Genetic Mapping And Differential Expression Analysis Of A Recessive Genic Male Sterile Gene In Sesame (Sesamum Indicum L.)

The hybrid vigor is widely existed in sesame (Sesamum indicum L.). One of the important ways for heterosis utilization is to use the genic male sterile lines for producing hybrid seeds. In China, several sesame recessive genic male sterile lines (RGMS) have been bred, which are characterized by male sterile stability, no negative cytoplasmic effects and broad restoring resources. Many hybrid varieties have been bred by producing F1seeds using RGMS lines. However, we have little knowledge about the genetic characterization and mechanism of sesame male sterility. Therefore, it is necessary to conduct further studies on these aspects. In this study,95ms-5AB, a two-type line, was used for studies on histology, mapping and differential expression by phenotypic, cytological and molecular biology methods. The main results and conclusions are as follows:1. The phenotypic observations showed that95ms-5A plants did not show any other obvious differences from the male fertile95ms-5B plants, except for having greenish, shriveled and slim anthers with few, small and degenerative pollens. The lengths of anther and style in fertile flowers are larger than those of sterile flowers, but no difference in corolla and filament length between fertile and sterile flowers. Compared with the RGMS lines transferring from86ms-1,95ms-5A had less mean pollens and smaller pollen size. The pollen grains of95ms-5A and86ms-1lines could not germinate in vitro.2. Histological observations revealed that abnormal inhibited male gametogenesis of95ms-5A appeared during the mononuclear microspore stage. It was characterized by collapsed microspores, adhesion with callose which did not fully degraded and the delayed degeneration of tapetum after the tetrad stage. The morphology of pollens was observed by scanning electron microscope. The sterile pollen wall was cupped and no germinal furrows and particles were observed on pollen exine.3. Genetic analysis of test cross and sib-mating of95ms-5indicated that the male sterility of95ms-5A was controlled by a single recessive genic male sterile gene Simsl {Sesamum indicum male sterility1). Allelic test with ms86-1confirmed that Simsl in95ms-5A is different from ms86-1.4. Amplified fragment length polymorphism (AFLP) markers linked to SiMsl were screened using bulked segregant analysis (BSA). A genetic linkage map of the SiMs1 gene was constructed using237plants derived from the sib-mating between the near-isogenic lines95ms-5A and95ms-5B. The SiMsl gene was found to be located in a region of8.0cM, with a distance of1.2cM from P06MG04and6.8cM from P12EA14, the closest markers on two sides. And marker P01MC08was identified to be co-segregated with SiMsl. The linkage map constructed in this study will be very useful for marker-assisted selection, and map-based cloning of SiMsl as well as for hybrid breeding in sesame.5. cDNA-AFLP technique was used to investigate the transcriptional profiles of the sterile flower buds in95ms-5A and the fertile flower buds in95ms-5B in order to elucidate the mechanism of this RGMS. A total of30reproducible differentially expressed transcript-derived fragments (TDFs) were detected, including27down-regulated and three up-regulated TDFs in sterile buds. The functional annotation analysis of27successfully cloned TDFs indicated that the corresponding genes of11TDFs may participate in the processes of energy metabolism, signal transduction and plant cell development. The other16TDFs were homologous to the proteins with unknown functions or with no homo logy in GenBank.6. Real-Time PCR analysis of21genes in buds at different stages was performed and their expression patterns were entirely the same with those identified by cDNA-AFLP. The expression patterns and functional annotation of above genes in different stages suggested that some genes, such as callose synthase catalytic subunit-like protein (TDF04), receptor-like protein kinase (TDF03) etc., related to the callose wall or tapetum degeneration, were suppressed in95ms-5A in a certain phase before the microspore mother cell or tetrad stage, which may lead to the delayed tapetum degeneration and blocked male gametogenesis. Some function unknown genes showed significant difference of expression level in certain stages between fertile and sterile buds, which may also be the reasons for male sterility. Semi-quantification Real-Time PCR was used to carry on tissue-special expressed analysis of8differential expression genes. One novel stamen-special expressed gene was identified, which showed significantly different expression level between fertile and sterile buds, and its function is unknown.