| As one of the most important crops in the world,rice provides a stable food supply for more than half of the population.Heterosis refers to the superior performance of hybrids over their parents.The utilization of heterosis significantly improves rice yield,which has made indelible contributions to food security.Although heterosis has been studied over two hundred years,a comprehensive research based on genetic regulatory networks is still missing.Shanyou 63（SY63）is the most widely cultivated three-line hybrid rice in China,as well as one of the model systems for rice heterosis study.Compared with the paternal parent Minghui 63（MH63）,SY63 showed higher SP with earlier flowering,which was referred to as optimal heterosis.This study attempted to reveal the genetic and genomic basis of optimal heterosis using recombinant inbred lines（RILs）and“immortalized F2s”（IMF2s）derived from SY63.The main results are as follows:1.Compared with MH63,SY63 showed higher yield but earlier flowering.The correlation analysis in RILs and IMF2s indicated that yield highly correlated with grain traits.The whole-genome additive and dominance effects tests were performed for grains per panicle,pikelets per panicle,heading date,plant height and seed setting rate.Grains per panicle can be attributed to two subcomponents,spikelets per panicle and seed setting rate.The significant additive effect loci of grains per panicle were contributed from the two subcomponent traits,while the significant dominance effect loci were determined by spikelets per panicle.2.Using SY63 and MH63 as the contrasts respectively,IMF2s showing higher SP but earlier flowering were selected.Genotype enrichment analysis using chi-square test in these IMF2 lines detected 14 and 7 enriched loci,including Ghd7,Ghd7.1,Hd1 and Hd3a/RFT1,which indicated that these genes might be the responsible genes.3.Ghd7 was the most significant locus for SP and HD in RILs.The RILs were divided into two subpopulations according to the genotype of Ghd7,referred to Ghd7-RIL and ghd7-RIL subpopulations,and QTL analysis was performed within each of the subpopulations to identify the genetic effects.The most significant locus in ghd7-RILs was Ghd7.1,indicating that the effect of this locus was somewhat suppressed by Ghd7.While the most significant locus in Ghd7-RILs was Hd3a/RFT1,demonstrating that Hd3a/RFT1-ZS97 interacted with Ghd7 and delayed flowering.Two NILs MH63（bin643-ZS97/ZS97）and MH63（bin1367-ZS97/ZS97）showed consistent genetic effects with RILs and IMF2s,suggested that the digenic interactions were reliable,although many of them may only had minor effects.The IMF2s were also divided into two subpopulations（Ghd7-IMF2s and ghd7-IMF2s）according to the genotype of Ghd7.Three and ten QTLs were detected for SP in two subpopulations,and four and eleven QTLs were detected for HD.4.Sequence analysis revealed that MH63 carried functional allele of Ghd7,Ghd7.1and Hd3a/RFT1-MH63,and nonfunctional allele of Hd1,while ZS97 possessed functional allele of Hd1,weak allele of Hd3a/RFT1-ZS97 and nonfunctional allele of ghd7 and ghd7.1.The phenotypic effects of the various combinations of these loci on SP and HD were further evaluated.Ghd7,Ghd7.1 and Hd3a/RFT1-ZS97 generally increase SP and delay flowering.The genetic effects of Hd1 depended on the existence of Ghd7and Ghd7.1:it delayed HD and increased SP when functional Ghd7 and Ghd7.1 were existed,while promoted flowering and decreased SP when Ghd7 and Ghd7.1 were absent.The genetic effects of each locus with heterozygous genotypes were evaluated by fixing the genotypes of the other three loci in IMF2s.In the background where Hd1 and Hd3a/RFT1 were heterozygous,IMF2s heterozygous for Ghd7 or Ghd7.1（or both）exhibited positive dominance effects（from incomplete dominance to overdominance）on both SP and HD,significantly increasing SP and delaying flowering relative to the mid-parent values.While in the double heterozygote of Ghd7 and Ghd7.1,Hd1 and Hd3a/RFT1 showed negative dominance effects on HD but opposite effects for SP in two years.The overall effects of heterozygous Hd1 and Hd3a/RFT1 were a decrease in HD in the hybrid with slightly fluctuating SP.5.We also searched the entire genome for pairwise interactions between loci by sequentially divided the RILs into two subpopulations according to the genotypes at each of the 1,628 bins.A total of 165 significant digenic interactions for SP and 101interactions for HD were detected in the whole genome.Among these interactions,Ghd8-MH63 allele could interacted with Ghd7 and delayed flowering.Relative to the functional Ghd8-Nip allele,the Ghd8-MH63 allele has a 1-bp deletion in the coding region,causing a frameshift and thus premature termination of the predicted protein.We inferred that the crippled Ghd8-MH63 might be able to promote flowering by partially blocking Ghd7with physical interaction.6.The Ghd7.1-MH63,Hd1-ZS97,Hd3a-ZS97,and RFT1-MH63 behaved as the predominant alleles in the wild rice.The Ghd7-MH63 allele was mostly preserved in Ind II and Ind III subgroups,while Ghd7.1-MH63 was retained and almost fixed in all cultivated rice groups.Hd1-ZS97 allele existed in all cultivated rice groups and was favored in the japonica subspecies and the Aus,Ind I and Ind III subgroups of indica subspecies.As for hybrid rice,a high frequency of MH63-type alleles of Ghd7,Ghd7.1,Hd1-MH63,Hd3a-MH63,and RFT1-MH63 were observed in the male parents（restoration lines）of three-line hybrid rice,and a considerable proportion of the ZS97-type alleles of ghd7,ghd7.1,Hd1-ZS97,Hd3a-ZS97,and RFT1-ZS97 were detected in the female parents（cytoplasmic male sterile lines）.In this study,we reanalyzed our data from RILs and IMF2s,incorporating the latest genomic information and functional genomics knowledge.The analysis showed that genetic interactions among the four major QTLs（Ghd7,Ghd7.1,Hd1,and Hd3a/RFT1）could provide an approximation to the explanation of the optimal heterosis of increased spikelet per panicle（SP）and early heading date（HD）in hybrid. |
