Fine Mapping Of Grain Length QGL1 And Analysis Of QTL For Important Agronomic Traits Based On Rice Chromosome Segment Substitution Lines Z688 And Z104

Rice（Oryza sativa L.）is the food on which people depend.As the global population has exceeded the 80 billion and the area of cultivated field has been decreasing,the demand for rice has become more urgent.The rice bowl of the Chinese people should be firmly in the hands of the Chinese people.Therefore,improving rice yield is the ardent expectation of every breeder.Plant height,grain size and other traits in rice are complex quantitative traits controlled by multiple genes,and the genetic basis is quite complex.Chromosome Segment Substitution Lines（CSSLs）in rice are ideal materials for creating natural variation,and also for genetic dissection of complex traits controlled by multiple loci.In particular,the quantitative trait loci（QTL）which were mapped by SSSLs can be directly applied to breeding practice,so they are ideal genetic materials for studying complex traits.Then,it is necessary to identify more favorable QTL to make whole-genome rice molecular design breeding into reality.However,the deveopment of rice single segment substitution lines（SSSLs）with target genes needs many years through advanced backcross and self-crossing combined by MAS of all markers in rice genome.Once the SSSLs covering all rice genome are completed,it is of very importance for both gene cloning and functional analysis and molecular breeding by design.In this paper,two rice chromosome segment substitution lines,Z688 and Z104,which were developed by advanced backcross,pollination combined by MAS of SSR markers using Nipponbare as recipient parent and indica restorer line Xihui 18 and Jinhui 35 as donor parents respectively,were used as materials to carry out a series of studies.The main results are as follows:1.Analysis of substitution segments and plant height and grain size traits of Z688Z688 was a chromosome segment substitution line developed with Nipponbare as the recipient parent and Xihui 18 as the donor parent.It contains 4 substitution segments from Xihui 18.The total estimated length of substitution segments is 8.53 Mb,and the average estimated length of substitution fragments is 2.13 Mb.Compared with the recipient parent Nipponbare,the grain length,length-width ratio and 1000-grain weight of Z688 were significantly increased;however the plant height of Z688 was significantly reduced;There was no significant difference between Z688 and Nipponbare in effective panicle number per plant,panicle length and grain width.The inner and outer epidermis of grain glume of Nipponbare and Z688 at heading stage were observed by scanning electron microscope.The results showed that the increase of grain size of Z688 was mainly caused by the increasing cell lengthof glume cells.2.Identification of QTL for plant height and grain size carried by the substitution segments of Z688Seven QTL for plant height and grain size traits were identified from the secondary F₂population produced by crossing of Nipponbare and Z688,including qPH1-1,qPH1-2,qGL1,qGL6,qGL12,qRLW1 and qGWT1,They were distributed on the substitution segments of chromosome 1,6and 12,and explained the phenotypic variation from 0.84%to 23.94%.Among them,two major qGL1 and qGL6 and minor qGL12 from Xihui 18 were linked with the long grain trait of Z688,and their additive effects increased the grain length of Z688 by 0.11 mm,0.09 mm and 0.06 mm,respectively,explaining the variation of grain length of 23.94%,15.36%and 8.83%.3.Development of secondary single and dual segment substitution lines and analysis of additive and epistatic effects of target QTL from Z688On the basis of QTL mapping,7 single-segment substitution lines（S₁-S₇）and 2 double-segment substitution lines（D₁-D₂）were developed in F₃ generation by molecular marker-assisted selection（MAS）.Then,seven QTL（qPH1-1,qPH1-2,qGL1,qGL6,qGL12,qRLW1 and qGWT1）identified in F₂ generation was further valided by seven single-segment substitution lines,indicating that these QTL are stable in inheritance,In addition,12 QTL（qPH6,qPH12-1,qPH12-2,qGL12-2,qGW1,qRLW2,qRLW6,qRLW12,qGWT2,qGWT6,qGWT12-1 and qGWT12-2）can be detected by these SSSLs.Therein,8 QTL as qGL1,qGL6,qGL12,qRLW1,qPH12-1,qGW1,qRLW2,qRLW6 and qGWT12-2 are novel identified from Z688 in the study.The epistatic effect analysis showed that pyramid of long grain qGL1（a=0.14）and qGL6-1（a=0.50）yielded-0.51 of epistatic effect,according to the genetic model of DSSL,the genetic effect of D₁ is 0.13,thus,the grain length（7.59 mm）of D₁was shorter significantly than that（8.33 mm）of S₄with qGL6,however,longer than that（7.33 mm）of Nipponbare and no significant difference with that（7.61 mm）of S₁carrying qGL1.Pyramid of long grain of qGL6（a=0.27）and qGL12-1（a=0.07）produced 0.04 of epistatic effect,thus,the grain length（8.11 mm）of D₂was longer significantly than those（7.88 mm and 7.48 mm）of S₅with qGL6and S₆with qGL12-1.So identifying additive and epistatic effects of different QTLs are important to predict the breeding potential of the target QTL.4.Genetic Analysis and fine mapping of grain length of qGL1In the secondary F₃ population which was composed of 735 individual plants developed by combinant plants of qGL1,the grain length displayed a bimodal distribution.According to Chi-square test,the long grain（565）:short grain（170）conforms to 3:1 separation ratio（χ²=0.45<χ²（0.05,1）=3.84）,indicating that the long grain controlled by qGL1 in S₁ and S₂ belong to a pair of dominant gene inheritance.qGL1 was fine mapped to the 336 Kb interval of SSR₂and SSR₃ on chromosome 1 using 170recessive plants（short grains）of the F₃ population developed by combinant plants of the qGL1.In this interval,three candidate genes of qGL1 were identified by gene prediction,DNA sequencing and qRT-PCR analysis between Z688 and Nipponbare.5.Analysis of substitution fragments and important agronomic traits of Z104Z104 was developed from the recipient parent Nipponbare and the donor parent Jinhui 35through advanced backcross and molecular marker-assisted selection.It contains 19 substitution segments which were distributed on chromosomes 1,2,3,4,5,6,7,8,10,11 and 12.The estimated total length of substitution fragments is 78.15 Mb,and the estimated average length of substitution fragments is 4.11 Mb.Compared with Nipponbare,the effective panicle number,spikelets number per panicle,secondary branch number,grain length,length to width ratio and 1000-grain weight,and yield per plant of Z104 were significantly increased,while panicle length,primary branch number,grains number per panicle and seed setting rate of Z104 were significantly decreased.6.Cytological analysis of glume of Z104 and NipponbareThe grain glume of Nipponbare and Z104 at heading stage was observed by scanning electron microscope.Compared with Nipponbare,the length of cells in the inner epiderm of the glume of Z104 was significantly increased,while there were no significant differences for the width of cells in the inner epiderm of the glume and the total number of cells in the outer epidermis of the glume of Z104 with that of Nipponbare.These results showed that the grain size of Z104 was mainly due to the increase in the length and no relationship with the width of cells as well as the number of cells of glumes.7.QTL for agronomic importance carried by substitution segments of Z104A total of 12 QTLs controlling plant height,grain type and yield traits of Z104 were identified in the secondary F₂ population constructed by crossing Nipponbare and Z104,which were distributed on the substitution segments of chromosomes 1,3,6,7,8,10,11 and 12,including 1QTL for plant height,1 for panicle length,2 for effective panicles per plant,2 for secondary branches per panicle,3 for spikelets number per panicle,1 for grains number per panicle,1 for grain length and 1 for yield per plant,which explained the phenotypic variation from 2.72%to 25.91%.Among them,one major QTL qGL3 from Xihui 18 was detected,and its additive effect increased the grain length of Z104 by 0.23 mm,explaining 25.91%of the grain length variation.Among them,9QTL as qPH12,qPL11,qPN1,qPN6,qNSB11,qNSB12,qSPP10-1,qSPP10-2 and qYD7 are novel detected from Z104.8.Development of secondary segment substitution lines of target QTL from Z104On the basis of QTL mapping,in total,12 secondary segment substitution lines were developed from Z104 by molecular marker-assisted selection（MAS）.They include one five-segment substitution line（FSL₁）,three seven-segment substitution lines（SNSL₁-SNSL₃）,seven eight-segment substitution lines（ESL₁-ESL₇）and five nine-segment substitution lines（NSL₁-NSL₃）.These secondary substitution lines laid a good foundation for further developing SSSLs of these target QTLs from Z104.