The test is based on the amplification of the orfB region of male fertile rapeseed mitochondrial DNA (mtDNA). The amplification reaction uses total nucleic acids of young seedlings, extracted in bulk. After the sequencing of the orfB gene region in the normal Brassica mtDNA, primers were designed for its amplification by PCR. Although the specificity of amplification for the male fertile (mf) rapeseed cytoplasm is partly impaired by the presence of tiny amounts of this fragment in the mtDNA of male sterile (ms) plants, this test proved to be applicable for the estimation of the level of contamination in seed lots in reconstituted mixes as well as in real lots.     

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.     

Genetic and cytological analysis of a new spontaneous male sterility in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.)

A male sterile plant appeared in the radish breeding program at the Hubei Academy of Agricultural Sciences, Hubei, China. In its progeny, a two-type (half of plants male sterile, the other half male fertile) line 01GAB was established. An F₂ population of 260 plants from a cross of male-sterile 01GAB and a male fertile line 9802H segregated for male fertility in a 3:1 ratio indicating that fertility was restored by a single dominant gene, here designated RsMs. A PCR-based DNA marker specific to the male fertility Rfob gene in 9802H was absent in 01GAB. Linkage analysis placed the RsMs locus 10.7 cM away from the Rfo locus. In an F₂ population of hybrids between 01GAB and male fertile 9802B, a co-dominant DNA marker for the RSultr3.2A (a radish sulfate transporter gene) locus was linked to the RsMs locus at 1.5 cM suggesting that fertility restoration in 01GAB was located in the region with known male sterility restorers in radish. However, no maintainer for the 01GAB source of male sterility has been identified so far. Cytological observations have shown that the abnormalities in male sterile anthers first appeared in tapetum at the tetrad stage, followed by a hypertrophy of the tapetal cells at the vacuolate microspore period. These results suggest that male sterility in 01GAB is likely to be genetic in nature, or it may represent a new type of the cytoplasmic male sterility.     

Production of fertile transgenic Brassica napus by Agrobacterium-mediated transformation of protoplasts

A protocol for Agrobacterium tumefaciens‐mediated transformation of Brassica napus mesophyll protoplasts is described. A strain with a neomycin phosphotransferase (nptII) gene and a KCS gene under control of a napin promoter was used at co‐cultivation. Transformed protoplasts were regenerated to fertile and morphologically normal transgenic plants. Transformants were confirmed by PCR of the nptII gene and NAP/KCS expression cassette, and Southern blot analysis. Seeds of the transformants showed a changed fatty acid profile: two transformants had a higher erucic acid level and differed significantly from that of B. napus. Genetic analysis of the progeny revealed that the kanamycin resistance introduced was inherited in a Mendelian fashion.     

PPR‐B‐31: a new maintainer allele in the male‐fertility restorer gene of radish (Raphanus sativus) Ogura cytoplasm

The PPR‐B gene is responsible for male‐fertility restoration of the Ogura‐type male‐sterile radish plants, and it is located in the complex Rfo locus in the vicinity of similar PPR‐A gene and PPR‐C pseudogene. The aim of this study was to identify PPR‐B alleles and understand the structure of the Rfo locus in radish breeding lines. Five lines of radish with normal male‐fertile cytoplasm were tested. The entire PPR‐B gene was amplified, sequenced and allelic PPR‐B sequences were identified. The results indicated that the maintainer lines 7, 15 and 21 contained a non‐restoring form of PPR‐B protein. A unique PPR‐B was found in lines 24/15 and 31 that are restorer and maintainer lines, respectively. The substitutions might be responsible for the loss of a restoring function of the PPR‐B‐31 allele. Amplification of the PPR‐A/PPR‐B and PPR‐B/PPR‐C intergenic regions allowed to identify rearrangements within Rfo locus. Obtained results confirm the wide allelic variation within the Rfo locus, as well as high genetic complexity of the fertility restoration mechanism in radish.     

Expression of male sterility in alloplasmic Brassica juncea with Erucastrum canariense cytoplasm and the development of a fertility restoration system

An alloplasmic mustard, Brassica juncea, has been synthesized by placing its nucleus into the cytoplasm of the related wild species Erucastrum canariense to express cytoplasmic male sterility. To achieve this, the sexual hybrid E. canariense (2n=18, E^cE^c) ×Brassica campestris (2n= 20, AA) was repeatedly backcrossed to B. juncea (2n= 36, AABB). Cytoplasmic male‐sterile (CMS) plants were recovered in the BC₄ generation. These plants are a normal green and the flowers have slender, non‐dehiscing anthers that contain sterile pollen. Nectaries are well developed and female fertility is > 90%. The fertility restoration gene was introgressed to CMS B. juncea from the cytoplasmic donor E. canariense through pairing between chromosomes belonging to B. juncea with those of the E. canariense genome. The restorer plants have normal flowers, with well‐developed anthers containing fertile pollen. Meiosis proceeds normally. Pollen and seed fertility averaged 90% and 82%, respectively. F₁ hybrids between CMS and the restorer are fully pollen fertile and show normal seed set. Preliminary results indicate that restoration is achieved by a single dominant gene. The constitution of the organelle genomes of the CMS, restorer and fertility restored plants is identical, as revealed by Southern analysis using mitochondrial and chloroplast probes atp A and psb D, respectively.     

A stable cytoplasmic male-sterile line of Brassica juncea carrying restructured organelle genomes from the somatic hybrid Trachystoma ballii+B. juncea

A cytoplasmic male-sterile (CMS) line of Brassica juncea has been developed by combining the cytoplasm originating from the somatic hybrid Trachystoma ballii+B. juncea, and the nucleus of B. juncea cv. Pusa Bold by repeated backcrossing. Male-sterile plants closely resembled the normal fertile B. juncea in general morphology, but had delayed flowering (5–7 days) when compared with fertile ‘Pusa Bold’ which flowered in 45 days. Stamens of the male-sterile line were transformed into petaloid structures. Pollen abortion occurred after tetrad formation. Female fertility of the male-sterile line was normal. Molecular analysis of organelle genomes indicated extensive mitochondrial DNA recombinations in the CMS line. Preliminary analysis of the chloroplast genome of the CMS line also indicated chloroplast DNA recombination.     

Development of PCR-based markers linked to a restorer gene for cytoplasmic male sterility in radish (Raphanus sativus L.)

A random amplified polymorphic DNA (RAPD) marker named OPC06-1900 was previously found linked to a fertility restorer gene (Rfw) for cytoplasmic male sterility (CMS) in radish (Raphanus sativus L.). The RAPD marker was converted to a dominant sequence characterized amplified region (SCAR) marker SCC06-1894 by molecular cloning and nucleotide sequencing. A BLAST search revealed that the SCAR marker SCC06-1894 showed significant homology to the corresponding regions of Arabidopsis and Brassica sulfate transporter genes. The presence of the intron and exon of the DNA fragment SCC06-1894 was demonstrated by comparing RT-PCR and PCR products. Thus, allele-specific oligonucleotide primers were designed to amplify the SCAR marker SCC06-415. PCR test with F₂ plants and sequence analysis showed that SCC06-1894 and SCC06-415 were allelic, linked to Rfw/rfw gene at 8.0 cM. Nine oligonucleotide primers were designed based on a single radish nuclear restorer gene mRNA. A survey of these primer combinations by bulked segregant analysis (BSA) identified three polymorphisms. The three PCR-based markers were co-segregant in the coupling phase and distant from the Rfw gene by 1.4 cM. These specific markers distributed on both sides of the Rfw gene and will be helpful for breeding new rapseed (Brassica napus L.) restorer lines.     

Assessment of cytoplasmic polymorphisms by PCR-RFLP of the mitochondrial orfB region in wild and cultivated radishes (Raphanus)

In order to determine the genetic relationship between wild and cultivated radish species, and those among the cultivated species, structural and sequence variations in the mitochondrial orfB gene region were studied in one cultivated and two wild species of Raphanus. Using PCR amplification patterns and RFLP of a PCR product of the region, 232 wild and 420 cultivated radish plants were classified into one of three types of orfB variation. The wild radish (especially the Japanese one) showed large polymorphism in each population with eight of 13 Japanese populations studied containing all three types, whereas cultivars were generally monomorphic. Although type 1 having Ogura male sterile cytoplasm was present with the highest frequency in Japanese wild radish, most cultivars were divided into type 2 or 3 with normal cytoplasm. Type 2 was widely distributed in European, Chinese and major Japanese varieties, while some Chinese varieties and several Japanese local radishes had type 3 cytoplasm. The comparison provides valuable information about the origin and differentiation of cultivated radishes and the relationship between cultivated and wild radishes.     

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.     

Effective Transfer of Cytoplasmic Male Sterility from Radish (Raphanus sativus L.) to Rape (Brassiest napus L.)1)

One of the CMS systems presently investigated by rape breeders is the alloplasmic system derived from a male sterile radish and transferred to rape. However, these male sterile lines of rape with the radish cytoplasm exhibit yellow leaf colour at low temperatures. Moreover, no restorer genes for this system are known to occur in rape. The present investigation was, therefore, directed to the formation of new cross combinations between radish and rape in order to eliminate the above mentioned problems by additional transfer of corresponding radish genes into the rape recipient. After the initial intergeneric crosses, male sterile hybrids were received by in-vitro embryo culture and colchicine treatment. These hybrids were not backcrossed to rape as usual, but to one of the monogenomic ancestral species of rape, in order to increase the probability of allosyndesis between radish and rape chromosomes. After subsequent back-crossing with rape, the desired male sterile plants with 38 chromosomes were recorded, forming normal green leaves even at low temperatures. In a parallel backcross programme, restorer genes from male fertile radish were also transferred into rape by the same backcrossing procedure. The first alloplasmic male fertile plants were found in the BC: generation. They were cross pollinated to male sterile plants to prove their potential of fertility restoration.     

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.