Genome-wide Variations And Embryo Sac Development Of Mutated Poly-embryonic(MPE)autotetraploid Rice

Autotetraploid rice is a new germplasm resource obtained by doubling of chromosomes by colchicine.There have been many studies on the reproductive characteristics of autotetraploid rice,but little is known about the effect of embryo sac development on autotetraploid rice.Therefore,an autotetraploid rice line,mutated polyembryonic(MPE),was used in this study,and its diploid counterpart was used as a control.First,high-throughput sequencing technology was used to re-sequence the whole genome of autotetraploid rice and its diploid counterpart,and pollen fertility and main agronomic traits of MPE were observed;second,the development and mature embryo sac fertility was observed through WE-CLSM(whole-mount eosin B-staining confocal laser scanning microscopy).The main findings are as follows:(1)Genome-wide variation analysis of MPE autotetraploid rice line.Whole-genome resequencing detected the genetic variations between tetraploid rice and its diploid counterpart at DNA level.There were 87,229 SNPs and 11,022 InDels in the whole genome of MPE versus E246(diploid rice),with an average of 23.37 SNPs and 2.95 InDels per 100 Kbp.A total of 17375 and 17171 structural variations(SVs)were identified in MPE and its diploid counterpart.In the exon,intron and intergenic regions,2330,3012 and 18070 SVs were detected in both types of rice,respectively.Compared with E246,there were 140 large-effects SNPs and InDels variants that might be related to the embryo sac development of MPE.The expression analysis revealed that 10 genes may have close relationship with the development of embryo sac.Of these,Os02g0292600 and Os06g0565200 were specifically expressed in ovary.The ten grain length,grain width,grain length-to-width ratio,and main panicle length of MPE were 11.62 cm,3.42 cm,3.40,and 22.64 cm,respectively,and pollen fertility of MPE was 25.38%.(2)Observation of embryo sac.The development process of MPE embryo sac was basically the same as that of normal diploid cultivar.It involves the formation of sporangial cells,the formation of megaspore mother cells,meiosis of megaspore mother cells,the formation of functional megaspore,and the formation of mono-nucleate embryo sac,eightnucleate embryo sac,and the maturation of embryo sac.There were also many abnormalities during the development of MPE embryo sac,including stagnation or degeneration before the formation of mono-nucleate embryo sac,the abnormal position or number of polar nuclei during two-nucleate and four-nucleate stage,and many other abnormalities during early,mid and late eight-nucleate embryo sac stages.Mature embryo sac abnormalities were also detected,such as egg apparatus degeneration,embryo sac without female germ unit,and abnormal numbers or position of polar nuclei.The abnormal position and number of cells/nuclei lead to poor fertility of the mature embryo sac.In summary,seed set and pollen fertility of the autotetraploid rice was low,and embryo sac abnormalities were more likely to cause the abortion of mature embryo sac.However,whole genome polymorphisms and genetic differences were high,and exhibited broad prospects for genetic improvement.Genetic mutations in genes associated with embryo sac development of polyploid rice require further studies.