Cytological Observation And Reserch On Sterility Genes Of A Thermo-sensitive Male Sterile Line SP2S In Brassica Napus L

Thermo-sensitive genic male sterile line of Brassica napus plays an important role intwo-line breeding. Because of it was controlled by factors of inheritance and environment, toreserch its inheritance law is much more complex than which controlled olny by inherility. Inthis study，the new thermo-sensitive genic male sterile line of Brassica napus were used andits near isogenic line SP2F as a control. Via growth chamber experiment, we contracted themateril SP2S which sterility temperature and its temperature-sensitive period. Simultaneously,we used SSR medhod to screen the relatived sterile molecule markers, by means ofCytological study Observed the degradation of callose in SP2F and SP2S anthers. The resultsindicated that：1. Photo-thermo-controlled chamber research: in the condition of14/10h (day/night),neither long nor shot photoperiod have effected on sterility. Fertility alteration of SP2S wascritical induced by temperature, and photoperiod to affect ignorance. Fretility alteration ofSP2S was in the temperature range11.8℃-14.8℃, and its temperature sensitive time was13-14days before flowering.2. Acetic carmine staining identficated Viability: Acetic carmine staining study showedthat the six level fertility of SP2S reached91.98%, pollens performanced of highly fertile.With the fertility level lower, the pollens vitality were lower. When the flower fertility levelwas reduced to3-1level, the pollens vitality trended to keep around55%, performancedsemi-sterility. Neither long stamens nor short stamens have no significant difference inviability.3. Histochemical staining observation: Via acetic carmine staining and microscopicobserved that the pollen grains shape of SP2S were deformity,retraction and deformation,mutual adhesions, not stained or stained light. The result showed a high degree of steritystatus; while pollens of SP2F were plump rounded, dark red after staining, indicating itsinclusion enrichment. The sradius of SP2F was significantly bigger than that of SP2S. anilineblue staining observed the callose of the SP2S was not degraded at tetrad stage, which madethe microspores adhesion with each other and then pollen aborted.4. Seperation of releatived genes controlling Thermo-sensitive Male Sterile and cloned of BnMSR66gene: We screened351pairs of SSR markers, and found6linkage markers,which after verified individual plant validation, the polymorphisms disappear. The clonedgene BnMSR66belongs to plant β-1,3-glucanase genes and share a high similarity with otherplants. The full length of BnMSR66is445bp,and the ORF is390bp, encoding130aminoacids, no difference between SP2S and SP2F.5. The expression of five genes, including BnMSR66and BnA6involved in callosedisssolution, and three QRT genes (BnQRT1、BnQRT2、BnQRT3) control microsporesseparation after meiosis and produce mature pollen tetrads, at four stages (PMC and meiosisstage, tetrad to the microspore released stage, uninucleat microspore, and the bicellularmicrospores stage, buds of0-1,1–2,2-3and3–4mm length) were quantatived by real-timePCR, showed that all the five genes were significantly up-regulated in SP2S than in SP2F.Results of both cytological observation and differential expression analysis exhibited thatdeformation and dysfunction of the tapetum lead to abnormal expressing of the genesinvovled in microspore separation and microspore abortion.