| Rice plays a central role in safeguarding the security of global food supplies and providing the staple food for most of the human population.With the research of molecular biology on genes related to agronomic traits of rice,more and more genes related to yield traits have been discovered.Modification of these genes with appropriate combinations is an effective way to obtain ideal highyielding new rice varieties.However,yield-related genes have pleiotropic effects on plant development in addition to their effects in regulating yield because of the genetic diversity among rice varieties.Therefore,in molecular design breeding,it is necessary to carefully analyze the gene function of a specific trait-improving gene in different varieties.The miR396-GRFs regulatory network plays an important role in the growth and development of rice.Previous studies have shown that miR396ef negatively regulates the expression of downstream genes OsGRF4/6/8 in rice,and increased expression of OsGRF4/6/8 confers larger grain size and panicles,respectively.Therefore,fully analyzing the functions of MIR396ef under different cultivated rice varieties is of great significance for future sustainable food security and the new green revolution.In this study,we selected eight high-quality japonica rice cultivars as genetic backgrounds to obtain the loss-of-function mutants of MIR396ef using gene editing technology.During the maturity period,yield tests were conducted on the mir396ef mutant,which fully assessed the differences in agronomic traits,grain quality,nitrogen use efficiency and root microbiota.The main research contents in this study were described as following:(1)CRISPR/Cas9 system is an efficient gene editing tool among japonica subspecies,with the highest frequency of homozygous mutation type 1 bp insertions,preference for insertion of T bases,and significant differences in mutation efficiency a under different genetic backgrounds.(2)mir396ef-HD5,mir396ef-NG9108,mir396ef-WYG27,mir396ef-XD3 and mir396ef-NG4 yield increased by 11.7%,10.3%,32.7%,4.7%and 6.1%,respectively.However,mir396efWYG29148,mir396ef-NG3 and mir396ef-ZD88 was decreased by 4.7%,12.0%,and 15.7%in yields,respectively.(3)The mir396ef mutant significantly increased flag leaf length,spike length,grain length,grain width and thousand grain weight in all cultivars,with an average increase of 19.0%,22.6%,17.6%,11.3%and 20.5%,respectively,indicating that miR396ef has a conserved function among j aponica rice subspecies.Plant height,culm strength,grain number per panicles and seed setting rate varied with variety.The decrease in the grain number per panicles or seed setting rate was the cause of the yield reduction.(4)The expression of OsGRF4,OsNIR2,OsAMT1.1,OsGS1.2 and OsNADH-GOGAT2 in roots and shoot was analyzed using qRT-PCR.The results showed that nitrogen-related gene expression was significantly up-regulated overall in the five yield-increasing mir396ef mutants,conferring higher nitrogen uptake and utilization to meet the nutritional requirements needed for larger grains.(5)mir396ef mutant did not change the amylose content and gel consistency.However,the percentage and degree of chalkiness were altered with genetic background.(6)The mir396ef-HD5 mutant enriches more Nitrospirae,mir396ef-NG3 enriched more Gemmatimonadota and Latescibacterota.In summary,using CRISPR/Cas9 technology to simultaneously knock out MIR396e and f in eight japonica rice cultivars,successfully obtained MIR396ef mutant with loss of function.The functional differences of MIR396efin agronomic traits,yield,grain quality,nitrogen use efficiency and root microbiota under multiple genetic backgrounds were fully evaluated.The study fully analyzed the functions of miR396-GRFs regulatory network,and provided breeding strategies for future sustainable food security and the new green revolution. |
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