Functional male sterility (ps-2) in tomato (Lycopesicon esculentum Mill.) and its application in breeding and hybrid seed production

Data and findings concerning the development of an effective male sterility system (as already applied in the practice of producing tomato hybrid seed) through the use of the positional sterility (ps 2) gene are reported. Undesirable selfing and the necessity of emasculation are discussed as the two main disadvantages that limit the use of ps 2-sterile seed parents in tomato hybrid seed production. The following specific characteristics in the performance of ps 2-lines were evaluated: 1) the percentage of selfing in the ps 2-lines varies within and between the years of growing, being forever lowest during the period of hybrid seed production; 2) the percentage of selfed seeds when using a ps 2-line as seed parent in producing hybrid seed is significantly lower than the percentage of selfing, observed on this same line; 3) the ps 2-gene expressivity varies depending on the genotype. This enables the breeding of ps 2-lines exhibiting very low percentage of selfing; 4) stamen emasculation at anthesis (as applied in ps 2-lines) is significantly easier and more rapid than emasculation in floral buds. The easy maintenance of the sterile lines – by artificial selfing it is possible to produce 100% ps 2 progeny – and the high yield of hybrid seed obtained are discussed as important advantages in using this type of sterility in tomato hybrid seed production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Conversion of the genic male sterility (GMS) system of bell pepper (Capsicum annuum L.) to cytoplasmic male sterility (CMS)

Non‐pungent bell pepper (Capsicum annuum L.) lacks the cytoplasmic male sterility (CMS) nuclear restorer allele, Rf, and CMS cannot be employed in its F₁ hybrid seed production. To demonstrate that the genic male sterility (GMS) system in non‐pungent bell pepper can be converted to the CMS male sterility system, the conversion of GMS to CMS for non‐pungent bell pepper line GC3 was conducted by introgression of S‐type cytoplasm and the Rf allele from tropical pungent donors. After morphological traits were evaluated, two lines from BC₁F₁ containing S‐type cytoplasm and four lines from BC₂F₂ containing Rf allele, phenotypically similar to GC3, were obtained and could be employed as CMS male sterile lines and restorer lines for non‐pungent bell pepper. Four molecular markers potentially linked to traits of interest were also evaluated in BC₁F₁ and BC₁F₂ populations. This is the first time that GMS has been successfully converted to CMS in bell pepper, a significant contribution for bell pepper hybrid seed production.     

Identification of AFLP markers linked to one recessive genic male sterility gene in oilseed rape, Brassica napus

The commercial utilization of heterosis in seed yield by means of hybrid varieties is of great importance for increasing oilseed rape production in China. This requires a functional system for the production of hybrid seed. The Brassica napus oilseed rape line 9012AB is a recessive epistatic genic male sterility (GMS) two‐type line, in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms1 and ms2) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms1ms1ms2ms2 plants. This study was conducted to identify molecular markers for one of the male fertility/sterility loci in the B. napus male sterility line 9012AB. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from male sterile and male fertile plants of the homozygous two‐type line 9012AB were subjected to amplified fragment length polymorphic (AFLP) analysis. A total of 256 primer combinations were used and seven markers tightly linked to one recessive genic male sterile gene (ms) were identified. Among them, six fragments co‐segregated with the target gene in the tested population, and the other one had a genetic distance of 4.3 cM. The markers identified in this study will greatly enhance the utilization of recessive GMS for the production of hybrid seed in B. napus oilseed rape in China.     

Molecular mapping of a dominant genic male sterility gene Ms in rapeseed (Brassica napus)

Rs1046AB is a genic male sterile two‐type line in rapeseed that has great potential for hybrid seed production. The sterility of this line is conditioned by the interaction of two genes, i.e. the dominant genic male sterility gene (Ms) and the suppressor gene (Rf). The present study was undertaken to identify DNA markers for the Ms locus in a BC₁ population developed from a cross between a male‐sterile plant in Rs1046AB and the fertile canola‐type cultivar ‘Samourai’. Bulked segregant analysis was performed using the amplified fragment length polymorphism (AFLP) methodology. From the survey of 480 AFLP primer combinations, five AFLP markers (P10M13₃₅₀, P13M8₄₀₀, P6M6₄₁₀, E7M1₂₃₀ and E3M15₁₀₀) tightly linked to the target gene were identified. Two of them, E3M15₁₀₀ and P6M6₄₁₀, located the closest, at either side of Ms at a distance of 3.7 and 5.9 cM, respectively. The Ms locus was subsequently mapped on linkage group LG10 in the map developed in this laboratory, adding two additional markers weakly linked to it. This suite of markers will be valuable in designing a marker‐assisted genic male sterility three‐line breeding programme.     

Identification of AFLP markers linked to the epistatic suppressor gene of a recessive genic male sterility in rapeseed and conversion to SCAR markers

A recessive epistatic genic male sterility (REGMS) two‐type line, 9012AB, has been used for rapeseed hybrid seed production in China. The male sterility of 9012AB is controlled by two recessive duplicate sterile genes (ms1 and ms2) interacting with one recessive epistatic suppressor gene (esp). Homozygosity at the esp locus (espesp) suppresses the expression of the recessive male sterility trait in homozygous ms1ms1ms2 ms2 plants. In this study, we used a combination of bulked segregant analyses and amplified fragment length polymorphism (AFLP) to identify markers linked to the suppressor gene in a BC₁ population. From the survey of 1024 AFLP primer combinations, eight markers tightly linked to the target gene were identified. The two closest markers flanking both sides of Esp, P9M5₃₇₀ and S16M14₇₈₀, had a genetic distance of 1.4 cM and 2.1 cM, respectively. The AFLP fragment from P4M8₁₉₀, which co‐segregated with the target gene was converted into a sequence characterized amplified region marker. The availability of linked molecular markers will facilitate the utilization of REGMS in hybrid breeding in Brassica napus.     

Generation of tribenuron‐methyl herbicide‐resistant OsCYP81A6‐expressing rapeseed (Brassica napus L.) plants for hybrid seed production using chemical‐induced male sterility

Chemical‐induced male sterility (CIMS) is a method for hybrid rapeseed (Brassica napus L.) production. Some sulphonylurea herbicides such as tribenuron‐methyl (TBM) are used as chemical hybridization agents (CHAs) in CIMS systems. However, the male parents must be protected from herbicide injury with a shield during spraying of the female parents with CHAs to induce male sterility. Thus, using herbicide‐resistant rapeseed lines as the male parents can significantly simplify the seed production procedure and reduce the cost in hybrid seed production. A rice cytochrome P450 hydroxylase, OsCYP81A6, has been previously characterized to confer resistance to bentazon and sulphonylurea herbicides. We demonstrate here that the introduction of OsCYP81A6 renders rapeseed plants resistant to TBM. Compared with wild‐type plants, the transgenic plants displayed normal stamen development and male fertility when treated with 0.05 mg/l of TBM, the dose used for inducing male sterility in hybrid seed production. These results indicate that the OsCYP81A6‐expressing rapeseed plants can be used as the male parents for hybrid rapeseed production using CIMS.     

Hybrid seed production in safflower (Carthamus tinctorius) following the induction of male sterility by gibberellic acid

The objective of the present research was to develop an effective and practical method for hybrid seed production in safflower, by inducing male sterility with gibberellic acid (GA₃). Seeds of the spiny line ‘5‐154’ and the non‐spiny variety ‘Dincer 5‐118’ were sown in alternate rows and the plants of the non‐spiny female variety were exposed to various concentrations of GA₃ for male sterility induction. Three successive sprays (75, 82 and 89 days after sowing) of 100 p.p.m. of GA₃ to safflower buds of <0.5 cm diameter at a pre‐meiotic interphase stage resulted in reduced pollen viability from 81.6‐6.7% compared with the control. An average hybrid seed percentage of 80.7% (87.5% in the secondary heads), identified by the use of the spiny‐capitulum marker variety ‘5‐154′, was obtained. The GA₃ treatments did not significantly affect the production of viable achenes. The results showed that GA₃ could be successfully used as a chemical hybridizing agent in hybrid safflower production.     

番茄ps-2雄性不育系01222的育性观察

对从保加利亚引入并选出的含有番茄ps - 2基因的雄性不育新品系 0 12 2 2进行了杂交制种试验。结果表明 ,该不育品系虽然会发生自交 ,但座果率极低 ,其中正常果率只占 2 9% ,其余均为僵果。通过开花后 1～ 3d人工去雄授粉和蕾期去雄杂交制种试验比较说明 ,即使 3d后的处理杂交种子纯度仍可达 99%以上 ,符合国际种子质量标准 ,但是杂交座果率、单果种子含量却随开花天数的增加而降低 ,以蕾期和开花后 1d处理最佳。表明该不育系是一个很有应用价值的新材料。     

A codominant SCAR marker linked to the genic male sterility gene (ms1) in chili pepper (Capsicum annuum)

As one of the genic male sterility (GMS) materials in chili pepper ( Capsicum annuum L.), GMS1 has been used for commercial F₁ hybrid seed production. The male sterility of GMS1 is controlled by a recessive nuclear gene, named ms ₁ . In this study, we developed DNA markers linked to the ms ₁ locus using a combination of bulked segregant analysis and amplified fragment length polymorphism (AFLP) in a segregating sibling population. From the screening of 1024 AFLP primer combinations, the AFLP marker E-AGC/M-GTG (514 bp) was identified as being linked to the ms ₁ locus at a distance of about 3 cM. Based on internal sequencing analysis of the E-AGC/M-GTG marker between male fertile and sterile plants, we identified three small deletions with a size of altogether 42 bp in the male-fertile plant and developed a codominant sequence characterized amplified region (SCAR) marker. This SCAR marker may be valuable for marker-assisted breeding in the hybrid seed production system of chili pepper using the GMS1 line.     

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS systems.     

Applications of Genie Male Sterility in Plant Breeding

The possible applications of genie male sterility (GMS) in plant breeding arc reviewed and discussed. The basic contribution of GMS is that it provides a means of genetic emasculation which can be applied for the massive production of hybrids. There are two main fields of application: (1) the production of hybrid varieties and (2) inter- and intraspecific hybridization and back-crossing programmes for the introduction of genetic variation into crop varieties. Several schemes have been proposed for using GMS in hybrid breeding in different crops. These are discussed under four headings: genetic methods, cy-togenetic methods, temporary restoration of fertility and manual pollination. The application of GMS in hybridization intended for the introduction of alien genes is discussed as well as factors affecting the pollination of genie male steriles.     

BS系列杂交小麦在陕西渭南地区制种可行性分析

高效制种技术是杂交小麦推广和产业化的重要影响因素,适宜制种区域的探索是制种技术研究的关键内容。以往基于光温敏不育的BS系列杂交小麦制种区域较为单一,多集中在安徽阜阳地区。为扩充杂交小麦的制种区域,本研究将陕西渭南地区与传统制种区域——安徽阜阳种子生产区进行了生态气候条件比较分析。渭南地区小麦生长期平均温度较阜阳低1.3℃,育性转化重要时期平均温度相差小于1℃;日可照时数与原阜阳制种区相近,从光温等生态气候条件上为BS系列杂交小麦制种提供了可能。同时,渭南地区在小麦种子生产关键时期降水量较阜阳低41.7%,种子质量更有保障。在此基础上,在陕西渭南地区开展了BS系列光温敏不育系育性鉴定和中试制种试验。结果表明,大部分BS系列骨干不育系自交结实为0.5粒/穗左右,不育度可达98%以上;各杂交种平均异交结实数可达14.4~20.7粒/穗,制种产量超过3 000kg/hm ²,并且种子纯度达到95%以上,符合杂交小麦制种生产要求。渭南地区可作为BS系列杂交小麦制种的备选区域。     

Genetic linkage between male sterility and non‐spiny trait in safflower (Carthamus tinctorius L.)

Genetic male sterility (GMS) exists naturally in safflower (Carthamus tinctorius L.). In the existing safflower GMS lines, sterile and fertile plants are distinguishable at flowering. This causes delay in fertile plants rouging and reduction in hybrid purity. In this investigation, a cross between a spiny GMS parent 13‐137 and a spiny non‐GMS parent ‘A1’ was effected. One sib cross, SC‐67, producing non‐parental‐type non‐spiny sterile and spiny fertile plants in F₃ was advanced to F₉ through sib crossing between non‐spiny sterile and spiny fertile plants. Mendelian digenic segregation was not observed for non‐spiny trait and male sterility. The results revealed strong linkage between these traits. The linkage was confirmed in F₂ generations of crosses between a non‐spiny marker‐linked GMS line (MGMS) and five elite lines. Male sterility–linked non‐spiny trait could distinguish sterile and fertile plants at elongation stage. The MGMS would be useful in production of pure F₁ hybrid seed and development of elite populations.     

Genetics, fertility behaviour and molecular marker analysis of a new TGMS line, TS6, in rice

The thermosensitive genic male sterility (TGMS) system has great potential for revolutionizing hybrid rice production through simple, less expensive and more efficient seed production technology. For the successful utilization of this novel male sterility system, knowledge of the breeding and fertility behaviour of a TGMS line is essential. In this study, the fertility transformation behaviour, the critical fertility and sterility temperatures and the mode of inheritance of male sterility were studied for a new TGMS line, TS6, identified at Tamil Nadu Agricultural University, Coimbatore, India. The pollen and spikelet fertilities recorded on plants raised at fortnightly intervals revealed that this line was completely sterile for 78 consecutive days (35/22 to 32/23°C, maximum/minimum temperatures) and reverted to fertile when the temperature was 30/18°C. It remained fertile continuously for 69 days and the maximum pollen and spikelet fertilities recorded were 75 and 70%, respectively. The fertility was highly influenced by daily maximum temperature followed by average and minimum temperatures. It was not influenced by relative humidity, sunshine hours or photoperiod. The critical temperature inducing sterility and fertility was 26.7 and 25.5°C, respectively. The male sterility in TS6 was inherited as a monogenic recessive in the F₂ and BC₁ populations of TS6 × MRST9 as well as TS6 × IR68281B. Using bulked segregant analysis on an F₂ population of TS6 × MRST9, an RAPD marker, OPC05₂₉₆₂, was identified to be associated with TGMS in TS6.     

The wi gene causes genic male sterility in Allium choenoprasum

Segregation studies following the transfer of the gene wi to different cytoplasm types, which have been distinguished by means of restriction fragment length polymorphism analyses using mitochondrial gene probes, revealed the formation of the wi‐sterility in each of the four cytoplasms examined. The male sterility is therefore only caused by the nuclear wi gene, i.e. an additional factor of a specific cytoplasm can be excluded. Hence, the wi‐sterility proved to be a genic male sterility (GMS) and not a cytoplasmic male sterility (CMS). The expression of the wi‐sterility appears to be stable, since it is not affected by high temperatures or tetracycline. Accordingly, a temporary pollen production, which would allow self‐fertilization for the maintenance of sterile lines, cannot be induced by controlling these environmental factors. In terms of hybrid breeding, this GMS therefore has no advantage over the previously described CMS system.     

Variations in chlorosis and potential usefulness of alloplasmic Brassica rapa with the cytoplasm of male sterile Brassica juncea

To select superior seed parents for vegetable hybrid seed production, we conducted interspecific crosses between male sterile Brassica juncea (2n = 36, AABB) and eight inbred lines of Brassica rapa (2n = 20, AA). Alloplasmic lines of B. rapa with the cytoplasm of B. juncea were developed from B. juncea × B. rapa hybrids by repeated backcrossing using B. rapa as the recurrent male parent until the BC₃ generation. Seed fertility, male sterility and chlorophyll content were investigated in these plants cultivated under four different temperature conditions (5, 10, 12 and 20°C). At 10°C, the alloplasmic lines of B. rapa with the cytoplasm of B. juncea were male sterile with partly chlorotic leaves. The alloplasmic B. rapa had lower chlorophyll a, chlorophyll b and carotenoid contents than those of the original B. rapa. The leaves recovered from chlorosis when the plants were cultivated at 20°C. An alloplasmic line of B. rapa (A6) is available as a seed parent for vegetable hybrid seed production and contributes seed fertility, slight chlorosis and stable male sterility.     

油菜隐性细胞核雄性不育的研究进展

油菜细胞核雄性不育是油菜杂种优势利用的重要途径。近年来,油菜隐性细胞核雄性不育研究取得了一些进展并育成了一批核不育杂交种。为了促进核不育研究的深入及油菜杂种优势利用水平,对双隐性核不育和隐性上位互作核不育2种类型的核不育系统的遗传模式、相关基因定位以及其不育分子机理方面的研究进行综述,并对其研究成果在将来油菜杂交育种中的利用进行展望。     

Cytoplasmic male sterility in Brassicaceae crops

Brassicaceae crops display strong hybrid vigor, and have long been subject to F₁ hybrid breeding. Because the most reliable system of F₁ seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted.     

质体转化开创作物杂种优势利用新途径

普通核雄性不育性能够满足对理想不育系选育的要求，是水稻等作物杂种优势利用的理想遗传元件。只要能解决其不育系繁殖问题，是理想的杂种优势利用方式。通过普通核雄性不育性的可育基因质体转化，将可育基因转移到存在于细胞质中的质体基因组中，可创造普通核雄性不育系的保持系，繁殖出普通核雄性不育系，从而实现三系法利用杂种优势；利用可产生雄性不育性的基因如TA29-barnase通过质体转化，向质体基因组中转移，产生由细胞质中转基因雄性不育基因控制的雄性不育系，任何常规品种都能作为其保持系，转barstar可育基因系(或常规品种)作为恢复系(或父本)，可同样实现三系法利用杂种优势。上述两种途径都是创造植物杂种优势利用的新途径。     

A comparative study of microsporogenesis and anther wall development in different types of genic and cytoplasmic male sterilities in chives

A new cytoplasmic and two genic male sterilities in chives are compared with male‐fertile counterparts for anther development and microsporogenesis. The genic male sterilities (wi and st1) fail to produce viable pollen, since microsporogenesis is stopped around the stage of tetrads. Compared with a previously described cytoplasmic male sterility, CMS₁, which also shows alterations during this stage, wi‐ and st1‐sterile plants fail to produce pollen walls. The last two male sterilities are very similar in development but can be distinguished by the missing breakdown of callose in st1‐sterile plants. In contrast to the sterilities described above, the second cytoplasmic male sterility, CMS₂, is caused by functional damages to sporophytic tissues, such as no degeneration of mesothecium cells and no thickening of the endothecial cell walls. These alterations prevent the release of pollen, although they are normally developed.