Preliminary Study Of The Mechanism For Leaf Morphology Variation Of Cuba Liuyecheng (Citrus Sinensis)

Bud mutation is one of the most important methods for fruit breeding.Leaf,the essential vegetative organ of plants,plays a decisive role in genetic improvement.The variation of leaf shape can provide a reference for new variety breeding.The research on the morphological mutation of citrus leaf will help elucidate mechanism of the leaf development,explore important functional genes,and provide some clues for genetic breeding as well.In this study,the wild type（N）of Cubu Liuyecheng（C.sinensis）and its mutant（W）with wider leaves were used as materials.The morphology of the leaves from both the wild type and the mutant was determined and their structure variations were identified with microsection technology.Endogenous hormones were detected,transcriptome and genome resequencing were used to elucidate the variation mechanism in physiology and molecular.The main results are as following:1.According to the morphology change during the developmental process,the development of both the leaves could be divided into 5 stages,named N1,N2,N3,N4,N5,W1,W2,W3,W4 and W5,respectively.There were significant differences in leaf width,leaf-shape index and leaf area in all stages between N and W based on the field observation and analysis.The significant difference in leaf width resulted in significant differences in leaf index and leaf area.Compared the leaves between the origin and the grafted,the trait of the mutation can be stably maintained through graft propagation.2.Paraffine section,semi-thin section and SEM（scanning electronic microscope）were used to observe the differences in anatomical structure between the wild type（N）and its mutant（W）.The main difference was that the ratio of vascular bundle tissue in main vein of N was higher than that of W at all stages of leaf development.The thickness of W5 was 5.60% thicker than that of N5 via semi-thin section.The parenchymal cells of palisade tissue in W5 were of loose arrangement,while those in N5 were tighter.The spongy tissue of W5 was relatively loose with much intercellular space,while that of N5 was with smaller space.Through SEM observation,we found that the vascular bundle structure,palisade tissue and spongy tissue of N5 were tighter than those of W5.3.Four endogenous hormones in the materials were detected by HPLC-ESI-MS/MS.Results showed that ZT in the mutant was significant higher than in the wild type with the same change trends,which decreased as the development of leaves;ABA in the mutant was also significant higher than in the wild type but with different trends.It deceased as the leaf development in the mutant.However,in the wild type it decreased firstly and then increased.IAA in the mutant was lower than in the wild type with different trends.It deceased as the development of leaves.However,in the wild type it decreased firstly and then increased.Little GA3 was involved in the process of leaf development,and it was only detectable in W3 and N3,which is higher in wild type than in the mutant.4.Leaves of N1,N3,N5,W1,W3 and W5 were used for RNA-seq and then analyzed with the genome of sweet orange（citrus.hzau.edu.cn）as reference.Results showed 74.4-78.7% clean reads were mapped onto genome,which covered 72.9-78.26% of the whole genome.30921 genes were obtained totally from the 6 samples.23728 genes expressed in the wild type,while 24089 expressed in the mutant.568 genes differentially expressed between N1 and W1,207 between N3 and W3,and 669 between N5 and W5.Among all the expressed genes between N and W,eight genes（Cs1g16650,Cs1g26650,XLOC₀05927,Cs3g17390,Cs6g08500,Cs7g27120,orange1.1t03155 and orange1.1t02879）were significantly expressed at all stages.WEGO analysis indicated that the differentially expressed genes were mainly involved in cellular components such as cell,cell part,membrane and membrane part.They participated in the activities of binding,catalytic,transcription regulator etc.and played essential roles in biological process such as biological regulation,cellular process,metabolic process,pigmentation etc.The expression trends of 9 genes from qRT-PCR were consistent with those of FPKM,which showed that the results were credible.qRT-PCR results of the 8 candidate genes mentioned above at the 5 developmental stages indicated that 5 of them were significantly expressed at 1st stage of the development of leaf polarity.5.More than 14.56 G data were obtained in both the wild type and the mutant by genomic resequencing.SNP,InDel,SV and CNV variations occurred in both of them when compared with the reference genome（http://citrus.hzau.edu.cn/orange /dow nlo ad/csi.chromosome.fa.tar.gz）.There were 5390241 SNP,537847 InDel,23171 SV and 6656 CNV in the wild type,and 5410179 SNP,538048 InDel,24872 SV and 6686 CNV in its mutant,respectively.By comparing the genome of wild type and its mutant,284495 SNP and 5800 InDel,31552 SV and 6679 CNV were found between them.SNPs were found in two of the candidate genes（Cs7g27120 and orange1.1t02879）,which needs further verification.In this research the stability of the mutation trait,the differences in morphology,structure,hormones and omics were studied.Results indicated that the trait of leaf morphology variation could be maintained stably by propagation,and there are significant differences in structure,hormone content,transcriptome and genome.Eight important candidate genes and the difference in genome between the wild type and the mutant has layed the foundation for further elucidate the mechanism of leaf morphology variation.