| Rice is one of the most important food crops in the world,providing the staple food for more than 1/3 of the population.The food security threat still looms to date.Improving rice yield is still an important goal of rice breeding.Innovative breeding methods are very necessary.Breeding by design is a new topic in the post-genomic era.The core of design breeding is the effective creation and utilization of naturally occur-ring variation.Therefore,the identification of favorable allelic variation is key.The rice grain size both affect the quality and yield of rice,so it plays an important role as an ag-ronomic trait in rice breeding.However,grain size are quantitative traits controlled by multiple genes,and their mechanisms of inheritance are complex and still not fully un-derstood.Rice chromosome segment substitution lines（CSSLs）differ from the recipient parent by only a few substitution segments,which are more effective for the identifica-tion of target QTLs and are suitable materials for genetic analysis.In particular,rice chromosome segment substitution lines can create abundant natural variation.Further-more,the identified favorable alleles can be directly applied to breeding practice.There-fore,CSSLs are good materials for genetic dissection and gene identification of com-plex traits,and also an important resource for design breeding.In this study,two short-wide grain CSSL-Z413 and CSSL of the donor parent Z436 were obtained through ad-vanced backcross and self-pollination,combined with the method of genome-wide mo-lecular marker-assisted selection,from an excellent indica restorer line Xihui 18 as the recipient parent and japonica variety Huhan 3 with strong stress tolerance as donor.In this paper,a series of studies have been carried out using these two CSSLs as research materials.The main results are as follows:1.CSSL-Z413 substitution lineZ413 was bred by advanced backcrosses and self-pollination combined with the whole genome molecular markers-assisted selection with the excellent indica restorer line Xihui 18 as the recipient parent and Huhan 3 as the donor parent.Z413 contained 6substitution segments,which were located on chromosomes 1,2,3,5 and 11,and the average substitution length was 3.38Mb.Compared with the recipient parent Xihui 18,plant height,number of primary and secondary branches,grain length,ratio of length to width,number of grains per panicle,number of spikelets per panicle and yield per plant of Z413 reduced significantly by 15.9%,34.2%,15.3%,11.8%,32.3%,34.2%,23.0%,and 12.8%respectively.The number of panicles per plant,grain width and 1000-grain weight of Z413 were significantly increased by 76.4%,29.90%and 7.28%,respectively.2.QTL detection of important agronomic traits carried by Z413A secondary F2 segregation population consisting of 150 individual plants was cre-ated with Xihui18/Z413 as a rice QTL mapping population.For QTL detection,P<0.05was the threshold for the presence of QTL.A total of 12 QTLs for important traits were identified,including 1 QTL for Plant height,1 QTL for panicle number,2 QTLs for number of primary branches,2 QTLs for grain length,2 QTLs for grain width,1 QTL for ratio of length to width,1 QTL for grain number per panicle,1 QTL for spikelet per panicle and 1 QTL for 1000-grain weight.3.Development of secondary segment substitution lines from Z413On the basis of primary QTL mapping,one single segment substitution line（SSSL）,7dual-segment substitution lines（DSSLs）,7 triple-segment substitution lines（TSSLs T1～T7）and 3 quadruple-segment substitution lines（QSSLs）were developed in F3 by MAS method.4.Identification of substitution segments and analysis of important agronomic traits of Z436.Z436 was bred with the excellent long-grain indica restorer line Xihui 18 as the re-cipient parent and the short-wide grain variety Huhan 3 as the donor parent.Z436 car-ries 8 substitution segments from Huhan3 with an average substitution length of 4.49Mb（indica 9311 is the reference genome）.Compared with Xihui 18 grain width and 1000-grain weight of Z436 increased significantly by 25.6%and 16.4%.The grain length,ratio of length to width,spikelet per panicle,grain number per panicle,plant height and the number of secondary branches of Z436 were significantly reduced by 7.6%,26.4%,22.1%,17.4%,19.6%and 21.0%respectively.There was no significant difference in panicle number per plant.5.QTL identification of important agronomic traits carried by the Z436The secondary F2 segregating population composed of 150 individual plants con-structed by the cross of Xihui1 and Z436 was used as the QTL mapping population.Taking P<0.05 as the threshold for the existence of QTL,A total of 18 QTLs for im-portant agronomic traits in rice were identified on the substitution segment of Z436,in-cluding 1 for plant height,1 for panicle length,1 for the number of primary branches,2for the number of spikelet’s per panicle,and 1 for seed setting rate,2 QTLs for grain length,4 QTLs for grain width,3 QTLs for the ratio of length to width and 3 QTLs for1000-grain weight.6.Breeding of secondary substitution lines with target QTL from Z436Based on the QTL mapping,the molecular marker-assisted selection was used to breed 15 secondary substitution lines the in F3 generation,including 7 single-segment substitution lines（and S1～S7）,8 dual-segment substitution lines（D1～D8）.7.Validation of target QTL using SSSLsCorresponding 7 SSSLs validated the genetic stability of seven QTLs including q GL11,q GW5,q GWT5,q RLW5,q RLW11,q SPP7,and q SSR7.In addition,S1～S7 re-vealed several minor QTLs that were not detected in the secondary F2 population of Xi-hui 18/Z436,including（q PH4,q PH5,q PL4,q NPB5,q NPB11,q SPP4,q SPP5,q SSR4,q SSR5,q GL6,q GL7,q GW2-1,q GW2-2,q GW6,q GW7,q RLW2-1,q RLW2-2,q RLW,6and q RLW7）,which indicated that the SSSLs shows a higher sensitivity for QTL detec-tion.8.Analysis of additive and epistatic effects of QTL using DSSLs and SSSLsPyramiding of different QTLs produced different epistatic effects and different phenotypes,while some QTLs displayed independent inheritance.The dual-segment substitution line（DSSL）D6 carried the substitution segment of chromosome 5 and 7,pyramiding of QTL-q GW5（additive effect=0.28）and q GW7（a=0.20）for grain width and QTL-q GWT5（additive effect=4.95）and q GWT7（a=4.5）for GWT yielded the epi-static effects of-0.32 and-5.55 respectively,indicating that the genetic effect of grain width of D6 increased the grain width and 1000-grain weight by 0.16 mm and 3.9 g,indicating that the pyramiding of q GW5,q GW7 and q GWT5,q GWT7 produced thinner and lighter grains.While pyramiding of q GWT2-1（a=1.45）and q GWT7（a=4.50）yielded the epistatic effect of increasing the 1000-grain weight of D2 genetically by2.05 g,Thus,1000-grain weight（30.7 g）of D2 was lighter than that（35.6 g）of S6（q GWT7）and heavier than that（29.5 g）of S1（q GWT2-1）.Pyramiding of q SPP4 and q SPP7 in,D4,q SPP5 and q SPP7 in D6 showed no epistatic effect,which indicated that they belonged to independent inheritance.Although there are many differences in phe-nomena,However,the performance after pyramiding of genes essentially depended on the comparison between the genetic effects（algebraic sum of the additive and epistatic effects of all QTLs）in the pyramidal line and the additive effect value of the single QTL.Consequently,we can predict the phenotype of pyramiding genome by the rule.These results are important in molecular breeding by design based on SSSLs. |
