Cloning And Functional Analysis Of SNG1,A Gene For Grain Size And Dissecting The Genetic Bases Of Appearance Quality Of Rice Grains

Rice is one of the most important food crops worldwide.High yield and good grain quality are among the most important goals in rice basic research and breeding.Appearance quality of the grain is a major problem of rice production around the world.It is mostly determined by grain shape and the translucency of the endosperm.Grain shape/size as specified by grain length,grain width,the length-width ratio and grain thickniss,is a major determinant of 1,000-grain weight,one of the three main components of grain yield.In addition,rice grain size is also a highly important quality trait,determining grain appearance quality and affecting grain milling,cooking and eating qualities.Grain chalkiness is a highly undesirable quality trait.It negatively affects the milling quality,decreases milled rice yield and market value of rice grain.In recent years,about a dozen of QTL genes for rice grain size have been isolated using map-based cloning approach.However,our understanding of the molecular regulation mechanism of grain size is far from enough.And only one QTL,Chalk5,controlling grain chalkiness has been cloned.Here we have successfully cloned a gene,SNG1,positively controlling grain size and weight.SNG1 has an important role in controlling sugar metabolism and gibberellin biosynthesis.We dissect the genetic bases of appearance qualiy of rice grains in a RIL population and validated the genetic effects of six minor QTLs for grain size or chalkiness in NIL-F₂ populations.The main results of our study are as fellows:1.We isolated a small grain mutant sng1（short and narrow grain 1）from T-DNA insertion library.However,the T-DNA flanking sequences could not be obtained.Genetic analysis showed that the small grain mutation was caused by a single locus.We tried to clone the gene using a map-based cloning approach.An F₂ population derived from the mutant and Zhenshan 97（an indica cultivar）was developed for preliminary mapping.And a QTL controlling grain length and grain width located at the end of chromosome 1was identified with bulked segregant analysis approach.The QTL was named as SNG1.NIL of SNG1 was constructed by backcrossing with the recurrent parent,Zhenshan 97.A BC₃F₂ population of 192 individuals was used to confirm the effect of SNG1.Linkage analysis showed the QTL could explain 64.8%,52.6%and 74.4%of the phenotypic variation of grain length,grain width and 1000-grain weight,respectively.In the BC₃F₂population,13 were recombinant plants.By combining the grain phenotypes of the progeny and marker genotypes for each recombinant,SNG1 was narrowed to a 480 kb region.To fine map the SNG1 locus,a large BC₃F₃ population of 7680 individuals was used to identify recombinants.Finaly,SNG1 was fine-mapped to a 5.5 kb region.Two open reading frames,LOC＿Os01g71310 and LOC＿Os01g71320,were present in this region.Sequencing of the entire region revealed only one SNP（T-A）between the sng1mutant and HW.The SNP locates in the last exon of LOC＿Os01g71310,changing an arginine codon（AGA）in HW to a termination codon（TGA）in sng1.This premature termination resulted in a 103 amino acids truncation in the C-terminus of the predicted protein in sng1.And genotype of the SNP in Zhenshan 97 was the same as HW.LOC＿Os01g71310 encodes a hexokinase.2.To confirm that LOC＿Os01g71310 is SNG1.We complement sng1 with HW SNG1c DNA driven by its native promoter.Transgene-positive plants in T₀ progeny could rescue the grain size,while transgene-negative ones could not.And cosegregation testing in T₁lines showed that the genotype correlated to grain length,grain width and 1000-grain weight.We over-expressed SNG1 in HW,and found that there was a remarkable overall increase in grain size and weight in the transgene-positive plants compared with the transgene-negative plants.And cosegregation testing in T₁ lines also showed that the genotype correlated to the phenotypes.Two additional knockout mutants（sng1c-1 and sng1c-2）were generated by CRISPR-Cas9.Their grain length,grain width and1000-grain weight were all significantly decreased.Taken together,these results confirmed that SNG1 positively regulate grain length,grain width and 1000-grain weight.3.SNG1 showed constitutive temporal and spatial expression patterns by using real-time PCR and examining the expression ofβ-glucuronidase（GUS）driven by the SNG1 promoter in transgenic plants.And the transcript was more abundant in young organs and connecting tissues.We transiently co-express SNG1 fused to yellow fluorescent protein（YFP）with mitochondrial marker in rice protoplasts,and showed that SNG1 was located on the mitochondrial outer membrane.4.To determine if SNG1 and the mutant alleles（sng1,sng1c-1 and sng1c-2）encode catalytic active enzyme,individual c DNA clones were transformed into the triple mutant YSH7.4-3C yeast（hxk1,hxk2,glk1）,which lacks endogenous HXK activity.Only the yeast strain transformed with wild type SNG1 grew on the selective medium that contained glucose as the sole carbon source.These findings demonstrate that SNG1 could phosphorylate glucose,while all the mutant alleles were loss of function.Compared with HW,glucose phosphorylation capacity of sng1,sng1c-1 and sng1c-2 in 8-cm young panicles were significantly decreased（-21.3%,-16.6%and-20.1%,respectively）,and glucose phosphorylation activity in two over-expression lines were significantly increased.These results indicated that SNG1 is a functional hexokinase and that other endogenous enzymes may also have important roles in glucose phosphorylation.5.To examine the functions of SNG1 in sugar sensing and signaling,we transformed SNG1 and sng1 into the Arabidopsis gin2-1 mutant,using the 35S promoter.After getting homozygous lines of transgenic plants with relatively high transgene expression levels,we sowed the progeny of selected transgenic plants onto 6%glucose-containing 1/2 MS media.Neither SNG1 nor sng1 could rescue the glucose-sensitive response in gin2-1mutant,suggesting that SNG1 did not function in sugar signaling.6.Scanning electron microscope analysis of the inner and outer surfaces of the middle part of lemma in HW and sng1 mutant showed that cell length of the inner and outer surfaces of sng1 mutant decreased by 9.8%and 10.0%,respectively.And cell width of the inner and outer surfaces of sng1 mutant decreased by 9.0%and 15.0%,respectively.We also detected the expression of 25 genes involved in the cell cycle,found that these genes were not down-regulated in 8-cm young panicles of sng1,suggesting that decreased seed size in sng1 might not result from cell division repression.Taken together,these data indicate that SNG1 regulates grain size by promoting cell expansion.7.sng1,sng1c-1 and sng1c-2 mutants all showed some gibberellin deficient or insensitive phenotypes,such as retarded seed germination and seedling growth,small and dark green flag leaves.We tested expression levels of genes involved in GA biosynthesis and signal transduction in 8-cm young panicles of HW and sng1 with real-time PCR,and found that the expression level of Os GA3ox2 was 8-fold higher in sng1 than that in HW,whereas expression level of Os GA2ox1 was 10-fold lower in sng1 than that in HW.And expression levels of genes involved in GA signal transduction（GID1,GID2 and SLR1）showed no differences between HW and sng1.We then measured GA concentrations in8-cm young panicles of HW,sng1,sng1c-1 and sng1c-2,found that concentrations of biologically active GAs（GA₁,GA₃ and GA₄）were all significantly decreased in the three mutants,and concentrations of upstream precursor of biologically active GAs(GA₁₂ and GA₅₃)were also significantly decreased.We analyzed the second leaf sheath length in response to exogenous GA₃ treatment.The mutants could respond to exogenous GA₃just like HW,and GA₃ could rescue the dwarf phenotype.These data indicate that GAs levels were reduced in the mutants.Up-regulation of Os GA3ox2 and down-regulation of Os GA2ox1 in 8-cm young panicle of sng1 should be homeostatic regulation mechanism tried to balance the levels of bioactive GAs in plants.8.Compared with HW,some other Os HXK genes like Os HXK1,Os HXK6,Os HXK8and Os HXK10,were up-regulated in 8-cm young panicless of sng1.And several genes involved in sugar transport were also up-regulated in sng1,such as GIF1,SUT1,GPT2-1,GPT2-2,GPT2-3 and BT1-3.These could be part of feedback regulation and compensatory mechanisms in response to deficiency of material and energy supply caused by loss of function of SNG1.9.In order to elucidate the genetic bases of appearance quality of rice grains,a recombinant inbred line population（RIL）derived from Beilu130（a japonica cultivar with extremly large grain and poor quality）and Jin23B（an indica cultivar with slender grain and relatively good quality）was developed for quantitative trait locus（QTL）mapping.We phenotyped grain length,grain width,length-width ratio,grain thickniss,1000-grain weight,white belly rate and white core rate.And a total of 51 QTLs were identified in two years.Most of them matched well with previous studies,and some QTLs have already been cloned,including GW2 and GW5/q SW5,which are major QTLs for grain width,length-width ratio,grain thickniss,1000-grain weight and white belly rate.We focused on newly found minor QTLs.Using marker assisted selection,NILs of six minor QTLs were constructed by backcrossing selected lines in the RIL with the recurrent parent,Jin23B,respectively.For each of the QTL,a NIL-F₂ population of 192 individuals was planted and the genetic effects were evaluated.The results showed that all the QTLs could be reproduced,and the genetic variation explained by each QTL in NIL-F₂population was elevated than that in RIL population.These results provided the bases for fine mapping and map-based cloning of the QTLs.In conclusion,SNG1 functions as a positive regulator of grain size and grain weight.It encodes a hexokinase with catalytic activity.And SNG1 did not act as a glucose sensor and function in sugar signaling.SNG1 mainly controls glume cell expansion by affecting sugar metabolism and GA biosynthesis,then influencing grain size and weight.We dissected the genetic bases of appearance qualiy of rice grains with a recombinant inbred line population,and evaluated the genetic effects of six minor QTLs with NIL-F₂populations,respectively.These results laid solid foundations for fine mapping and map-based cloning of the QTLs.