| Hybridization and polyploidization are the common mode of evolution in flowering plants.Precise control of chromosome behavior and favorable development of anther structure are vital for conferring reproductive stability during hybridization and polyploidization.Hybridization and polyploidy result in rapid changes at genetic level including the influence of meiosis process in reproductive cells and expression pattern in relevant regulatory genes.Therefore,it is significant to explore the effects on male gametes development and regulation mechanism at the initial stage of formation in hybrids and polyploids.The cultivated Brassica plant,such as Brassica rapa,Brassica oleracea and Brassica napus,are important materials in hybridization and polyploidization research.In this study,to reveal the effect on male gametes development and gene expression pattern after hybridization and ploidy changes,omics sequencing technology and molecular cytology were used to analyze the autotetraploid Brassica rapa,haploid Brassica napus(AnCn)and hybrid Brassica napus(ArCo).The main findings are as follows:1.The autotetraploid B.rapa was artificially induced from diploid B.rapa with colchicine treatment.Compared with diploid B.rapa,a cluster of gigantism flower buds were obtained and the pollen stainability was reduced slightly in autotetraploid B.rapa.Cytological investigations demonstrated that chromosome behaviors were aberrant during meiosis,including univalent,multivalent and chaotic alignments at metaphase,aberrant segregation and lagged chromosomes at telophase in autotetraploid B.rapa.yH2AX dynamic process indicated that DSBs formation occurred normally but DSBs repair progression was failed at subsequent stage in autotetraploid B.rapa.The transcriptome and proteomic analysis showed that a total of 4580 differentially expressed genes(DEGs)and 562 differentially expressed proteins(DEPs)were identified in floral buds between autotetraploid and diploid B.rapa.The functional analysis showed that DEGs and DEPs assigned to "Cell cycle control,cell division,chromosome partitioning" were preferentially up-regulated,which suggested a robust regulation of cell division in autotetraploid B.rapa.However,DEGs(DMC1,CHR34,EME1B)and DEPs(PARP2,LIG1,BARD1)related to DNA repair and homologous recombination were significantly down-regulated in autotetraploid B.rapa,which indicated DNA repair pathways were more likely affected during meiosis in autotetraploid B.rapa.In combination with the floral RNA-seq data and proteomic data,a number of DEPs were positively correlated with their transcript abundance,but posttranslational modification of proteins might also play a role in regulating meiosis course after polyploidization.2.Haploid B.napus(AnC n)was successfully achieved through microspore culture from allotetraploid B.napus.The floral organ of haploid B.napus(AnCn)was morphologically similar to the donor B.napus,whereas the pollen grains were infertile and tapetum was disintegrated in advance.The process of meiosis was disordered with incomplete synapsis and unpaired univalent,and the distribution of tubulin was irregular with dotted fibers in haploid B.napus(AnCn).The transcriptome analysis indicated that 8831 DEGs were identified between allotetraploid and haploid B.napus(AnCn),among which "Translation,ribosomal structure and biogenesis" and"Posttranslational modification,protein turnover,chaperones" were the two largest groups of DEGs,which implied that translation and posttranslational modification might be susceptible by ploidy change.In addition,TUBA3,TUBB1,TUBB9 assigned to "Cytoskeleton" and DEGs referred to "pollen exine formation","sporopollenin biosynthetic process","anther dehiscence" and "anther wall tapetum development"were down-regulated in haploid B.napus(AnCn),which suggested that microtubule dynamics and pollen development were affected in haploid B.napus.The inferences of transcriptome analysis were positively correlated with the molecular cytology results.3.Hybrid B.napus(ArCo)was produced via crosses between natural diploid B.rapa and B.oleracea.Compared with paternal lines,the morphological characterization of hybrid B.napus was an obvious intermediate phenotype,but the anthers remain close and tapetum was detached from the inner wall of anther.The pollen grains were irregular with only 1%stainability,and the meiosis process was highly abnormal for incomplete synapsis,univalent,chromosome bridges and fragments in hybrid B.napus(ArCo).The microtubule localization showed that tubulin signal was unstable with deficient perinuclear microtubule and disordered configuration of spindle in hybrid B.napus(ArCo).The transcriptome analysis revealed that 25339 and 26908 differently expressed genes were found in hybrid B.napus(ArCo)compared with B.rapa and B.oleracea respectively,which suggested that the profound effects on gene expression appeared to be caused more by hybridity than genome doubling.The DEGs involved in "Chromatin structure and dynamics","Cell cycle control,cell division,chromosome partitioning","Replication,recombination and repair" and "Cytoskeleton" were tended to be up-regulated,and tubulin genes TUBA3,TUBA6 and TUBA7 were transgressive up-regulated expression in hybrid B.napus(ArCo).In addition,ubiquitin-related genes MIEL1 and SINAT1 in "Ubiquitin mediated proteolysis" pathway and FAR in "Wax biosynthesis" pathway were transgressive down-regulated expression in hybrid B.napus(ArCo),which implied that the processes of meiosis and sporopollenin were damaged,meanwhile,ubiquitin may play an important role during male gametes development at the initial stage after hybrid B.napus formation.4.According to the abnormal statistics of meiosis process,the percentage of cells exhibited diploid-like meiosis in hybrid B.napus(ArCo)were higher than haploid B.napus(AnCn).Compared with the RNA-seq data between hybrid B.napus(ArCo)and haploid B.napus(AnCn),meiosis-related genes were more likely up-regulated in haploid B.napus(AnCn),including SPO11,NBS1,RAD51,TOP3A.However,most of pollen development related genes were up-regulated in hybrid B.napus(ArCo),which implied that the regulation of defect in meiosis process and pollen development were diverse during different evolution.In general,the abnormal meiosis and microspore caused by the change of ploidy and hybridization will lead to sterility pollen,which was positively correlated with the results of omics analysis,and many of candidate genes contributed to the abnormal phenomenon were found.This study provides a detailed cytology and omics landscape of male gametes development during hybridization and ploidy changes,which definitely affords a better understanding of genetic alteration at the initial stage of formation in hybrid and polyploid. |
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