Genetic Analysis And Cloning Of The Related Genes Of A Rolled Leaf Mutant In Peanut(Arachis Hypogaea L.)

Peanut is one of the most important oil crops and economic crops in the world.It is important to improve the yield of peanut.As the main organ for photosynthesis,leaf morphology is one of the most important agronomic traits of peanut.As the focus of plant-type breeding,change of leaf morphology can affect the physiological function of photosynthesis and stress resistance,and then affect the growth and yield of peanut.Researches show that moderate leaf curl is beneficial to keep leaf upright without drooping,raise light transmission rate of individual and group in the lower part,increase the photosynthetic area and improve the light use efficiency,which lays a good foundation for plant yield improving.Therefore,the study of the genes associated with leaf curl is of great significance for the breeding of ideal plant type,high yield and breed improvement.In this study,a semi-dominant rolled-leaf mutant,named rl1（rolled leaf 1）,obtained from peanut cultivar Fenghua 1（FH1）mutagenized by ⁶⁰Co-γrays,was used as the material for analysis.The characteristics of rl1 were analyzed from genetics,cytology,physiology and biochemistry,and RNA-Seq.The main results were as follows:1.Hybridization was made between rl1 and FH1 for genetic analysis.All the leaves of the F₁ plants showed half-rolled,while the F₂ generations showed phenotypic segregation,with rolled,half-rolled and normal leaf morphologies.The ratio of these three phenotypes was consistent with the 1:2:1 ratio,which indicated that the traits of rl1 maybe controlled by a single semi-dominant nuclear gene.2.The leaves of rl1 rolled inward extremely during the whole growth period,with plant height shortening and plant-type compact.In addition to leaf,the petiole,gynophore,pedicel and root also showed some degree of bending.From the flag to the third,leaf rolling index of the main and lateral stem of rl1decreased gradually.The phenotype of F₁ generations showed an intermediate form between FH1 and rl1.2.By comparison of plant morphology of rl1 and FH1,we found that the height and stem diameter of rl1 were significantly lower than that of FH1.Yield statistics analysis results showed that the rl1 kernel rate,full fruit rate were slightly higher than that of FH1,while 100-kernel weight,100-pod weight and average yield per plant were significantly lower than that of FH1.4.The tissue structure of rl1,F₁ generation and FH1 were observed by paraffin section.The results showed that,compared to FH1,the aquiferous tissue cell of rl1 was thicker,cells with different sizes and loosely arranged,palisade aquiferous ratio was minimum,leaf vein arranged in disorder and veinlets dense.Results of cross section of petiole showed two small vascular bundles next to two larger vascular bundles at the petiole port of rl1 and F1 generation,and disappeared alternately along the direction of petiole bending,while in FH1 this phenomenon did not appear.Cross section of gynophore showed that the vascular bundles of rl1 were not uniform,and the size of vascular bundles on both sides of bending site was different.Cross section of shoot vascular bundles showed that rl1 had the thinnest phloem and xylem.However,there was no significant change in the cross section of roots.5.Results of scannig electron microscopy showed that the upper epidemis of FH1 and rl1 leaves were coverd with waxes,while there was no wax in the lower epidermis.The waxes of FH1 are long and dense,and the waxes of rl1 were significantly shorter and formed vertical sheets.The density of stomata and lower epidermis trichomes of FH1 were significantly higher than that of rl1,and the size length and width of epidermis trichomes was slightly larger than that of rl1,while the stomatal aperture of rl1 slightly larger than that of FH1.6.The analysis of chlorophyll content showed that the content of chlorophyll a,chlorophyll b,chlorophyll a+b and carotenoid of rl1 were significantly higher than that of FH1,while the ratio of chlorophyll a to b was significantly lower than that of FH1.7.Transcriptome sequencing（RNA-seq）data of rl1 and FH1 were analyzed.Among the differentially expressed genes in four tissues,the expression level of c95764 transcript in FH1 was significantly higher than that in rl1.Sequence analysis showed that c95764 had no open reading frame,and located on 5519366to 5519900 of peanut Aradu.A03 chromosome,and there were several copies in the whole genome with similarity exceeding 90%.By blast in peanut CDS database,we found that the similarity between c95764and Araip.6T6S8 reached 90.48%.It is speculated that there may be some relationship between them,so we selected the c95764 transcript for further study and named as c95764 gene.8.The c95764 gene was cloned and the sense and antisense plant expression vector were constructed and transformed into rl1 and FH1 respectively.The transgenic plants were confirmed by kanamycin selection and PCR verification.One overexpressing transgenic plants has been obtained.The overexpressing transgenic peanut partially restored the rolled phenotype of FH1.