Cytogenetics And Identification Of The Genes Associatted With High Fertilty In Neo-tetraploid Rice Using Directed Inbred Lines

Autotetraploid rice is new germplasm from artificial polyploidization of diploid rice.They has obvious biology advantages,but low fertility limited the yield advantage and the application in production.In recent years,high fertility tetraploid rice lines have been selected in our lab using hybridization of different types of autotetraploid lines,namely neo-tetraploid rice.The seed setting rate of neo-tetraploid rice can reach 80%,and the F₁hybrids of neo-tetraploid rice and autotetraploid rice has obvious yield advantage,which showed a broad prospect of use.In order to reveal the mechanism lies in fertility of neo-tetraploid rice,directed inbred lines have been selected from the hybridization between neo-tetraploid rice and autotetraploid rice.High and low fertility directed inbred lines were crossed,the high and low fertility individuals in the offspring were used in this study.The main results were as follows:（1）Reproductive characteristics and cytogenetics of the directed inbred lines and their offspring.The fertility of high-fertility directed inbred line（XJG）is significantly higher than the low-fertility directed inbred line（XJD）,the seed setting of XJG and XJD were75.14%and 9.51%,respectively.The fertility were observed at different season,the seed setting of high-fertility offspring（FG）and low-fertility offspring（FD）were 85.85%and15.23%,respectively.The pollen fertility of high-fertility materials is significantly higher than the low-fertility materials,the pollen fertility of XJG and FG were 87.40%and83.38%,while pollen fertility of XJD and FD were 51.77%and 54.40%,respectively.All of the tetraploid rice have experienced 9 periods in meiotic process in this study,the abnormal frequency of low-fertility directed inbred line and their low-fertility offspring were obvious higher than high-fertility directed inbred line and their high-fertility offspring,especially in anaphaseⅡand telophaseⅡ,XJD were 53.47%and 47.12%,XJG were31.63%and 8.86%,FD were 53.76%and 45.13%,FG were 25.60%and 7.36%.The mean meiotic configurations of XJG,XJD,FG and FD at diakinesis were（0.44±0.10）Ⅰ+（7.56±0.35）Ⅱ+（0.13±0.04）Ⅲ+（8.01±0.41）Ⅳ;（0.36±0.07）Ⅰ+（9.58±0.35）Ⅱ+（0.20±0.04）Ⅲ+（6.95±0.36）Ⅳ;（0.50±0.09）Ⅰ+（9.07±0.43）Ⅱ+（0.24±0.06）Ⅲ+（7.18±0.41）Ⅳ;（0.35±0.08）Ⅰ+（9.15±0.40）Ⅱ+（0.17±0.04）Ⅲ+（7.19±0.40）Ⅳ,the mean meiotic configurations at metaphaseⅠwere（0.37±0.09）Ⅰ+（8.98±0.60）Ⅱ+（0.26±0.08）Ⅲ+（7.22±0.36）Ⅳ;（0.68±0.14）Ⅰ+（9.98±0.58）Ⅱ+（0.32±0.08）Ⅲ+（6.60±0.32）Ⅳ;（0.35±0.10）Ⅰ+（8.29±0.34）Ⅱ+（0.17±0.05）Ⅲ+（7.64±0.46）Ⅳ;（0.63±0.12）Ⅰ+（9.83±0.48）Ⅱ+（0.38±0.08）Ⅲ+（6.64±0.34）Ⅳ.Investigated the embryo sac fertility of this four materials,the abnormal behavior mainly includes:degeneration of embryo sac,degeneration of female germ cells,degeneration of eggs,small blastocyst,polar nucleus anomalies and double ovary,the most common anomalies were embryo sac degeneration,degeneration of female germ cells and polar nuclear translocation.The normal frequency of XJG,XJD,FG,FD were 91.52%,85.71%,91.72%,86.92%.It is suggested that the difference in fertility of mature embryo sac is not the main reason leading to difference in seed setting rate.（2）The whole genome sequencing variation of the directed inbred lines.After quality assessment of resequencing data,we obtained 657289 SNPs and 134465 InDels in XJG,and 687503 SNPs and 150139 InDels in XJD.In order to screen for fertility-related genes,we should remove the two coexisting mutations,the unique SNPs of XJG was 21627 and InDels was 45823,there were 21627 NON_SYNONYMOUS_CODING SNPs and 1342FRAME-SHIFT InDels,the number of genes involved were 5939 and 1102,as the same,the unique SNPs and InDels were 232128 and 54017,there were 23560NON_SYNONYMOUS_CODING SNPs and 1342 FRAME-SHIFT InDels,the number of genes involved were 6154 and 1168.Gene ontology showed that some gene related to DNA binding and meiotic process.（3）Screening differential genes based on DNA-BSA of the directed inbred lines and their offspring.We have gotten 20 genes located Chr1,97 genes located Chr2,131 genes located Chr7,142 genes located Chr8,a total of 390 genes by 50X50 BSA-seq which was based on whole genome sequencing,81 genes include Non_Synonymous_Coding SNPs or FRAME-SHIFT InDels among them,these 81 genes are maybe likely related to fertility.（4）Transcriptome sequencing analysis of the directed inbred lines and their offspring.The transcriptome sequencing analysis used the anther and embryo Sac in meiotic process of XJG,XJD,FG,FD.Compared the transcriptome result of XJG with XJD,we have obtained 1118 genes those were significantly differentially expressed,412 genes upgrading,706 genes down,and 659 genes were significantly differentially expressed from FG VS FD,363 genes upgrading,396 genes down.Gene ontology showed that some genes play important roles in DNA binding and transcription factor activity.Combined the results of transcriptome sequencing analysis,re-sequencing and DNA-BSA analysis,three genes have been found.LOC_Os07g22680 plays a critical role inmeioticchromatinreconstruction,telomereformationandrecombination,LOC_Os07g38630isunannotated,thefunctionneedsfurtherverification,LOC_Os08g33190 is about linker histone H1 and H5 family protein,the histone H1 and H5are closely related DNA binding,those three genes are most likely related to fertility.