Study On The Cellular And Molecular Genetics Of Low Fertility In Autotetraploid Rice

Autotetraploid rice is a new germplasm resource obtained by chromosome doubling with strong hybrid vigor?Indica × japonica?,higher grain weight and higher nutritional quality compared with diploid rice,however,the poor seed set limits its utilization.Therefore,it is significance to carry out the researches on the cellular and molecular genetic mechanism of low fertility in autotetraploid rice.Although some information about the cytogenetics of low fertility?meiosis in pollen mother cells?in autotetraploid rice is available,the molecular genetics of sterility,especially the expression patterns of small RNAs in autotetraploid rice are still poorly understood.In the present study,we planned to investigate the cytogenetics,sequence variations,methylations and the expression levels of small RNAs,long non-coding RNAs and genes during pollen and embryo sac development by using the autotetraploid rice and its diploid counterpart.Finally,we sought to elucidate the cellular and molecular genetic mechanisms of the spikelet sterility in autotetraploid rice by these comprehensive analyses.The main results are as follows.?1?Cytological observations on pollen and embryo sac development in autotetraploid riceThe stages of pollen and embryo sac development were same in 02428-4x and 02428-2x by cytological observations,and they could be divided into different stages;however,many abnormalities were found in 02428-4x.Degeneration of pollen mother cells?PMC?,multiple nucleoli,abnormal tetrads and abortive pollens at bi-cellular pollen stage,were observed during pollen development in 02428-4x,which result in pollen sterility?43.3%?.Different kinds of abnormalities were observed during the development of embryo sac in 02428-4x,including functional megaspores degeneration,abnormal mononucleate embryo sac,embryo sac degeneration,abnormal polar nuclei and double ovules with unsynchronized development,which leaded to embryo sac sterility?55.48%?.In addition,abnormal chromosome behavior and chromosomal configurations were found during PMC meiosis of 02428-4x.The chromosomal configurations were I?1.03±1.59?+II?11.18±5.76?+III?0.81±1.14?+IV?4.83±2.45?+V?0.19±0.49?+VI?0.36±0.90?in diakinesis of 02428-4x.What?s more,many abnormalities were observed in 02428-4x during anther development,such as anther locule degeneration,tapetum pre-degeneration and imbalance tapetum size.Partial pollen and embryo sac sterilities were the two important factors that leaded to low seed set?15.65%?in 02428-4x.?2?Analysis of DNA polymorphisms in autotetraploid riceA total of 34416 SNPs and 6993 InDels were detected in 02428-4x compared to 02428-2x by re-sequencing.In total,2320 genes associated with missense mutation,of which four were the homozygosity loci,including LOC_Os01g37780,LOC_Os09g37270,LOC_Os11g27670 and LOC_Os11g35756.Besides,412 genes associated with major impact polymorphisms,of which four were the homozygosity loci,such as LOC_Os03g06260,LOC_Os05g10440,LOC_Os08g24060 and LOC_Os11g36880.Furthermore,841 genes along with 2777 SVs in 02428-4x were enriched in DNA replication,chromatin assembly or disassembly,chromatin binding and chromatin,and 84 regions performed differential CNVs in 02428-4x.?3?Analysis of transcriptome and methylome during PMC meiosis in autotetraploid riceIn total,313 and 352 genes were specifically showed up-regulation and down-regulation in meiosis of 02428-4x.The down-regulated genes were enriched in transcription factor activity.Meanwhile,a total of 12,457 and 47,408 differentially methylated regions?DMR?were showed hypermethylation and hypomethylation in 02428-4x compared to 02428-2x.A total of 7749 DMR relating to transposable elements?TEs?were exhibited hypomethylation in 02428-4x.Additionally,162 differentially expressed genes displayed the relationship with meiosis in autotetraploid rice,including 13 genes related to DNA polymorphisms and 149 genes associated with DNA methylation.What?s more,42 genes related to DNA polymorphisms and DNA methylation,and four genes associated with DNA polymorphisms,DNA methylation and differentially expression patterns.Abnormal expression of these 208 genes might lead to pollen sterility in autotetraploid rice,such as LOC_Os03g58600?MEL1?,LOC_Os03g44760?OsAM1?,LOC_Os08g03682?CYP703A3?and LOC_Os10g35180?OsABCG26?.?4?Analysis of small RNAs during pollen and embryo sac development in autotetraploid riceIn total,748 miRNAs were found in 02428-4x and 02428-2x,of which,43 miRNAs were specifically detected during the pollen development in autotetraploid rice and 49 miRNAs were specifically related to the development of embryo sac in autotetraploid rice.A total of 321 and 368 differentially expressed miRNAs?DEM?were detected during pollen and embryo sac development in 02428-4x,respectively.Gene Ontology enrichment analysis on the targets of DEM associated with embryo sac and pollen development revealed many prominent functional gene classes,such as cell differentiation,cell death and signal transduction were enriched during embryo sac development,while flower development and transcription factor activity were detected during pollen development in 02428-4x.Of these DEM,osa-miR1436_L+3₁ss5CT and osa-miR167h-3p were associated with the female meiosis,while osa-miR159 a.1 and osa-MIR159a-p5 were related with the male meiosis.Furthermore,miR2118 and miR2275 families,the trigger of 21nt-phasiRNAs and 24nt-phasiRNAs,were accumulated during pollen development of autotetraploid rice.24nt-phasiRNAs displayed down-regulation in pollen development of autotetraploid compared to diploid rice and their spatial-temporal expression patterns were similar to osa-miR2275 d.We speculated that down-regulation of miR2275 may result in the low expression of 24nt-phasiRNAs,resulting the abnormal pollen mother cells development in autotetraploid rice.Various kinds of differentially expressed 24 nt siRNAs associated with transposable elements?TEs-siRNAs?were also detected in autotetraploid rice.Moreover,down-regulated TEs-siRNAs were associated with pollen development,whereas up-regulated 24 nt TEs-siRNAs were related to embryo sac development of autotetraploid rice,indicating that reduction of 24 nt TEs-siRNAs of autotetraploid rice may induce genome instability and infertility.?5?Analysis of long non-coding RNAs associated with meiosis in autotertaploid riceA total of 431 and 32 lncRNAs displayed differential expression patterns?DEL?during meiosis of anther and ovary in Taichung65-4x,respectively.Of these DEL,59 DEL could serve as small RNA precursors,and 55 of them displayed down-regulation in autotetraploid rice.Moreover,328 DEL were associated with the transposable elements,which showed low expression during meiosis in autotetraploid rice.Among the potential targets of the anther DEL,flower development,DNA binding and nucleus were significantly categorized,whereas response to abiotic stimulus was significantly enriched among the ovary DEL.In addition,a total of 237 and 20 DEL were associated with meiosis in autotetraploid rice pollen mother cells and embryo sac mother cell?EMC?,respectively.Down-regulation of some meiosis-related target and its DEL regulator were found in Taichung65-4x,such as MEL1 regulated by TCONS₀0068868,and meiotic asynaptic mutant 1 regulated by TCONS₀0057811.The low expression levels of lncRNAs,associated with small RNAs,transposable elements and meiosis-regulated targets,may cause great harm to meiosis process that lead to low fertility in autotetraploid rice.In summary,low feritility of autotetraploid rice not only cause by the cytogenetics,such as the abnormal chromosomal behaviors and abnormalities in tapetum development,but also the DNA polymorphic,differentially methylated and differentially expressed genes associated with meiosis.Meanwhile,abnormal expression of the non-coding RNAs during pollen and embryo sac development in autotetraploid rice could also result in sterility of autotetraploid rice.Taken together,the reason of sterility in autotetraploid rice was complexity,and might be affected by multiple factors.