QTL Analysis On Rice Appearance Quality Traits And Fine Mapping And Cloning Of Its Related Genes

Rice(Oryza sativa L.)is the staple food in China.Rice yield increase and quality improvement are very important for food security and meeting the strong requirement of high quality rice in Chinese nowadays.Grain shape(GS)and chalkiness both belong to rice appearance qualities(RAQ).GS exhibits strong correlation with kilo-grain weight(KGW),which contributes to rice yield.Chalkiness negatively affects rice appearance,processing quality and eating quality,and is the most important factor that limits rice quality improvement in China.In this research,rice appearance qualities were studied using Jiafuzhan,an early indica rice cultivar developed by Rice Genetics and Breeding Group in Xiamen University.Using various genetic populations,QTL(Quantitative trait loci)analysis on RAQ was carried out.One gene associated with chalkiness,Chalk5-2,was fine mapped.Additionally,a GS mutant,W60-4,was identified in fast neutrons mutagenized Ubi-Kitaake in Professor Pam's Lab in University of California,Davis.A candidate gene,GS9,was cloned.The main results in this study are summarized as the following:(1)Guanglu'ai/Jiafuzhan recombinant inbred line(GJ RIL)was developed and a genetic map was constructed previously by our research group.The RAQ phenotyping data of GJ RIL in a total of 6 growth seasons were used for genetic analysis,QTL mapping,epistasis and environmental effect analysis.Genetic analysis showed that grain length(GL),grain width(GW),length/width(L/W)exhibited the inheritance of typical quantitative ingeritance.Percentage of grains with chalkiness(PGWC)and degree of endosperm chalkiness(DEC)showed skewness distribution,and may be affected by both major and minor genes together.Correlation analysis revealed that GL echibits high negative correlation with GW in 5 growth seasons,and GW highly significantly positively correlated with PGWC and DEC respectively.In QTL mapping,3,1,4,3 and 3 QTLs were detected for GL,GW,L/W,PGWC and DEC respectively,and the explained phenotypic variation(H2)is 2.02%?12.7%.There is one shared QTL for the latter 4 traits,located in RM516-XM12 on chromosome 5 and the H2 were 4.11%?12.7%.In QTL epistasis analysis,3,5,3,6 and 4 pairs of epistasis QTLs were identified for GL,GW,L/W,PGWC and DEC traits respectively.The H2s ranges between 0.70%?6.17%.Four QTLs,qGL3,qGW5,qL/W8 and qDECl,took part in the epistatic interactions of GL,GW,L/W and DEC,respectively.A new stable QTL,qL/W8,was identified.Environmental effect analysis showed that the interaction between all the QTLs/epistasis QTLs and environment was not significant.(2)Amyloplast structure in rice endosperm was observed by scanning electron microscope(SEM).The results showed that some small starch grains and round or elliptic amyloids were stacked together loosly which led to many cracks in chalky area,while in the area with no chalkiness,there is no small,single starch grain,and polyhedral amyloids are packed together closely.(3)Backcrossing population and Near-isogenic lines(NILs)were generated by crossing using Zhenshan97B(ZB)as a recurrent parent,and ZhenjiaB(ZJB)as a donor parent.1 QTL for GW,2 QTLs for GW,2 QTLs for L/W,1 QTL for PGWC and 1 QTL for DEC were detected in a BC6S2.An interval RM18076—RM18035,located within the RM516—XM12 interval mapped in Chapter 2,harbored the QTLs for GW,L/W,PGWC and DEC,and the H2 was more than 40%.This interval was a major QTL for RAQ and exhibited pleiotropism.The major QTL was named Chalk5-2 here.Chalk5-2 was fine mapped to a 61-kb interval,RM 18035—CH5-37,using the NILs.It includes 7 predicted genes,including a hexokinase gene and a GW5 gene.Sequence analysis revealed that there was a 15-bp deletion in GW5 of ZB,which resulted in a 5-amino acid deletion in protein,suggesting GW5 is one of candidates for Chalk5-2.LOC_Os05g09500's sequence analysis showed that the sequences(from-1331 bp to+1248 bp)in ZB were greatly different from that in ZJB,therefore,LOC_Os05g09500 was also one of the candidate for Chalk5-2.(4)A GS mutant,W60-4,was identified in a M2 population derived from fast neutron-mutagenized Ubi-Kitaake seeds.Genetic analysis in F2 and BC172 population showed that W60-4 harbored a recessive homozygous mutation,which reduced about 1 mm in GL.The gene is semi-dominant.SEM result revealed that the longitudinal length of glume shell cells in W60-4 were slightly shorter than those of its parents,suggesting that this gene may affect both the length and number of glume shell cells.Whole-Genomes sequencing on pooled mutants and Ubi-Kitaake were conducted.16 mutations were identified through sequence alignment,among which 6 mutations located on known genes.Co-segregation analysis revealed a candidate gene,named GS9,which is a brittle culm gene,BCl2,cloned in 2010.