| Rice is the staple food of more than 3 billion people worldwide.The annual global rice cultivation area is approximately 160 million hectares distributed in 117 countries with an average yield of 4.4 tons per hectare.With a continuous increase in population and a decrease in cultivated land yearly,it is an inevitable choice to significantly increase yield per unit area of rice to ensure food security for billions of people.China has a 40-year history of hybrid rice cultivation.These planting experiences demonstrated that the effective utilization of heterosis is a strategy to increase rice yield.In hybrid rice seed production,the farmers manually cut off one-third or half of the flag leaf blade of the parents at the initial heading stage to remove barriers to cross-pollination.This procedure is not only labour-intensive but also requires high operating skills to avoid injuring the young panicle.Therefore,breeding a male sterile line through flag leaf inclination not only omits the procedure of cutting flag leaves but also facilitates the mechanization of hybrid rice seed production.Focusing on this scientific issue,the following two studies were conducted in this paper.The first study was to fine mapping of the qFLA8-2-L that was initial location in the predecessor.Allelic FLA1 gene was cloned to control the large FLA,and functional analysis of FLA1 was performed.The second is to investigate the phenotypic data of FLA of 441 accessions.Combined with the SSR molecular marker genotype data of this population,a mixed linear model was used for correlation analysis to mine the superior allelic variation of FLA.The variety of the carrier predicts the parental combinations that can be used to increase the FLA.The main findings are as follows.1.Functional analysis of rice flag leaf angle function gene FLA1（1）The qFLA8-2-L locus was located within the 93.5 kb range between InDel36 and RM23071 on the long arm of chromosome 8.In this study,using 863B as the recipient parent and A7444 as the donor parent,the main effect QTL qFLA8-2-L controlling the rice FLA with only flag leaves inclination was obtained（the nearest isogenic line was named fla-R）.Using 4382 hybridization-derived secondary F2 segregating populations,the qFLA8-2-L locus was located within 93.5 kb between InDel36 and RM23071 on the long arm of chromosome 8.（2）The allele controlling the large flag leaf angle was FLA1（LOC08g31690）.Within the fine maping range,there are 4 Open Reading-Frames（ORFs）.Through sequencing and comparison,we found that the ORF1,ORF2,and ORF4 were different betwen fla-R and 863B.ORFS was no difference between fla-R and 863B,which excluded the possibility that ORF3 was a candidate gene.qRT-PCR analysis of ORF1,ORF2,ORF4 from different tissues showed that ORF1 and ORF2 were not expressed in flag leaves and flag leaf lamina joints,but ORF3 was highly expressed in flag leaf and leaf lamina joints.There was a significant difference between fla-R and 863B（P<0.5）.FLA1（LOC08g31690）was identified as a candidate gene in conjunction with gene function annotation.The qRT-PCR and GUS activity analysis showed that FLA1 had the highest expression in flag leaves and flag leaf lamina joints.At the same time,the expression level was also higher in vigorously growing stem sections,root tips,and young panicles.Transgenic complementation（CV-FLA1863B）and transgene overexpression（OE-FLAlfla-R）tests further demonstrated that FLA1 can cause the flag leaf inclination.（3）FLA1 encodes the F-BOX family protein.Through bioinformatics analysis,we found that FLA1 encodes the F-BOX family of proteins.Two amino acid differences in the position of F-BOX between 863B and fla-R were found by sequencing alignment,namely valine-leucine,glycine to alanine（P-L,G-A）.The amino acid difference in the F-BOX domain may be a major factor in the difference in the function of the protein FLA1.Subcellular localization results show that FLA1 protein localizes in cell membrane,nucleus and chloroplast.The yeast two-hybrid result shows that its interaction protein is ARP1.（4）The GA3 content of young panicle and flag leaf and flag leaf lamina joints of fla-R was higher than 863B.The young panicles and flag leaves and their leaf occipital parts of fla-R and 863B young panicle differentiation were extracted respectively.The endogenous hormones were measured by liquid chromatography and mass spectrometry.The results showed that the content of GA3 in fla-R young panicles and flag leaves and flag leaf lamina joints was significantly higher than that of 863B.Exogenous hormone sensitivity test results showed that the amount of endogenous GA3 synthesis was the main reason for the flag leaf inclination.（5）The fla-R flag leaf lamina joints longitudinal cell length increased,and the number of cell layers at the distal end decreased.The flag leaf lamina joints of fla-R and 863B were cut out respectively,and the transverse sections and vertical sections were obtained by using frozen sections and paraffin sections.The results of light microscopy showed that the large flag leaf angle near-isogenic line fla-R showed an increase in longitudinal cell length and a decrease in the number of apical cell layers compared to 863B.（6）FLA1 can increase the outcrossing rate by 8.62%.The combination of fla-R,863B,and CV-FLA1863B with purple rice can be used to prove that large flag leaf angles can be used as seed parents to increase hybrid rice Seed production efficiency provides convenience for hybrid rice mechanization.It was found that the flag leaf angle material as a parent can increase the outcrossing rate by 9.6%（fla-R）,8.62（.CV-FLA1863B）.2 Miningof Favourable Marker Alleles for Flag Leaf Inclination in Oryzasativa by Association Mapping（1）Genetic variation for the FLA trait in rice exists in natural populations.A marker analysis of the 441 accessions using 262 SSRs resulted in the detection of 2354 alleles.The average number of alleles per locus was 8.9847,with a range of 2-24.The average genetic diversity for all loci was 0.6960,with a range of 0.0204（RM433）to 0.9328（RM7545）.The average PIC value was 0.6645,ranging from 0.0202（RM433）to 0.9288（RM7545）.Two hundred markers showed PIC values more than 0.55,46 markers showed PIC values between 0.25 and 0.55,and 16 markers showed PIC values less than 0.25,indicating that the population has high genetic diversity and rich genetic information and is suitable for genome-wide association analysis.（2）The natural population of 441 accessions is divided into 8 subgroups.The 441 accessoins was divided into 8 subgroups.The numbers of accessions from SP1 to SP8 were 47,81,47,38,36,75,35,and 46,respectively.The decay rate of D’ in each subpopulation follows the regression equation Y=bln（X）+c.The LD decay distance from SP1 to SP8（P<0.05）was 23.93cM,29.78 cM,17.99cM,26.52cM,29.81cM,46.66cM,29.25cM and 25.95cM.This result indicated that the LD decay velocity varied among these subpopulations.Among the eight subpopulations,the LD decay distance in SP3 was the shortest（17.99 cM）while that in SP6 was the longest（46.66 cM）,.indicating that accessions in Jiangsu and Heilongjiang（SP6）have been subjected to more extensive artificial selection than those in Vietnam（SP3）.（3）A total of 7 SSR marker loci were detected in the marker-trait association analysis in the two years,of which 4 were newly discovered markers that were significantly associated with the FLA.Seven SSR loci were detected in both 2015 and 2016.The seven loci were RM6266 on chromosome 3,RM348 and RM4835 on chromosome 4,RM2530 on chromosome 7,RM6215 on chromosome 8,RM258 on chromosome 10,and RM7303 on chromosome 11（Table 4）.The lowest PVE was 5.65%（RM6215）and the largest PVE was 14%（RM4835）.Among them,three markers,RM6215,RM4835,and RM2530,have been reported.The four markers RM6266,RM348,RM258,and RM11 are new markers that are significantly associated with the flag leaf angle.（4）Among the 27 favourable alleles detected,four alleles had phenotypic effects greater than 100.A total of 53 alleles were detected in both years,of which 27 alleles were positive favourable alleles.The positive favourable phenotypic effect value ranged from 0.10 to 44.5°.There are four phenotypic effect values greater than 10°,which are RM348-130（44.5°）,RM7303-90（25°）,RM258-180（14.8°）,and RM4835-230（11“）.（5）Nine parental combinations were predicted for improving FLA via crossing the accessions carrying the 4 alleles.Based on the distribution of favourable alleles and the PVE values of the seven marker-trait association loci detected in the two years,we predicted the parental combinations that could improve the flag leaf angles by crossing.We found seven rice accessions with more excellent alleles associated with FLA,namely,HKWGT,Longgeng 26,WMZD,Huakenuo,Kangzhounuo,Buxienuo and Hongganlizhihong（HGLZH）.The top nine parental combinations were predicted for improving the FLA in rice via cross breeding.Nine combinations,which increased FLA by 46° to 95° through pyramiding the favourable alleles contained in seven typical accessions,were predicted.This study cloned the functional gene FLA1,which controls the change of flag leaf angle,and used genome-wide association analysis to find four superior alleles with phenotypic effect values greater than 10°,and predicted nine excellent hybrid combinations.These results can not only provide the genetic resources and theoretical basis for the research of rice flag leaf angle mechanism,but also can be used to improve the flag leaf angle of F1 hybrid rice and solve the problem of cutting leaves in the hybrid rice seed production process. |
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