Fine-mapping And Functional Analysis Of QTLs Controlling Nitrogen-mediated Growth Response And Yield-related Traits In Rice

Rice is one of the most important cereal crops in the world,which is widely cultivated all over the continents,and the annual acreage for rice exceeds 1.6 million hectare.In adaption to different geographical and climate conditions,rice is slowly evolved into various cultivars,showing significant divergence in plant architecture,panicle architecture,grain shape,nutrient efficiency,stress tolerance and so on.Nitrogen(N)is one of the essential macronutrients for plant growth and development.Under N starvation conditions,application of N fertilizers significantly increases rice yield.But N fertilization efficiency is graduately declining along with increasing N fertilization,which not only raises costs but also leads to many environmental problems.Therefore,it is theoretically and practically meaningful to develop new rice cultivars with superior N efficiency,for which the cultivars could not only obtain higher yield with less N fertilization,but also avoid the environmental problems caused by over fertilization.This research firstly evaluated the genetic variations of N responsiveness using different rice varieties,and then mapped the QTLs controlling yieldrelated and N-mediated growth responses in rice through linkage analysis.The main results were as follows:1.We selected two cultivars,NJ6 and QZL2,significantly contrasting in plant architecture,panicle architecture and N responsiveness from 40 natural rice materials in hydroponics.We further re-sequenced the RILs developed from NJ6 and QZL2 by implementing nextsequencing technology,and constructed a new genetic map with high resolution.The total length of the map was ~1421.8 cM,and its average length between adjacent markers was 0.51 cM.2.The results from field experiments in three locations of three years showed: plant height,flag leaf length,panicle length and grain length significantly positively correlated with each other,but negatively correlated with leaf colour;flag leaf width significantly positively correlated with grain number,but negatively correlated with tiller number.QTL mapping in multiple environments for multiple traits showed: NAL1,DTH8 and DEP1 were pleiotropic genes;SD1,GS3 and GW5 were stable dominant genes responsible for plant height,grain length and grain width,respectively.NAL1 was strongly artificially selected in the tropical japonica subpopulation,and it might improve yield to introduce the R type of NAL1 protein into O.glaberrima.OsSPL7 and OsGRAS27 might be the two candidate genes to the QTLs responsible for panicle development in the Chromosome 4.3.The results from N application experiments in both field and hydroponics showed: tiller number and grain number were the two most sensitive agronomic traits to N fluctuation.The results from correlation analysis indicated that N responsiveness traits tightly related to yield traits in rice.qNGR_TN9,was a key QTL controlling N-mediated tillering in hydroponics,which was allelic to DEP1.SD1 was the candidate gene for the key QTL controlling N responsiveness of plant height in field;NAL1 might be the candidate gene for the major QTL controlling N responsiveness of plant height and tiller number;DTH8 might be the candidate gene for the QTL controlling N responsiveness of flag leaf length,flag leaf width and leaf color.Combining with RNA-seq data,we found that the expression of OsTCP19 was induced by N starvation,which was located in the QTL region controlling N-mediated tillering.4.A total of 68 QTLs were detected for yield-related traits under three N supply conditions.Among them,9 QTLs were detected at all the three N levels;one was at both LN and MN levels;and one was at LN and HN levels;six were detected at both MN and HN levels;9,6 and 10 QTLs were specifically detected at LN,MN and HN levels,respectively.On the chromosome 3,a new stable QTL region of 400 kb controlling grain number was mapped at three N levels,explaining 7.15%,5.50%,6.65% phenotype variation at HN,MN and LN levels,respectively.At LN level,3 QTLs controlling tiller number and primary panicle branches were identified,responsible for 10.32%,9.82%,8.00% phenotype variance.Analysis on excellent allelic genes indicated that pyramiding DEP1 and NAL1 might coordinate nutrient and yield traits,and subsequently reach high yield and high nutrient efficiency.5.In order to validate the function of the candidated gene DEP1 in controllling tiller number response to N supplies,we constructed a pair of near isogenic lines under the WYJ7 background.Preliminary results from hydroponics indicated that dep1 suppressed the regulatory role of N on rice tillering through different mechanisms at different N levels.At LN level,dep1 mainly maintained normal growth through increasing N absorption as well as influencing on N redistribution.At HN level,dep1 increased N efficiency through enhancing N redistribution.Above all,our study systematically analyzed important QTLs controlling yield traits and N responsiveness traits in rice through next-sequencing technology together with yieldrelated phenotypic and N response data.The results showed that SD1,NAL1,DTH8 and DEP1 were involved in the N-mediated rice plant growth and development.Pyramiding of excellent allelic genes could contribute to higher yield and higher N efficiency.On chromosome 3 and 4,we fine-mapped the QTLs for grain number,and functionally analyzed the candidate genes.Under low and high N conditions,dep1 might functioned in different molecular pathways to improve N efficiency.This study revealed the molecular and genetic mechanism of cooperating with yield and N utilization in rice,which might provide theoretical foundation and candidate genes for breeding new ‘high yield and high efficiency' rice cultivars.