Molecular And Cytological Analyses Of A Synthetic Allotriploid Brassica Hybrid (ABC) Between B. Carinata (BBCC) And B. Rapa (AA)

Interspecific hybridization was an important way for gene exchange in plant and the formation of new germplasm resources.Hybridization among three diploid basic species of Brassica has resulted in a series of rich germplasm resources,such as Brassica napus,Brassica juncea,Brassica nigra and their subspecies.Brassica has become an important source of vegetables and oilseeds.At present,synthetic Brassica allopolyploidy has become an excellent model material for studying the recombination and stability of hereditary.In this study,a new synthetic allotriploid hybrid ABC was successfully obtained by artificial hybridization of allotetraploid B.carinata(BBCC)as female parent and B.rapa(AA)as male parent at flowering stage.The phenotypic characteristics of allotriploid and its parents were observed and counted,and the meiosis processes of allotriploid hybrids and their parental lines were studied.The expression of specific proteins regulating synaptosome formation during meiosis was studied by immunofluorescence staining technology.The dynamic location of cytoskeleton during meiosis was studied by immunofluorescence localization technology.The chromosomes pairing within and between heteroploid genomes were analyzed by genome in situ hybridization technology.Paraffin section technique was used to observe the anther development of hybrids and their parents.Fluorescence quantitative PCR was used to analyze the different expression levels of the key genes about meiosis and anther development in order to analyze the relationship between three genomes at the cellular and molecular levels,and to explore the effects of interspecific hybridization on the formation and development of gametophytes,as well as on their flowers.The study results are as follows:1.Allotriploid Brassica hybrids were successfully obtained by hybridization and embryo rescue techniques.The hybrids were basically parental intermediate type.Compared with the parents,the morphological characteristics of the hybrid had no significant difference.The plant height,leaf color and shape,flower color all showed the intermediate type of parents.Some traits were closer to the female parent,probably because there was an additional genomic copy of the female parent in the hybrid ABC,but its pollen fertility was significantly lower than that of the parents,and the plant was sterile.2.Compared with parental lines,the meiosis process of hybrids showed serious abnormalities.During the prophase of meiosis,some synaptonemal complexes appeared on the chromosomes of hybrids.At the end of meiosis,there were different numbers of univalent and multivalent.At the metaphase of the first and second meiosis,a number of scattered univalent surrounded the equatorial plate,chromosome bridges appeared at the anaphase of meiosis,and lagging chromosomes and fineness appeared at the end of meiosis.The phenomenon of unequal cell segregation led to the difference of microspore size and morphology from meiosis to microsporogenesis,and produced different numbers of microspores,which formed different pollen grains at later stage.3.The fluorescence localization results of ZYP1 immunoprotein showed that at the pachytene stage,some homologous paired on the chromosomes of hybrids.The fluorescence signals of ZYP1 were found at the sites where synapsed took place.Compared with the parents,the intensity and trend of the signals were more consistent,which indicated that the hybrids might promote meiosis by regulating the expression of ZYP1,a transverse filament protein in meiosis.Some synapses of homologous segments of chromosomes in the process of meiosis were regulated.4.Immunofluorescence localization of cytoskeleton?-tubulin showed that the cytoskeleton of hybrids did not exist in the form of normal spindle microtubules in the metaphase of meiosis compared with their parents,and the existence of membrane microtubules in the anaphase was abnormal compared with their parents,which might be one of the internal factors leading to abnormal chromosome arrangement and separation in hybrids.5.The results of genomic in situ fluorescence hybridization showed that in hybrid ABC,there were not only synaptic pairings within genome,but also synaptic pairings between genome B and genome A/C.This synaptic pairing within and between groups may affect the meiosis process and produce a little fertile pollen grains.6.The results of anther paraffin section showed that the tapetum of the hybrids could not degrade in the tetrad stage during the anther development,and this phenomenon continued until the mononuclear microspores,indicating that the hybridization process may affect the normal development of the anthers.In turn,affecting the maturity of pollen.7.Quantitative real time PCR results showed that the expression levels of seven key regulatory genes of meiosis and two key regulatory genes of anther development in hybrids were significantly down regulated compared with B.carinata.Among these,ASY1,DMC1,SPO11-1,RAD51 and TPD1 were the five genes with the greatest reduction.This result mostly accounted for the abnormal meiosis and anther development of allotriploid Brassica hybrids.