Fine-mapping And Candidate Gene Analysis Of QTL QTGW10-20.8,A QTL For Thousand Grain Weight In Rice

Thousand grain weight（TGW）is one of the most important determinants of grian yield in rice.It is closely related to grain length（GL）and grain width（GW）that are also crucial traits for grain quality.Investigation of the genetic basis of grain weight variation among parental lines of hybrid rice would facilitate not only the utilization of beneficial alleles in rice breeding,but also the identification of more key regulators controlling this important trait.In previous study,three recombinant inbred line（RIL）populations,Teqing/IRBB lines（TI）,Zhenshan 97/Milyang 46（ZM）and Xieqingzao/Milyang 46（XM）,had been constructed.In present study,firstly,the three RIL populations were used to identify QTL for TGW that were commonly segregated in different populations and located apart from the QTL cloned.Finally,three QTL were separated in a 4.2 Mb region on chromosome 10 and one of them was fine-mapped into a 70.7 kb region.The main results are as follows:1.QTL analysis in three RIL populationsA total of 27 QTL for TGW were identified using the MET functionality of QTL IciMapping.Seventeen of them were found in two or all the three populations,and the remaining ten were identified in one population.Eight of the 17 QTL commonly segregated in different populations were located in regions where QTL for grain weight have been cloned.Among the remaining nine QTL,qTGW10.1 and qTGW10.2 were two closely-linked QTL.The qTGW10.1 commonly segregated in ZM and XM populations.The qTGW10.2 showed the highest LOD,proportion of phenotypic variance（R²）and additive effect in the TI and ZM populations.Therefore,these two QTL were taken for validation.2.Dissection of three QTL for grain size on chromosome 10One F_9:10 family was constructed for validation of qTGW10.1.This population segregated in four regions,including one each on chromosomes 1,9,10 and 11,respectively.A total of seven QTL for grain size were identified.In qTGW10.1 region,only one QTL only for GL was identified,explaining 9.00%of the phenotypic variance.Since this QTL mainly regulated GL,it was named as qGL10.The IRBB52 allele increased GL by 0.020 mm.One near isogenic lines（NIL）-F₂ and five F_9:10 NIL populations were constructed for validation and dissection of qTGW10.2.Two QTL mainly regulating TGW were dissected,namely,qTGW10.2a and qTGW10.2b,respectively.The additive effects for TGW,GL and GW estimated from the two NIL populations segregating in qTGW10.2a region were similar,with the TQ allele increasing TGW by 0.66 and 0.59 g,GL by 0.110 and 0.098 mm,and GW by 0.019 and 0.016 mm,respectively.The additive effects for TGW and GW of qTGW10.2b were much smaller than that of qTGW10.2a.In the two NIL populations which only segregated for qTGW10.2b,the TQ allele increased TGW by 0.13 and 0.12 g,and GW by 0.012 and0.011 mm,respectively.Finally,three linked QTL,qGL10,qTGW10.2a and qTGW10.2b,were dissected in a 4.2 Mb region.These results provide a new evidence to explore the genetic regulation for a quantitative trait,that is,multiple QTL controlling the same trait could be closely linked in a chromosomal region.3.Fine-mapping and candidate gene analysis of qTGW10.2aFive F_11:12 NIL populations with sequential heterozygous segments covering the interval of RM25766–RM5352 were constructed for fine-mapping of qTGW10.2a.The region of qTGW10.2a was narrowed down to a 70.7 kb interval flanked by Te20881 and Te20882.Because qTGW10.2a mainly regulated TGW and the segregating marker Te20811 was located at 20.8 Mb on rice chromosome 10,we renamed it as qTGW10-20.8.Then,one F_12:13 NIL population only segregated in the qTGW10-20.8 region was constructed for validation of the genetic effects on TGW,GL,GW and heading date（HD）,respectively.The additive effects were similar for the NIL populations planted at Lingshui and Hangzhou,with the TQ allele increased TGW by 0.54 and 0.52 g,GL by0.055 and 0.078 mm,GW by 0.016 and 0.021 mm,and promoted HD by 1.9 and 1.2 d,respectively.Sequence and gene function prediction analysis suggested that ORF7encoding the MIKC-type MADS-box protein OsMADS56 is very probably candidate for qTGW10-20.8.As compared to NIL^IRBB52,the transcript level of ORF7 was higher in the leaf and young panicle of NIL^TQ.Then,the 8 cm young panicle was taken for RNA sequencing.A total of 556 differentially expressed genes between NIL^TQ and NIL^IRBB52were identified.As compared to NIL^IRBB52,the numbers of differentially expressed genes up-and down-regulated in NIL^TQ were 272 and 284,respectively.The GO term and KEGG pathway enrichment suggested that qTGW10-20.8 might regulate grain weight through the plant hormone signal pathway.These results lay a foundation for the molecular mechanism of qTGW10-20.8,and provide a new gene resource for rice breeding and an important direction for understanding of the regulation mechanism of seed development.