SSR and SCAR markers linked to the fertility-restoring gene for a D2-type cytoplasmic male-sterile line in wheat

‘Yi 4060’ is an elite restorer line of a non‐photoperiod‐sensitive D²‐type cytoplasmic male‐sterile (CMS) line of wheat. Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were employed to map one major fertility‐restoring gene (D²Rf₁) in ‘Yi 4060′. The sterile and fertile DNA pools were established from individuals in BC₆, based on bulked segregant analysis. One RAPD marker E09, linked to D²Rf₁, was converted to a SCAR marker and designated as E09‐SCAR₈₆₅. The genetic distance between E09‐SCAR₈₆₅ and D²Rf₁ is 9.5 cM. Two SSR markers, Xgwm11 and Xgwm18, were also linked to a D²Rf₁ and co‐segregated with E09‐SCAR₈₆₅. The three molecular markers are useful in marker‐assisted breeding of the elite restorer lines for D² ‐type CMS lines in wheat.     

Inheritance and gene tagging of male fertility restoration of cytoplasmic-nuclear male-sterile line NJCMS1A in soybean

The F₁, F₂ and F_2:3 of the NJCMS1A × 'Zhongdou 5' cross were used to analyse the inheritance of the male fertility restoration of the cytoplasmic-nuclear male-sterile line NJCMS1A in soybean. The results of genetic analysis showed two pairs of dominant genes conferring the male fertility restoration of NJCMS1A, which further confirmed previous results. The F₂ population from the NJCMS1A × 'Zhongdou 5' cross was used for tagging the restorer genes for NJCMS1A with 664 pairs of simple sequence repeat primers selected randomly from the genetic linkage map of soybean published by Cregan et al. (1999) . Satt626 on linkage group M and Satt300 on linkage group A1 of the integrated linkage map by Song et al. (2004) were found to link to the two restorer genes of NJCMS1A. The maximum-likelihood estimates of the genetic distance between the two markers, Satt626 and Satt300, and the two restorer genes of 'Zhongdou 5' were 9.75 and 11.18 cM, respectively.     

Floral initiation in field-grown forage peas is delayed to a greater extent by short photoperiods, than in other types of European varieties

Frost is one of the main climatic stresses which has to be overcome by a winter pea crop. Some forage lines show a delayed floral initiation, which helps them to escape the main winter freezing periods, as a higher susceptibility to frost is observed after floral initiation commences. Frost tolerant forage peas have been used in all winter pea breeding programs in France and our main purpose was to evaluate to what extent those lines and the current winter varieties are variable for the date of floral initiation in field conditions. A field experiment was carried out during two years at the INRA experimental station of Mons (northern France). Different genotypes (9 in 1995–1996 and 12 in 1996–1997) were sown at approximately monthly intervals between September and June to provide a range of photothermal conditions. The date of floral initiation was determined by destructive sampling. We observed variability for the date of floral initiation among the different genotypes and sowing dates. Our data particularly highlighted the different reactions to photoperiod. Three varieties had no detectable reaction. Six varieties exhibited a quantitative response, with varied intensities. Lastly, the three forage varieties showed a qualitative, high response to photoperiod, which indicated the presence of the Hr allele, already described in Pisum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pollen Characteristics and Genetics of Induced and Spontaneous Genetic Male-Sterile Mutants in Rice

Pollen characteristics, inheritance and allelism of 23 genetic male-sterile (ms) mutants of rice (Oryza sativa L.) were investigated. Ten of the mutants were induced by ethyl methane sulfonate, one induced by ethyleneimine, seven induced by gamma rays, one induced by streptomycin, one derived from tissue culture and three were spontaneous mutants found in the field. Four pollen abortion types were observed among these ms mutants: 3 no-pollen (NP) type, 6 complete pollen abortion (CPA) type, 13 partial pollen abortion (PPA) type, and 1 stainable pollen abortion (SPA) type. Progeny tests over two years indicated that each of the mutants was inherited as a monogenic recessive. A partial diallel cross among the 23 ms mutants indicated that mutant E2 was allelic to G4, E5 was allelic to E9, N2 was allelic to N3, and that three other mutants, E3, G6 and T1, shared the same ms locus. Gene symbols ms-46 (t) through ms-63 (t) were assigned to these mutants.     

Heritability and gene effects for root characteristics in peas measured at flowering

Summary Heritability and gene effects for root weight, root volume and root to shoot weight ratio were determined in peas (Pisum sativum L.) at flowering. The populations used were developed from four crosses between lines and cultivars differing in size of the root systems. Broad-sense heritability was between 0.41 and 0.81 for root weight, and between 0.44 and 0.77 for root volume. Additive and dominance effects were important in the genetic control of root weight and volume in all populations, while epistatic effects were important only in two populations. The importance of the genetic parameters in the control of root to shoot weight ratio was unclear. Assuming high correlations between root growth in soil-filled pots in the greenhouse and growth in the field, with the presence of large additive effects and high heritability estimates for root weight and volume, selection for superior pure lines with large root systems should be effective.     

The distribution of normal and male-sterile cytoplasms in Chinese sugar-beet germplasm

Forty-two Chinese sugar-beet breeding lines were evaluated for the presence of normal and male-sterile (Owen) cytoplasms using polymorphisms in the chloroplast petG-psbE region as well as in the mitochondrial minisatellite loci. The polymorphisms detected allowed the distinction of three cytoplasm types over the whole sample, one being associated with Owen cytoplasm, a second with the maintainer inbred ‘TK-81mm-O’-type cytoplasm (termed normal-1 cytoplasm) and a third with another maintainer inbred ‘NK-310mm-O’-type cytoplasm (normal-2 cytoplasm). Western blot analysis was carried out to confirm that expression of the male-sterility-associated protein (preSATP6) occurred in plants with Owen cytoplasm but not in plants with either normal-1 or normal-2 cytoplasm. Of the 42 breeding lines examined, 14 had exclusively normal (normal-1 and/or normal-2) cytoplasm and 11 had only Owen cytoplasm. The remaining 17 lines possessed both normal and Owen cytoplasms, and noticeably, some of these 17 lines have been expected to become the source of superior maintainer lines. The results thus show that molecular identification of the cytoplasm is required to avoid wasting resources on account of attempting to develop the maintainer genotype from plants with Owen cytoplasm.     

Male gamete development and early tapetal degeneration in cytoplasmic male-sterile pepper investigated by meiotic, anatomical and ultrastructural analyses

Cytoplasmic male‐sterile (CMS) pepper (Capsicum annuum L.) is a useful germplasm for heterosis breeding, however, the process of male gamete development and the sterility mechanism is unclear. In the present study, the developmental sequence in CMS pepper and the causative factors responsible for pollen abortion were investigated through combined meiotic analysis, paraffin section observations, and transmission electron microscopy. Results from meiotic analysis showed that meiosis in a male‐sterile inbred line, CMS 21A, was normal, and uni‐nucleate pollen could be released from the tetrads of both CMS 21A and the fertile maintainer line 21B. However, only the uni‐nucleate pollen from CMS 21B could develop into functional gametes, while the uni‐nucleate pollen from the 21A went through a process of rupture, and the cellular components were released into the anther sac. Observations of paraffin sections viewed with the light microscope showed that the tapetum of CMS 21A at the uni‐nucleate stage swelled abnormally and was pressed against pollen grains of the locule. Further observations with transmission electron microscopy revealed that the mitochondria in CMS 21A tapetum were highly vacuolated, and there was no accumulation of sporopollenin on the surface of CMS 21A pollen, suggesting a close relationship between early degeneration of tapetum and CMS.     

Male sterility in populations of Daucus and the development of alloplasmic male-sterile lines of carrot

A collection of Daucus species and subspecies, as well as alloplasmic carrot lines were evaluated for expression of male sterility. More than 100 male‐sterile plants were identified and their phenotypes and partial genetic background were characterized. Three potentially new cytoplasmic male‐sterile systems based on the cytoplasm of Daucus carota (D. c.) gummifer, D. c. maritimus and D. c. gadecaei were selected. Genetic analyses suggested that a nuclear‐cytoplasmic interaction determined the male sterility of the three sources. A clear differentiation between the new sources and carrot cultivars was possible based on the phenotypic and molecular genetic relationships.     

Development of dominant nuclear male-sterile lines with a blue seed marker in durum and common wheat

In order to develop genie male‐sterile lines with a blue seed marker, male‐sterile plants, controlled by a dominant nuclear gene Ms2, were used as female parents against a 4E disomic addition line ‘Xiaoyan Lanli’(2n= 44, AABBDD+4EII) as the male parent to produce monosomic addition lines with blue seed. Male‐sterile plants from the monosomic addition lines were pollinated with durum wheat for several generations and in 1989 a male‐sterile line with the blue grain gene and the male‐sterile gene Ms2 on the same additional chromosome was detected and named line 89‐2343. Using this line, the blue seed marker was successfully added to a short male‐sterile line containing Ms2 and Rht10. The segregation ratios of male sterility and seed colour as well as the chromosome figurations of different plants indicated that the blue grain genes, Ms2 and Rht10 were located on the same additional chromosome. Cytological analysis showed that the blue marker male‐sterile lines in durum wheat and common wheat were monosomic with an additional chromosome 4E. The inheritance ratio for blue seed male‐sterile plants and white seed male‐fertile plants was 19.7% and 80.3%, respectively, in common wheat. The potential for using blue marker sterile lines in population improvement and hybrid production is discussed.     

Restriction analysis of the mitochondrial DNA of male-sterile and maintainer lines of pearl millet, Pennisetum americanum L.

Mitochondrial DNA from two pairs of cytoplasmic male-sterile (cms) and maintainer lines of pearl millet was investigated by restriction-enzyme analysis and Southern-blot hybridization using three mitochondrial gene probes. Each pair of male-sterile and maintainer lines was of a different nuclear origin. The objective was to distinguish differences in the DNA base-sequence organization of the mitochondrial genomes of cms and maintainer lines from the two sources. Restriction-enzyme analysis revealed differences between the different cms and maintainer lines. Southern-blot hybridization experiments using cloned mitochondrial gene probes further distinguished differences between different lines. It is expected that the restriction-fragment-length polymorphisms revealed in the Southern-blot-hybridization experiments will be useful in distinguishing and classifying cms and maintainer lines obtained from different nuclear backgrounds.     

Recurrent selection using male sterility and hydroponic tiller culture in pedigree breeding of wheat

A procedure for recurrent selection based on the male sterility gene, Ms3, was implemented. To facilitate the production of large numbers of hybrid progeny, a simple hydroponic system was developed in which male‐sterile tillers cut at the flowering stage can be pollinated and maintained for about 8 weeks‐long enough to produce a large quantity of viable hybrid seeds. The recurrent selection steps were integrated with a pedigree breeding programme employing different selection cycles for male and female plants. F₁ female plants are subjected to a single screening for seedling resistance. In addition to F₁ seedling screening, F₂‐F₄ male families are field‐selected for disease resistance, agrotype and quality in a pedigree programme before being used in crosses.     

Male Sterility in Barley

The anther form and development of eight two-rowed genie male sterile barley mutants are abnormal. The male sterility in each mutant is conditioned by single non-allelic recessive genes. These mutant genes cause reductions in anther size and lumen diameter. Though in seven mutants, the male meiosis is normal until microspore liberation, the micro-spores abort in all cases after release from the PMCs. In one mutant, the microspores degenerate at the tetrad stage before release from the PMCs. In four mutants, the tapetal development and disintegration are normal, in four others they are abnormal. Despite these variations, the male sterility in all the eight mutants is complete.     

Stability of male-sterile sources and fertility restoration of their hybrids in pearl millet

Genetic background has a significant effect on the expression of cytoplasmic-nuclear male sterility (CMS) in pearl millet (Pennisetum glaucum (L.) R. Br.). Therefore, a reliable characterization of CMS sources requires the use of near-isonuclear lines and their hybrids. We used this approach to characterize five CMS sources (A₁, A₂, A₃, A_v, and A₄). Male sterility of 81A₄ was the most stable, followed by 81A₁ and 81A_v, indicating the relative merits of these CMS sources in breeding stable male-sterile lines. Male-sterile lines 81A₂ and 81A₃ were highly unstable, indicating their minimal value. Differential male fertility restoration patterns of hybrids made on 81A_V and 81A₄ suggest that the A_v and A₄ cytoplasms represent CMS systems that are different from each other and from A₁, A₂ and A₃. An evaluation of topcross hybrids of 15 diverse populations made on 81A₁ and 81A₄ showed that each population had restorers and maintainers of both cytoplasms and that the frequency of maintainers of A₄ was as high as, or higher than, that of the A₁ cytoplasm. Thus, use of the A₄ cytoplasm can substantially increase the probability of breeding stable male-sterile lines based on inbreds developed from diverse genetic backgrounds, and also provide the opportunity for breeding restorers from each of these diverse genetic sources.     

Cultivar × rhizobial strain interactions in peas with respect to early symbiosis, nodule initiation and N uptake

Sixteen pea cultivars differing in their phenotypic characteristics were studied in order to characterize the cultivars' variability in symbiotic characters using three Rhizobium leguminosarum biovar viceae strains. The peas did not show any cultivar × strain interactions with the rhizobial strains used with respect to root hair infection frequency or day of nodule initiation. Three cultivars had high root hair infection frequencies, and five had only a few infected root hairs. Four of five leafless/semi-leafless varieties investigated and one cultivar with normal leaves formed nodules quickly, whereas three varieties with normal leaves formed nodules slowly. Ten of the cultivars showed cultivar × strain interactions with respect to N uptake, indicating that the performance of the N2-fixing symbioses depends on the rhizobial partner. Six cultivars showed cultivar × strain interactions with respect to dry matter production and the nodule proportion of the dry matter. Of the cultivars investigated, we conclude that some of the leafless/semi-leafless varieties (‘Capella’, ‘Filby’ and ‘Solara’) are suitable for inclusion in a future breeding programme, since these cultivars combined several important symbiotic characters.     

豌豆抗白粉病资源筛选及分子鉴定

由豌豆白粉菌引起的白粉病是豌豆生产上的重要病害,利用抗病品种是防治该病害最经济有效的方法。本研究在控制条件下苗期接种鉴定了396份豌豆资源对2个不同地理来源的豌豆白粉病菌分离物EPBJ和EPYN的抗性,用4个与豌豆抗白粉病基因er1连锁的SCAR标记对66份免疫或抗病资源进行标记基因型鉴定。结果表明,在鉴定的396份资源中,有101份资源表现免疫或抗病,其中对分离物EPBJ和EPYN免疫的资源分别为59份(14.9%)和60份(15.2%),对2个分离物均免疫的资源有54份(13.6%);在鉴定的82份中国资源中,有8份对2个分离物均表现免疫。分子标记将66份免疫或抗病资源鉴定为13个标记基因型,同一地理来源的抗性资源分属不同的标记基因型,其中8份来自中国云南的抗性资源分属7个标记基因型。研究表明,中国存在有效的豌豆白粉病抗源,抗性资源具有丰富的遗传多样性。     

Further cytological characteristics of a male-sterile mutant in soybean affecting cytokinesis and microspore development

A spontaneous male‐sterile mutation affecting microspore development has been found in BR97‐13509H line of the official Brazilian soybean breeding programme. Slightly abnormal and irregular chromosome segregation occurred in both meiotic divisions, albeit insufficient to explain total pollen sterility. The main cause of male sterility was the absence of cytokinesis after telophase II. Instead of the typical tetrads of microspores, four‐nucleate coenocytic microspores were formed. Coenocytic microspores resulting from the absence of cytokinesis have also been described in ms1 and ms4 male‐sterile soybean mutants, although with certain differential characteristics. In the BR97‐13509H mutant line, after interphase the four nuclei were rejoined in one or two metaphase plates and progressed through the first pollen mitosis. Pollen then underwent a progressive process of degeneration characterized by continuous cytoplasmic shrinkage. The second pollen mitosis failed to occur, and pollen sterility became total.     

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.     

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.     

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.