Study On The Effect Of The Sepal Of Pear On Fruit Shape And Analysis Of Its Related Genes

China’s total pear output ranks first in the world,but the poor fruit shape has affected its competitiveness in global high-level trade.‘Yuluxiangli’is favored by consumers because of their excellent internal quality,but the persistence of sepals causes the appearance and intrinsic quality of the fruit to decrease.Therefore,it is important to explore the mechanism of persistence and shedding of pear fruit sepals.Based on the previous research on the regulation of ‘Yuluxiangli’ sepals,this experiment explored the effects of ‘Yuluxiangli’ sepals regulation on the appearance of quality of the fruit surface,from physiobiochemistry,histomorphology and transcriptomics.Based on whole-genome resequencing,DNA-BSA mixed pool resequencing,KASP genotyping technology and published pear-related sequencing data,genes at the molecular that associated with sepal shedding be mined;finally,members of the OFP gene family were identified,biological information was analyzed and their characteristics of expression were checked.These would provide theoretical basis for the study of sepal shedding at the molecular level and its research on fruit surface and fruit shape,as well as reference genes for early screening of high-quality pear varieties without sepal.The main results were as follow:1.Paclobutrazol PP₃₃₃（1000 mg / L）treated ‘Yuluxiangli’fruits before and after shedding of the sepals,compared with the persistence sepals,the IAA and GA decreased significantly,while the abscisic acid（ABA）increased significantly.In PP₃₃₃-treated shedding sepals,PP2C（Pbr012909.1）containing the abscisic acid negative regulatory gene of GA cis-response element（GRE）,receptor-type kinase gene RLK（Pbr025207.1）,and IAA cis-response element（ARE）genes（Pbr012908.1）and ARF（Pbr025194.1）genes were down-regulated;ABA cis-response element（ABRE）containing ethylene synthesis negative regulation gene ETO1（Pbr012910.1）was down-regulated,while ABA response elements（Pbr016952.1）,the expression of OFP negatively regulated transcription factors,increased compared with GA-treated controls.The flatness of fruit surface within calyx was significantly higher than that of wihtout calyx fruit,and there was a positive correlation between the ratio of abscised sepals in pear.That is to say,the higher the ratio of abscised calyx,the better the flatness of fruit surface was.2.After PP₃₃₃ treatment,the content of IAA in the fruit surface of the sepal shedding fruit was reduced,and the lignin distribution in the cross-section of the pulp was balanced.The cells of the pulp were densely regular,and the cells were round and small;the cells of pericarp fruit were loosely arranged and irregular,and the cells were elongated in calyx persistence fruits.Transcriptomics analysis revealed differential expression with IAA signaling pathway genes,transcription factors,and lignin anabolic pathway genes.Specifically,the IAA efflux carrier protein genes PIN（Pbr028379.1 and Pbr038852.1）,the IAA influx carrier protein gene LAX（Pbr009498.1）,and the IAA negative regulatory gene GH3（Pbr030587.1,Pbr030571.1,and Pbr021158.1）,transcription factor genes OFP4（Pbr017273.1）and OFP8（Pbr016952.1）gene expression were up-regulated,MYB（Pbr015587.1）and b HLH（Pbr001646.1）gene expression were down-regulated,expression of 4CL（Pbr024635.1）and COMT（Pbr013510.1）was down-regulated.It was showed that PP₃₃₃ affected the development of some fruit cells by affecting IAA signaling pathway genes and possibly alone or in combination with other transcription factors,thus affecting genes such as lignin biosynthesis genes and cell division-related genes.3.It was found that the ethylene signal pathway gene ERF109（Pbr004946.1）was down-regulated in the raised fruit surface of ‘Yuluxiangli’ sepal persistence pears,and transcription factors that related to the ethylene signal pathway such as WRKY41（Pbr041477.1）,MYB4（Pbr017740.1）were down-regulated;the abscisic acid synthesis gene NCED1（Pbr020310.1）was down-regulated in the bulged parts of the fruit surface.In addition,the hormone signaling pathway auxin negative regulators SAUR71（Pbr041494.1）and GH3.1（Pbr034943.1）were down-regulated in the bulged parts of the fruit surface of the sepal persistence fruit,while the ubiquitin-degrading metabolic pathway gene F-Box（Pbr013022.1）expression was down-regulated in the bulged parts of the fruit surface.At the same time,expansin EXPA8（Pbr042694.1 and Pbr014595.1）,which were related to cell division,was highly expressed in the bulged parts of the fruit surface.It was speculated that the genes of ethylene signal pathway and auxin and ABA synergistically regulated the bulged fruit surface with sepal persistence.4.WGCNA analysis of the sepal persistence fruit of ‘Yuluxiangli’and ‘Kuerlexiangli’ and the sepal shedding fruit ‘Xuehuali’ showed that some OFP genes（Pbr017273.1）that regulated fruit shape and TALE family members of BLH（Pbr005910.1,Pbr022850.1 and Pbr008379.1）and KNOX（Pbr004512.1 and Pbr016430.1）sub-family members and GA synthesis and degradation pathway genes GA3ox（Pbr032502.1 and Pbr016989.1）,GA2ox（Pbr009085.1,Pbr020983.1,Pbr025274.1,Pbr012220.1,and Pbr032502.1）in the same module.Gene expressed analysis revealed that OFP2 and OFP4 genes were highly up-regulated in the similarly fruit-shaped ‘Xuehuali’ and ‘Yuluxiangli’,and the expression of BLH1,BLH2 and KNOX genes in young fruits of ’Yuluxiangli’ and the paternal ‘Xuehuali’were higher than that of the female parent ‘Kuerlexiangli’ with persistence sepals.And the expression of GA degradation gene GA2 ox was much higher than gibberellin synthesis gene（GA3ox）.Dynamic changed gibberellin anabolic metabolism in‘Kuerlexiangli’with higher fruit shape index.Studies have shown that genes from members of the transcription factor family,such as OFP4,could participate in fruit shape development alone or in combination with other regulatory factors through the GA signal pathway.5.Analysis of the whole genome structure of sepals persistence fruit ‘Yuluxiangli’ and its male parent sepals shedding fruit revealed four structural variants of SNP,InDel,SV and CNV,corresponding to 2895,151,239,and 218 mutant genes,respectively.Focusing on GO and KEGG analysis of non-synonymous mutation SNPs occurring in ‘Xuehuali’ and ‘Yuluxiangli’,mutations in genes involved in growth hormone,gibberellin,and ethylene signal pathways such as auxin response factors ARF3 and ARF5（Pbr000415.1 and Pbr005854.1）,auxin response proteins IAA13 and IAA27（Pbr002447.1 and Pbr032907.1）,gibberellin signaling pathway DELLA gene（Pbr012085.1）,ethylene metabolic pathway ethylene inactivation genes EIL2 and EIL3（Pbr008360.1 and Pbr000646.1）,ethylene receptor protein gene ETR1（Pbr025056.1）,ethylene response transcription factor gene ERF,and ABA response element OFP negatively regulated transcription factor genes.These non-synonymous SNP genes could play a biological role in the sepal shedding through transcript levels by cis-acting elements or in combination with other transcription factors.In addition,it was found that structural mutations of InDel,SV and CNV might affect sepal development.6.The persistent sepal and shedding sepal F1 generation which hybridized by ‘Kuerlexiangli’and ‘Xuehuali’were analyzed by DNA-BSA mapping,and 29 genes that could affect sepal development were screened.Prediction of the promoter elements of the above genes revealed some hormones such as GA,IAA,and ABA,and other response elements.The analysis of the structure of the above-mentioned identified genes revealed that most contained introns and some contained no introns.By analyzing the functional annotations of databases such as COG,GO,KEGG,SWISS-PROT,real-time PCR,and public sequencing data analysis of sepal development,14 genes that might be involved in sepal shed process were screened.And KASP technology was successfully used to type SNP non-synonymous mutation sites within 7 candidate genes.7.From the identified candidate genes,analyze the OFP gene family that related to sepal development and affected fruit shape development.Twenty-eight OFP family members were identified from the white pear genome,and biological information such as gene structure,evolutionary relationship collinearity,and selective evolution of the members of the pear OFP family were analyzed.Most of the members of the pear OFP family had no intron structure,and they had a close relationship with western pear.Most members of the pear OFP gene have been purified selection during the evolution process,and their Ka / Ks <1 was relatively stable.The expression results showed that it was expressed in various tissues of pear,different growth,development and stress response,indicating that members of the OFP gene family might not only participate in sepal and fruit shape development,but also play multiple biological functions in pear growth and development.