Analysis Of QTL For Grain Size Based On Rice CSSL-Z66 And Z668 And Fine Mapping Of QGL3.2 For Grain Length

Grain size is an important factor affecting yield,appearance and processing quality of rice.Therefore,it is of great significance to explore genes controlling grain size in rice.However,its genetic mechanisms are complex and belong to quantitative inheritance controlled by polygenes with minor effects.Chromosome segment substitution lines are ideal materials for natural variation creation,QTL dissection,functional analysis and pyramid breeding of favorable alleles.In this study,two CSSLs-Z66 and Z668 containing different substitution segments were used as materials,which was developed by excellent indica restorer line R225 as the donor parents based on the genetic background of japonica Nipponbare.Firstly,the secondary F₂ segregating population were constructed from the recipient parents Nipponbare coossed with two CSSLsto map QTL for rice grain size.Then MAS method was used to further dissect these target QTLs into different single and dual segment substitution lines（SSSLs and DSSLs）.And then the additive and epistatic effects among these QTLs were analyzed using SSSLs and DSSLs.Finally,fine mapping and candidate gene analysis of major QTL qGL3.2 for grain length was performed.This study laid a good foundation for mapping cloning,functional analysis and intelligent design breeding of these QTLs.Specific research results are as follows:1.Identification of substitution segments and analysis of grain size of Z66Z66 was developed using advanced backcrossing and self-crossing combined with MAS of SSR markers by Nipponbare as the recipient parent and R225 as the donor parent.Z66 combined 12chromosome substitution segments from R225.The total length of all the substitution segments was39.81Mb,among which the longest was 13.90 Mb,the shortest was 0.25 Mb,and the average substitution length was 3.32 Mb.Compared to Nipponbare,Grain length,length-width ratio and 1000-grain weight of Z66 increased significantly,while grain width of Z66 had no significant difference with that of Nipponbare.2.Identification of grain size QTL in F₂ isolated populations of Z66/NipponbareA total of 12 grain size QTLs were identified from F₂ segregated populations of Z66/Nipponbare,including 3 QTLs for grain length（qGL3.1,qGL7.1,qGL10）,3 QTLs for grain width（qGW6,qGW9-1,qGW10）,3 QTLs for ratio of length to width（q RLW3.1,q RLW9-1,q RLW10）,and 3 QTLs for 1000-grain weight（qGWT3,qGWT9-2,qGWT10）.They are distributed on the chromosomes 3,6,7,9 and10.3.Construction of single and dual segment substitution lines and analysis of additive and epistatic effects of Z66 target QTLsThe above QTLs were further dissected into five single-segment substitution lines（S1-S5）and four double-segment substitution lines（D1-D4）by using MAS method.9 QTLs（qGL3.1,qGL7.1,qGL10,qGW6,qGW10,q RLW3.1,q RLW10,qGWT3,qGWT10）could be verified by single segment substitution lines,indicating that these QTLs were genetically stable.In addition,6 novel grain size QTLs（qGL9-2,qGW9-2,q RLW6.1,q RLW7.1,q RLW9-2,qGWT7.1）were identified by single segment substitution lines.Compared with previous studies,qGL9-2,q RLW9-1,q RLW9-2,qGW9-2 and qGWT9-2 may be novel QTLs identifiedin the study.Two-non-allelic QTL pyramid analysis showed that the pyramid of different QTLs produced different epistatic effects.For example,the pyramids of q RLW3.1（a=0.21）and q RLW9-2（a=0.08）produced an epistasis effect of 0.10,which made the ratio of grain length to width in D2 significantly larger than that in Nipponbare,S1（q RLW3.1）,and S4（q RLW9-2）.The pyramid of qGWT3（a=3.99）and qGWT10（a=3.98）yielded an epistasis effect of-5.35,and its genetic effects in D3（2.62 g）significantly increased 1000-grain weight of D3 than in Nipponbare,but significantly decreased than that in S1（qGWT3）and S5（qGWT10）.Understanding the interaction effects between target QTLs can predict the phenotype of futural QTL pyramid genotypes,which is important for intelligent design breeding in rice.4.Substitution segment identification and grain size analysis of Z668Similarly,Z668 was developed by Nipponbare as the recipient parent and R225 as the donor parent,containing 8 substitution segments.The total length of chromosome substitution segments was 29.075 Mb,and the average length of chromosome substitution segments was 3.63 Mb.The longest substitution segment was 15.7 Mb and the shortest was 0.625 Mb.Compared with Nipponbare,Z668 plants exhibited significant dwarf,the grain length,ratio of length to width and 1000-grain weight of Z668 was increased by 39.9%,31%and 25.9%,respectively,and grain width of Z668 was decreased by 3.3%.5.Cytological analysis of the glume of Nipponbare and Z668The morphology of outer epidermal cells and inner epidermal cells of Nipponbare and Z668 were observed.The results showed that compared with Nipponbare,the length of inner epidermal cells of Z668 was significantly longer than that of Nipponbare,and the cell width was significantly reduced,while the number of outer epidermal cells was not significantly different.These results indicated that the long-narrow grain of Z668 was caused by the increase of cell length and decrease of cell width of glume.6.Grain size QTL identification of F₂ segregated populations of Z668/NipponbareA total of 8 grain size QTLs were identified from F₂ populations segregated from Nipponbare/Z668,including 3 for grain length（qGL3.2,qGL12-1,qGL12-2）,3 for ratio of length to width（q RLW3.2,q RLW12-1,q RLW12-2）,and 1 for 1000-grain weight（qGWT7.2）,1 QTL（q SSD3）were found on chromosomes 3,7 and 12,respectively.7.Construction of single and dual segment substitution lines and analysis of additive and epistatic effects of target QTLs from Z668Using MAS,these target QTLs were further dissected into 5 single-segment substitution lines（S6-S10）and 1 dual-segment substitution line（D5）.Among them,qGL12-2,qGWT7.2,q RLW12-2could be verified by single segment substitution lines,and 10 novel QTLs（qGL1,q RLW1,qGL6,q RLW6.2,qGWT6,qGL7.2,qGW7.2-1,q RLW7.2-1,q RLW7.2-2,qGW7.2-2）were detected into single segment substitution lines.The pyramid analysis of allelic QTLs in the D5 showed that the pyramid of qGL6 and the substitution segments of chromosome 7 in D5 produced longer grains,and the pyramid of qGW7.2-2（a=–0.04）and the substitution segments of chromosome 6 produced 0.05epistatic effect,which made no significant difference between the grain width of D5 and Nipponbare,however was significantly higher than that of S9.However,q RLW6.2（a=0.08）and q RLW7.2（a=0.04）belong to independent inheritances,as are qGWT6（a=1.57g）and the substitution segment of chromosome 7.Consequentely,understanding the inheritance model of different allelic QTLs are important for design breeding.8.Genetic analysis of major qGL3.2In the F₃ population consisting of 499 individual plants developed by combinant plants of qGL3.2（whose other loci of GL was consistent with that of its recipient Nipponbare）,long grain and short grain basically displayed bimodal distribution.142 plants with short grains were distributed between7.01 mm to 8.05mm.The Chi-square test showed that the long grain（142）:long grain（357）matched the 3:1 segregation ratio（χ2=0.079<χ2（0.05,1）=3.84）,indicating that the long grain controlled by qGL3.2 was dominant and the long grain was dominant to the short grain.9.Fine mapping and analysis of candidate genes of major qGL3.2qGL3.2 was the major QTL that contributed the most to the grain length of Z668.Then 27 pairs of SSR primers were designed in the substitution interval RM5864 and RM5626 where qGL3.2 was located,and 5 of them showed polymorphism.qGL3.2 was further fine mapped into the 300kb region between SSR3 and SSR4 on chromosome 3 using 142 recessive plants with short-grains.A comparison of the genes in this region revealed 7 genes that might be associated with grain size,and through DNA amplification and sequencing,Finally,5 genes were considered as possible candidate genes for qGL3.2.The 5 candidate genes were analyzed by timed quantitative fluorescence PCR in the root,stem,leaf,sheath and ear of Nipponbare and Z668 respectively,and the results showed that the expression levels of these 5 candidate genes in different parts were significantly different from those of Nipponbare.Therefore,these 5 genes may be candidate genes for qGL3.2.