| Rice tillering,one of the most important factors that influences plant architecture and grain yield,is controlled by multiple factors such as genetic factors,endogenous and exogenous hormones,and environmental factors.Mining more genetical factors underlying tiller number and the further genetic improvement of rice tillering are important directions for the rice ideal plant architecture breeding.The current study identified two rice tiller suppression mutants tsl,ts2 by EMS mutagenesis.By the positional cloning strategy,we fine mapped two major genes ts1 and ts2 responsible for the tiller suppression trait in the both mutants.Based on the bioinformative analysis,the most possible candidate genes for tsl and ts2 were identified.We also investigated the performances of plant architecture-related and yield-related traits of the ipal-derived new rice conventional lines under different plant densities and paclobutrazol treatments,and analyzed the breeding potential of the superior allele ipal.The main results were represented as below:1.The rice tiller suppression mutant ts1 exhibited significantly lower culm numbers when compared with the wild type(WT)LY95.During the whole growing stage,the tsl muatant could produce 2.2 ± 0.48 culms at most,while LY95 could produce 5.3±0.58 culms.Morphorlogical and histological studies revealed that the abnormal formation of the tiller buds might result in the tiller suppression phenotype of tsl.Genetic studies demonstrated that the dominance degree of the tsl allele relative to TS1 allele was-0.79,which indicated that the mutant allele ts1 was incompletely recessive to its WT allele TS1 in cv.Wushansimiao.2.By utilization of the bulked segregant analysis and recessive-class analysis,we fine mapped the major gene ts1 in the marker interval ID8378-SSR6884 of the distal region of the short arm of rice chromosome 2,and found RM3340 co-segregating with tsl in the fine mapping population.By utilization of the NCBI-Blast online tool,we found the corresponding physical position of tsl was 318,378 bp-426,884 bp,which was a～108.5 kb region on the short arm of Chr 2.3.Through the MSU Rice Genome Annotation Project database(http:/rice.plantbiology.msu.edu/index.shtml),a total of nineteen ORFs were found in the candidate physical interval.None of the nineteen ORFs have been reported to be involved in rice tillering regulation.Therefore,ts1 could be a novel rice tillering regulator.Based on the functional prediction and physical position,genomic DNA sequences of seven candidate ORFs(LOC_Os02g01590,LOC_Os02g01610,LOC_Os02g01700,LOC_Os02g01710,LOC_Os02g01720,LOC_Os02g01730,LOC_Os02g01740)were sequenced.It was found that only one polymorphism occurred between the WT and mutant type.At +733 site of the only one exon for the candidate gene LOC_Os02g01610,there harbored a C→T nucleotide substitution.Gene expression analysis revealed that the relative expression level of LOC_Os02g01610 in the tsl mutant shoot apexes(40DAS)was significantly decreased by 47.8%when compared with that of WT.Keeping above results in view,LOC_Os02g01610 was the strongest candidate gene for tsl.In addition,expressions of MOC1 and HTD1 in the tsl mutant shoot apexes(40DAS)were significantly decreased by 77.3%and 31.4%when compared with that of WT,indicating that ts1 gene may regulate rice tillering through MOC1 and HTD1 associated pathways.4.Based on the C.+733C→T point mutation of LOC_Os02g01610 harbored in the mutant type,a co-dominant molecular marker cd-733C/T was designed by the ARMS-PCR method so as to specifically recognize the three kinds of genotypes(mutant homozygous type,heterozygous type,wild homozygous type)of LOC_Os02g01610.SNP assay results revealed that molecular marker cd-733C/T has great specificity and high sensitivity in the ts1/Wushansimiao F2 population,their both parents and F1 plants.Furthermore,based on the genotyping with marker cd-733C/T,all segregants of the fine mapping population were mutant type,which indicated that the c.+733C→T point mutation was co-segregated with the tiller suppression phenotype.All the amplicons of 276 morden rice varieties or rice breeding materials belonged to wild type,the result suggested that the point mutation c.+733C→T probably resulted from EMS mutagenesis rather that natural variation.5.The tiller suppression mutant ts2 exihibited a different tiller suppression phenotype when compared with the WT LY95 and the tsl mutant.The ts2 mutant kept monoculm till 53 days after sowing(DAS),while WT produced up to 3.88 ± 0.34 culms,ts1 produced 1.73 ± 0.70 culms.After 53 DAS,the ts2 mutant began to produce tillers,but the difference on the culm number trait between WT ’and the ts2 mutant remains significant till 67 DAS.At 74 DAS,the ts2 mutant produced 4.75±0.54 culms,and showed no significant different compared with that of WT LY95(4.88 ±0.63 culms)(p=0.76).By contrast,the tsl mutant kept its significant different on culm number trait during the whole growing season when compared with that of WT.Therefore,the ts2 mutant showed different culm number variation from WT before 67 DAS,which was different from that of the tsl mutant.Morphorlogical and histological studies revealed that the abnormal formation of the ts2 tiller buds might result in its tiller suppression phenotype before 53 DAS.Genentic analysis of the ts2 mutant demonstrated that,on the culm number trait,the dominance degree of the ts2 mutant relative to wild type cv.Wushansimiao was-0.53(53DAS),which suggested that the ts2 mutant was incompletely recessive to Wushansimiao.6.By utilization of the BSA and RCA methods,we fine mapped the major gene ts2 responsible for the muant phenotypic variation of the ts2 mutant onto the short arm of rice chromosome 8,The ts2 gene was localized into marker interval SSR9192-SSR1856.It was tightly linked to InDel marker ID5605-2.Based on the NCBI database,we obtained the physical interval information of the ts2 gene,which was a-182.7 kb fragment from 3,839,192 bp to 4,201,856 bp on the short arm of Chr8.By querying the potential candidate genes based on the physical interval information in two rice reference genomic databases(Nipponbare version):The MSU Rice Genome Annotation Project(http://rice.plantbiology.msu.edu/index.shtml)and The RiceGAAS(http://ricegaas.dna.affrc.go.ip),a total of 29 and 15 candidate ORFs were found respectively.Combined the gene function prediction and the recombinant frequencies between the tightly linked marker ID5605-2 and ts2 gene,we deduced that LOC_Os08g07010 and LOC_Os08g07060 were the most possible candidates for ts2.7.Field expreiments demonstrated that,under plant three seedlings per hill ×applied ddH2O treatment(D1P1),the ipa1-derived new rice conventional lines exhibited its plant architecture with large panicle but low tillering ability.By contrast to D1P1 treatment,plant five seedlings per hill × appiled paclobutrazol treatment(D2P2)could significantly promote panicle number of the ipa1-derived new rice conventional lines.In addition,the D2P2 treatment could also keep or reduce spikelets per panicle,increase theoretical and harvest yield of the ipa1-derived new rice conventional lines.Two-way ANOVA analysis suggested that,between practical factors plant density and paclobutrazol level,the paclobutrazol factor played a major role.The application of paclobutrazol could averagely promote 16.95%,3.43%and 17.20%of panicle number、thousand grain weight and harvest yield of the ipal-derived new rice conventional lines,respectively.Under D2P2 condition,the theoretical yield of K1757(11.80 t/hm2),K1758(12.05 t/hm2)have reached that of N301(12.01 t/hm2)and Hefengzhan(11.97 t/hm2)under D1P1 condition.The ipal-derived new rice conventional lines K1757 and K1758 showed their great potential in rice high yield breeding program. |
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