Genetic and molecular analysis of the restoration of fertility ( Rf ) genes for Ogura male-sterility from a Japanese wild radish ( Raphanus sativus var. hortensis f. raphanistroides Makino)

Ogura male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. In this study, in order to gain better understanding of the variation and evolution of the restoration of the fertility (Rf) gene for Ogura male-sterile cytoplasm, the nucleotide sequence of the orf687 homologue in the Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides Makino) was analyzed using an F₂ population made with a cross between a Japanese wild radish plant containing the Rf gene and ‘Uchiki-Gensuke’ (a maintainer of Ogura-male sterility). Segregation of male-fertile/-sterile plants in the F₂ generation suggested that another unidentified Rf gene unlinked to orf687 exists in the Japanese wild radish. The genotype of orf687 was determined for each F₂ plant by Southern hybridization with an orf687 gene probe, mismatch-specific endonuclease digestion of PCR products, and direct sequencing of a PCR product. Genotyping revealed that some fertility-restored plants are homozygotic for the ‘Uchiki-Gensuke’ type orf687 allele, supporting the idea that another gene different from orf687 also functions as an Rf gene for Ogura male-sterility. Protein analysis using an antibody raised against the Ogura-specific ORF138 protein suggests a mechanism of fertility restoration by the unidentified Rf similar to that by orf687. Sequence analysis of orf687 from a Japanese wild radish plant and ‘Uchiki-Gensuke’ revealed that both orf687 regions encode a mitochondrially-targeted protein consisting of 687 amino acids with 16 PPR motifs. Comparison of the deduced amino acid sequences with those of the known orf687 sequences from ‘Yuan hong’ and ‘Kosena’ containing Rf and recessive one (rf), respectively, showed that three unique amino acid replacements are present in ORF687 of the Japanese wild radish. Two of the three replacements, that from lysine to isoleucine at position 232 and from asparagine to asparate at position 240, confer negative charges to the protein. Since the Rf of ‘Yuan hong’ was reported to have a unique replacement that confers a negative charge to ORF687 (from asparagine to aspartate at position 170), it is proposed that the amino acid replacements conferring a negative charge to ORF687 are important for determining the status of the Rf/rf gene.     

Molecular and biological studies on male sterile cytoplasm in Cruciferae. II. The origin of Ogura male sterile cytoplasm inferred from the segregation pattern of male sterility in the F1 progeny of wild and cultivated radishes (Raphanus sativus L.)

Summary To determine the origin of Ogura male sterile cytoplasm in radish (Raphanus sativus L.), wild and cultivated radishes were crossed. Three types of progeny resulted from the F₁ hybrids between the wild radish from Kushikino with Ogura-type mtDNA and the cultivars (Uchiki-Gensuke or Comet). The segregation patterns of the male sterility were compared with those of Ogura cytoplasm. The male sterility induced in the F₁ hybrid was maintained by crossing with Uchiki-Gensuke, that maintains Ogura male sterility. In the two types of progeny, in which Comet (a restorer of Ogura cytoplasm) was used as one of the parents, both fertile and sterile plants segregated at the predicted ratio on the assumption that a single dominant fertility restoring gene exists in the restorer. From these results, we concluded that the Ogura cytoplasm is identical to that of the wild radish, and the former originated in a population of Japanese wild radish.     

Discovery of new male-sterile cytoplasm sources and development of a new cytoplasmic-nuclear male-sterile line NJCMS3A in soybean

Most of the cytoplasmic-nuclear male-sterile (CMS) lines of soybean were developed only from a limited cytoplasm sources and performed not as good as required in hybrid seed production, therefore, to explore new male-sterile cytoplasm sources should be one of the effective ways to improve the pollination and hybridization for a better pod-set in utilization of heterosis of soybeans. In the present study, total 80 crosses between 70 cultivated and annual wild soybean accessions and three maintainers (N2899, N21249, and N23998) of NJCMS1A were made for detecting potential new sources with male-sterile cytoplasm. The results showed that in addition to the crosses with N8855.1 (the cytoplasm donor parent of NJCMS1A) and its derived line NG99-893 as cytoplasm parent, there appeared three crosses, including N21566 × N21249 and N23168 × N21249, with male-sterile plants in their progenies. According to the male fertility performance of backcrosses and reciprocal crosses with the tester N21249, the landrace N21566 and annual wild soybean accession N23168 were further confirmed to have male-sterile cytoplasm. Accordingly, it was understood that the source with male-sterile cytoplasm in soybean gene pool might be not occasional. The results also showed that the genetic system of male sterility of the newly found cytoplasm source N21566 was different from the old cytoplasm source N8855.1, while N23168 was to be further studied. Based on the above results, the derived male-sterile plants from [(N21566 × N21249) F₁ × N21249] BC₁F₁ were back-crossed with the recurrent parent N21249 for five successive times, and a new CMS line and its maintainer line, designated as NJCMS3A and NJCMS3B, respectively, were obtained. NJCMS3A had normal female fertility and stable male sterility. Its microspore abortion was mainly at middle uninucleate stage, earlier than that of NJCMS1A and NJCMS2A. The male fertility of F₁s between NJCMS3A and 20 pollen parents showed that 7 accessions could restore its male fertility and other 13 could maintain its male sterility. The male sterility of NJCMS3A and its restoration were controlled by one pair of gametophyte male-sterile gene according to male fertility segregation of crosses between NJCMS3A and three restorers. The nuclear gene(s) of male sterility in NJCMS3A appeared different from the previously reported CMS lines, NJCMS1A and NJCMS2A. The development of NJCMS3A demonstrated the feasibility to discover new CMS system through choosing maintainers with suitable nuclear background.     

Male sterility in alloplasmic Brassica rapa L. carrying Eruca sativa cytoplasm

For the development of a new cytoplasmic male sterility (CMS) system in Brassica rapa (2n= 20, AA), intergeneric hybridization was performed in Eruca sativa (2n= 22, EE)×B. rapa. The original amphihaploid F₁ plant (2n= 21, EA) generated via embryo rescue produced a sesquidiploid F₁ plant (2n= 31, EAA), from which the alloplasmic F₃ plants were generated. In F₃, some progenies with malformed anthers were maintained as male-sterile lines up to the F5 generation. In the F₆ and F₇ generations, the alloplasmic male-sterile plants were bred by backcrossing to several B. rapa genotypes and were then classified into the three distinctive types: petaloid, antherless and brown anther, in addition to three intermediate types between them. By southern blot analysis, each plant of the petaloid and antherless types was shown to carry the cytoplasm genome of E. sativa. These male-sterile plants produced as many viable seeds as the corresponding male-fertile plants, although their nectary gland development was minimal. Consequently, CMS lines of the petaloid and antherless types with enhanced seed fertility and nectary gland development could provide promising plant materials for F₁ seed production in B. rapa.     

Moricandia arvensis cytoplasm based system of cytoplasmic male sterility in Brassica juncea: Reappraisal of fertility restoration and agronomic potential

Cytoplasmic male sterility (CMS) system based on the cytoplasm from Moricandia arvensis (mori) was investigated for fertility restoration and agronomic potential. Fertility restorer gene for mori CMS was introgressed from cytoplasm donor species as all the evaluated Brassica juncea genotypes (155) acted as sterility maintainers. The allosyndetic pairing between M^a and the A/B genome chromosomes in the monosomic addition plants (2n= 18II + 1M^a) facilitated the gene introgression. Partial fertility restoration (43–52% pollen grain stainability) in F₁ hybrids and absence of segregation for male sterility in F₂ progenies suggested gametophytic control of fertility restoration. The pollen fertility in the F₁ hybrids was, however, sufficient to ensure complete seed set upon bag selfing. Introgression from M. arvensis also helped in correction of chlorosis associated with mori cytoplasm in CMS and fertile alloplasmic B. juncea plants. Yield evaluation of thirty F₁ hybrids having the same nuclear genotype but varied male sterilizing cytoplasms (mori, oxy, lyr, refined ogu), in comparison to respective euplasmic hand bred control hybrids, allowed an estimate of yield penalty associated with different CMS systems. It ranged from 1.8% to 61.6%. Hybrids based on cytoplasmically refined ogu were most productive followed by those based on cytoplasmically refined mori CMS. The male sterility systems emanating from somatic hybridization were found superior than those developed from sexual hybridization.     

Effect of Ogura male-sterile cytoplasm on the performance of cabbage hybrid variety

Summary Cabbage hybrid seeds are commercially produced by means of self-incompatibility. This system may show some instability mainly under tropical conditions, where cytoplasmic male sterility can be an alternative approach for hybrid seeds production. However, cabbage hybrids holding Ogura male-sterile cytoplasm show some irregularities during development. By assessing some characteristics during the growing cycle of male-sterile cabbage hybrids and comparing them to genomic similar male-fertile ones and to the most common cabbage hybrid cultivated in Brazil, it was observed that the male-sterile hybrids had the same vigour, uniformity, number of leaves, resistance to Xanthomonas campestris pv. campestris, and earliness as their male-fertile counterparts and performed better than the commercial check hybrid for some of these characteristics. Although male-sterile hybrids showed yellowing of leaves, some parental combinations succeeded in overcoming or strongly reducing this cytoplasmic effect.Abbreviations dat days after transplanting - CMS Cytoplasmic Male Sterility - CNPH National Centre for Vegetable Crops Research     

Development of cytoplasmic-genic male sterility in safflower

An interspecific cross was made between Carthamaus oxyacantha and the cultivated species C. tinctorius to develop a cytoplasmic‐genic male sterility (CMS) system in safflower. C. oxyacantha was the donor of sterile cytoplasm. The 3: 1 segregation pattern observed in BC₁F₂ suggested single gene control with dominance of male‐fertility over male‐sterility. The information obtained from crossing male sterile X male fertile plants in BC₁F₃ and BC₁F₄ generations showed statistically significant single gene (1: 1) segregation for male sterility vs. male fertility. The results demonstrated that C. tinctorius possesses a nuclear fertility restorer gene and that a single dominant allele restored fertility (Rf) in progeny carrying CMS cytoplasm of C. oxyacantha. Male sterility occurred with the homozygous recessive condition (rfrf) in a sterile C. oxyacantha cytoplasm background and not in the normal cytoplasm of C. tinctorius. The genetic background of different restorer lines of C. tinctorius having normal cytoplasm did not effect fertility restoration. The absence of male sterile plants in C. tinctorius populations ruled out the possibility of genetic male sterility. Normal meiosis in F₁ and BC₁F₂ ruled out a cytogenetic basis for the occurrence of male sterility.     

Identification of Maintainer Genes in Brassica napus L. for the Male-Sterility-Inducing Cytoplasm of Diplotaxis muralis L.

Male fertility of F¹ interspecific hybrid plants derived from crosses between cytoplasmic male-sterile Brassica campestris in Diplotaxis muralis cytoplasm and 147 B. napus cultivars was Investigated. F¹, plants obtained, from crosses with the B. napus cultivars‘Mangum’and‘Hinchu’were male-sterile while F¹ plants derived from all other crosses were male-fertile. This indicated that these two cultivars carried maintainer genes far the male-sterility-inducing cytoplasm of D. muralis. Sterility was stable In plants derived from backcrosses of male-sterile F; plants with‘Mangun and‘Hinchu’but the seed set of backcross plants was low. With restorer genes readily available in B. napus, these findings could lead to the development of a new cytoplasmic male sterility system for the breeding of B. napus hybrid cultivars.     

Identification of a sequence-tagged site (STS) marker linked to a restorer gene for Ogura cytoplasmic male sterility in radish (Raphanus sativus L.) by non-radioactive AFLP analysis

To identify DNA markers linked to a fertility restorer (Rf) genefor Ogura cytoplasmic male sterility in radish (Raphanus sativus L.),a non-radioactive, amplified fragment length polymorphism (AFLP) analysiswas performed on bulked DNA samples from male-sterile and male-fertileradishes. Ten male-fertile and 10 male-sterile plants selected arbitrarilyfrom an F₂ population made by selfing of F₁ plant from a crossbetween a male-sterile (`MS-Gensuke') plant and a restorer (`Comet') plantwere used as material. Using 32 AFLP primer pairs, one AFLP fragment(AFLP190) which is specific to the bulked DNA samples from male-fertileF₂ plants was identified. AFLP190 was characterized by molecularcloning and nucleotide sequencing, and was converted to a sequence-taggedsite (STS) marker, STS190. A linkage analysis performed in 126individuals of two independent F₂ populations showed tight linkageof STS190 to the Rf gene. The rate of recombination between themarker and Rf was estimated to be less than 1%, making STS1901.2 cM from the gene.     

Effect of Ogura male-sterile cytoplasm on the performance of cabbage hybrid variety. II. Commercial characteristics

Summary The main constraint of using Ogura male-sterile cytoplasm in Brassica is the induction of leaf yellowing at low temperature and the low seed set. But whether or not the cytoplasm can disturb the general commerical performance of the plants is not well-known. Therefore, this work was carried out with the purpose of evaluating Ogura cytoplasmic male-sterile cabbage hybrids and compare them to genomic similar male-fertile ones and to the most popular cabbage hybrid cultivated in Brazil. Harvest data showed an effect of the cytoplasm on reducing plant and head weight, core length and head length and width, although not altering heading index, shape and core indexes, nor head compactness. On the other hand, it was observed also that a careful selection of the parental lineages can produce male-sterile hybrids as good as their male-fertile analogues. All the evaluated hybrids were similar or better than the check for the characteristics under study.Abbreviations dat - days after transplanting - CNPH - National Center for Vegetable Crops Research     

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.     

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.     

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.     

Genetic studies on the interspecific cytoplasm substitution lines of japonica varieties of Oryza sativa L. and O. glaberrima Steud.

Summary Interspecific cytoplasm substitution lines of Oryza sativa and O. glaberrima, i.e. (sativa)-glaberrima and (glaberrima)-sativa, have been bred by means of successive backcrosses, using three japonica varieties of sativa and two glaberrima strains.In all the six substitution lines with the cytoplasm of the glaberrima strains, the fertility increased with succeeding backcrosses, and eventually completely fertile plants whith the characteristics of the parental japonica variety appeared. This indicates that the glaberrima cytoplasm exerted no effect on the genome manifestation of these japonica varieties. Of the five substitution lines with the cytoplasm of each of the japonica varieties, four lines produced male sterile (M.S.) plants only in the backcross generations. In the remaining substitution line with the cytoplasm of the japonica variety Akebono, there was simultaneous segregation for male sterile (M.S.) and pollen fertile plants bearing indehiscent anthers (ID.M.F.) in the backcross generations. In the compulsively selfed progeny of ID.M.F. plants, pollen fertile plants with dehiscent anthers (D.M.F.) occurred with M.S- and ID.M.F. plants. Morphologically, these three types were supposed to have the same genetic background as the glaberrima parent. It was established that D.M.F.-and ID.M.F. plants were homozygous and heterozygous for a dominant nuclear gene restoring pollen fertility, respectively, and the M.S. plants and the two glaberrima strains used in this study carried a recessive gene for pollen sterility in homozygous condition. The restorer gene was assumed to derive from the japonica variety Akebono. The expression of the restorer gene was of the sporophytic type. The pollen sterility of the substitution lines that possessed the cytoplasm of the japonica varieties was of cytoplasmon-genic nature.     

Intergeneric hybridization of Diplotaxis erucoides with Brassica napus. I. cytogenetic analysis of F1 and BC1 progeny

Summary Intergeneric hybrids (F₁) Diplotaxis erucoides (DeDe) x Brassica napus (AACC) and the first backcross to B. napus (BC₁) have been obtained through in vitro culture of excised ovaries. The chromosome numbers of F₁ and BC₁ plants proved the occurrence of unreduced gametes. The study of metaphase I chromosome pairing showed that autosyndesis in De genome and allosyndesis between De and A/C genomes might exist. The male fertility of the F₁ plants was low. Some male-sterile plants were found in F₁ and BC₁ progeny. The possibilities of creating addition lines B. napus-D. erucoides and of obtaining a new cytoplasmic male sterility in B. napus are discussed.     

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.     

Identification and characterization of important sterile and maintainer lines from various genotypes for advanced breeding programmes of onion (Allium cepa)

Onion is one of the major vegetable crops in terms of production as well as consumption. In the current research, available onion genetic stock was evaluated to identify male-sterile lines and produce high-yielding F₁ hybrids for future breeding programmes. A mitochondrial DNA-based marker was mapped and correlated with phenotypic traits to isolate male-sterile plants. Based on the floral and pollen structure, nine putative male-sterile lines were identified. On the other hand, for nuclear marker identification at Ms locus, two sets of primers were used, one for Ms dominant allele and another for sterile and maintainer plants. Results revealed that 70% of open pollinated varieties (OPVs) possess plants with sterile cytoplasm coupled with genetic sterility at Ms locus, called sterile “A” line. Approximately 20% of plants in some genotypes were identified with normal (N) cytoplasm having recessive fertility gene at Ms locus, called maintainer “B” line. Based on the present findings, “A”, “B” and “R” (restorer line), future F₁ hybrid seed production systems in onion is discussed.     

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。     

Identification of Rfd1, a novel restorer-of-fertility locus for cytoplasmic male-sterility caused by DCGMS cytoplasm and development of simple PCR markers linked to the Rfd1 locus in radish (Raphanus sativus L.)

Previously, novel cytoplasmic male-sterility (CMS) caused by DCGMS cytoplasm was discovered in radish (Raphanus sativus L.) introduced from Uzbekistan. We performed extensive progeny tests and identified two fertility restorer lines (‘R171’ and ‘R121’) for this new CMS. Two F₁ hybrid populations were self-pollinated and backcrossed to produce F₂ and BC populations. Inheritance patterns of male-sterility in segregating populations varied depending on paternal lines. Segregation of male-sterility in F₂ populations originating from the cross between MS19 and R121 showed that a single locus was involved in fertility restoration. However, populations originating from the cross between MS15 and R171 showed the involvement of more than one restorer-of-fertility genes. The single fertility restorer locus identified in the cross between MS19 and R121 was designated Rfd1 locus. Bulked segregant analysis was performed using RAPD and AFLP, which identified one marker each. Both RAPD and AFLP markers were converted into simple PCR-based co-dominant markers after their isolated flanking sequences were analyzed. Indels 773-bp and 67-bp in length were identified between two Rfd1 allele-linked flanking sequences of the RAPD and AFLP fragments, respectively, then utilized to develop simple PCR markers. In addition, we prove that the newly identified Rfd1 locus is independent of the Rfo locus, another radish fertility restorer for CMS caused by Ogura cytoplasm.