Improvement Of Rice PTGMS Line Feng39S For Resistance To Diseases And Insect-pest By Whole Genome Marker Assisted Background Selection

Rice blast,bacterial blight(BB)and brown planthopper(BPH)are the most crucial diseases and insect pest of rice planting area in our country and even over the world,causing serious damages to rice yield and quality.Two-line hybrid rice is one of the main approaches to make use of heterosis for indica rice in southern China,and it is also the developmental direction of heterosis utilization of rice worldwide.The relevant resistance performances of photo-thermo-sensitive genic male sterile(PTGMS)lines directly affect the resistance level of their derived two-line hybrid combinations,and the most cost-effective and environmental-friendly way is to stack the major resistance(R)genes for genetically improving the resistance to diseases and insect pests.Feng39 S was an elite indica PTGMS line developed by Hefei Fengle Seed Co.,Ltd,it shows good characteristics with stable sterility,compact plant type,strong tillering ability and good rice quality,and the derived hybrid combinations have been widely spread,while they were susceptible to rice blast,BB and BPH.The objective of this study was to precisely introgress blast resistance gene Pi2,BB resistance genes Xa7 and Xa23,and BPH resistance genes Bph14 and Bph15 into Feng39 S through backcrossing and whole-genome marker-assisted background selection.Firstly,a set of monogenic chromosome segment substitute lines(CSSLs)harboring different R genes were developed,and then a series of newly constructed rice PTGMS lines conferring resistance against blast,BB and BPH were obtained by rational gene pyramiding.The main results are as follows:1.For improving the blast resistance of Feng39 S as soon as possible,Pi2 was introgressed from Hua1201 S into the genetic background of Feng39 S by molecular marker-assisted backcrossing,two breeding lines bearing homozygous Pi2,designated as DB16206-34 and DB16206-38,were finally obtained.Artificial inoculation of 57 rice blast strains indicated that the blast resistance frequency(RF)of DB16206-34 and DB16206-38 were both 94.70%,while the RF of Feng39 S was 18.30%;the field evaluation of blast resistance at two blast epidemic sites,Yichang and Enshi,showed that the improved lines and partial derived hybrids were moderately resistant or better resistant to both leaf and neck blast,which were significantly higher than that of Feng39 S and its derived hybrids.In addition,DB16206-34 and DB16206-38 exhibited similar features of sterility-fertility alteration,main agronomic traits and rice quality to that of Feng39 S,as well as the yield performance of the derived hybrids.Thereinto,DB16206-34 was named as ‘Hua634S’,which was technically verified as a blast-resistant PTGMS line by the Hubei Provincial Crop Variety Certification Committee,and hybrid combinations ‘Hua634S/9311’ and ‘Hua634S/Fengxianghui No.1’,were tested in the Hubei and National Rice Regional Trials as blast-resistant two-line hybrid combinations,respectively.2.Resistance genes,Pi2 from DB16206-172(a sib line of DB16206-34),Xa7 from Hua1228 S,Xa23 from Hua1015 S,Bph14 and Bph15 from Hua1165 S,were introgressed into the recurrent parent(RP)Feng39S by backcrossing and whole genome marker assisted background selection,the monogenic line DBQ18071-414-3-3 was developed with the introgression fragment of 567 kb containing Pi2,and the residual genetic background was identical to that of the RP,with a genetic background similarity percentage of 99.85%,in the same way,other monogenic lines were also obtained,which harbored Xa7,Xa23,Bph14 or Bph15,respectively,the introgression fragments of each line were ranging from 688.4 kb to 1574.9 kb,with genetic background similarity percentage of 99.82%-99.60%.Resistance evaluation results showed that DBQ18071-414-3-3(Pi2)was resistant to rice blast,DB17174-111-2(Xa7)and DB17207-217-244-8(Xa23)were resistant to BB,DBQ18077-3-2-1(Bph14)and DBQ18080-61-407-1(Bph15)were moderately resistant to BPH.Besides,the growth duration,main agronomic traits,rice quality and the characteristics of fertility-sterility alteration of the five monogenic lines were similar with that of Feng39 S.The established introgression lines were used for developing the pyramided lines in the research.3.Four pyramided lines(PLs)were constructed by rational crossing among the five monogenic lines followed by molecular detection for the R genes.Among them,two PLs carrying ‘Pi2+Xa7+Bph14+Bph15’ were designated as DB18128-19-164-2 and DB18128-19-361-1.The total size of four introgression fragments containing R genes were 3689.4 kb,with a genome background similarity percentage of 99.05% to that of Feng39 S.Other two PLs bearing ‘Pi2+Xa23+Bph14+Bph15’ were designated as DB18129-34-268-38 and DB18129-34-303-6.The total size of four introgression fragments containing R genes were 3974.2 kb,with a genome background similarity percentage of 99.05% to that of Feng39 S.These PLs were used in identification for diseases and insect resistance,main agronomic traits,rice quality and cross-testing in next experiments of the research.4.Artificial inoculation of rice blast strains in the Plant Protection Institute of Guangdong Academy of Agricultural Sciences showed that the RF of the four PLs were ranging from 94.12% to 97.06%,while the RF of Feng39 S was only 35.29%.The field evaluation of blast resistance at epidemic site in Wangjia village,Yuan’an county,Yichang city of Hubei province,showed that the leaf blast scores of the PLs were 0-2 and neck blast incidence of PLs were 0%-6%,while Feng39 S was highly susceptible to blast,with leaf blast score of 7 and neck blast incidence of 76%.Using Huanghuazhan(HHZ)as male parent crossing with four PLs,the derived hybrid combinations also showed enhanced blast resistance,with leaf blast score of 0-3 and neck blast incidence of 4%-9%,whereas the corresponding leaf blast score and neck blast incidence of ‘Feng39S/HHZ’were 5 and 51%,respectively.The field evaluation of blast resistance at other epidemic site in Lianghe village,Enshi city of Hubei province showed that leaf blast scores of PLs were 2 and neck blast incidences of PLs were 9%-15%,while Feng39 S was highly susceptible to blast,with leaf blast score of 8 and neck blast incidence of 100%.5.Seven strains of Xanthomonas oryzae pv.oryzae(Xoo),PXO61,PXO99,ZHE173,GD1358,Fu J,YN24,and He N11 were inoculated to four PLs and their hybrid combinations for BB resistance evaluation in the disease nursery of Huazhong Agricultural University(HAU).The results showed that the PLs,DB18129-34-268-38 and DB18129-34-303-6 harboring Pi2+Xa23+Bph14+Bph15 genes,and the derived hybrids by crossing with male parent HHZ and Wushansimiao(WSSM)were highly resistant to all seven Xoo strains.Other two PLs,DB18128-19-164-2 and DB18128-19-361-1 carrying Pi2+Xa7+Bph14+Bph15 genes,were resistant to PXO61,ZHE173,GD1358,Fu J and He N11,but susceptible to PXO99 and YN24,and the corresponding hybrid combinations were resistant to PXO61,ZHE173,Fu J and He N11,but susceptible to other three Xoo strains.However,the RP Feng39 S was susceptible to six Xoo strains and just moderately resistant to He N11,and the derived hybrids using HHZ and WSSM as male parents were susceptible to all inoculated Xoo strains.6.BPH bioassay results at seedling stage indicated that the monogenic line DBQ18077-3-2-1(Bph14)was moderately resistant to BPH,DBQ18080-61-407-1(Bph15)and four PLs bearing both Bph14 and Bph15 were resistant to BPH.The hybrid combinations,which derived from the PLs by crossing with the male parent bearing both Bph14 and Bph15,were resistant to BPH.However,the hybrids derived from male parent without Bph14 and Bph15 conferred moderate resistance to BPH.The evaluation results at adult-plant stage showed that the monogenic lines,the PLs and the corresponding derived hybrids were resistant to BPH.7.The characterization results of fertility-sterility alteration in plant growth chamber revealed that the critical sterility temperature points of five monogenic lines and four PLs were located between 22℃ and 23℃ of daily mean temperature,and the stable sterile period under natural fields at Wuhan were 81 d-86 d,which were identical to that of Feng39 S.8.Field trials of fertile period in winter season of Hainan showed that the average days to heading(DTH)of the improved PTGMS lines were 99 d-101 d,the grain yield per plant were 20.9 g-24.8 g,the plant height(PH)were 82 cm-88 cm,the panicle number(PN)were 9-10,the panicle length(PL)were 19 cm-20 cm,the spikelets per panicle(SPP)were 138-162,seed fertility were 60%-73%,and 1,000-grain weight were 25 g-26 g,in addition,results of rice quality traits showed that the amylose content of the improved lines were 12%-13%,the gel consistency were 89 mm-91 mm,chalky rice percentage were 0.0%-0.5%,head rice length/wide ratio were 2.7-2.9,and the head rice rate of the improved lines were 66%-69%,which were similar with those of the RP Feng39S;the characterization results at sterile period in summer season of Wuhan showed that the average DTH of the improved lines were 84 d-86 d,leaf number of main stems were14.0-14.4,the PH were 85 cm-87 cm,the PN were 8-9,the PL were 24 cm-25 cm,the SPP were 175-192,and stigma exsertion rate of the improved lines were 24%-39%,likewise,there was no obvious difference between the improved lines and the RP in these investigated traits.9.Yield tests of the derived hybrids from the four selected PLs and Feng39 S as female parents,by crossing with four male parents,WSSM,HB17004-7-88,HHZ and HB17010-180-171-1 indicated that the average yield per plant were 33 g-39 g and 49g-51 g by a randomized block trials with three replications in the rice breeding station of HAU in Hainan and Wuhan,respectively.The yield performance and other main agronomic traits were consistent with those of the derived hybrids from Feng39 S by the analysis of variance(ANOVA).The general combining ability(GCA)analysis of yield showed that the GCA of the PLs were identical to that of the RP Feng39 S.The above research results demonstrated that the R genes,Pi2,Xa7,Xa23,Bph14 and Bph15 were successfully and precisely introduced into the genetic background of Feng39 S by whole genome marker assisted background selection,and the developed PLs were highly similar with Feng39 S in genetic background.The PLs conferred significantly enhanced resistance to rice blast,BB and BPH,with barely changes in original features,including patterns of fertility-sterility alteration,growth characteristics,main agronomic traits,flowering habits,rice quality and GCA of yield performance.The developed PLs could be used for developing two-line hybrid rice varieties with multi-resistance,replacing Feng39 S in two-line hybrid rice breeding in future programmes.This study was the first breeding case to improve multiple traits with multiple genes precisely introgressing by using genome-wide background molecular selection techniques.