The Cellular And Molecular Mechanisms Of Pome Size Domestication In Eriobotrya Japonica

The genus Eriobotrya Lindl.belongs to Maloideae and contains more 26 speciees or formas,and it sets pome fruit as apple does.Among all these species,Eriobotrya japonica is the very specieces domesticated for fruit production,and it weights from 5 g to 200 g randomly according to diverse improvement-utilization levels.There is large size cultivars famine in loquat,which restraints the fruit production.To date,the cellular inhibitions of fruit growth are remained to be elucidated,so does the molecular regulation of cell progressions and pome size domestication.Lacking of these studies impedes human knowledgement on loquat fruit growth and is disadvantage to large size cultivar breeding and fruit production management.The cellular measurements of developmental and mature fruits in this study aim to find out the cellular causes of fruit size formation and pome size domestication during loquat evolution.Function analyses of homologous genes,RNA-sequencing and whole genome resequencing of diverse loquat plants were takan to screen seleted loci which contribute to fruit size enlargement of E.japonica as was gradually domesticated.The main results were as follows:(1)Whole developmental measurements of fruit growth and cell progression showed that low cell proliferation rate during early development resulted into few cell layers in coretex which inhibited coretex thickening and fruit enlargement.A weekly cellular scenario for loquat fruit development was established in this study as well.The cellular scenario showed that fruitlet started and continued peak cell division for about 6 weeks soon after fertilization and then stop cell production after that.Cells began enlargation from 14 days past anthesis,and a peak cell expansion phase started at 84 days past anthesis.Transcription analyses of homologous genes demonstrated that the expression patterns of EjCDKB2;2 and other 4 core cell cycle genes correlated to cell division intensity in fruits,meanwhile the exprssionn of EjEXPA1 and EjEXPA15 were excellent markers to show cell expansion intensity.In addition,EjFWLs seemed to be repressors of cell division while EjWEE1 and EjKRP3 were supposed to be envolved in cell cycle exit.(2)Two fw2.2 homologs,EjFWL1 and EjFWL2,were isolated from ‘Zaozhong-6' cortex.The expression patterns of these genes negatively correlated to that of cell division marker genes mentioned aboved in cell division phase receptacles.Overexpression of EjFWL1 in tomato,inhibited cell division and ovary and fruit size,as ovary diameter decreased by 5.92-18.05%,cell layers decreased by 4.56-31.23% and fruit diameter and fruit weight decreased by 8.04-19.05% and 19.83-40.56%.And all the size changes were related to down regulating core cell cycle gene expression.Gene expression comparation of EjFWLs at early fruit development revealed that expression level cooperated with peak transcriting time in fruit size determination via repressing cell division.Treatments like CPPU and bagging verified that advancing of EjFWLs expression promoted cell division in both receptacle and petal as large size fruit did.Located to membrane,the EjFWLs were shown to interact with EjAGL2 and EjCK?,which located to both nuclear and membrane,at membrane.(3)Leave ploidy and fruit cell detection of ZP44 and ZP65 lines,revealed that both lines were diploid plants,not polyploidization took place on large-fruited ZP65.Cell number and cell size co-operate one another regulating loquat fruit growth,with cell size contributes more than cell number.(4)10 developmantal fruit samples of ZP44 and ZP65 were taken for RNA-seq.A total of 37598 unigenes were assembled with the clean reads.For these unigenes,the sequence reached a length of 1213 bp on average.Differential exprrsion analyses figured out a set of 17541 DEGs(differential expression genes)among these samples.Common expression clustering via k-means algorithm classified these DEGs into 30 clusters,according to their expression patterns.Among these,cluster 6,cluster 14,cluster 23 and cluster 25 correlated greadly to fruit growth.Function annotation demonstrated that these DEGs were involved in pathways and cell precesses like plant hormone signal transduction,brassinolide biosynthesis,plant-circadian rhythms,zeatin biosynthesis,ribosome biogenesis,RNA polymerase and SCF-ubiquitin ligase complex.(5)95 homologs,which were involved in fruit or organ size regulation in tomato or Arabidopsis,were identified in loquat.The cell division related genes such as AHP1,TCP14 and ANT showed higher expression levels in ZP65 during early development,while cell size regulation genes like S6 K,CYP85A,CYP90A1,bHLH137,SAUR and IAA16 all up regulated expression during later fruit development.The expression of size regulation genes implied that Auxin signal transduction,brassinolide biosynthesis,ribosome biogenesis as well as transcript factors like TCPs and bHLHs played crucial roles in fruit size regulation.(6)80 DELLA,31 GID and161 genes being involved in Auxin signal transduction were identified from loquat genome database.Expression profiles of these genes showed that 8 GID and 10 DELLA were supposed to be correlated to fruit size.Obvious different exprrsion were showed in 8 Aux/IAAs,4 ARFs and 11 SAURs,like Eri037021.1,Eri022289.1 and Eri015123.1,among ZP44 and ZP65.All these data implied that both Gibberellin signaling and Auxin signal transduction play vital roles in fruit cell size regulation.(7)The correlationships of fruit size and cellular characteristics were analyzed on 9 Eriobotrya plants,which contained 5 wild loquat species like Eriobotrya deflexa and 4 common loquat(including wild grown loquat from Ruyuan,large size ZP65,small size ZP44 and their female parent Zaozhong-6).And we found that cell number and cell size act together in pome size regulation,while cell size contributed more greatly to fruit growth.This suggested that larger cell size generally gave rise to large size fruits,and implied that fruits with more cell and larger cell size were under artificial selection during loquat domestication.(8)Whole genome resequencing of 29 Eriobotrya plant divided plants into a wild loquat cluster(with 6 species including Eriobotrya henryi,Eriobotrya prinoides,Eriobotrya daduheensis,Eriobotrya elliptica,Eriobotrya deflexa and Eriobotrya deflexa f.koshunensis)and a common loquat cluster.The common cluster was further classified into a wild group(including 9 plants such as Yunnangushu,Puye and Doupipa)and a cultivated group(including Zaozhong-6 and some other 13 cultivated plants).These identified the hypothesis that cultivated loquat was singly domesticated around the world in China.119 loci under selection were identified by genome comparisions on wild grown and cultivated loquat.These loci were involved in physiological progressions like Plant-pathogen interaction,Auxin biosynthetic process,Carbohydrate biosynthetic process,Cell redox homeostasis,Cellular response to abscisic acid stimulus,Regulation of transcription,Auxin polar transport,Cellular response to cold,Cell cycle regulator,Photosynthesis,Ribosome biogenesis,Carbohydrate metabolic process and Auxin signal transduction.(9)According to the phylogenetic tree drawn via SNPs comparison,all the resquencing plants were classified into small and big fruit size groups and association analyses were taken to detecte loci correlate with fruit size.A total of 14 genes were co-arase in selection analyses and these association analyses,and were supposed to be involved in pome size domestication in loquat.Expression analyses implied that 8 of these genes,IAA16,IAA3,SAUR15 A,UBP13,AAE18,MPK9,TMV and WAT1,were supposed to correlate with cell division or cell enlargement.(10)Stable gene variations were detedted in Eri036655.1,Eri036656.1 and Eri015123.1 among the small size-wild grown loquat and the large size-cultivated loquat.The expression patterns of these genes in wild and cultivated loquat fruits demonstrated that the gene changes especially that in Eri036655.1 and Eri036656.1 resulted into higher expression of downstream genes of Auxin signal transduction pathway in cultivated ZZ than the wild plant,which may contribute to cell enlargement as fruits grown.This implied the crucial roles these genes may play in fruit development and domestication.