Fine-Mapping Of Heading-Date QTL QHD7b And Functional Analysis Of OsMTS1 Regulating Heading In Rice

Heading date(HD)is an agronomic trait influencing maturity,regional adaptability,and grain yield.In this study,we identified a major QTL controlling HD under both long-and short-day conditions and named it qHD7b.Firstly,we used a map-based cloning method to clone qHD7b.Then we used CRISPR/Cas9 method to validate the function of the candidate gene and also performed some function analysis of qHD7b.In addition,we studied the function analysis of OsMTS1 by constructing CRISPR/Cas9 knockouts and overexpression transgenic plants in Indica and Japonica rice.Finally,we analyzed the function of OsMTS1.The main results are as follows:A set of high throughput genotyped chromosome segment substitution lines(CSSLs)comprises of 75 lines derived from the cross between Zhonghui9308(ZH9308,late HD)and XieqingzaoB(XQZB,early HD)were used to identify QTL for heading date under long-day and short-day conditions.A total of fourteen QTLs named qHD2a,qHD4a,qHD4b,qHD5a,qHD6a,qHD6b,qHD7b,qHD7c,qHD8a,qHD10a,qHD10b,qHDlla,qHD12a,and qHD12b were identified from BC4F6 between 134 RILs and recurrent parent ZH9308.However,only qHD6a and qHD7b were consistently detected in all four environments.The phenotypic variance explained by qHD6a and qHD7b varied from 10.1%to 36.1%(mean 23.1%)and 8.1%to 32.8%(mean 20.5%),respectively.Therefore,qHD7b was selected for fine mapping,which may be used in molecular marker-assisted breeding.One of the CSSL lines(CSSL52),which harbored a segment from the early heading XieqingzaoB(XQZB)parent at the qHD7b locus in the genetic background of ZH9308 was selected as starting material to validate,fine map,and evaluate the effect of qHD7b on heading date trait under long-day and short-day conditions.CSSL52 showed a significantly earlier heading date than ZH9308 under longand short-day conditions.To confirm the existence of QTL qHD7b,a secondary F2(BC5F2)population obtained from the cross of CSSL52 and genetic background parent ZH9308 was used.qHD7b was mapped between the markers InD4373 and InDe13 with an interval of 1,245.9-kb on chromosome 7 using the F2(BC5F2)population.Using a backcross population evaluated for four seasons under different day lengths and temperatures,Then BC5F2:3 population was used for fine mapping.The qHD7b interval was delimited to a 912.7-kb region,which is located between RM5436 and RM5499.These results contribute to the broader adaptability of marker-assisted breeding to develop high-yield rice varieties.Twenty-nine putative candidate genes were annotated in the delimited region,of which Os07g0261200 had 5.984-kb deletion between the two parents.We regarded Os07g0261200 as the candidate gene of qHD7b.Expression analysis suggests that Os07g0261200,which encodes CCT(CO,CO-LIKE,and TIMING OF CAB1)domain protein,is a positive regulator of heading date and is the most likely candidate gene for qHD7b.CRISPR/Cas9 gene editing was used to validate the function of the candidate gene.We regarded Os07g0261200 as the candidate gene of qHD7b.The ghd7 mutant generated through CRISPR/Cas9 gene editing exhibited early heading under long-day and short-day conditions.We also identified Os10g30860 and Os12g08260 as candidate partner proteins of Ghd7 from Y2H library screening.O-Methyltransferase 1(OsMTS1)encoding a methyltransferase functions as a suppressor of heading date in Zhonghui8015(ZH8015)and Nipponbare(NIP)rice under both under long-day and short-day conditions.Here,we constructed knockouts and overexpression transgenic plants of OsMTS1 in ZH8015 and NIP for the first time to validate its function in rice subspecies Oryza sativa ssp.Indica and O.Sativa ssp.Japonica for heading date.The loss-of-function of Methyltransferase 1(MTS1)affects melatonin biosynthesis,promoting rice flowering time.OsMTS1 represses the heading date in ZH8015 and NIP,with a stronger effect in ZH8015 than NIP and more in long-day than short-day conditions.The overexpression of OsMTS1 lines delayed the heading date in both ZH8015 and NIP and more in long-day than short-day conditions.OsMTS1 represses the heading date through up-regulating Heading date 1(Hd1)and down-regulating Early heading date 1(Ehd1),and the florigen gene Heading date 3a(Hd3a).The OsMTS1 protein interacted with OsHCT1 proteins using a yeast two-hybrid(Y2H)assay.Overexpression of OsHCT1 headed 4.7 days later under long-day.Taken together,CRISPR/Cas9,genetic complementation,and overexpression results validated that OsMTS1 represses heading in rice under both long-and short-day conditions.Collectively,our qHD7b results contribute to the broader adaptability of marker-assisted breeding to develop high-yield rice varieties.In addition,OsMTS1 is a valuable target for breeding early maturing rice varieties via CRISPR/Cas9 gene editing of the functional allele.