| After evolution and domestication,Asian cultivars of rice(Oryza sativa L)have shown high adaptibility to diverse ecological regions and cropping seasons.Heading date(HD)is the key to rice regional adaptibility and has great impacts on rice grain yield.Due to the phenotype variation of HD,rice cultivars distribute world-wide,but a proper flowering performance could enable the completion of the normal life cycle as well as the optimization of yield in a given planting region.During the breeding process via marker-assisted selection(MAS),negative traits,like flowering delay,could be introduced together with the aim trait.Flowering delay would decrease the adaptibility of rice varieties,then threaten the food security.After the twice “leap”,rice has achieved a high yield production.The utilization of inter-subspecies heterosis would be the improtant way to increas the rice yiled production.However,“late flowering” and “hybrid sterity” are the twao main problems for indica-japonica hybrid breeding.Using wide compatible genes like S5-n,could improve the hybrid fertility.In order to reveal the genetic mechanism of rice regional adaptibity determined by HD,then targeted improve the haeding date for rice breeding,and utilize the inter-subspecies heterosis,we started the related research on rice via re-sequencing,gene diagnosis and gene editing.The main results are as follows:1.We conducted haplotype(hap)analysis by re-sequening the coding sequences of major flowering genes Ghd7,Ghd8,and Hd1 in a rice core permplasm of 328 cultivars,and 49 wild rice accessions.In total,163 varieties were used for analysis and 13 combinations were detected.Generally,cultivars with different combinations showed different phenotypes of HD and grain yeild per plant(YD)when grown in Wuhan under long-day conditions(LD).WWN was the dominant one in japonica,and cultivars with WWN headed at 84.3 d and produced YD of 18.7 g.SNN was dominant in indica,and cultivars with SNN showed the HD of 84.8 d and YD of 25.9g on avearge.Plants with SSN were associated with the HD of 95.2 d and the highest YD of 30.7 g per plant.While cultivars with SSF headed latest at 115.4 d and produced a lower YD of 12.2 g.Different combinations' ecogeographic distribution showed their critical role in the adaptability and diverse ecotypes(rice cropping seasons).In South and Central China,NNF was mainly used as early rice under a double-season cropping system,while SNN,SNF,SWN and SSN were found in most single-season crooping cultivars.WWN plants had a wide distribution,and acted as single-season rice in Northeast China.SSF was only appeared in low-latitude regions due to it's highest sensibility to photoperiod.The YD performances in Nanning,Guangxi(subtropical area)and Lingshui,Hainan(tropical area)showed that SSF was the superior combination in tropical area for more YD.2.Gene diagnosis of major flowering genes in Kongyu 131(KY131)showed that the combination of KY131 was Hd1NHd2NHd4NHd5WHd6NHd16 F,only with functional alleles of Hd5/Ghd8 and Hd16/EL1.We chose Ghd8 as the target gene for targeted improvement of the heading date in KY131 introgressed Pi1(KP1)via gene editing.We then constructed the CRISPR-Cas9 vector for Ghd8 with multiple target sites,and obtained mutants with a frequency of 91.7% when transformed into KP1 callus.The frequency of mutants contained large deletionas(? 100 bp)was 63.6%.As expected,the ghd8 mutant lines headed later than the control KP1 under SD.When planted to Harbin,Heilongjiang under LD,all the “transgene clean” lines headed earlier than the control KP1,even than KY131.Theses results indicated that targeted improvement of heading performance could be achieved via gene editing.Evaluation of blast resistance in natural field showed that the heading date improved lines still possessed the resistance to rice blast as KP1.3.Via gene editing,we developed mutants targeted S5-i in indica background.The results showed that wide compatible S5-n alleles could be induced by destruction of SP coding sequence in S5-i via gene editing based on multiple targets.The spikelet fertility(SF)comparision between the experimental(F1 of S5-n line with japonica)and control group(F1 of hd1 NIL with japonica)showed that SFs of experimental groups were much higher than that of control groups.Among all the F1 s,hybrids from crosses with Wuyugeng 7 showed the highest SFs both in the experimental and control group,the SF inceased up to 80.9% from 46.3% with an increase of 34.6% when sowed on May 9th.All these results indicated that SF in indica-japonica hybrid could be markly increased by using S5-n allele induced by gene editing,which supplied with a new strategy as well as genetic resources for utilization of inter-subspecies heterosis. |
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