Fine mapping and candidate gene analysis of a novel fertility restorer gene for C-type cytoplasmic male sterility in maize (Zea mays L.).

KEY MESSAGE: A novel strong fertility restorer gene Rf12 for C-type cytoplasmic male sterility of maize was finely mapped on chromosome 2. Its best candidate gene Zm00001d007531 is predicted to encode a p-type PPR protein. The lack of strong restorer gene of maize CMS-C greatly limits its application in hybrid seed production. Therefore, the cloning of maize CMS-C novel strong restorer genes is necessary. In this study, a strong restorer line ZH91 for maize CMS-C was found, and the novel restorer gene named Rf12 in ZH91 had been mapped in a 146 kb physical interval on maize chromosome 2. Using the third-generation high-throughput sequencing (ONT), the whole genome sequence of ZH91 was got, and with integrating the annotation information of the reference genome B73_RefGen_v4 and B73_RefGen_v5, four candidate genes were predicted in ZH91 within the mapping region. Then using gene cloning, stranded specific RNA sequencing, qRT-PCR analysis and subcellular localization, Zm00001d007531 was identified as the most likely candidate gene of Rf12. Zm00001d007531 encodes a p-type PPR protein with 19 PPR motifs and targets mitochondria and chloroplast. Stranded specific RNA sequencing and qRT-PCR results both show that the expression of Zm00001d007531 between anthers of near-isogenic lines C478Rf12Rf12 and C478rf12rf12 was significantly difference in pollen mother cell stage. And the result of sequence alignment for Zm00001d007531 gene in 60 materials showed that there are twelve SNPs in CDS region of Zm00001d007531 were tightly linked to the fertility. The finding of a novel strong restorer germplasm resource ZH91 for maize CMS-C can greatly promote the application of maize CMS-C line in maize hybrid seeds production, and the identification of candidate gene Zm00001d007531 can accelerate the backcrossing process of maize CMS-C strong restorer gene Rf12 to some extent.

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.

The characterization and candidate gene isolation for a novel male-sterile mutant ms40 in maize.

KEY MESSAGE: A novel genic male-sterile mutant ms40 was obtained from EMS treated RP125. The key candidate gene ZmbHLH51 located on chromosome 4 was identified by map-based cloning. This study further enriched the male sterile gene resources for both production applications and theoretical studies of abortion mechanisms. Maize male-sterile mutant 40 (ms40) was obtained from the progeny of the ethyl methanesulfonate (EMS) treated inbred line RP125. Genetic analysis indicated that the sterility was controlled by a single recessive nuclear gene. Cytological observation of anthers revealed that the cuticles of ms40 anthers were abnormal, and no Ubisch bodies were observed on the inner surface of ms40 anthers through scanning electron microscopy(SEM). Moreover, its tapetum exhibited delayed degradation and then blocked the formation of normal microspores. Using map-based cloning strategy, the ms40 locus was found to locate in a 282-kb interval on chromosome 4, and five annotated genes were predicted within this region. PCR-based sequencing detected a single non-synonymous SNP (G > A) that changed glycine (G) to arginine (A) in the seventh exon of Zm00001d053895, while no sequence difference between ms40 and RP125 was found for the other four genes. Zm00001d053895 encodes the bHLH transcription factor ZmbHLH51 which is localized in the nucleus. Phylogenetic analysis showed that ZmbHLH51 had the highest homology with Sb04g001650, a tapetum degeneration retardation (TDR) bHLH transcription factor in Sorghum bicolor. Co-expression analysis revealed a total of 1192 genes co-expressed with ZmbHLH51 in maize, 647 of which were anther-specific genes. qRT-PCR results suggested the expression levels of some known genes related to anther development were affected in ms40. In summary, these findings revealed the abortion characteristics of ms40 anthers and lay a foundation for further studies on the mechanisms of male fertility.

Identification and characterization of the TCA cycle genes in maize.

BACKGROUND: The tricarboxylic acid (TCA) cycle is crucial for cellular energy metabolism and carbon skeleton supply. However, the detailed functions of the maize TCA cycle genes remain unclear. RESULTS: In this study, 91 TCA genes were identified in maize by a homology search, and they were distributed on 10 chromosomes and 1 contig. Phylogenetic results showed that almost all maize TCA genes could be classified into eight major clades according to their enzyme families. Sequence alignment revealed that several genes in the same subunit shared high protein sequence similarity. The results of cis-acting element analysis suggested that several TCA genes might be involved in signal transduction and plant growth. Expression profile analysis showed that many maize TCA cycle genes were expressed in specific tissues, and replicate genes always shared similar expression patterns. Moreover, qPCR analysis revealed that some TCA genes were highly expressed in the anthers at the microspore meiosis phase. In addition, we predicted the potential interaction networks among the maize TCA genes. Next, we cloned five TCA genes located on different TCA enzyme complexes, Zm00001d008244 (isocitrate dehydrogenase, IDH), Zm00001d017258 (succinyl-CoA synthetase, SCoAL), Zm00001d025258 (α-ketoglutarate dehydrogenase, αKGDH), Zm00001d027558 (aconitase, ACO) and Zm00001d044042 (malate dehydrogenase, MDH). Confocal observation showed that their protein products were mainly localized to the mitochondria; however, Zm00001d025258 and Zm00001d027558 were also distributed in the nucleus, and Zm00001d017258 and Zm00001d044042 were also located in other unknown positions in the cytoplasm. Through the bimolecular fluorescent complimentary (BiFC) method, it was determined that Zm00001d027558 and Zm00001d044042 could form homologous dimers, and both homologous dimers were mainly distributed in the mitochondria. However, no heterodimers were detected between these five genes. Finally, Arabidopsis lines overexpressing the above five genes were constructed, and those transgenic lines exhibited altered primary root length, salt tolerance, and fertility. CONCLUSION: Sequence compositions, duplication patterns, phylogenetic relationships, cis-elements, expression patterns, and interaction networks were investigated for all maize TCA cycle genes. Five maize TCA genes were overexpressed in Arabidopsis, and they could alter primary root length, salt tolerance, and fertility. In conclusion, our findings may help to reveal the molecular function of the TCA genes in maize.

Comparative transcriptome analysis of isonuclear-alloplasmic lines unmask key transcription factor genes and metabolic pathways involved in sterility of maize CMS-C.

Although C-type cytoplasmic male sterility (CMS-C) is one of the most attractive tools for maize hybrid seed production, the detailed regulation network of the male sterility remains unclear. In order to identify the CMS-C sterility associated genes and/or pathways, the comparison of the transcriptomes between the CMS-C line C48-2 and its isonuclear-alloplasmic maintainer line N48-2 at pollen mother cell stage (PS), an early development stage of microspore, and mononuclear stage (MS), an abortive stage of microspore, were analyzed. 2,069 differentially expressed genes (DEGs) between the two stages were detected and thought to be essential for the spikelet development of N48-2. 453 of the 2,069 DEGs were differentially expressed at MS stage between the two lines and thought to be participated in the process or the causes of microspore abortion. Among the 453 DEGs, 385 (84.99%) genes were down-regulated and only 68 (15.01%) genes were up-regulated in C48-2 at MS stage. The dramatic decreased expression of the four DEGs encoding MYB transcription factors and the DEGs involved in "polyamine metabolic process", "Cutin, suberine and wax biosynthesis", "Fatty acid elongation", "Biosynthesis of unsaturated fatty acids" and "Proline metabolism" might play an important role in the sterility of C48-2. This study will point out some directions for detailed molecular analysis and better understanding of sterility of CMS-C in maize.

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.