Cytology And Gene Expression Profiling Of Four Types Of Male Sterility In Cabbage(B. Oleracea Var. Capitata)

Cabbage (Brassica oleracea var. capitata) is an important leafy vegetable crop with a widely growing area only second to Chinese cabbage (Brassica rapa) in China. Utilization of male sterile lines are becoming the trends for cabbage heterosis breeding. Much effort has been made in recent years to put the four cabbage male sterilities into practice in China. However, little progress has been made because the developmental pathway and mechanism of these male sterilities have not been well explored. In the present study, light microscopy and transmission electron microscopy (TEM) techniques were conducted to systematically investigate the anther development and microsporogenesis of NiCMS, RGMS, OguCMS and DGMS. Based on the cytological result here, we employed high through-put transcriptome analysis techniques such as cDNA-AFLP and microarray to analyze the comprehensive gene expression profilings of four kinds of male sterilities and to further explore the developmental pathway and mechanism of these male sterilities. Main conclusions are listed as follows:1. Cytological analysis clarified the main sterile stages and characteristics of four male sterilities. NiCMS occurred at the sporogenesis cell stage with abnormal tapetal cell differentiation and development The first sign of abnormal anther development of RGMS happened at pollen mother cell stage and its main sterile characteristics were observed in abnormally developed tapetal cells and middle layer cells. OguCMS often occurred during early tetrad stage and its abnormal activities of tapetal cells were observed after meiosis. Most of microspores in OguCMS could be released from tetrads and then aborted after being squashed by hypertrophic tapetum cells. DGMS abortion often occurred during late tetrad stage. The most important characterisitics were that the pollen mother cell primary wall surrounding the developing microspores in DGMS remained intact till very late pollen stage.2. Four types of male sterile cabbage lines and their corresponding fertile lines were used to analyze the gene expression by means of cDNA-AFLP. In total, we got 113 differentially expressed TDFs, which were classified into 12 groups. Among them, 76 TDFs( 67.25% of the total) were classified into group A, B, C and D, which were possibly involved in Sporogenesis cell, Pollen mother cell, Pre-tetrad and Post-tetrad stage anther development, respectively and sequentially interrupted in four male sterile lines. The sequentially interruption model revealed that most of the genes involved in anther development expressed as a streamline. Sequence analysis of 23 representative TDFs from each group showed that 16.7% of the differentially expressed genes regulated cell wall modification.3. we compared genomic-wide gene expression profiles of four male sterilities with use of Arabidopsis whole genome microarray and identified 1209 stamen preferentially expressed gene. Expression profile cluster analysis and Venn diagram overlap relationship analysis showed that 65.27% of the totally down-regulated genes which conformed to the sequentially interruption model referred as the result of cDNA-AFLP. We had obtained further knowledge about the sterile mechanism of four kinds of male sterility at the level of gene expression. The present study here confirmed that themechanism of all the four male sterilities was that abnormal tapetum cell development interferred the normal microsporegenesis. The present study revealed novel insights into mechanism of cybrid CMS as conferred by unique gene expression profile of three kinds of overlap relationships of two CMS types. The result here revealed that NiCMS showed much more interference in organelles related genes in cytoplasm than OguCMS, and the abnormal mitochondrial gene expression of two CMS indicated similar molecular mechanism of the decrease or loss of the function of stress response. And we also identified 89 anther wall specific expressed genes (mainly tapetum specific genes). Further analysis revealed that genes showing transcription factor activity were overrepresented, while genes with kinase activity were drastically underrepresented in anther wall. 4. The present study has further explored the molecular mechanism of DGMS. Many of the down-regulated genes (8.7%) fuctioned as cell wall modification and metabolism. However, Only few genes which encode beta-l,3-glucanase were down regulated with a slighter suppress level. It suggested that slight suppressed beta-l,3-glucanase genes might be the reason of delayed-degradation of callose, but delayed-degradation of callose might not be (at least not main) the reason of arrestment of microspore releasing. DGMS controlled a PMC primary wall metabolism pathway different from QRT3. This pathway included five of the detected significantly down-regulated polygalacturonase genes, At3gO782O, At3gO784O, AtlgO279O , At2g23990 and At5g48140.Out of the 277 down regulated genes, 251 (90.6%) were overlapped with ATH1 affymatrix Arabidopsis chip. Among the overlapped genes 238 were expressed in pollen, and only 13 were not expressed in pollen. They were most likely tapetum specific. We inferred that the molecular mechanism of the dominant cabbage male sterility was that DGMS blocked few genes expressed in tapetum cells which prevented the timing degradation of PMC wall and finally resulted in the suppression of a large number of genes in pollen grains. So, we inferred that the 13 putative tapetum-specific genes should be the upstream genes in the function network of DGMS. Maybe all of them were commonly regulated by DGMS, or even one of them, itself, was the true DGMS that we were pursuing.