Exploration Of Heterosis Loci For Yield Components Using CSSLs And Functional Analysis Of The Spermine Synthase Gene OsSPMS1 In Rice

Rice is one of the most important global foodstuffs,providing food and nutrition to more than half of the world's population.Improving of rice grain yield has always been one of the most important goals for breeders.There have been two breakthroughs in the history in high-yield rice breeding in China.The first one is the utilization of sdl,reducing plant height and increasing grain yield in 1960s.The second is the utilization of cytoplasmic male sterility,promoting the development of hybrid rice in 1970s.Despite the great success achieved by the exploitation of heterosis in rice,the genetic basis of heterosis is still not well understood.In recent years,more modern technologies,provided by molecular biology,have been added to traditional breeding process in rice.With the development of CRISPR/Cas9,grain yield could be improved by editing the target genes,which regulate yield and yield components in rice.In this study,two parts of reasearch work had been carried out,and the main results are as follows.Part 1 Exploration of heterosis loci for yield components using CSSLs in rice128 chromosome segment substitution lines(CSSLs),derived from a cross between indica rice(Oryza sativa L.)inbred 9311 and japonica rice inbred Nipponbare,had been constructed to investigate the genetic mechanism of heterosis.Three photo-thermo-sensitive-genic male sterile lines(PTGMS,Guangzhan 63-4s,036s and Lian99s)were selected to cross with each CSSLs to produce four testcross populations(TC 1,Guangzhan 63-4s×CSSLs in 2009;TC 2,Guangzhan 63-4s×CSSLs in 2011;TC 3,036s×CSSLs in 2015;TC 4,Lian99s×CSSLs in 2015).The main results obtained are as follows:1.The means of most traits,except for seed setting rate(SSR)and thousand-grain weight(TGW),in the TC 1 population were higher than the corresponding values in the CSSLs.In addition,the mean values of panicle length(PL),panicle per plant(PPP),and grain yield per plant(GYPP)in TC 2 were higher than those in the CSSLs in 2011.Only PL and PPP showed better performance in the TC 3 and TC 4 populations than in the CSSLs.The mean values of spikelet per panicle(SPP)and GYPP in the CSSLs were higher than those in the TC 3 population,and lower than those in the TC 4 population.These results suggested that different PTGMS may have different combination abilities and that Guangzhan 63-4s and Lian 99s have higher general combination abilities than 036s for yield production.2.Except for PPP and SSR,the other five traits were significantly correlated between the TCs and CSSLs.That is,the better parents,the better hybrids.3.Based on the bin-maps of CSSLs,we totally detected 62 quantitative trait loci(QTLs)for yield and yield factors and 97 heterotic loci(HLs).They were all identified in certain intervals,and some of these loci were located together as a cluster.4.The result of heritability analysis indicated that plant height(PH),PL,GYPP,and TGW showed high heritability(>50%).This data indicated that genetic factors played dominant roles in these four traits.The lowest broad sense heritability was for PPP(21.47%),suggesting that it could be strongly affected by environmental factors.Additionally,PH,PL,SSR,TGW and GYPP showed partial dominance and SSR showed overdominance.Part 2 Functional analysis of OsSPMSl,a spermine synthase-encoding gene in ricePolyamines are a class of low molecular weight aliphatic cations with strong biological activities found in a wide variety of prokaryotic and eukaryotic organisms.The polyamines in higher plants mainly include putrescine(Put),spermidine(Spd),and spermine(Spm).In this study,we characterized the a spermine synthase-encoding gene,OsSPMSl(LOC_Os06g33710).Two homozygous trangenic RNAi lines(RNAil and RNAi2)and two overexprssion lines(OE1 and OE2)were generated and used to investigate the roles of OsSPMSl in rice plant architecture and grain yield.1.Bioinformatics analysis showed that OsSPMS1 encodes a spermine synthase with a Spermine_synth domain.Its homologs are found widely in green plants,from green algae to angiosperms.2.Quantitative real-time PCR(qRT-PCR)analysis indicated that OsSPMS1 constitutively expressed in all examined tissues,including leaf,node,sheath,stem,and panicle.The highest transcript level was detected in leaf,and the lowest was detected in panicle.We also compared the OsSPMS1 expression levels at different stages of panicle development,and found that OsSPMSl expression was highly variable and peaked at the middle stage of panicle elongation.These results were further confirmed by GUS staining.To investigate the subcellular localization of OsSPMSl,its coding sequence was fused with green fluorescent protein(GFP)to generate a fusion construct,which was then transformed into rice protoplast and tobacco epidermal cells.Fluorescence signal showed that OsSPMS1 was located in both nucleus and cytoplasm.3.Several yield traits of the transgenic lines were measured at mature stage in 2016 and 2017.The OE lines showed depressed plant height,decreased grain weight,lessened grain number and substantial reduced grain yield per plant.By contrast,down-regulation of OsSPMS1 had positive effects on multiple yield traits,and the grain yield per plant of RNAi lines increased by 16.47%and 19.54%in 2016,and by 16.84%and 21.16%in 2017,respectively.These results suggest that OsSPMS1 acts as a negative regulator for grain yield and yield components.4.Histomorphological analysis indicated that OsSPMS1 negatively modulates cell size in stems.Scanning electronic microscopy analysis suggested that the grain size of transgnic lines were mainly determined by cell length of spikelet hulls.We can conclude that OsSPMSl negatively regulates rice organ size by cell expansion.5.We measured the endogenous poly amine contents of wild-type(WT),RNAi and OE transgenic lines,and found that down-regulation of OsSPMS1 lead to increased Spd and reduced Spm,while evevated expression of OsSPMS1 over-consumed the Spd but had litter effects on Spm accumulation.We demonstratesd that OsSPMS1 is a spermine synthase that plays roles in the synthesis of Spm.Additonally,we found that OsSPMS1 interacts with OsSPMS2 in yeast two-hybrid system.6.Overexpression of OsSPMS1 inhibited seed germination,but the application of exogenous Spd failed to restore its germination rate.However,exogenous ethylene precursors SAM and ACC could significantly restore the germination rate of OE seeds.We further measure the contents of ACC and ethylene in transgenic lines and found that both of them were increased in RNAi lines but decreased in OE lines.Taken together,we proposed a working model of OsSPMS1 regulating plant architecture and grain yield in rice.7.Two homozygous mutants of OsSPMS1 in SK118(Suken 118,a high-yield variety released in 2016)background were obtained using a CRISPR/Cas9 system.Both of them showed increased plant height and grain weight,which were similar to those of RNAi lines in Nipponbare background.The grain yield per plant of the osspmsl mutants increased by 14.39%and 13.08%,respectively,suggesting that OsSPMS1 is a key target site for rice high-yield breeding.