| ALK and Wx are two key genes affecting rice grain quality,controlling the gelatinization temperature(GT)and amylose content(AC)in rice,respectively.The natural allelic variation of these two major genes is the main cause of the diversity of eating and cooking quality in rice.The study on the effects caused by the combinations of different alleles of these two genes could make deep insight into the formation of rice quality and provide guidance for the practice of quality improvement.In this study,the main allelic variation of ALK gene was identified by genomic association analysis,and the functional differences of the main three alleles of ALK gene on grain quality were verified by constructing near-isogenic lines(NILs)as well as transgenic lines.Besides,the rice NIL lines containing different main allelic variants of ALK and Wx genes were constructed to clarify the effects of different combinations of these two genes’ alleles on rice grain quality.Finally,the response of rice quality containing different ALK and Wx allelic combination to high-temperature was investigated by high-temperature treatment during the grain filling stage.These studies could provide important basis for molecular design breeding under high-quality rice during global warming.The main results are as follows1.The allelic variation of the ALK gene that controls the low gelatinization temperature of rice could be subdivided into ALKa(737-Met,781-Leu)and ALKb(737-Val,781-Phe),which were classified as the same class as SSⅡd.In this study,the distribution of ALKa and ALKb in 399 rice varieties was analyzed,of which 63.2%of ALKa(A-GC)existed in japonica rice,while 91.3%of ALKb(G-TT)existed in indica rice.Evolutionary analysis of ALK alleles showed that it is likely that the two low GT alleles with less effect on the GT would have derived from their progenitor gene ALKa,which controlling the high GT.Although the amino acid substitution in ALK gene of both low GT alleles led to the decrease of gene expression,the gelatinization temperature of ALKb was lower than that of ALKa,that is to saya the effect of Phe/Leu variation type in ALK allele on the decrease of gelatinization temperature was more significant than that of Met/Val mutation type.The thermodynamic parameters such as To、Tp、Tc、△Hgel that reflecting the gelatinization temperature were affected by the fine structure of starch crystallization zone.The results of DSC analysis showed that the thermodynamic properties were related to the content of short amylopectin(DP 6≤11),but independent of the proportion of crystallization zone corresponding to the content of amylopectin.Starch fine structure analysis based on gel permeation chromatography(GPC)and high-efficiency anion exchange chromatography(HPAEC)showed that the short chain(DP 6-12)and intermediate chain(DP 13-21)of NIL(ALKb)starch was significantly reduced relative to the Nip(ALKa)and NIL(ALKc)rice lines.In addition,the amylopectin structure of DP>10 could form a double helix together with proteins and lipids and result in high gelatinization properties.2.The NILs containing different combination of Wx alleles and ALK alleles were constructed to identify the different effects of Wx alleles and ALK alleles on rice quality.The gel consistency(GC)value of the Nip-Wxb/ALKc rice was significantly higher than that of the Nip-wx and Nip-Wxa,and the gel consistency of the rice from high AC lines Nip-Wxa/ALKa and Nip-Wxa/ALKc was remarkably reduced.3.Two SNP variants at the C-terminal of ALK gene were selected as the two alleles of ALK gene,ALKc(G-GC)controlling high GT and ALKa(A-GC)controlling low GT,respectively.The GT of ALKc rice line was significantly higher than that of ALKa under the same Wx background There was no significant difference in GT between wx and Wxb rice lines under the same ALK background,and the GT of Wxa rice line was significantly lower than that of wx and Wxb NILs with the same ALK allele.It indicated that there was a certain negative correlation between GT and AC in rice grains with altered amylose content.4.The results of RVA analysis of rice samples showed that the lower breakdown viscosity and higher setback viscosity of Nip-Wxa background resulted in poor tasting value,while the higher breakdown viscosity and lower setback viscosity of Nip-Wxb and Nip-wx resulted better tasting value.ALK allele with the same Wx genotype mainly caused the different pasting temperature during rice gelatinization,but merely had effect on the breakdown viscosity and setback viscosity in rice samples.These results further clarified the effect of the Wx alleles and the ALK alleles in the formation of rice quality,that is,the AC and the GC were mainly controlled by the Wx gene,while the GT and the viscosity of the starch were controlled by the ALK and Wx genes5.Rice samples grown under high temperature condition during grains filling generally showed the characteristics of harder gelatinization,which indicates that the change of thermodynamic characteristics during rice grain filling stage may be one of the main reasons for the decrease of cooking and eating quality of rice planted under high temperature.The results showed that the GT of ALKc rice lines was higher than that of ALKa rice lines,showing a negative correlation between GT and AC,which may be resulted from the competition for the substrate used in the synthesis of both amylose and amylopectin.The AAC of ALKNILs planted under high temperature was significantly lower than that planted under normal temperature under the background of Nip-Wxb rice containing low ang middle AC.However,under the same background of glutinous Nip-wx rice,the AC of ALK NILs planted under high temperature condition was higher than that at normal temperature.By comparing the difference of AC in the same ALK rice lines planted under different temperature conditions,it was clear that although the AC remarkablychanged in ALKc rice lines than that of ALKa rice lines caused by high temperature,the AC in ALKa rice lines was higher than that of ALKc rice lines.The GC of the ALKc rice lines remarkably reduced than that of ALKa rice lines planted under high temperature than normal condition.The GC and the GT of these rice lines had a positive correlation in rice lines grown at same temperature condition.High temperature during grain filling stage had major effect on the chain length distribution and starch grain morphology of rice starch.AC and the content of A chain and short B chain in amylopectin decreased,and the proportion of long B chain increased under high temperature.These changes in starch content and structure were the main reasons for the increase of GT of rice lines grown under high temperature. |
