Cytological Studies And Gene Mapping Of A Rice Rolled Leaf Mutant And A Pistile Sterile Mutant

Rice leaf is not only a vital site for photosynthesis,but also an important factor for ideal plant architecture in rice.A mutant rl16(t)(rolled leaf 16)with rolled leaves was derived from the indica type rice 9311(wild-type WT)via the radiation of 60Co-y.From the cytological observation and genetic mapping,we had the following conclusions:1.Compared with wild-type,the mutant had not only typically adaxial rolled leaves,but also decreased height and panicle length,as well as the seed setting percentage.The whole plant of rl16(t)appeared leaf longitudinally rolled into the approximate tubular phenotype from seedling stage(three leaves and third true leaf folded)and kept it at the rest of time.Oppositely,the plant of WT had flat leaves of the entire life.Paraffin section of flag leaf indicates the number and area of bulliform cell of rl16(t)were lower than WT.The number of bulliform cell in WT was 385.1 ±43.6 cells/mm2,while therl16(t)was 1059.5 ±254.4 cells/mm.In addition to the bulliform cell,there were no significant and obvious changes of rl16(t)compared with WT.The carotenoid content of rl16(t)is lower than that of WT,but the chlorophyll-a,chlorophyll-b and total chlorophyll content were significantly higher than WT.2.Hybridization F1 from the cross between rl16(t)and WT had flat leaves.Meanwhile,there were 97 plants with rolled leaf and 326 plants with flat leaf of the 423 F2 plants.As a consequence,separation ratio conformed to 3:1(chi-square = 0.86<chi-square 0.05 = 3.84).Genetic analysis indicated that rolled leaf character was controlled by a recessive nuclear gene.Rl16(t)was initially mapped in the region between the RM23769 and RM23916 on the long arm of chromosome 9.Furthermore,with enlarged population and more developed InDel markers,Rl16(t)was finally delimited to a 51 kb region governed by the InDel marker DF70 and SSR marker RM23818.There were three coding genes(LOC_Os09g09320,LOC_Os09g09360,LOC_Os09g09370)of known structure domain.There was only half of the WT in rl16(t)on the expression of LOC_Os09g09360.As for quantitative expression analysis of the eight genes that contributed to the rolled leaf,seven of them had a decline,while OSZHD1 appeared a rise.3.The rolled leaf of rl16(t)was due to reduced number and area of bulliform cells.Rl16(t)gene would be a novel rolled leaf gene.As the result of quantitative gene expression.LOC_Os09g09360 may be the target genes.Our research was useful in map-based cloning of target gene and marker-assisted transferring rolled gene in rice breeding programs.Seed setting rate of rice is an important factor of production,and the process of pollination is a matter of seed setting.In this study,we detected a rice sterility mutant from the cytological observation and genetic mapping,we had the following conclusions:1.Mutant was planted for several generations and we found that there was an extremely significant difference in seed setting rate between mutant(11.13 ± 1.16%)and WT(94.69 ± 0.55%),respectively.To observe the fertility of mutant pollen,our research used 12-KI staining and found there was a decline of mutant pollen fertility(66.30± 3.08%)compared with WT(90.32 ± 1.70%).As for the survey of pollen sterility effect on spikelet low-fertility,WT and mutant had a cross with the same male sterile line as male parent,respectively.Even though the mutant pollen could meet the requirement of the fertilization of male sterile line as the wild-type pollen(t = 0.97,t0.05,4 = 2.77,t<tO.O5,4).So,pollen was not the cause of low seed setting rate in mutant.So,we found the mutant(93.88 ± 1.19%)and WT(96.87:± 1.12%)both had normal embryo sac with complete structure of seven cells and eight nuclears in embryo sac so that the embryo sac was not the reason of low seed setting rate in mutant by laser scanning confocal microscopy.At the same time,we also had a look at the mutant stigma development by scanning electron microscope.The ovary was blossomed by FAA and dehydrated by series of alcohol,then softened by KOH,finally stained by aniline blue to observe pollen germination in stigma.To observe the development of fertilized egg in different periods after flowered,we made use of the way that a combination of soaking by hydrochloric acid and staining by I2-KI.But,the mutant had a broken and lagged stigma compared with the WT.The study found that there was also no significant difference in fertility rate of embryo sac 1 d,7 d,30 d after flowering between WT and mutant.It means that the process of fertilization was in charge of the low seed setting rate.The experiment of pollen germination on stigma found that few pollen adhered to the mutant stigma and the pollen tube hardly reached the micropylar end.These prevented the double fertilization in the mutant on germinate and couldn't form mature seeds.2.Meanwhile,separation ratio conforms to 3:1(?2 = 0.71,?20.05,1=3.84,?2<?20.05,1).There were 205 F2(mutant×02428)plants for the primary mapping of the target gene.Using SSR markers and InDel markers,target gene was mapped a 1387 kb region between the RM5622 and WD-17 on the short arm of chromosome 2.3.Our research found that the low seed setting rate of mutant was that the failed germination of pollen tube resulted in failed double fertilization.There was no discovery of related genes,so the target gene was a new sterility genes.This study made a good foundation for fine-mapping and map-based cloning of the target gene.