Genetics and cytology of a new genic male-sterile soybean [Glycine max (L.) Merr.]

 Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F₁ or F₂ generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains. Received: 13 May 1996 / Revision accepted: 19 August 1996     

Identification of a single nucleotide deletion causing frameshift mutation in OsDFR2A in a genic male sterile mutant of rice and its possible application to F1 hybrid breeding

Stable genic male sterility (GMS), which is not influenced by environmental factors, has not been used for F₁ hybrid seed production because male-sterile inbred lines cannot be developed and male-sterile plants must be selected from segregating populations every time. However, the stability of male sterility may provide a reliable system for F₁ seed production without contamination of selfed seeds. A genic male-sterile mutant in rice (Oryza sativa L.), C204, which was selected from progeny of the cultivar ‘Koshihikari’ irradiated by gamma rays, has shorter and whiter anthers than those of ‘Koshihikari’ and has no pollen grains. Segregation analysis of C204 suggested the male sterility of this mutant to be controlled by a recessive allele of a single gene. Linkage analysis of a mutated gene responsible for the male sterility revealed the gene to be in a region of ca. 75 kb on the long arm of chromosome 9. The nine genes predicted in the 75-kb region were sequenced, and compared with the published Nipponbare genome sequences. A single-base deletion was found in the first exon of a C204 allele of Os09g0493500, which encodes an NAD-dependent epimerase/dehydratase family protein, resulting in a frameshift causing a premature stop codon. A dot-blot single nucleotide polymorphism marker for detection of the single-base deletion in Os09g0493500 was developed. We herein propose an F₁ hybrid seed production system using stable GMS with a simple selection method of GMS plants.     

Cytochrome P450 mono-oxygenase CYP703A2 plays a central role in sporopollenin formation and ms5ms6 fertility in cotton

The double-recessive genic male-sterile(ms) line ms5 ms6 has been used to develop cotton(Gossypium hirsutum) hybrids for many years, but its molecular-genetic basis has remained unclear.Here, we identified the Ms5 and Ms6 loci through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 gene editing.Ms5 and Ms6 are highly expressed in stages 7–9anthers and encode the cytochrome P450 monooxygenases CYP703A2-A and CYP703A2-D. The ms5 mutant carries a single-nucleoti...     

玉米隐性核不育突变体ms14的遗传分析与基因定位

玉米雄性不育材料是一种宝贵的种质资源,不育基因的遗传分析与定位研究对玉米分子育种和杂种优势利用具有重要价值。通过对从美国引进的玉米雄性不育突变体材料ms14进行雄花育性鉴定和花药I₂-KI染色,表明该突变体是无花粉型雄性不育;通过不育突变体ms14与正常自交系郑58、昌7-2杂交获得F₁,然后自交构建两个F₂遗传分离群体（ms14×郑58和ms14×昌7-2）,并进行雄花育性调查、数据统计和遗传分析,发现可育株数与不育株数的分离比是3∶1,表明该突变体由隐性单基因控制;通过SSR等分子标记与不育位点的连锁分析,将ms14基因定位在玉米第1染色体的SSR标记umc2025和umc1676之间,遗传距离分别是2.2cM和0.3cM。对玉米不育基因ms14的遗传分析和初步定位,为该基因的精细定位和克隆、不育机理的解析及其产业化应用奠定了基础。     

Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen

Reversible male sterility and doubled haploid plant production are two valuable technologies in F₁-hybrid breeding. F₁-hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F₁-hybrid seed technology based on the metabolic engineering of glutamine in developing tobacco anthers and pollen. Cytosolic glutamine synthetase (GS1) was inactivated in tobacco by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters. Pollen in primary transformants aborted close to the first pollen mitosis, resulting in male sterility. A non-segregating population of homozygous doubled haploid male-sterile plants was generated through microspore embryogenesis. Fertility restoration was achieved by spraying plants with glutamine, or by pollination with pollen matured in vitro in glutamine-containing medium. The combination of reversible male sterility with doubled haploid production results in an innovative environmentally friendly breeding technology. Tapetum-mediated sporophytic male sterility is of use in foliage crops, whereas microspore-specific gametophytic male sterility can be applied to any field crop. Both types of sterility preclude the release of transgenic pollen into the environment.     

MALE STERILITY 3 encodes a plant homeodomain-finger protein for male fertility in soybean

Male-sterile plants are used in hybrid breeding to improve yield in soybean (Glycine max (L.) Merr.). Developing the capability to alter fertility under different environmental conditions could broaden germplasm resources and simplify hybrid production. However, molecular mechanisms potentially underlying such a system in soybean were unclear. Here, using positional cloning, we identified a gene, MALE STERILITY 3 (MS3), which encodes a nuclear-localized protein containing a plant homeodomain (PHD)-finger domain. A spontaneous mutation in ms3 causing premature termination of MS3 translation and partial loss of the PHD-finger. Transgenetic analysis indicated that MS3 knockout resulted in nonfunctional pollen and no self-pollinated pods, and RNA-seq analysis revealed that MS3 affects the expression of genes associated with carbohydrate metabolism. Strikingly, the fertility of mutant ms3 can restore under long-dconditions. The mutant could thus be used to create a new, more stable photoperiod-sensitive genic male sterility line for two-line hybrid seed production, with significant impact on hybrid breeding and production.     

Differential proteomic studies of the genic male-sterile line and fertile line anthers of upland cotton (Gossypium hirsutum L.)

Pollen development is disturbed in the microspore development stage of the double-recessive nuclear male-sterile line ms5ms6 (Gossypium hirsutum L.). This study aimed to identify differentially expressed anther proteins and their potential roles in pollen development and male sterility. We compared the proteomes of sterile and fertile anthers of the double recessive nuclear male-sterile line ms5ms6. Approximately 1,390 protein spots were detected by two-dimensional differential gel electrophoresis. Proteins with altered accumulation levels in sterile anthers compared with fertile anthers were identified by mass spectrometry and the NCBInr and Viridiplantae EST databases. Down-regulated proteins in the sterile anthers included cytosolic ascorbate peroxidase 1 and glutaminyl-tRNA synthetase (glutamine-tRNA ligase). Several carbohydrate metabolism- and photosynthesis-related enzymes were also present at lower levels in the mutant anthers. By contrast, ATP-dependent RNA helicase eIF4A-13, NADH dehydrogenase subunit 1, enolase, gibberellin 20-oxidase, gibberellin 3-hydroxylase 1, alcohol dehydrogenase 2d, 3-ketoacyl-CoA synthase, and trehalose 6-phosphate synthase were expressed at higher levels in sterile anthers than in fertile anthers. The regulation of upland cotton pollen development involves a complex network of differentially expressed genes. This study provides the foundation for future investigations of gene function in upland cotton pollen development and male sterility.     

Alloplasmic male-sterile <Emphasis Type="Italic">Brassica juncea</Emphasis> with <Emphasis Type="Italic">Enarthrocarpus lyratus</Emphasis> cytoplasm and the introgression of gene(s) for fertility restoration from cytoplasm donor species

A new cytoplasmic male sterility (CMS) source in Brassica juncea (2n = 36; AABB) was developed by substituting its nucleus into the cytoplasm of Enarthrocarpus lyratus (2n = 20; E(l)E(l)). Male sterility was complete, stable and manifested in either petaloid- or rudimentary-anthers which were devoid of fertile pollen grains. Male sterile plants resembled the euplasmic B. juncea except for slight leaf yellowing and delayed maturity. Leaf yellowing was due mainly to higher level of carotenoids rather than a reduction in chlorophyll pigments. Female fertility in male-sterile plants varied; it was normal in lines having rudimentary anthers but poor in those with petaloid anthers. Each of the 62 evaluated germplasm lines of B. juncea was a functional maintainer of male sterility. The gene(s) for male-fertility restoration ( Rf) were introgressed from the cytoplasm donor species through homoeologous pairing between A and E(l) chromosomes in monosomic addition plants (2n = 18II+1E(l)). The percent pollen fertility of restored F(1) ( lyr CMS x putative restorer) plants ranged from 60 to 80%. This, however, was sufficient to ensure complete seed set upon by bag selfing. The CMS ( lyr) B. juncea compared favourably with the existing CMS systems for various productivity related characteristics. However, the reduced transmission frequency of the Rf gene(s) through pollen grains, which was evident from the sporadic occurrence of male-sterile plants in restored F(1) hybrids, remains a limitation.     

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.     

Inheritance of spontaneous male sterility in pigeonpea

 A male-sterile plant was observed in the UPAS-120 cultivar of pigeonpea (Cajanus cajan). The plant was about 5–7 days late-flowering and had white translucent anthers with complete pollen sterility. The inheritance of this spontaneous male sterility was studied in a cross involving the mutant and fertile UPAS-120, including their F₁, F₂, BC₁F₁ and BC₂F₁ generations. The results suggested that the male sterility was genetic and due to a recessive gene. Received: 12 November 1996/Accepted: 17 January 1997     

Genetics of a new male-sterility locus in pigeonpea (Cajanus cajan [L.] Millsp.).

A natural male-sterile mutant was found in the population of a short-duration pigeonpea (Cajanus cajan[L.] Millsp.) cultivar ICPL 85010. This mutant is characterized by light yellow anthers of reduced size that are devoid of pollen grains. This mutant was crossed with two pigeonpea cultivars to study its inheritance. The F1, F2, and test cross data of the two crosses suggested that this male sterility trait is genetic in origin and is controlled by a single recessivegene. The F1 (mutant x ICPL 85010) plants were crossed with translucent (ms1) and arrowhead type (ms2) genetic male steriles reported earlier to study their allelic relationships. Segregation in the three-way cross F1 and F2 populations revealed that the mutant male-sterile gene was nonallelic to ms1 and ms2 loci and it is designated ms3. The new male sterility sources in pigeonpea will help in producing high-yielding hybrids and populations in diverse phenological groups.     

A straight-forward seed production technology system for foxtail millet (Setaria italica)

For autogamous crops, a precondition for using heterosis is to produce sufficient pure male-sterile female parents that can be used to produce hybrid seeds. To date, cytoplasmic male sterility(CMS)and environment-sensitive genic male sterility(EGMS) have been used commercially to exploit heterosis for autogamous species. However, neither CMS nor EGMS has been established for foxtail millet(Setaria italica). Here, we report on the establishment and application of a seed production technology(SPT)...     

MS1 is essential for male fertility by regulating the microsporocyte cell plate expansion in soybean

Male sterility is an essential trait in hybrid seed production, especially for monoclinous and autogamous food crops. Soybean male-sterile ms1 mutant has been known for more than 50 years and could be instrumental in making hybrid seeds. However, the gene responsible for the male-sterile phenotype has remained unknown. Here, we report the map-based cloning and characterization of the MS1 gene in soybean. MS1 encodes a kinesin protein and localizes to the nucleus, where it is required for the male meiotic cytokinesis after telophase Ⅱ. We further substantiated that MS1 colocalizes with microtubules and is essential for cell plate formation in soybean male gametogenesis through immunostaining. Both ms1 and CRISPR/Cas9 knockout mutants show complete male sterility but are otherwise phenotypically normal, making them perfect tools for producing hybrid seeds.The identification of MS1 has the practical potential for assembling the sterility system and speeding up hybrid soybean breeding.     

茄子功能性雄性不育的遗传及其与果紫色基因连锁关系的研究

采用新育成的茄子功能性雄性不育系uGA 1-MS和2个栽培品种,进行双亲本杂交世代遗传试验,发现F_1和B_2代植株皆雄性正常,B_2和F_2代可育株和不育株呈1:1和3:1分离,表明茄子功能性雄性不育性状由单隐性基因支配,用基因符号fms表示。连锁测验数据表明基因+/fms与果紫色基因X/x紧密连锁。预期该雄性不育性可在茄子杂种优势育种和种子生产上加以利用。     

Expression of Engineered Nuclear Male Sterility in Brassica napus (Genetics,Morphology, Cytology,and Sensitivity to Temperature)

A dominant genetic male sterility trait obtained through transformation in rapeseed (Brassica napus) was studied in the progenies of 11 transformed plants. The gene conferring the male sterility consists of a ribonuclease gene under the control of a tapetum-specific promoter. Two ribonuclease genes, RNase T1 and barnase, were used. The chimaeric ribonuclease gene was linked to the bialophos-resistance gene, which confers resistance to the herbicide phosphinotricine (PPT). The resistance to the herbicide was used as a dominant marker for the male sterility trait. The study presented here concerns three aspects of this engineered male sterility: genetics correlated with the segregation of the T-DNA in the progenies; expression of the male sterility in relation to the morphology and cytology of the androecium; and stability of the engineered male sterility under different culture conditions. Correct segregation, 50% male-sterile, PPT-resistant plants, and 50% male-fertile, susceptible plants were observed in the progeny of seven transformants. The most prominent morphological change in the male-sterile flowers was a noticeable reduction in the length of the stamen filament. The first disturbances of microsporogenesis were observed from the free microspore stage and were followed by a simultaneous degeneration of microspore and tapetal cell content. At anthesis, the sterile anthers contained only empty exines. In some cases, reversion to fertility of male-sterile plants has been observed. Both ribonuclease genes are susceptible to instability. Instability of the RNase T1-male sterility trait increased at temperatures higher than 25[deg] C. Our results do not allow us to confirm this observation for the barnase male-sterile plants. However, the male-sterile plants of the progeny of two independent RNase T1 transformants were stably male sterile under all conditions studied.     

Linkage mapping of a dominant male sterility gene Ms-cd1 in Brassica oleracea.

The dominant male sterility gene Ms-cd1 (c, cabbage; d, dominant) was identified as a spontaneous mutation in the spring cabbage line 79-399-3. The Ms-cd1 gene is successfully applied in hybrid seed production of several Brassica oleracea cultivars in China. Amplified fragment length polymorphism (AFLP) technology was used to identify markers linked to the Ms-cd1 gene in bulks of male-sterile and male-fertile individuals of a segregating BC3 population and in a near-isogenic population of 25 male-sterile plants. Twelve markers within a 20-cM interval proximal to the Ms-cd1 gene were identified, 5 of which can be used to select homozygous male-sterile Ms-cd1/ Ms-cd1 plants. Three AFLP markers and 3 sequence characterized amplified region markers that were linked to MS-cd1 mapped onto linkage group O9, corresponding to chromosome 3 of B. oleracea. This region corresponds to the top of chromosome 5 in Arabidopsis thaliana.     

Genetic analysis and molecular mapping of a nuclear recessive male sterility gene, ms91(t), in rice.

Mutations that result in plant male sterility provide means not only to probe reproductive development but also to facilitate commercial heterosis application and hybrid seed production. In this study, we report a novel male sterility gene, ms91(t), in a spontaneous mutant line (SH38) from a Chinese rice cultivar (Oryza sativa subsp. japonica 'Jijing14'). The sterility of SH38 was studied by examining its progenies derived from crosses with 6 japonica cultivars. Corresponding F2 populations were obtained by selfing each of the 6 F1s and a backcross population was produced by crossing SH38 to the F1 of SH38 x C18. Our results revealed that SH38 has normal agronomic traits but produces no pollen grains. Segregations of male-sterile and male-fertile progenies in the F2 and backcross populations fit well with ratios of 3:1 and 1:1, respectively, indicating that ms91(t) is a single recessive gene. Amplified fragment length polymorphism (AFLP) analysis of SH38 and Jijing14 plants showed the presence of a unique band in SH38. Simple sequence repeat (SSR) analysis of the bulked and individual progenies of the F2 population of SH38 x C18 showed linkage of ms91(t) with the SSR marker RM5853 on chromosome 1. Subsequently, ms91(t) was fine-mapped to the interval between markers RM7075 (3.75 cM) and RM5638 (3.57 cM). Our results would facilitate the isolation of ms91(t) and male sterility in heterosis application.     

丹参雄性不育系Sh-B的鉴定与花粉发育过程的解剖学研究

在显微水平上对新发现的丹参雄性不育系Sh-B花药发育过程进行了解剖学观察,并对其花粉活力和结实率进行了鉴定。结果显示:根据花器官及花药的形态、大小以及花丝的长度,可以将Sh-B不育株分为3个不育类型,即Sh-B1、Sh-B2和Sh-B3。这3种不育类型均属于雄性不育,其花丝不到正常可育株的1/2,花药干瘪而瘦小,内无花粉粒或花粉无活力;其根、茎、叶以及种子形态结构与正常可育植株基本相似。产生雄性不育的主要原因有:花粉囊药室内壁纤维层加厚,影响花药壁开裂;小孢子母细胞周围不产生胼胝质或产生的胼胝质很少;绒毡层细胞延迟解体;花粉粒畸形。在其花药发育的小孢子母细胞时期、四分体形成前期、单核期、双核期均可能产生雄性不育的小孢子或花粉粒。