Construction Of High-density Genetic Maps Of Pear And Expression Analysis Of Sugar Metabolism-related Key Genes

The objects of this study were constructing higher density pear genetic linkage maps and understanding expression patterns and evolutionary mechanisms for sugar metabolismrelated keys genes.This study focused on 1)construction of two sets of high-density genetic maps based on the restriction site-associated DNA sequencing(RADseq)technology and applied to QTL identification,2)construction of an integrated genetic map using the strategy of merging maps by common markers and improvement of the anchoring of 'Bartlett' genome using genetic maps,3)analysis of gene expression patterns of sugar metabolism related genes in 'Dangshansuli'(Pyrus bretschneideri)fruits and leaves;and 4)investigation of the evolutionary mechanism for three key genes in sorbitol metabolism pathway using bioinformatics methods.The results were as below.1.A high-density pear genetic map consisted of 3143 SNP markers and 98 SSR markers that localized in 17 linkage groups was constructed based on the 'Bayuehong'(P.bretschneideri x P.communis)x 'Dangshnasuli' population using the RADseq technology.The genetic map spanned 2243.4 cM of pear genome with an average marker interval of 0.70 cM.Based on this high-density genetic map and two-years of fruit phenotyping data,32 potential QTLs for 11 traits were identified,which included the length of pedicel,single fruit weight,soluble solid content,transverse diameter,vertical diameter,calyx status,fresh color,juice content and skin smooth.Among them,some important fruit-related traits were the first time identified,such as calyx status,the length of pedicel,and flesh color.Furthermore,reliable localization of QTLs was verified repeatable.2.An integrated SSR and SNP-based consensus genetic map was constructed for pear based on common SSR markers between nine published maps.The map consisted of 5,085 markers,including 1,232 SSRs and 3,853 SNPs,and spanning 3,266.0 cM in total,with an average marker interval of 0.64 cM,which represents the highest density consensus map of pear to date.Using three sets of high-density SNP-based genetic maps with European pear genetic backgrounds,291.5 Mb of the 'Bartlett'(P.communis)genome v1.0 scaffolds were anchoring into 17 pseudo-chromosomes.These scaffolds accounted for 50.5%of the genome assembly,which was a great improvement compared with the 29.7%achieved originally.Intra-genome and inter-genome synteny analysis of the new 'Bartlett' genome v1.1 assembly with the Asian pear 'Dangshansuli' and apple(Malus x domestica)genomes uncovered four new segmental duplication regions.3.Based on transcriptome analysis of four stages of 'Dangshansuli' fruits and leaves,eight key genes were identified highly related to sugar metabolism.The expression pattern analysis revealed showed that sorbitol-6-phosphate(S6PDH)were not higher expressed than sucrose biosynthesis genes in leaves,whereas two aldolase(ALD)genes were highly expressed,indicating potential important roles of ALD in sugar biosynthesis in leaves.Two sorbitol transporters(SOT)were found highly expressed in leaves,indicating they were potentially responsible for sorbitol export from leaves.Four sorbitol dehydrogenase(SDH)genes,one alkaline/neutral invertase(ANINV)and one sucrose synthase(SUS)gene were higher expressed in fruits,indicating they were potential key genes responsible for sink unload.Alternative splicing(AS)analysis revealed that AS was related to higher expressed genes.The ratio of AS genes in sugar metabolism-related genes was higher than the average level of the whole genome,indicating a role for AS in sugar metabolism.Additionally,partial genes were verified by quantitative real-time PCR.4.The genome-wide identification of three key functional genes encoding S6PDH,SDH,and SOT genes were conducted in four Rosaceae species showed dominant sorbitol bio-pathway(named sorbitol present group,SPG,peach,mei,apple,and pear)and another three related species showed non-sorbitol bio-pathway(named sorbitol absent group,SAG,including tomato,poplar and Arabidopsis)were used as reference.The results showed that the sorbitol pathway genes in SPG expanded mainly through dispersed and tandem gene duplications.Branch-specific model analysis revealed SDH and S6PDH clade A were under stronger purifying selection in SPG.A higher frequency of optimal codons was found in S6PDH and SDH than that of SOT in SPG,confirmed the purifying selection effect on them.Besides,branch-site model analysis revealed SOT genes were under positive selection in SPG.Expression analysis showed diverse patterns of sorbitol-related genes.