| Three-line hybrid rice has made a great contribution to increase the yield of rice globally.Three-line indica hybrids which have been extensively used in commercial rice hybrids have been developed mainly on wild abortive（WA）-type cytoplasmic male sterility（CMS）.Although WA-type sterile cytoplasmic has been used widely in indica hybrids,the genetic basis of fertility restoration of WA-type CMS lines has not been explained clearly.Meanwhile,some WA-type CMS lines having,under the influence of the external environment,the selfed seeds phenotype,and the poor gene resources in restorers influence the purity of hybrid seeds and the utilization of heterosis.Compared with three-line indica hybrids,japonica hybrid cultivation which has been developed mainly on Chinsurah Boro Ⅱ（BT）-type CMS.Because of the single source of cytoplasm and lacking yield competitive advantage in comparison to japonica inbred lines,BT-type japonica hybrids have been developed relatively slowly.With the consideration of the male sterility stability,flowering habit and recoverability of japonica CMS lines with iso-nuclear allo-cytoplasm,we provided an idea to develop Honglian（HL）-type japonica hybrid rice.HL-type japonica CMS lines can be easily obtained,along with BT-type japonica maintainers,by backcrossing,but there was few HL-type japonica restorer lines existed in BT-type japonica restorer lines.Therefore,researches on discovering new WA-type fertility restoring genes（Rfs）and HL-type japonica Rfs will be assist in breeding three-line hybrid rice.In the present study,the Rf for HL-type japonica CMS lines in BT-type japonica restorer line SiR2982 was studied,and the ability of the Rf to restorer the fertility HL-type japonica CMS lines was studied by constructing high generation backcross populations.Additionally,a new Rf for WA-type indica CMS lines was mapped.The results are as followed:1.800 and 1,200 plants in the HL-LqxA//Lqx/SiR2982 F2 and F3 populations were used to refine the position of the Rf in BT-type japonica restorer line SiR2982.Ultimately,the location of the Rf gene was delimited to a region of 一100 kb,between the markers STS 10-65 and STS 10-63 on the long arm of chromosome 10 according to the information of recombinants,within which Rfla（Rf5）is located.The Rfs in BT-type japonica restorers have been derived from a single source,and only the Rfl locus has been shown to exist in most BT-type japonica restorers.Thus,we inferred the dominant Rf in SiR2982 was Rfla（Rf5）,and sequencing the Rfla allele in SiR2982 demonstrated the inference.2.The natural spikelet fertility levels of fertile plants in HL-LqxA/Lqx/SiR2982 population showed a continuous distribution,and the average natural spikelet fertilities of these plants were significantly lower than that of the HL-LqxA/SiR2982 F1 plants,indicating that Rf5（Rf1a）had only a partial capability of restoring fertility to HL-type japonica CMS lines,and that other Rf genes and/or modifiers were required for the normal fertility of HL-type hybrid F1 plants in japonica.Using HL-LqxA as the recurrent parent,the HL-LqxA//Lqx/SiR2982 BC4F2 population was structured.The average natural spikelet fertility of plants harboring Rf5Rf5 were significantly higher than that of the plants harboring Rf5rf5,indicating that the dosage effect of Rf5 exists on the fertility restoration of HL-type CMS japonica lines.3.TFA,a WA-type CMS line,bred by Guangdong Academy of Agricultural Sciences,had the high combining ability,but there were selfed seeds on TFA plants under high temperatures,indicating that TFA may carry minor Rfs.To improve the sterility stability of TFA plants,TFA/ZS97B//TFB and TFA/ZS97B//ZS97B populations were constructed.There were a few plants with different levels of stainable pollen grains and spikelet fertility in these two populations,indicating that there were Rfs in TFB and ZS97B,and the complementation and dosage effect of Rf on the fertility restoration of WA-type CMS might exist.The number and fertility level of the fertile plants in TFA/ZS97B//ZS97B population was lower than that of the fertile plants in TFA/ZS97B//TFB population,showing TFB might harbor more minor Rfs.4.Selecting a fertile plant（Number P2-1014）as the male parent and TFA as the recurrent parent,the TFA/ZS97B//TFB BC4F1 and BC4F2 populations were constructed.During the years of 2014 and 2015,we observed he pollen sterility and natural spikelet fertility of plants in BC4F1 and BC4F2 populations,by which the fertility restoration to WA-TFA in the mentioned populations was inferred to be controlled by one dominant gene,temporarily named Rf19（t）.5.Two phenotypically contrasting bulks,one sterile and one fertile were formed by mixing the DNA from the 50 corresponding plants in BC4F1 population.Using SLAF-seq technology combined with three correlation analysis methods of SNPindex,ED algorithm and bayesian algorithm,Rfl9（t）was preliminary mapped to a candidate region from 9.93～13.32Mb on chromosome 10.Using plants in BC4F2 population,Rfl9（t）was mapped to a region from 13.08～13.84Mb,between 25300S-3/Dral and RM25384,with the genetic distance was 0.23cM and 0.30cM,respectively.6.qRT-PCR assays confirmed that Rfl9（t）restored the fertility of WA-type CMS lines via reducing the mitochondrial sterility gene WA352 transcripts,which was similar to that of Rf4,a cloned major Rf for WA-type CMS.Rf4 encoded pentatricopeptide repeat（PPR）motif containing proteins,and according to the information from a web site RGAP（Rice Genome Annotation Project）,we speculated that Os10g26070,an ORF encoding PPR proteins within the position region,might be the candidate gene of Rf19（t）.7.Sequencing results revealed that TFA and ZS97 had the same encoding sequence of Os10g26070,but there were 5 bases differences between TFA and fertile plants(Rfl9（t）Rfl9（t）,indicating that Rfl9（t）was not derived from ZS97B.However,Rfl9（t）was a new Rf for WA-type CMS.These results will lay a foundation for cloning Rfl9（t）and provide a reference for breeding WA-type three-lines. |
