Phenotypic Identification And Gene Mapping Of A Drooping Leaf Mutant Dl(t) In Rice(Oryza Sativa L.)

Leaf blade is a main organ of photosynthesis and responsible for over ninety percent of rice yield. Therefore, increasing utilization efficiency of light energy is significant in crop breeding. Rice leaf is long sword-shape, the midrib is formed by blade cell proliferation at intermediate zone. Compared with parallel vein, plays a major role in mechanical support of the blade, thus rice midrib is an important factor to ensure blade erect. Drooping leaf is mainly due to partial loss of leaf midrib.In this study, the progeny of rice(Oryza sativa L.) restorer line Jinhui 10 has been induced by EMS,in which we found a drooping leaf mutant named dl(t)tentatively.According to many generations selfing observation,they mutant traits inherited steady.This paper studied dl(t) phenotypic identification, paraffin slice microscope observation, genetic analysis, gene mapping and prediction of the candidate gene, quantitative PCR expression analysis.The main results were as follows:1. dl(t) phenotypic identificationAfter compared the rice drooping leaf mutant plant morphology and flower organ shape and structure with the wild type, the rice drooping leaf mutant showed severe drooping. From the base to the tip about 2/3 blade had midribs and they were thick and short, the rest 1/3 blade had not midrib and showed drooping. Midrib was obvious occured at the flag leaf; second upper leaves all showed partial drooping. However, ithad normal spikelets, included six stamens and a pistil, inside and outside glume each one.However, the leaves of the wild type strain were showing upright, from the base to the tip blades were having midribs, from coarsing to tapering. On the other agronomic traits, mutant plant was not significantly different from the wild type.2. dl(t) paraffin slice and microscopic observationAt the tillering stage, leaves of the wild type and mutant were used to observe paraffin slice by microscope. Compared with the wild type, the mutant was missing midribs at the middle and the upper of the plant, there are many vascular bundles,but the central area of the blade was lacking of a central vascular bundles and two distinct cavities. midrib structure was completely similar to lateral vein, indicated the mutant did not form a clear midrib structure.3. dl(t) genetic analysisReciprocal hybrid between the drooping leaf mutant Xinong 1A and dl(t) was done for genetic analysis.In mature period, observed the leaves traits of each plant. F1 plants were phenotypically normal, While there was a clear separation in F2 generation,appeared normal and mutant two types, erect normal strain had 1173, drooping leaf mutant had 376, accorded to the chi-square measurement,it conformed to 3: 1segregation ratio(χ2=0.40<χ20.05=3.84). Genetic analysis used F1 and F2 populations,which suggested that the traits of dl(t) were controlled by a single recessive nuclear gene.4. dl(t) gene mappingUsed Xinong 1A/dl(t) of the F2 recessive drooping leaf plants as mapping population, used SSR and Indel molecular markers to do gene mapping, gene was finally located in the short arm of chromosome 3,the primers between 3-5 and 3-3, the physical distance is 170 kb.5. dl(t) candidate gene predictionAccording to the rice genome annotation website RGAP(http:www.gramene.org/Oryza_sativa/Location)provided information on the range,which contained 20 annotated genes. Mainly encoded proteins associated with mitochondria and chloroplasts, DUF domain protein, expression protein and so on. It also contained the LOC_Os03g11600 gene, which had been reported drooping leaf gene encoding YABBY domain protein.Sequenced the drooping leaf gene LOC_Os03g11600 of dl(t) positioned within the interval, and found that it compared to wild type, dl(t) in YABBY domain proteincoding gene LOC_Os03g11600 was not detected differences in the coding region. The promoter region in front of ATG at 3676 base occurred T to C nucleotide substitutions.The promoter region in front of ATG at 5943 base occurred C to G nucleotide transversion, These might caused changes in cis-regulatory elements,resulting in changes of gene expression.Therefore, we initially assumed LOC_Os03g11600 is the dl(t) candidate gene.6.DL gene expression analysisDraw the wild and the mutant blade materials at tillering period, extracted and purified RNA, reversed transcription cDNA, qPCR quantitative analyzed of DL gene.The result showed that the expression of the mutant was significantly lower than the wild-type. Speculated the drooping leaf trait was associated with low expression of the gene.