Exploration and utilization of maize male sterility resources.

Male sterility refers to the defective development of male reproductive organs, which led to plants incapable of producing normal and functional pollens. Maize (Zea mays L.) is one of the most important food crops, as well as one of the earliest crops to utilize heterosis in breeding. Single cross hybrid has been the main type of maize heterosis utilization for a long time. The planting area of maize hybrid in China has been stable at about 620 million mu. More than one billion kilograms of commercial hybrid seeds are needed each year, and the annual seed production area has been stable at about 2.5 million mu in recent years. So far, manual emasculation has been the major way of maize hybrid seed production in China, which is laborious and time consuming. Generally, spatial isolation is necessary for maize hybrid seed production, this requirement results in only some regions in the country suitable for maize hybrid seed production. Manual emasculation requires seasonal demand of labors. At present, with the urbanization of a large number of rural laborers, the seed production regions experience a serious labor shortage. Accordingly, the cost of seed production increases with the rising of land rent and labor costs. In addition, it is difficult to guarantee the seed purity with manual or mechanical emasculation for hybrid seed production. However, incorporating male sterility into maize hybrid seed production could reduce its cost and ensure hybrid seed purity. It can also avoid the difficulties of manual or mechanical emasculation in field operation under extreme weather conditions. Therefore, it is the inevitable trend of development in the maize seed industry. In this review, we summarize the exploitation and creation of maize cytoplasmic male sterility (CMS), maize genic male sterility (GMS) resources in China, and the developing process from natural discovery to targeted creation of male sterility resources in plants, and the research progress of maize male sterility. We then analyze the application status and existing problems of maize male sterility, based on the development trend of maize seed industry, as well as the research and application status of male sterility in China. We also identify seven aspects that need to be further strengthen, thereby providing the reference for the creation, research and utilization of maize male sterility in the future.

[Genetics of fertility restoration in the isocytoplasm allonuclear C-group of cytoplasmic male sterility in maize].

Maize is one of the first crops to produce hybrids using cytoplasmic male sterile lines. The C-type cytoplasmic male sterile (CMS-C) line is vital for hybrid seed production, and the fertility-restoration reaction along with its stability has a direct bearing on its applications. However, fertility restoration mechanism of CMS-C is complex and is still not clear so far. To further explore the factors affecting the fertility restoration of maize CMS, a series of test crosses were carried out by pollinating the isocytoplasm allonuclear CMS-C lines C48-2, Chuangzaosi and C478 with the test lines 18 bai, zi 330, 5022 and the restorer line A619, respectively. Four F2 populations and six double-cross combinations were obtained from the self-cross of fertility restored F1 and pollinating male-sterility-maintained F1 with the male-fertility-restored F1, respectively. Meanwhile, we developed the incomplete diallel-cross combinations using the isocytoplasm allonuclear male sterile lines as maternal parents and their respective maintainer lines 48-2, huangzaosi and 478 as paternal parents. All the F1, F2 and double-cross populations were planted at distinct locations in different years, and the fertility-restoration reaction was scored by field investigation and pollen staining with I2-IK. The results were as follows: 1) The same test line could restore the CMS-C line at a certain genetic background, but failed to restore the CMS-C line at the other genetic backgrounds, suggesting that the genetic background of CMS-C lines plays an important role in the fertility restoration. 2) The fertile-to-sterile segregation ratio of (C48-2×A619) F2 population planted in both Sichuan and Yunnan fited well to 15:1 by the χ 2 test. However, the fertility level of individuals in Yunnan mainly belonged to the 3 and 4grades, but which in Sichuan mainly belonged to the 5 grade, indicating the environmental factors had effect on the fertility-restoration reaction of (C48-2×A619) F2. 3) In our study, 18 bai could not restore C478, and 48-2 could not restore C478, but the fertile and sterile segregated plants were unexpectedly found in their double-cross population [(C478×18 bai) F1S×(C48-2×18 bai) F1F]. The similar case was also observed in the double-cross population [(C48-2 × zi 330) F1S × (C478 × zi 330) F1F]. Therefore, we speculated that there are minor fertility restorer genes not only in the nuclear background of C48-2 and C478, but also in zi 330 and 18 bai, and when these minor genes were gathered by hybridization, they could restore the fertility of C478 and C48-2. This conforms to the restorer genes dose-effect for fertility restoration in the plant CMS system. These results not only contribute to our understanding of the complexity and diversity of CMS-C restoration mechanism, but also provide an important reference for the practical applications about maize CMS-C.

Research progress on mechanisms of male sterility in plants based on high-throughput RNA sequencing.

Male sterility is defined as failing to produce functional pollen during stamen development in plants, and it plays a crucial role in plant reproductive research and hybrid seed production in utilization of crop heterosis. High throughput RNA sequencing (RNA-seq) has been used widely in the study of different fields of life science, as it readily detects all the mRNA and non-coding RNA in cells. Recently, RNA-seq has been reported to be applied in different species and kinds of pollen abortion types in plants, which has contributed to the understanding of the molecular mechanism and metabolic networks of male sterility at the transcription level. In this review, we summarize research progress on the mechanisms of male sterility in plants, focusing on RNA-seq analysis encompassing strategies of RNA library construction, differentially expressed genes and functional characteristics of noncoding RNAs involved in stamen abortion. Furthermore, we also discuss application of transcriptome sequencing technology to elucidate pollen abortion mechanisms and map fertility-related genes. We hope to provide references to the study of male sterility in plants.

[Research progress of the bHLH transcription factors involved in genic male sterility in plants].

Male sterility exists widely in the spermatophytes. It contributes to the study of plant reproductive development and can be used as an effective tool for hybrid seed production in heterosis utilization. Therefore, the study on male sterility is of great value in both theory and application. As one of the largest transcription factor families in plants, basic helix-loop-helix proteins (bHLHs) play a crucial role in regulating plant growth and development. This paper introduces the mechanism of bHLH regulating stamen development in several important model plants. Furthermore, we discuss the molecular mechanisms of genic male sterility resulting from bHLH dysfunction to provide references for crop breeding and theoretical studies.

[Combining ability analysis for SP4 lines of maize from space flight].

Three maize (Zea mays L.) inbred lines 08-641, RP125, and 18-599 were carried into cosmic space by recoverable satellite "Shijian 8". Some mutant lines were selected from SP4 and combinations were made according to the NC II genetic design. The materials were planted in Sichuan and Yunnan separately to analyze combining ability based on the incomplete diallel cross design. The results showed that space flight affected the combining ability of mutant lines, and the GCA value of mutant lines were different in two kinds of environmental condition. The GCA of ear length, row per ear, kernel per row, and yield per plant for the mutant line C03 showed substantial increase compared with the control 08-641; the GCA of row per ear, kernel per row, and other yield component traits for the mutant lines C01 and C04 were significantly higher than those of the control. The SCA of yield and yield components for the combinations derived from the mutant lines C06, R18, and S22 were higher than others. These results laid a material foundation for maize breeding and provided some important references for improving and utilizing the mutant lines.

[Genetic analysis of maize cytoplasmic male sterile mutants obtained by space flight].

Three maize male sterile mutants were obtained from the offsprings of two maize inbred lines 18-599 and 08-641, which were carried into space by the Shijian 8 Satellite. The stability of male sterile expression was observed in different locations, years, and seasons. In order to analyze the genetic characteristic of male sterility, testcross, backcross and reciprocal cross were made with these male sterile plants. The results showed that the male sterility character was stable in different locations, years, and seasons, and the sterility was inheritable. Because the maintainer lines and restorer lines for these sterile materials were found, and there was no male sterile plant separated among the reciprocal cross F2. Thus, we concluded that these mutants could be cytoplasmic male sterile. Combining the results of male fertility restoration test and PCR analysis, we could conclude that the three male sterile mutants were classified into the CMS-C type in maize. Owing to their difference in fertility restoration, these mutants may belong to different subgroups of CMS-C type. The discovery of the three male sterile mutants increased the genetic diversity of CMS-C type, improved the tolerance to Bipolaris maydis, and laid a foundation for extensive application of CMS-C in seeds production.

RNA editing of mitochondrial functional genes atp6 and cox2 in maize (Zea mays L.).

RNA editing of two mitochondrial or organs genes, atp6 and cox2, in different tissues were analyzed using homonucleic but alloplasmic, and homoplasmic but heteronucleic maize (zea mays L.) as experimental materials. A total of 18 and 26 editing sites for atp6 conservative region transcript were identified by direct and clone sequencing, respectively. By direct sequencing 23 and 22 editing sites for cox2 transcript were identified in 48-2 and Huangzaosi nuclear backgrounds, respectively. From the direct sequencing results, the occurrence rates of different transcripts generally increase in sterile lines. It is concluded that RNA editing of atp6 and cox2 might have a certain relationship with maize CMS.

[The editing sites in the transcripts of mitochondrial atp6 gene of maize sterile lines and maintainer line].

Using maize (Zea mays L.) cytoplasmic male-sterile lines T Huang Zao Si, C Huang Zao Si, S Huang Zao Si and maintainer line N Huang Zao Si as the plant materials, editing sites in the conservative area of mitochondrial atp6 gene transcripts of the 4 experimental materials' tassels, of which microspores had developed to uni-nucleate stage, were analyzed. The results showed that DNA sequences of the T, C and S male-sterile cytoplasms were completely unanimous, while being compared with the N-cytoplasm, all the sequences were similar except for the 27th and 28th nucleotides. However, the cDNA sequences of each cytoplasm were not always the same. By comparing DNA and cDNA sequences, we found that within the conservative area of atp6 gene transcripts there were 19, 22, 20 and 19 editing sites in the N, T, C and S cytoplasms, respectively. The 4 cytoplasms also shared 18 sites. The majority of the editings occurred at the 1st or the 2nd position of codons, which might alter the amino acid type. Most the shared editings were fully editing, and the 1st and the 19th sites were partially edited in nearly all cytoplasms, except for the 19th sites editing in the N-cytoplasm. The specific editings in each cytoplasm occurred in the form of partially editing. Thus the editing of atp6 gene in maize was not only sequence specific but also affected by cytoplasmic background. Furthermore, plant RNA editing was inclined to improve the predicted protein's hydrophobicity and enhance the conservation among species.

[Cytological observation on pollen abortion of genetic male sterile mutant induced by space flight in maize].

In order to understand the cytological mechanism of pollen abortion of genetic male sterile mutant induced by space flight in maize, the sister cross population were used for sterility analysis and cytological observation. Intact anther observation, isolated cells observation and paraffin section were adopted in this research. The results showed that pollen abortion occured mostly in dyad stage of meiosis in genetic male sterile mutant. The dyad were degenerated with abnormal shape. In late anther developing stage, the tapetal cells were giant vacuolated and delayed degeneration. The pollen mother cells (PMC) began to dissolve and degenerate in a few anther before meiosis.

[The discovery of a specific DNA fragment associated with maize cytoplasmic male sterility and its differential display].

Three pairs of PCR primers were designed according to the mitochondrial DNA sequence. PCR amplification was applied to 3 sets of isonuclear alloplasm materials and 3 sets of isoplasm allonuclear materials. Multiplex PCR and general PCR protocol were adopted with total genomic DNA. As for the primers having detected polymorphsim between male sterility and its maintainers, differential display was conducted with mRNA from different development stage of microspore. The results showed as follows: with total genomic DNA template, primer P1-P2 has amplified a specific fragment only in all the male sterile materials, primer P5-P6 has amplified a specific fragment only in maintainer Huangzaosi, primer P3-P4 has no amplification in all the experiment materials. So primer P1-P2 can be used to distinguish male sterile cytoplasm and normal cytoplasm. RT-PCR was conducted with primer P1-P2 in inbred line huangzaosi and 48-2 with male sterile cytoplasm and normal cytoplasm, mRNA was separately isolated from tetrad stage, uninucleate stage and binucleate stage of microspore development, cDNA was obtained with random hexanucleotide primers. With the cDNA template, specific amplified fragments were also detected by primer P1-P2 in the male sterile materials at different development stage of microspore, but there was no amplification by primer P1-P2 in the 2 maintainer lines. This result indicated that primer P1-P2 can be transcripted at 3 development stages of microspore in all male sterile materials, and same transcript was produced by primer P1-P2 among all male sterile materials include 3 sets of isonuclear alloplasm and 3 sets of isoplasm allonuclear. It was suggested from this experiment that the specific DNA sequence detected by primer P1-P2 in all male sterile material total genomic DNA might be related to the cytoplasmic male sterile character.

[Locating maize male sterility gene induced by space flight using microsatellite markers].

Two F2 populations with different kinds of spike derived from maize male sterility materials RP(3)195 (A) x S37 (inbred line) ,which had been sib-bred for many generations,were used for sterility analysis and gene location. There were 138 and 247 plants in the two F2 populations respectively. Among the 326 pairs of microsatellite primers selected,56 were found polymorphic. Linkage analysis of F2 populations with the 56 pairs of primers showed that microsatellite markers bnlg197 and umc1012 were linked with the male sterility gene. The genetic distance between marker bnlgl97 and the male sterility gene in the two different F2 populations were 7 cM and 14.5 cM respectively. The genetic distance between marker umc1012 and the male sterility gene in the 138 plants was 28.5 cM. Thus the male sterility gene was located on chromosome 3L.

[The production and multi-color genomic in situ hybridization identification of maize-Z. perennis substituted material].

Genus Zea. L was composed of two sections: sect. Luxuriantes Doebley & Iltis including Z. dipperennis, Z. luxurians and Z. perennis, and sect. Zea. mays consisting only one species, annual Z. mays. To improve the biodiversity of germ-plasm in maize breeding, the study of transferring maize relatives gene into common maize were performed. Firstly, interspecific hybrids between maize (Zea. L) (2n = 20) and Z. perennis (2n = 40) were produced with the aim of transferring desirable horticultural traits from Z. perennis to maize. The F1 of maize x Z. perennis (2n = 30) plant had the most frequent configuration of 5 III + 5 II + 5 I, which were sterile and difficult to produce progeny because of genomic affinities. However,few F2 individuals of maize x Z. perennis could be obtained by some specially treatments, and one maize-like F2 plant were obtained, which were used as a female parent in backcrossing with maize parent. Twelve F2 x P1 ( BC1 F2 ) plants were obtained and then self-crossed to produce self-crossing generation of F2 x P1 (2n = 20) ( BC1 F3). The phenotypic characters of parents, F1 (2n = 30) hybrids, F2 and F2 x P1 were investigated, such as plant height, flowering, leaf shape and tillers. To further verify the genomic organization of maize-Z. perennis material, maize (inbred line 48-2) and BC1 F3 chromosomes, the root tip cells were analyzed by Multi-color GISH. We probed maize and BC1 F3 chromosomes with the probe mixture containing biotin-labeled Z. perennis genomic DNA and digoxigenin-labeled maize genomic DNA, the maize spread exhibited 10 chromosomes with yellow signals and the other 10 chromosomes carried green fluorescing bands. However, although the BC1 F3 was 2n = 20 in all cases. Multi-color GISH images revealed that 17 chromosomes had uniform signals similar to maize chromosomes, but dispersed red signals over the remaining three chromosomes were observed, which indicated that the 3 chromosomes originated from Z. perennis, and they were smaller than maize chromosomes. Data obtained from multi-color GISH images indicated that BC1 F3 was probably a substituted material from maize-Z. perennis.

[Genetic analysis about maize male sterile mutant obtained by space flight].

The seeds of maize single hybrid Chuandan No. 9 were carried into space in 1996 by satellite. After the seeds were planted in field in comparison with travel in space seeds which was not carried into space. Fortunately, male sterile plants were discovered in one of the ear rows. The stability of male sterile expression was observed in different years, different locations and different generations. In order to analysis the genetic characteristic of male sterility, test cross, sister cross, back cross, reciprocal cross and self-pollination were conducted with these male sterile plants. The results showed that the male sterility was stable in different years and different locations, it is inheritable from generation to generation. The sterility is controlled by a single nuclear recessive gene. The appearance of male sterile mutant is the conclusion of gene mutation which happened in nuclear by space flight. This mutant material always accompanies with lower plant height.

[A study of genetic stability of male sterile mutant of maize obtained from space flighted seeds].

OBJECTIVE: To select a male sterile mutant of maize through space flight for production application. METHOD: Air dried seeds of maize (Chuan Dan No. 9) were carried to space for 15 d. After returned to the earth, a male sterile mutant was selected and a sterile line was obtained through direction breeding. RESULT: It was found that the sterile material was thoroughly abortive. The sterile was trail stable and expressed a genetic feature of single recessive gene controlled nucleus sterility. CONCLUSION: The appearance of male sterile mutant was due to gene mutation caused by space conditions.