Abnormal Behavior Of Pollen Mother Cells Induced By High Temperature In Camellia Oleifera

High temperature,as a means of physical mutagenesis,can induce abnormal cytological behavior during sporogenesis,resulting in gametophyte doubling.This study focused on the effects of high temperature treatment on the meiosis behavior of pollen mother cells of Camellia oleifera by cytological behavior observation and transcriptome sequencing analysis.Conducted C.oleifera pollen mother cells meiosis time node under the instant discriminant,high temperature treatment of pollen mother cells meiosis chromosome behavior,cell plate during the period of the formation,cytokinesis observation and cell cycle protein,tubulin,screening of cytokinesis the differentially expressed genes,functional annotation and fluorescent quantitative analysis and research.The main results are as follows:(1)The immediate identification of meiosis time nodes of pollen mother cells of C.oleifera is the premise of microspore chromosome doubling induced by high temperature treatment.Cytological observation showed that the anther development of flower buds at different positions on the branch and the anther development of single flower bud were obviously different.There was no significant correlation between the meiosis stage of long stamen microsporogenesis and that of short stamen microsporogenesis,indicating that there was no correlation between the meiosis process and filament length.(2)Pollen mother cells of C.oleifera were closely related to style length and external morphology of flower buds.Although different meiosis stages could be observed within the fixed style length,the ratio of pollen mother cells at different developmental stages was significantly different.When the style grew to more than 1.97mm,pollen mother cells entered meiosis.When the style grew to more than 2.87mm,pollen mother cells ended meiosis and were in microspore stage.With the development of spore-mother cells,the flower bud width-length ratio showed an "S"shape,increasing from 0.53 to 0.56,and then decreasing to 0.54 at microspore stage.There was no obvious elongation at the end of meiosis and before the formation of tetrad,and the ratio of width to length of flower buds reached a maximum of 0.56 at the tetrad stage.(3)At the meiosis stage of pollen mother cells,it was found that high temperature could effectively induce the formation of 2n pollen in C.oleifera.Leptotene to pachytene and diploidy to diakinesis were both effective treatment periods for producing large pollens under high temperature treatment,and there was a significant positive correlation between the flower bud ratio in these two periods and 2n pollen yield(r=0.8632,P=0.0123 and r=0.8644,P=0.0121).The pollen yield of 2n was significantly different at different temperatures.The 2n pollen induction rates were 9.44%and 6.19%at 45℃ for 4h and 5h,at 47℃ for 4h and 5h,respectively.High temperature treatments at 45℃ and 47℃ are better than those at 43℃ and 49℃.(4)High temperature treatment had important effects on chromosome behavior,cytoplasmic division and cell plate formation of C.oleifera.Through microscopic observation,it was found that there were abnormal phenomena in chromosome behavior and cell plate formation of pollen mother cells after high temperature treatment,including univalents,chromosomal condensation defects,extra-equatorial chromosome,early segregation,backward chromosome and asynchronous meiosis.During the tetrad period,uneven distribution of cytoplasm was observed,and diad,triad and polyad were formed.The cell plate is monoporous’ or bisects the cytoplasm,or herringbone,or cruciform.(5)Illumina Hiseq high-throughput sequencing platform was used for transcriptome sequencing analysis of the control group and the experimental group that were continuously treated at high temperature for 5h.A total of 100,202 Unigene genes were detected.A total of 20 genes related to cyclin,tubulin and cytokinesis were screened by COG database,Swiss-Port database and KOG database.Cluster heat map analysis of expression levels showed that most genes in 45℃ and 47℃ high temperature treatment groups showed downregulated expression trend.qPCR showed that MKK6,MMD1,MOR1 and TPX2 genes were very sensitive to high temperature,and were significantly down-regulated or extremely down-regulated at 43℃,45℃ and 47℃.The relative expression levels of MKK6,MMD1,MOR1 and TPX2 genes at 49℃ were higher than those at other temperature treatments.