| Autotetraploid rice is a new germplasm resource obtained by diploid rice chromosome doubling.The germplasm have obvious biological advantages,however,the low fertility limits its utilization in production.After years of efforts,high fertility tetraploid rice lines have been selected in our lab,namely neo-tetraploid rice.The F1hybridized between neo-tetraploid rice and low-fertility autotetraploid rice not only is high fertility,but also has obvious yield advantages,which showed a broad application prospects.In order to clarify the genetic mechanism of the high-fertility neo-tetraploid rice,the hybrid F1 was obtained by hybridization of high-fertility neo-tetraploid rice Huaduo 3（H3）and low-fertility autotetraploid rice T452,F1 self-crossing to obtain F2,then the high-fertility directed inbred line JG and low-fertility directed inbred line JD were continuously directed breeding according to the criteria of the seed setting more than 80%or less than 20%from F2.The following studies were carried out:one is the cytological study of reproductive processes by using the WE-CLSM technology（whole-mount eosin B-staining confocal laser scanning microscopy）in JG and JD and their parents;the other is fertility-related gene localization by BSA-Seq and BSR-Seq,and further verification by q RT-PCR.The main results are as follows:（1）Reproductive characteristics of high-fertility directed inbred line JG and low-fertility directed inbred line JD and their parentsThe high-fertility directed inbred line JG and the high-fertility parent H3 were compared with the low-fertility oriented inbred line JD and the low-fertility parent T452,respectively.The mature pollen fertility of the former two was significantly higher than the latter two.The mature pollen fertility of JG,H3,JD and T452 was 88.53%,87.45%,25.08%and 23.70%,respectively.The main abortive pollen types of JD were typical abortive pollen（18.44%）,empty abortive pollen（14.85%）,no fully stained pollen（55.17%）and small pollen grains（1.23%）.Statistics of fertilized 3 days of embryo sac based on WE-CLSM,the results showed that the embryo sac fertility of JG and JD and their parents was basically normal,and there was no significant difference.However,the frequency of unfertilized embryo sacs was significantly different in four materials.The frequencies of unfertilized embryo sacs of JG,H3,JD and T452 were 7.62%,12.30%,53.76%and64.38%,respectively.The frequency of unfertilized embryo sacs of JD and T452 is significantly higher than that of JG and H3.It shows that the effect of pollen fertility on embryo sac fertilization is significant.（2）Meiotic chromosome behavior and configuration of pollen mother cells of high-fertility directed inbred line JG and low-fertility directed inbred line JD and their parentsThe meiosis process of pollen mother cells of JG and JD and their parents are basically the same.However,there are differences in the types of chromosomal behavior abnormalities in different periods,such as metaphase chromosome drag,anaphase chromosome lag,telophase micronucleus,spindle abnormality,and dyad undivided.It is worth mentioning that the abnormal frequencies of the metaphase II and the anaphase II of the four materials are higher than other periods.Among them,JG was 43.85%and 41.44%,JD was 52.35%and 56.67%,H3 was 22.27%and 44.51%,and T452 was 44.93%and44.59%,respectively.They have significant differences in the chromosome configuration in diakinesis and metaphase I.Whether it is in diakinesis or metaphase I,the number of bivalents of JG and H3 is higher than that of JD and T452,and the number of JG in diakinesis and metaphase I bivalents are 5.8±0.14 and 9.43±0.02;JD is 4.74±0.26 and8.52±0.18;H3 is 5.76±0.05 and 9.28±0.02,;T452 is 3.47±0.17 and 8.50±0.51,respectively.The above results indicate that the high fertility of JG and H3 may be related to the number of bivalents higher than that of JD and T452.（3）Mapping of high fertility genes by BSA-seq and BSR-seq in Neo-tetraploid RiceMapping of fertility genes is carried out in two steps:The first is to use the F2 and F3of the JG and JD groups to construct the extreme trait mixing pool and perform BSA-seq.We obtained a total of 1346124 high-quality SNPs and 294650 In Dels in F2.Correlation analysis was performed on these mutation sites,and one region on chromosome 7（18180000-19500000）was mapped,and 185 genes were annotated in this region.Among them,non＿synonymous mutant genes existed between the progeny or the parent.114,there are 26 frame-shift mutant genes.A total of 1343958 high-quality SNPs and 295,480 In Dels were obtained in F3.The correlation analysis also mapped to a region on chromosome 7（18400000-22120000）,in which a total of 536 genes were annotated.There are 33non＿synonymous mutation genes and 97 frame-shift mutant genes.The second is to use the meiotic period anthers of the JG and JD populations for BSR-seq analysis in F4.We obtained 65202 SNP,correlation analysis mapped 107 genes located in chromosomes18915234-19692348 and 19736756-19748279,all of which had non-synonymous mutations or frameshift mutations between parents or between progeny.Comprehensive result of BSA-seq and BSR-seq found that the three results were mapped to the segment18915234-19500000 in chromosome 7,which contained 78 genes that may be related to the fertility of tetraploid rice.（4）q RT-PCR verification of high fertility related genes in neo-tetraploid riceThe functions of 78 fertility-related genes screened by BSA-seq and BSR-seq were analyzed and found that 9 of them may be related to pollen mother cell meiosis and pollen fertility.The nine genes are LOC＿Os 07g31870,LOC＿Os 07g32010,LOC＿Os 07g32020,LOC＿Os 07g32040,LOC＿Os07g32170,LOC＿Os07g32406,LOC＿Os07g32480,LOC＿Os07g32650,LOC＿Os07g32660.q RT-PCR was used to verify the difference in expression of these 9 genes in JG and JD and their parents.The gene of LOC＿Os07g32480,LOC＿Os07g32406,LOC＿Os07g32040,LOC＿Os07g318704 were differentially expressed between high fertility and low fertility materials,and both were down-regulated in high fertility materials.In addition,q RT-PCR expression studies on 24 meiotic-related genes,such as LOC＿Os07g32480,showed significant differences in the expression levels of LOC＿Os06g41050,LOC＿Os11g40150 and LOC＿Os09g35000 between high-fertility and low-fertility materials.These results preliminarily indicated that the above seven genes may be related to the fertility of new tetraploid rice.In summary,the reasons for affecting the fertility of neo-tetraploid rice are various.From the aspect of cytogenetics,the meiosis terminal phase of pollen mother cells and the high number of bivalents in diakinesis and metaphase I of pollen mother cells are one of the reasons for the high fertility of neo-tetraploid rice;From the aspect of molecular genetics,the sequence variation and expression difference of meiosis-related genes may determine its high fertility,which deserves further study. |
