Study On Molecular Cytology Of Fertility And Heterosis In Autotetraploid Rice T449

Autotetraploid rice is a germplasm developed from diploid rice through chromosome doubling by colchicine.The autotetraploid rice hybrid(indica x japonica)showed obvious biological advantages and yield increasing potential,but low fertility limited its commercial utilization.Our lab previous study demonstrated that the interaction of pollen sterility genes(Sa,Sb and Sc)is one of the important reasons for the pollen sterility of hybrid in autotetraploid rice hybrid,and found that the hybrids sterility could be overcome by double neutral genes,Saⁿ and Sbⁿ,in autotetraploid rice hybrids.Therefore,we used an autotetraploid rice line(T449)harboring double neutral genes for pollen fertility at Sa and Sb loci as materials.Firstly,the fertility of T449 was studied by cytology and genomics,including(1)analysis on the agronomic traits and pollen development of T449;(2)analysis on the whole genome re-sequencing variation of T449 and characteristics of gene expression profile at pollen development.Secondly,study on the cytology and transcriptome of overcoming hybrid sterility by developed hybrids,which were different genotypes of pollen fertility loci interaction.Finally,the characteristics and molecular genetic basis of heterosis were studied by crossing with neo-tetraploid rice.The main results are as follows:1.The agronomic traits and cytology of pollen development in T449In the agronomic traits,the mean value of effective panicle number,seed setting rate and pollen fertility was 84.5cm,4.25,34.47%and 54.09%in T449,respectively;and was96.50 cm,7.35,89.38%and 94.43%in E249,respectively.There was significant phenotypic difference between T449 and E249.Cytological observations showed that meiotic stages in T449 were consistent with E249,but T449 showed that chromosome straggling at metaphase stage,chromosome lagging at anaphase and telophase stages,and asynchronous division.The semi-thin section of saccharide distribution showed abnormal accumulation of saccharides in T449 and the contents of fructose and glucose were markedly higher in T449 than E249 at the single microspore stage.The above results indicate that abnormal chromosome behavior and saccharide distribution are two important reasons for the low fertility of T449.2.Study on whole genome variation and gene expression profile at pollen development in T449A total of 81 SNPs and 182 In Dels were identified in T449 compared to E249 by re-sequencing,and we detected only three non-synonymous SNPs and six large-effect In Dels,which were associated with three and six genes,respectively.Total of 195 genes associated with 1794 SVs in T449 were enriched in cell death,apoptosis,and programmed cell death.A total of 60 CNVs were detected between E249 and T449,which were associated with 617 genes,and two meiosis-related genes were identified,including Os AM1 and RAD17.In addition,we detected 1645 and 3299 genes at the meiosis and single microspore stages in T449 compared to E249 by RNA-seq,respectively.In meiosis stage,75meiosis-related and meiosis-specific differentially expressed genes(DEGs)were detected,including Os MTOPVIB and Os MOF.In single microspore stage,transcriptome analysis revealed down-regulation of a sucrose transporter(Os SUT5)and two monosaccharide transporters(Os MST1 and Os MST8)in T449.These results suggested that polyploidy not only induces abrupt expression changes in the meiosis-related genes that lead to abnormal chromosome behaviour,but also causes changes in the saccharide distribution and expression patterns of saccharides-related genes,which explain the complexity of the reason for low pollen fertility in autotetraploid.To verify the function of the sucrose transporter Os SUT5 in pollen development,we knocked out Os SUT5 by CRISPR/Cas9.The phenotypic investigation of the T1 generation revealed that the loss of Os SUT5 gene led to decrease pollen fertility.This result indicates that Os SUT5 has important functions in pollen development,and deserves further study.3.Analysis of T449 carrying double neutral genes,Saⁿ and Sbⁿ,overcome indica x japonica hybrids sterility in autotetraploid rice.Three different autotetraploid rice hybrids(T449×T431,T449×T435 and T449×T438)with different gene?interactions?at Sa,Sb and Sc loci were prepared by crossing T449harboring double neutral genes,Saⁿ and Sbⁿ,with autotetraploid near-isogenic lines.Firstly,the pollen fertility results showed high pollen fertility(>70%)was found in three types of autotetraploid rice hybrids(T449×T431,T449×T435 and T449×T438),which had double neutral genes at Sa and Sb pollen sterility loci,but the pollen fertility of autotertraploid hybrid(T431×T438),which was without Saⁿ and Sbⁿ double neutral genes,only was12.17%.There was a significant difference between harbording Saⁿ and Sbⁿ double neutral genes and without Saⁿ and Sbⁿ double neutral genes.Further analysis of three aototetraploid hybrids(T449×T431,T449×T435 and T449×T438)the meiotic chromosome behavior of pollen mother cells revealed that there were significant differences in chromosome abnormalities,and the abnormally chromosomal frequency of three autotetraploid hybrids(T449×T431,T449×T435 and T449×T438)are lower than the hybrid(T431×T438).In addition,transcriptome analysis showed that the number of DEGs in the comparison group(T449×T431)vs(T449×T435),(T449×T431)vs(T449×T438)and(T449×T438)vs(T449×T435)were only 110,85 and 6,respectively.There was not significant difference in the number of DEGs between different autotetraploid rice hybrids,which had double neutral genes at Sa and Sb pollen sterility loci.The intersubspecific autotetraploid rice hybrids sterility could be overcome by double neutral genes,Saⁿ and Sbⁿ,in autotetraploid rice hybrids.4.Heterosis evaluation and molecular genetic mechanism of T449.All autotetraploid rice hybrids,which harbored double neutral genes,Sa-n and Sb-n,displayed highest mid-parent heterosis(MPH)and high-parent heterosis(HPH)for grain yield per plant,average of value are 170.89%and 68.67%,respectively.The hybrid F₁(T449×H1)displayed significant positive MPH for the plant height,effective panicle number,grain length,grain width,1000-grain weight,filled grain number,total grain number,grain yield per plant and seed setting,and the highest MPH is 182.36%;and also showed positive HPH values for plant height,filled grain number,total grain number,grain yield per plant and seed setting,and the highest HPH is 79.72%.Meanwhile,the embryo sac fertility was more than 90%and pollen fertility was 77.01%in F₁(77.95%).In cytological result showed the average frequency of normal chromosome behavior at metaphase I,anaphase I,telophase I,metaphase II,anaphase II and telophase II were83.82%,87.95%,96.53%,65.57%,32.00%,92.25%and 98.99%in F₁,respectively;and remarkably higher than T449.In chromosome configuration,the bivalent chromosomes were the most frequent in F₁,which was significantly higher than the bivalent frequency of the parent;and each pollen mother cell were 9.07 and 12.57 bivalents at diakinesis and metaphase I in F₁,respectively.Transcriptome analysis revealed that 15938 DEGs from 927 to 2693,which is uniquely belonging to F₁compared to parents(DEG_FPU),were found in the nine tissues.The DEG_FPU significantly enriched carbohydrate metabolism in the nine tissues,and many saccharides metabolism and starch synthase related genes were up-regulated in hybrid,such as Os BEIIb and Os SSIIIa were up-regulated in grain after three days flowering in F₁compared to both parents.In addition,many meiosis-related and meiosis-specific genes were up-regulated in F₁ compared with the autoteteaploid rice of low seed setting in anther during meiosis stage,such as DPW and CYP703A3.Moreover,many DEG_FPU were identified in the yield-related quantitative trait loci(QTLs)regions as possible candidate genes.In summary,the low fertility of T449 not only causes abnormal chromosomal behavior and the abnormal distribution of saccharide in anthers by cytological observation,but also induces abrupt expression changes in the meiosis-related and saccharide-related genes.Meanwhile,the T449,which carrying double neutral genes,Saⁿ and Sbⁿ,could overcome autotetraploid rice hybrids sterility,and the hybrids have obvious heterosis by T449crossing neo-tetraploid rice.Our study provides new germplasm resources for polyploid rice breeding and provides a theory for their molecular breeding.