Evaluation Of Self-incompatibility Of Fortunella Hindsii And Exploration Of Its Tetraploid Germplasm

Fortunella hindsii belonging to Rutaceae,originates in China and is distributed in Guangdong,Guangxi,Hunan,Zhejiang,Fujian and Jiangxi province.Because of its short juvenile phase,it can be regarded as a model plant of citrus,which is of great significance in scientific research.This study identified and evaluated the ploidy and self-incompatibility for the exploration and application of F.hindsii germplasm;as provided a reference for the creation of triploid seedless genotype in the future.This study explored and obtained one plant of tetraploid through ploidy identification.After evaluating the traits of the leaf,flower,fruit,and seed,it was found that the leaf index and leaf stomatal density of the tetraploid plant were significantly reduced compared to the diploid one;leaf width,area,thickness,weight,stalk and leaf stomata were significantly increased with less leaf wax.This study found that the wax of F.hindsii leaves was in turn represented by fatty acids,alcohols,terpenes and alkanes through GC-MS detection;and the content of each component in the tetraploid was less than than in the diploid,which was proved by the water loss rate experiment that the reduction of wax was not conducive to the water retention of the leaves.The overall flower length,diameter,petal length,petal thickness,filament length,and pistil length of the tetraploid floral organs were significantly greater than that of diploid,but the petal width and pedicel length were not significantly different from the diploid.The germination in vitro test showed that the germination time of tetraploid pollen was longer with lower germinating rate than the diploid respectively;the germination rate of diploid pollen was 92.46%at 40 h,while tetraploid was only 80.56%for the same time.The vertical diameter and peel thickness of tetraploid fruit were obviously greater than that of diploid,while the transverse diameter of the peel was opposite.The fruit was narrow and long and the pedicel length was not significantly different from that of diploid.In addition,there was no significant difference in the color of the peel and the diameter of the seeds of the mature fruits between these two types,and the seeds were narrower and longer.Phenotype identification of embryos revealed that tetraploid plant is polyembryony.In the self-pollination experiment,self-compatibility tetraploid of F.hindsii was identified,and nine strains of self-incompatibility F.hindsii were obtained,which were divided into three genotypes based on the PCR amplification,including PN01(S₇S₁₂),PN02?S₂S₈?and PN03?S₄S₇?.The detection results of specific expression showed that the S gene was specifically expressed in the style.Subsequently,with PN01 and PN03 as the male and female parents,mutual cross were used to generate the progeny population.When PN01 was used as the male parent,only pollen carrying the S₁₂ genotype passed on to the offspring;when PN03 as the male parent,only pollen carrying the S₄genotype passed on to the offspring,which verified the function of the S-RNase gene and laid the foundation for future genetic transformation verification of S-locus with F.hindsii.