The Molecular Mechanism Research Of Abnormal Meiosis In ‘Wuzi Ougan'

‘Wuzi Ougan',a bud variation of Ougan(Citrus suavissima),has all of the same excellent characters as Ougan and its fruits can be stored for half a year remaining the same flavor under the room temperature.Seedless fruits now occupy an important position in the domestic and international market,specially the parthenocarpy.It is meaningful to study the seedless molecular mechanism so as to provide theoretical guiding for the cultivation of seedless citrus varieties.The anthers of ‘Wuzi Ougan' and its wild type were squashed to observe the development of microspores and the abnormal meiotic process of ‘Wuzi Ougan'.RNA-Seq was carried out using the anthers during the period of microsporocyte,from which we obtained a large amount of information.23 differentially expressed genes and 31 meiosis special genes were selected to detect the relative expression by RT-qPCR,the main research results were as follows:1.According to the pressing plate method,the relationship between the diameter of flower buds and the stage of microspore development was determined.(1)the period of microsporocyte : about2.0-2.4 mm;(2)The beginning of meiosis: above 2.4mm;(3)The period of tetrad: about 2.8mm-3.1mm;(4)The period of single-celled: about 3.5-4.5mm.(5)The period of dual-core:about 4.5-6.2mm;(6)The muture period:about 6.6-6.9mm.In addition,a lot of abnormal meiotic phenomenons of ‘Wuzi Ougan' were observed,including chromosome bridge,failed separation in meiotic metaphase?,disappearance of meiosis ?,desynchrony of meiotic anaphase ?,unequal separation,severe fragmentation of chromosome and the formation of polyad.Through the paraffin section of anthers during the tetrad,we found that the microspores of ‘Wuzi Ougan' had different sizes,irregular shape and distinctly different dyeing depth.2.A total of 29.68 Gb clean data was achieved by RNA-Seq,which was assembled into 174325 Transcripts and 92023 Unigenes.Through BLAST,we got 41366 Unigene annotations,8244 SSRs,144703 SNPs and 251 DEGs whose expression in ‘Wuzi Ougan' was more than 1 times higher(18)or lower(214)than Ougan.The GO annotation of DEGs showed as follows:(1)In biological process,the metabolic process(23.5%)and oxidation-reduction process(22.4%)were in the majority;(2)In molecular function,the combine(40.7%)and transfer function(29.7%)were in the majority;(3)In cellcomponent,the component of membrane(33.3%)and membrane(33.3%)were in the majority.For the KEGG anonation,15 DEGs were located on 30 KEGG pathways that contained 6(20%)amino acid metabolism pathway,5(17%)carbohydrate metabolic pathways and 4(13%)lipid metabolic pathways.3.This research selected 18 down-graduated and 5 up-graduated DEGs to do the RT-qPCR experiment,and the results showed that the expression trends of the 23 DEGs were the same as the transcriptome data in spite of the quantitative value,indicating the reliability of RNA-Seq.4.31 meiosis special genes genes were selected from the RNA-Seq results to detect their relative expressions in 5 pollen development stages,namely,the microsporocyte(?),tetrad(?),single-celled(?),dual-core(?)and the muture period(?).20 genes(64.5%)mainly expressed in period?and ?,5 genes(16%)had an almost relative expressione in the 5 periods.In period ?of‘Wuzi Ougan',20 genes' relative expressions were significantly lower than Ougan,5 genes' were significantly higuer than Ougan and 6 genes' had no significant difference.After exploring and analysing the function of the 33 genes,we could speculate that the cause of polyad during tetrad period was the failed formation of normal DSBs.The abnormal DSBs prevented the synapsis and homologous chromosomes separated randomly in meiotic metaphase,which led to the appearance of abnormal tetrads.