QTL Analysis Based On A Rice Short-wide Grain CSSL-Z414 And Fine Mapping Of QGL11 And QGW5

Rice grain size is an important agronomic trait,which can affect the yield and quality of rice.However,grain size is controlled by multiple genes and has a complex inheritance.Genetic analysis of rice grain size traits is of great significance for rice grain size improvement.Chromosome Segment Substitution Line（CSSL）can dissect inheritance of complex traits into qualitative inheritance and hence are ideal materials for genetic research.Except for a small amount of substitution segments from the donor parent,each CSSL has the same genetic background as that of the recipient parent.It is beneficial to analyze and study these complex traits.In this study,a rice short-wide grain CSSL-Z414 with excellent restorer line Xihui 18 as the recipient parent and Huhan 3 as the donor parent was constructed by a combination of advanced backcrossing,selfing and molecular marker-assisted selection（MAS）all over the whole rice genome.Then comprehensive analysis for both phenotype and molecular identification of Z414 were carried out.Finally,QTL mapping for the agronomic traits were conducted using the secondary F₂segregation population from Xihui 18/Z414 and carried out.Based on the QTL mapping,the single-segment substitution line（SSSL）and dual-segment substitution lines（DSSL）containing the target QTLs were developed in the F₃generation.Then,the additive and epistatic effects of the target QTLs were performed using the corresponding SSSL and DSSL,together with the genetic effects and phenotypes of novel genotypes after pyramiding of the target QTLs.Finally,fine-mapping of major QTLs was done by the substitution mapping method.The main results were as follows:1.Z414 is a chromosome segments substitution line developed from Xihui 18 as the recipient and Huhan 3 as the donor parent.Z414 contained 4 substitution segments from Huhan 3.The total estimated length of substitution segments of Z414 was 12.17 Mb,the average length was 3.04 Mb,the shortest substitution length was 1.42 Mb,and the longest substitution length was 5.58 Mb.Compared with Xihui 18,Z414 displayed significant increase in grain width,1000-grain weight,brown rice rate and chalkiness degree.While there was significant decrease in panicle length,grain length and ratio of length to width of Z414.and there were no significant differences in the other traits.2.At the completion of the booting stage and before the heading period,the inner and outer epidermal cells of the glume in Xihui18 and Z414 were investigated using scanning electron microscope.The cell length in the inner epidermis of the glumes of Z414 exhibited no significant difference compared with that of Xihui 18,and the cell width in the inner epidermis of the glumes of Z414 was significantly increased by 22.23%than that of Xihui 18,and the number of cells of Z414was significantly decreased by 13.52%than that of Xihui 18.The results indicated that the short-wide grain of Z414 was mainly caused by decrease of glume cell number and increase of glume cell width.3.In the F₂population of Xihui 18/Z414,which consisted of 121 individuals,the grain width basically showed a bimodal distribution,the narrow grain was distributed in 2.90-3.46 mm,a total of84 plants,and the wide grain was distributed in 3.46-3.70 mm,a total of 37 plants.The ratio of narrow grain to wide grain fitted to a 3:1 segregation ratio by Chi-square test（χ²=2.01<χ²（0.05,1）=3.84）.The result indicated that the wide grain of Z414 was mainly controlled by a recessive major QTL in the genetic background of Xihui 18.The remaining traits displayed continuous and normal distribution characteristics,indicating that the inheritance of the remaining traits were still controlled by many loci,and QTL mapping can be performed.4.A secondary F₂segregation population constructed by the Xihui 18/Z414 was used to map QTL for 7 traits,including panicle length,grain length,grain width,Ratio of Length to width,1000-grain weight,chalkiness,and brown rice rate.A total of 7 QTLs for agronomically important traits were identified on 3 chromosomes 1,5and 11,including q PL3,qGW5,qGL11,q RLW5,q RLW11,qGWT5 and q BRR11.The allele qGW5 from Huhan 3 increased the grain width,explaining50.39%of the phenotypic variation.The allele qGL11 from Huhan 3 decreased the grain length,explaining 9.96%of the phenotypic variation.5.On the basis of primary QTL mapping,6 single segment substitution lines（S1-S6）were developed in F₃.Six QTLs（q PL3,qGW5,qGL11,q RLW5,q RLW11,qGWT5）could be verified by novel developed single segment substitution lines（S1-S6）.these 6 QTLs were shown to be genetically stable.In addition,4 QTLs（qGL3,qGL5,q CD3 and q CD5）were novel detected by S1and S5.The short panicle of Z414 was harbored by q PL3,the short grain of Z414 was controlled by qGL11,qGL3 and qGL5,the wide grain of Z414 was responded by qGW5,the lower ratio of length to width was controlled by q RLW5 and q RLW11,larger grain of Z414 was in charge of qGWT5,higher brown rice rate of Z414 was explained by q BRR1.The q CD5 and q CD3 answered for the higher chalkiness degree of Z414.Among them,q BRR11 was greatly affected by the environment,cannot be stably inherited,and cannot be repeatedly detected in the corresponding single-segment substitution lines.6.On the basis of primary QTL mapping,2 dual-segment substitution lines（D1,D2）were developed in F₃.Pyramid of qGL3（a=0.22）and qGL11（a=-0.19）yielded an epistatic effect of-0.31in D2.The result suggested that pyramid of qGL3 and qGL11 resulted in shorter grains than S6（containing qGL11）,indicating that qGL11 displayed epistatic to qGL3.Pyramid two substitution loci without QTL for 1000-grain weight on chromosomes 3 and 11 in D2 produced an epistatic effect of-2.94,1000-grain weight of D2 displayed significantly lower than that of S1,S6 and Xihui18.Other trait loci in the 2-substitution fragments of D1 and D2 were inherited independently.7.Based on the above results,we further analyzed qGL11 using S6 with 4 polymorphic SSR markers from 28 newly designed markers,whose the maximum length was 1.66 Mb.To perform fine mapping of qGL11,we developed five novel secondary SSSL（S7–S11）by crossing Xihui 18 and S6.Based on the theory of substitution mapping,qGL11 was delimited to 810 Kb of the maximum length between RM1812 and RM6085.In the 810 Kb interval,there were forty genes with specific functional description,we found only Cyc T1;3 might be the candidate gene of qGL11 according to the possible signaling pathway regulating grain size.By DNA sequencing between Xihui 18 and S6,there were 6 SNP differences and a 25-base insertion in the 5’UTR and 1 SNP difference in the3’UTR,and 1 SNP difference in the CDS which did not cause amino acid change,and the protein structure displayed no difference between S6 and Xihui18.But the expression levels of Cyc T1;3 was significantly higher in sheath and panicle in S6 than in Xihui 18.Again the short grain of Z414 was mainly caused by decrease of glume cell number,which was consist with the function description of Cyc T1;3.Thus,Cyc T1;3 might be the candidate gene for qGL11.8.Using overlapping substitution mapping of three single segment substitution lines S3,S4 and S5 from the F₃populations of Xihui 18 and Z414,qGW5 was located in the substitution of RM405to RM3328.In the substitution interval of S5,Three polymorphic markers from 21 newly designed markers were found,and qGW5 was delimited to 1.430 Mb of the maximum length between RM405and RM17984.Within the interval,we found 6 genes involved in the reported signaling pathway for grain size.By DNA sequencing of these genes,only GS5 and Os TAR1 were found existing differences between Xihui 18 and S5.Concerning GS5,（GGC）₇repeat after the 90th base of the CDS were found in S5,while only（GGC）₅repeat in Xihui 18.Moreover,the protein structure displayed some differences between S5 and Xihui18,q RT-PCR analysis also displayed that expression levels of the GS5 were significantly higher in stem,leaf,sheath and panicle in S5 than in Xihui18.For Os TAR1,as an IAA biosynthesis gene,there were 4 SNP differences in the CDS between Xihui18 and S5,of which 3 caused amino acid mutations and 1 nonsense mutation.While q RT-PCR analysis showed there are no significant expression differences of the Os TAR1 were found in all organs between Xihui 18 and S5.Thus,GS5 should be the prior candidate gene for qGW5 and Os TAR1 was potential one for qGW5.