Map-based Cloning And Functional Analysis Of QHD5,a QTL Controlling Heading Date In Rice(Oryza Sativa L.)

Heading date(HD)is an important ecological trait that determines growing seasons and regional adaptability of plants to specific natural environments.HD also has effects on yield.In this study,we identified a major QTL controlling HD under both long-day and short-day condition and named it as qHD5.Firstly,we used map-based cloning method to clone qHD5.Then we used CRISPR/Cas9 and genetic complementation method to validate the function of candidate gene.Finally,we analyzed the function of qHD5.The main results are as follows:1.Fine-mapping of qHD5We have identified a major-effect locus named qHD5 using a RIL population derived from a cross between BigGrain1(BG1)and XiaoLiJing(XLJ).To study the genetic effect of qHD5 on heading date,a near-isogenic line(NIL)carrying a segment containing qHD5 of BG1 in XLJ background was developed.NIL(BG1)and NIL(XLJ)have a minimum of 10-day difference in HD under both long-day and short-day conditions in rice.NIL(BG1)headed earlier than NIL(XLJ)under all conditions.Genetic analysis indicated that qHD5 behaves as a semi-dominant Mendelian factor.Using 2,200early-heading individuals and newly developed InDel markers,we narrowed qHD5 down to a 52.59kb genomic region.Five putative ORFs were predicted in this region.Of these,ORF5(LOC_Os05g03040)is predicted to encode an AP2-like transcription factor.Sequencing resultsshowed that Os05g03040 has some difference between NIL(BG1)and NIL(XLJ).There were a 60-bp insert/deletion and six SNPs in the coding region between the two NILs.We regarded Os05g03040 as the casual gene of qHD5.2.Cloning and functional analysis of qHD5CRISPR/Cas9 and genetic complementation method validated the function of candidate gene.We regard Os05g03040 as the causal gene for qHD5.Under the XLJ background,qHD5~BG1G1 allele can promote HD and qHD5~XLJLJ allele can delay HD.Transient expression of qHD5-XLJ-GFP fusion protein was exclusively colocalized with a nuclear marker OsMADS3-mCherry in rice leaf protoplasts.Transactivation activity assays showed that qHD5 had a strong transcriptional activation activity in yeast.Deletion analysis showed that neither the two AP2 domains nor the middle region between them was essential for its transactivation activity.These results support a role of qHD5 in transcriptional regulation of gene expression in the nucleus.We examined its expression levels of qHD5 in various NIL(XLJ)tissues collected from plants grown in CLD conditions by RT-qPCR.qHD5 transcripts were detected in all tissues examined,but its expression was most abundant in leaf blade.The expression of qHD5 is not controlled by single light or dark but controlled by regular light and dark change.We compared the expression of several photoperiod-related genes in NIL(BG1)and NIL(XLJ)plants under CLD and CSD conditions.We found that the mRNA levels of Hd3a,RFT1,Ehd1,Hd1,and Ghd7were different in NILs.Together with the data in RNA-seq,we conclude that qHD5 controls heading date through Ehd1 pathway.We grow NIL(BG1)and NIL(XLJ)in Hangzhou natural long-day condition at four different time.The results suggested that the HD difference between the NIL(BG1)and NIL(XLJ)was not influenced by the light length and the temperature change.Moreover,there were significant difference between NIL(BG1)and NIL(XLJ)in yield-related traits.We collected 105 rice core germplasm and sequenced the qHD5 in those materials.The results showed that the 6 SNPs can also be detected.And the 105germplasm can be divided into 9 haplotypes.80%core germplasm belongs to Haplotype 1 and Haplotype 4.Evolutionary origin analysis results showed that qHD5 has obvious differentiation phenomenon between Indica and Japonica.