Cytogenetic And Genetic Mechanisms Analysis At Meiosis In Haploid Brassica Rapa

Brassica rapa(2n=20)is an evolutionarily typical mesohexaploid,which underwent whole genome triplication(WGT),thus leading to a more complicated genomic complements in flowering plants.Meiosis is a specialized cell division and chromosome segregation process used to produce gametes in which the ploidy level is half that of the progenitor cell.In this study,the objective is to investigate the chromosome behaviors during meiosis and pollen fertility at cytogenetic and transcriptomic levels in haploid B.rapa(Brassica rapa).Ploidy was confirmed with flow cytometry and chromosome counts.The flower and pollen development,in especial homoeologous behavior analysed through the microscopic observation in haploid B.rapa.The changes of gene expression were assessed by the method of RNA-seq and quantitative real-time PCR.1.Through microspore culture successfully obtained the haploid B.rapa,the flower size were tiny to the diploid,and the fertility and viability of pollen grains were reduced significantly,with aberrant meiosis course in haploid B.rapa.2.The scanning electron microscopy(SEM)indicated that the most pollen grain of haploid B.rapa was seriously empty and shrunken.Transverse sections of anthers from diploid and haploid B.rapa display the tapetum is left with only a single layer of cells with less dense,vacuolated cytoplasm.This is a reason for the pollen grains of viability reduced.3.The cytological result showed an aberrant chromosome behaviors with abnormal cytoskeleton distribution during meiosis in haploid B.rapa.Non-homologous chromosome likely paired with.The immunofluorescence analyses showed the synapsis was occurred between non-homologous at meiosis I in haploid.The frequent bivalent and chaotic univalent were observed at diakinese,this abnormalities lead to subsequent improper chromosome segregation and unsynchronized segregation,which chromosomes number in nuclei of microspores were different.This is another reason for the pollen grains of viability reduced.4.The changes of gene expression were assessed by the method of RNA-seq and quantitative real-time PCR.The transcriptomes in flowers showed meiotic genes were differentially expressed in haploid compared with diploid B.rapa,with repaired genes(DMC1,MSH2)down-regulated,but homologous(also involved in somatic)recombination genes(RAD51,RAD54)up-regulated,indicating a disrupted meiotic recombination pathway in haploid B.rapa.Such profound alterations may make for the non-homologous chromosome pairing and recombination in haploid B.rapa,that would reduce pollen fertility in haploid B.rapa.In general,this study was investigated the genetic mechanisms of pollen fertility reduced at cytogenetic and transcriptomic levels in haploid,which gives us a better understanding of the variability for the effect in absence of homologous chromosomes at meiosis in different genome haploidization.