Population Genetic Analysis Of Peach Germplasm And The Construction Of Digital Gene Expression Profilings Of Fruit

Peach （Prunus persica （L） Batsch） is one of the most prevalent stone fruits in the Rosaceae family, genus Prunus, due to its excellent taste and richness in nutrients. More than2000new commercial cultivars have been released using traditional breeding approach in the past20years, however, the genetic diversity is not increased with the growing number of cultivars because of a limited number of founders selected and narrow genetic background in breeding programs. Many research has been done on peach genetic diversity, but the result are mainly adapted for the tested samples, since the genotypic data are not capable to be joint for comparison analysis due to the different markers, plant material and analytical methods selected. Being the center of origin of peach, China has the longest history of peach cultivation （more than4000years） and the richness of genetically diverse germplasm after a long term natural selection and artificial domestication. It will be great helpful for peach breeding program to select the most powerful parentage material.In our research,48highly polymorphic SSR markers were selected for genotyping434peach genetic resources with ABI3130system, and results were combined with the genotypic data of224occidental peach resources obtained with the same method. The comparison analysis was conducted to evaluate the difference of genetic diversity between different peach groups. Finally, a peach genotype database was constructed with15fluorescence-labeled SSR markers selected. Two hundred and eighty one Chinese peach resouces were chosen to construct Illumina Infinium SNP chips according to information provided the linkage disequilibrium （LD） results in different populations. SNP loci which were related to seven qualitative traits （leave gland, flower showy, flesh color, flesh texture, fruit hairness, flesh adhesion and fruit shape） were defined based on genome wide association study （GWAS）.Aroma and texture are two important factors to evaluate the peach fruit quality, and both of them are complex traits regulated by multiple gene families. A great progress has been made on aroma and texture functional related genes based on candidate genes, EST library and microarray, however, the molecular mechanism is still unclear because of the limited number of detected genes. In this study, twelve digital gene expression profilings were constructed with the RNA samples from4commercial peach cultivars （’Hu Jing Mi Lu’,’Yu Lu’,’Jin Xiu Huang Tao’and ’Zhong Hua Shou Tao’） and three ripening stages of ’Hu Jing Mi Lu’ using high-throughput Illumina RNA-seq technology. The molecular mechanisms both for aroma compounds biosynthesis and fruit texture change were analyzed from a whole transcriptome view. The main results were as follows:1. Analysis of genetic diversity of658peach accessions based on48SSR markersIn total, five hundred and eighty eight alleles were obtained with48SSRs on653peach accessions, giving an average of12.25alleles per locus. The observed heterozygosity （Ho） ranged from0.13（PMS02） to0.63（UDP96-005）, with an average value0.47. Genetic diversity in Oriental group significantly was significantly higher than Occidental group, and the inclusion of Orientalaccessions into the occidental group can significantly increase genetic diversity. Genetic distance clustered the658accessions into two main groups according to the geographic origin, domestication history and fruit traits:non-persica species （wild related species） were separated from the Prunus persica accessions, old landraces were separated from cultivars, Oriental accessions were separated from Occidental accessions, and Occidental nectarines were separated from Occidental peaches. Two hundred and eighty one accessions were chosen for the construction high density SNP chips. Linkage disequilibrium （LD） analysis in each of these subpopulations showed that LD decayed faster in Oriental （3.36cM） than in Occidental （5.50cM） samples. In’Yu Lu’ peaches considerable LD extension was observed, while no variation of LD extension with linkage map distance was observed in the landraces. Within all three sub-populations from the first STRUCTURE result, LG1had the greatest proportion of allele pairs in LD.2. The construction of peach genotype database with peach genotype database construction with fluorescent-labeled SSR markers and its applicationFifteen selected SSRs could be grouped into3multi-plex groups and used conveniently under the ABI3130system. The current fingerprint database comprised of669accessions, among which426（95%） of peach cultivars from Chinese germplasm have their own specific genotype. One hundred and fifty nine core collections with specific genotype, including8Chinese reference cultivars, had been listed in preliminary peach SSR marker fingerprint database. The pedigree information of seven groups of Chinese cultivars were confirmed to be coincide with known reference, but for those Japanese cultivars, the pedigree information was deviated from our analysis.3. Genome wide association study （GWAS） based on9K high density SNP chips.Using the9K SNP chips of281Chinese peach accessions,3781SNPs with the minor allele frequency higher than0.03were selected. One hundred and fifty nine core collections were selected for germplasm saving, keeping and GWAS after SNP genotyping. Phylogenetic dendrogram with Neighbor joining algorithm showed high agreement with Structure assignment, and also verified the results obtained from48SSR markers. The results from GWAS showed that SNP IGA544512located on Scaffold5was the most significant SNP which were associated with the shape of leave gland. SNP_IGA₁03771and SNP_IGA₆8612located on Scaffold1and Scaffold6, respectively, were significantly associated with flower showy type. SNP IGA93479and SNP IGA93768located on Scaffold1were associated with flesh color. Three SNPs （SNP_IGA₄77941、SNP_IGA₄77945、SNP_IGA₄77951） on Scaffold4were associated with fruit texture melting/non-melting. SNP_IGA₅96063on Scaffold5were significantly associated with fruit skin hairness. SNP IGA610889and SNP IGA124466located on Scaffold6and Scaffold1, respectively, showed highly association with flesh adhesion. SNP_IGA₆98099, SNP_IGA₆97021and SNP_IGA₆97026on Scaffold6were significantly related to fruit shape.4. Volatile and texture related genes identification using digital gene expression profilingsTwelve DGE profiles, constructed using three ripening stages of’Hu Jing Mi Lu’ and three other peach cultivars, generated approximately89881875reads with average read lengths of100bp. Around78.5%clean sequence reads were mapped to the peach reference genome. The qRT-PCR validation with15aroma and texture-related genes showed highly correlation with RNA-seq results. The expression patterns of4431similar functional genes were clustered into24groups during the ripening process, while5541differentially expressed genes were grouped into14clusters among four different cultivars. Significantly different expression levels of lipid transfer protein（LTP）, acyl-CoA oxidase （ACX1） and epoxide-hydrolase （EPH1） was observed during fruit ripening stages, which associated with lactone formation. The transcript abundance of LTP increased from S1to S2, then decreased at the final ripening step S3. LTP may also contribute to fruit softening. A new member of the ACX gene family, ACX2, was inferred to be associated with y-decalactone formation due to its specific C18substrates. Among all the KEGG pathway, large number of differential expressed genes were detected in flavone and flavonol biosynthesis both in different ripening stages and different cultivars.