Study Of EQTL And Gene Co-Expression Network On Stone Cell In Pear

Pear(Pyrus)belongs to the Malinae subtribe of the Amygdaloideae subfamily within Rosaceae.It is one of the important temperate fruit trees,favored by the public,and has high economic value.However,different from other Rosaceae species,pear accumulates a large amount of stone cells in the flesh of its fruits;these thickened cell clusters of secondary wall materials cause a gritty texture and contribute to poor fruit taste and negative consumer satisfaction.At present,traditional local varieties are the main cultivated varieties in China.The high stone cell content of these varieties leads to a lack of market competitiveness and has become one of the important factors restricting the healthy development of pear industry in China.Therefore,it is urgent to understand the regulatory process during stone cell formation.However,many previous studies have focused on the regulation of a small number of structural genes or transcription factors on the lignification process.Our understanding of the gene regulatory networks of stone cell in pear fruit remain limited,and its metabolism pathways are known to be complex.In this study,the multidimensional strategies,including expression quantitative trait loci(eQTL)mapping,gene co-expression network,comparative transcriptomic,or differentially expressed gene analyses,were used to determine the core factors controlling the stone cell formation based on gene expression data in fruits.The mains results are as follows:1.We collected a total of 206 sand pear cultivars(P.pyrifolia)that collectively represent broad genetic diversity and large phenotypic variability.Global gene expression profiling using RNA-seq was carry out on fruit flesh samples collected from each variety at 49 days after full blooming(DAF).A total of 35,897 genes and identifying 494,844 high-quality SNPs.Using weighted gene co-expression network analysis(WGCNA),we identified 9 stone cell formation modules and constructed one lignin and cellulose co-expression network consisting of 159 transcription factor genes and 72 structure genes.Expression quantitative trait loci(eQTL)analyses identified 609 local eQTLs and 75,179 distant eQTLs for 609 and 3,223 genes from stone cell modules,respectively.According to the eQTL distribution,35 master genes from 85 distant eQTL hotspots were identified,and seven co-regulated gene pairs were found.Combining WGCNA and eQTL analyses,genetic regulatory networks was constructed,and in which bHLH169 could regulate lignin and cellulose biosynthesis pathway,while MYB84 could regulate lignin biosynthesis pathway.This study uncovered major insights about the complex gene regulatory network controlling stone cell formation,and providing candidates for mining important functional genes.2.We collected ’Dangshansuli’ pear and ’red Fuji’ apple fruits at different developmental stages for transcriptome(RNA-seq)sequencing.We showed a global comparative transcriptomics analysis between lignified pear and un-lignified apple fruits,identifying specific biological processes and functional genes during stone cell formation.A total of 28,195 orthologous gene groups consisted of 38246 pear and 34396 apple genes were indentified,providing a basis for comparison of gene expression patterns.RNA-seq-based WGCNA co-expression networks were established in the two Rosaceae species.For pear co-expression networks,we associated modules with lignin content,and discovered a specific module and many specific biological processes for stone cell formation including light response,hormone response,induction of programmed cell death,and plant defense.The comparative analysis of gene co-expression network across the two species was performed based on independent co-expression networks and orthologous gene pair,and 32 transcription factors within the core cross-species modlue were identified as potential genes involved in stone cell formation.PbrMYB24 appears to be a transcriptional activator of lignin biosynthesis genes 4CL1,CCR1,CCR2 and CCOMT2 and regulates secondary wall formation of stone cells in pear fruit.This study presents the power of comparative transcriptomics analysis across species to uncover the regulatory candidates associated with the biosynthesis and accumulation of the stone cell.3.We performed a genome-wide identification,structural and evolutionary analyses for the MADS-box gene family,and identify differentially expressed MADS-box genes related to stone cell formation through transcriptome data.A total of 95 MADS-box genes were identified in the pear genome,and classified into two types by phylogenetic analysis.Type Ⅰ MADS-box genes were divided into 3 subfamilies and type Ⅱ genes into 14 subfamilies.Synteny analysis suggested that whole-genome duplications have played key roles in the expansion of the MADS family,followed by rearrangement events.Purifying selection was the primary force driving MADS-box gene evolution in pear,and one gene pairs presented three codon sites under positive selection.Full-scale expression information for PbrMADS genes in difererent organs was provided and proved by reverse transcription PCR analysis.Furthermore,the 7 MADS-box genes were predicted as important candidate genes related to lignin biosynthesis during stone cells formation based on differential expression analysis.