Mapping Of QTLs Controlling Clum Diameter,Leaf Width,Grain Number Per Panicle And The Leaf Lesion Mimic And Leaf Rolling Mutnat Llmrm1 In Rice

With the population growth and arable land area reduction,the food security situation is gradually rigorous.Rice is one of the most important food crop in China,therefore,improving yields and resistance of rice is significant to ensure national food security and societal stability.It is the most direct and effective way to improve the rice yield by mapping new yield and resistance related genes and applying them in breeding practice.QTLs mapping of qCD11,qFLW11 and qSPPll for clum diameter,leaf width and spikelets per panicle,respectively.The combination of improving plant architecture and harnessing heterosis is an important way to increase rice yield.Thick clum,wide leaf and big panicle are the significant indicators of high-yielding rice.To map and clone new imporant genes or QTLs controlling clum,leaf and panicle characters,we conducted QTL analysis using F2 population derived from CLP characterized by thick clum,wide leaf and big panicle,and the Nipponbare.Based on the primary mapping,the advanced backcross population is developed to narrow down the intervals of target fragment which control stem diameter,leaf size and spikelets per panicle.The main results in this study are present below:1.There are great differences in many agronomic traits between CLP and Nipponbare.The phenotypic value of CLP are about two times than those of Nipponbare in stem diameter,flag leaf width,number of primary branches,number of secondary branches,spikelets per panicle and seed setting density.2.We have constructed a genetic linkage map including 110 markers using the F2 population comprising 265 plants derived from CLP and Nip.The genetic linkage map covered 1526.6 cM of the rice genome,and the average distance is 13.88 cM between markers.Additionally,we found 24 distorted markers in F2 population.A total of 42 QTLs for 16 traits were detected in the F2 population.Among the 42 QTLs,the number of QTLs controlling tiller number,plant height,culm diameter,flag leaf length,flag leaf width,flag leaf length-width ratio,panicle length,number of primary branches,number of secondary brances,spikelets per panicle,seed setting density,grain length,grain width,grain length-width ratio,grain area and thousand grains weight were 1,2,2,1,4,1,2,2,4,4,3,3,4,3,3 and 3 respectively.3.68 two-points interactions for 15 traits were detected from CLPxNip F2 population using ICIM-EPI mapping.The types of interactions are primarily between non-QTL loci,and the styles of interactions rely on agronomi traits.4.Among the 42 QTLs detected in F2 population,some QTLs explained more of phenotypic variation,and some others were associated with plant type.Hence we developed BC,F2 population to detect and analysis these QTLs,and the results showed that the contribution rate of most QTLs decteced in BC,F2 increased.Based on the literature,we speculate that GL3.1 or GS3 is responsible for the qGL3,qGLWR3,qGLWR3 and qTGW3 located on chromosome 3 between RM15281 and Ind9,GW5 or GS5 is responsible for qGW5 and qGLWR5 located on chromosome 5 between RM169 and RM5140,IPA1 is responsible for qCD8,qFLW8 and qSPP8 located on chromosome 8 between Ind22-Ind23.5.We found that the QTLs controlling clum diameter,flag leaf width and spikelets per panicle located on chromosome 11 between RM206 and RM224 were detected in different environment and generations,and that the contribution rates of the QTLs became more and more high with the increase of backcross generation.qCDll,qFLW11,qSPP11 explained 5.46%,5.68%and 5.22%of phenotypic variation in F2 population,respectively,explained 12.04%,12.26%and 11.42%of phenotypic variation in BC1F2 population,explained 33.29%,29.48%and 28.34%of phenotypic variation in BC2 F2 population.6.According to the above results,wefocused on the QTLs controlling clum diameter,flag leaf width and spikelets per panicle located on chromosome 11 between RM206 and RM224.Then CLP were used as the donor parent and the Nipponbare used as the recipient parent to develop NILs.In BC6F2 population,among the sixteen traits,culm diameter,flag leaf width and spikelets panicle showed a bimodal distribution,however,the other traits still showed a continuous distribution of variation.Therefore,we developed polymorphic markers between RM206 and RM224.Finally,the QTLs controllingculm diameter,flag leaf width and spikelets panicle were narrowed to the region between M11-3 and M11-4.7.According to the literature,there are eight kinds of proteins involved in the stem,leaf and panicle in the region between M11-3 and M11-4.These proteins include F-box domain containing protein,leucine rich repeat family protein,laccase precursor protein,transferase family protein,armadillo/beta-catenin-like repeat family protein,lipase class 3 family protein,20G-Fe oxygenase family protein,lipase precursor and kinesin motor domain containing protein.Gene mapping of a leaf lesion mimic and rolling mutant llmrml in riceGrain yield reduction caused by a variety of plant diseases in rice production is a serious threat to the food security.The lesion mimic is one of plant diseases which display similar to hypersensitive responses lesions without pathogen invasion.It could provide theoretics support for elucidating the genetic base of resistance by developing and identifying lesion mimic mutants and by cloning and functional analysis of the lesion mimic mutant genes.We could develop rice varieties with high resistance by resistive breeding to ensure food security.In this study,we identified a mutant with lesion mimic and abaxially rolled leaves by screening a rice ethyl methane sulfonate(EMS)breeding population.Then 93-11 used as the recipient was back crossed with the mutant by several generations until the traits were stable,and named the mutant llmrm1(leaf lesion mimic and rolling mutnat 1).We study the llmrml byphenotype observation,histological analysis,genetic analysis and gene mapping.The main results in this study are are present below:1.The llmrml is characterized by lesion mimic and abaxially rolled leaves and plays pleiotropic effects on agronomic traits,such as plant height,seed setting rate.There was little difference between llmrml and wild type before the fourth leave stage.After that,however,the mutant llmrml started to develop reddish-brown lesion spots on the leaves,and the number of spots proceeds to increase,spreading from the tip to middle part of leaves along with development.Following the lesion spots,leaves gradually rolled toward the abaxial side and become more evident during plant growth.2.We found that the photosynthetic pigment contents,including chlorophyll a,chlorophyll b,total chlorophyll and carotenoid,were almost the same as wild type before the initiation of the lesion spots.After lesion spots presented,however,the photosynthetic pigment contents in llmrml were significantly reduced.Compare to wide type,the contents of chlorophyll a,chlorophyll b,total chlorophyll and carotenoid were reduced 70%,51%,67%and 53%,respectively.Additionally,after lesion spots presented,the Pn,Gs,Ci and Tr of llmrml reduced 76%,56%,14%and 53%.3.The observation byTEM showed that the llmrml mutant exhibited irregularly and abnormal chloroplasts,in which the thylakoid lamella was loosely distribute,demonstrating that chloroplasts developde abnormally in llmrm1.Additionally,trypan blue staining assay on the leaves of llmrml mutant and wild type revealed that lots of navy blue spots were observed on llmrml mutant leaves after staining,andno navy blue stained spots were detected in wild type,illustrating the formation of lesion mimic spots were accompanied by the cell death.4.We found no difference between the llmrml mutant and wild type in the organization of the sclerenchyma and vascular tissues by analysing paraffin sections.Statistical analysis indicated that the number of bulliform cells in the rolled regions starting in the llmrml mutant was 7.7±0.48,in the wild type was 5.6±0.70,indicating that the leaf rolling phenotype was caused by the increased number of bulliform cells.5.Genetic analysis revealed that llmrml was controlled by a single recessive nuclear gene by analysing the F2 populations of llmrml ×93-11 and llmrml ×Nip.Gene mapping and sequencing showed that the llmrmlmutant carried a single base substitution(C to T)at nucleotide 3086 nt of LOC_Os05g31530,resulting in a change from Proline to Serine.LOC_Os05g31530 encodes NBS-LRR protein,and has NBS and LRR structural domain.6.Quantitative Real-time PCR analysis of pathogenesis-relatedgenes revealed that the relative expression of Betvl,nPR10 and PR1a raised 29 times,18 times and 4 times compared with wide type,respectively.Quantitative Real-time PCR analysis of leaf-rolling genes showed that the relative expression of NAL7,NRL1 and RL14 increased 5 times,3 times and 2 times,respectively,and the relative expression of SRL1 reduced 1/2 compared with wide type.These results suggested that the llmrm1 mutant resistance might be enhanced and LLMRM1 might be in the same regulatory pathwauly with NAL7,NRL1,RL14 and SRL1.