Genome Variation And Functional Analysis Of Fertility Related Genes In Neo-tetraploid Rice

Autotetraploid rice is a new germplasm by diploid rice genome duplication.Compared with diploid rice,autotetraploid rice and intersubspecific(indica x japonica)autotetraploid rice hybrids F₁ showed stronger biological advantages.However,low seed setting is the major barrier in commercial utilization of polyploid rice.Over the past 20years,our group has developed neo-tetraploid rice lines,which have the characteristics of high fertility and heterosis when crossed with autotetraploid rice having low fertility.And two lines of the new germplasm,Huoduo No.1 and Huaduo No.2 obtained the plant variety rights protect.So far,there are a lot of studies on autotetraploid rice.But there are few researches on Neo-tetraploid rice,especially DNA genome structural variations and gene expression in neo-tetraploid rice compared to autotetraploid parents.Therefore,morphological,cytological and molecular biology analysis reveal agronomic traits,DAN variations and gene expression profile at meiosis stage between neo-tetraploid rice and autotetraploid rice parents.The main results were as follows:(1)The three neo-tetraploid rice lines displayed significant differences in agronomic traits,including plant height,number of filled grains per plants,and 1000-grain weight.Importantly,the seed setting and pollen fertility of neo-tetraploid rice lines were significantly higher than their parents.Three neo-tetraploid rice lines produced 68.3%,68.1%and 71.8%seed set,while 31.5%and 25.4%seed set yielded by autotetraploid parents.Pollen fertility of neo-tetraploid rice lines was more than 92%,while parents produced 65.83%and 76.75%pollen fertility.Non-significant differences were detected in embryo sac fertility between neo-tetraploid rice lines and autotetraploid parents.Furthermore,the seed setting of hybrid lines F₁ generated by crossing with various autotetraploid(indica x japonica)were fertility.It indicates neo-tetraploid rice can overcome the sterility of intersubspecific(indica x japonica)F₁ hybrid.(2)Chromosome behavior observation revealed that the meiotic stages of neo-tetraploid rice were similar to their autotetraploid parents.Many abnormalities,including chromosome straggling,chromosome lagging,abnormal spindle,asynchrony of the chromosome and abnormal cell shape,were observed in autotetraploid parents.However,few abnormalities were detected in neo-tetraploid rice compared with their parents.Moreover,there are significant differences in the chromosome configuration between neo-tetraploid rice and their autotetraploid parents.The mean chromosome configuration of neo-tetraploid rice lines were(0.53±0.09)I+(9.79±0.46)II+(0.16±0.04)III+(6.85±0.23)IV?(0.40±0.08)I+(9.27±0.34)II+(0.11±0.03)III+(7.18±0.17)IV and(0.11±0.03)I+(9.73±0.28)II+(0.37±0.03)III+(7.01±0.13)IV,respectively.The mean chromosome configuration of their autotetraploid parents were2n=48=(0.34±0.09)I+(5.55±0.40)II+(0.04±0.02)III+(8.68±0.19)IV+(0.22±0.05)VI+(0.05±0.03)VIII and 2n=48=(0.55±0.1)I+(7.93±0.27)II+(0.07±0.02)III+(7.16±0.14)IV+(0.03±0.01)V+(0.27±0.05)VI+(0.12±0.03)VIII,respectively.(3)A total of 4109881 SNPs and 640592 In Dels were detected in the three neo-tetraploid rice lines relative to T44 and T45,respectively.A total of 9397 and 6980genes,harbored at least SNP and In Dels,exhibited mutations in neo-tetraploid rice lines compared to T44 or T45,respectively.These genes including meiosis related genes,transcription factor,protein kinase,epigenetic related-genes and calcium signal system gene.A total of 4121 genes,harbored at least SNP and In Del,exhibited mutations in neo-tetraploid rice lines compared to T44 and T45.These genes were significantly enriched in post-translational protein modification,protein phosphorylation,regulation of gene expression,RNA metabolic process,and recognition of pollen,DNA repair,ubiquitin ligase complex,nc RNA metabolic process and t RNA metabolic process.Os12g0211400 and Os10g0357800 had strong direct interaction relations with fertility related genes.But the m RNA level of these genes,interactive genes with mutated gene and 31 meiosis related genes were not significant changes for Neo-tetraploid rice.342 genes showed specific mutations in neo-tetraploid rice lines compared to T44 and T45.In additional,about 600specific PAVs were detected in neo-tetraploid rice lines compared to autotetraploid parents,and these PAVs influenced the length of chromosome in varying degrees.(4)Transcriptome sequencing was used to identify differences in genes expression between neo-tetraploid rice lines compared to autotetraploid parents at meiosis stage.The results showed that 1473 and 766 genes were common and differentially expressed in neo-tetraploid rice lines compared to T44 and T45.In comparison to T44,1473 genes were differentially expressed in neo-tetraploid rice lines,including 132 nc RNA,41 transposon elements and 306 specific expressed genes.766 genes were differentially expressed in neo-tetraploid rice lines compared to T45,including 12 transposon elements,113 nc RNA and 197 specific expressed genes.Combining Re-sequence and transcriptome sequencing,m RNA level of 23 mutant genes exhibited significant changes in neo-tetraploid rice compared to T44.Of these,23 fertility related genes were found to be differentially expressed in meiosis stage.Only the function one gene(Os12g0572800)was described.Moreover,4 epigenetics related genes showed differentially expressed pattern,including Os03g0226901,Os01g0719100,Os04g0572600 and Os05g0392400.In comparison to T45,m RNA level of 12 mutant genes depicted significant changes for Neo-tetraploid rice.Among these genes,8 fertility related genes were differentially expressed,but their functions are unknown during meiosis stage.Moreover,3 epigenetics related genes showed differentially expressed patterns,including Os01g0719100,Os04g0572600,and Os05g0392400.(5)Knockout vectors were constructed for targeting t RNA methyltransferase?protein phosphatase 2C?calmodulin dependent protein kinases and DNA glycosylase by CRISPR/Cas9.Transgenic plants were obtained by agrobacterium-mediated transformation of rice callus with the knockout vectors.The mutation corresponding to target gene were detected at T0 generation.The mutation types were deletion,insertion and mismatch.The phenotypic investigation of the T1 generation revealed that the loss of DNA glycosylase gene and protein phosphatase 2C gene led to decrease seed setting.This indicates that t RNA methyltransferase gene and protein phosphatase 2C gene have important function in plant fertility.Our results suggested that three neo-tetraploid rice lines displayed significant differences in agronomic traits genomic,especially seed setting.Structural reprogramming,DNA variations,differential expressions of some important meiosis and epigenetics related genes were associated with high fertility in neo-tetraploid rice.