Development Of SSR And SNP Markers Based On Whole-genome Variants And Their Potentional Application In Peach

Peach(Prunus persica L.)is a diploid species and model plant of the Rosaceae family which is widely cultivated around the wold.Peach germplasm resources are the material base of genetic improvement and scientific research,and the quantity and quality of peach can affect the sustainable development of peach directly.With the completion of peach genome and high-throughput sequencing technology,providing genome-wide information and identifying a large number of DNA variants.With the rapid development of sequencing technology and reduced sequencing cost,more and more peach genome data were obtained.How to filter and apply the sequencing data effectively are big challenges for researchers.In this study,based on the re-sequencing data of 366 peach accessions,we analyzed the genome-wide variations,screened SSR and SNP markers with high polymorphic to assess their application of genetic diversity analysis,association analysis for important agronomic traits and variety identification on peach.We also expect that our results will provide new insights into the theoretical basis for landrace and improve varieties.Overall,we have obtained the following results:1.we analyzed the variations for peach.Through re-sequencing of six distantly related peach accessions,1,166,551 single nucleotide polymorphisms(SNPs),44,245 structure variants(SVs),12,302 copy number variants(CNVs)and 141,895 simple sequence repeats(SSRs)were detected.Based on the identified SSR markers,we found mono-nucleotide and di-nucleotide repeats were abundant,accounting for 47.36%and 39.87%,respectively.We also analyzed the SSR motifs in different regions.Of all SSRs located in UTR regions,di-nucleotide repeats were the most abundant,accounting for 46.36%of all motifs.AG/GA(290)and CT/TC(266)were the most and the next was AT/TA(54),AC/CA(30),and GT/TG(23),No CG/GC repeats.Based on the whole genome annotation information,we investigated the tissue-specific expression of the genes contained more than ten SSR loci and large-effect SNPs,then a total of 9,13,22,17,and 8 genes that showed higher expression in leaves,fruits,seeds,root,and phloem than the other tissues,respectively,and these founds will help the development of molecular markers and gene mining.2.We developed 15 high polymorphic SSR markers.we filtered SSR for polymorphic,SSR types and distribution.Finally,194 SSR markers were screened for primer design,and 187 specific SSR primers were obtained.These primers were amplified in 21 peach accessions,and 15 high polymorphic SSR primers were selected.Based on the 15 high polymorphic SSR primers,we analyzed the genetic diversity of 221 accessions,constructed a neighbor-joining phylogenetic tree based on the genetic distances calculated from the genotypes at all 15 SSR positions,and three clusters were delineated.We also constructed SSR fingerprint for 200 edible peach varieties,65 ornamental peach varieties and 80 registration varieties.We also carried out purity identification for offspring.These will protect intellectual property rights for breeders and provide theoretical basis of molecular identification.3.We developed 775 high polymorphic SNP markers.Based on whole-genome re-sequencing of 360 peach accessions,16,658,391 SNPs were identified.By filtering out missing data and SNPs with a sequencing depth<10×,quality score<1000,allele number>2 and π<0.48,775 high-quality SNP markers with high polymorphic were selected.Using the sequencing data for the 775 SNPs in the population,we constructed a neighbor-joining phylogenetic tree based on the genetic distances,which classified the 360 peach accessions into two major groups.we also performed association studies for flesh adhesion,flesh color,fruit hairiness,fruit flavor,fruit shape,pollen fertility traits and maturate date.We identified a SNP(Pp04:24,189,168 bp)that was the most strongly associated with flesh adhesion,a SNP(Pp01:29,561,885 bp)for flesh color,a SNP(Pp05:13,889,348 bp)for fruit hairiness,a SNP(Pp05:494,049 bp)for fruit flavor,a SNP(Pp06:28,389,195 bp)for fruit shape,and a SNP(Pp06:28,389,195 bp)for pollen fertility.Next,we developed a minimal SNP set for variety identification and constructed SNP fingerprint for 80 edible peach varieties,29 ornamental varieties and 70 registration varieties.In addition,we identified a genome region with the highest SNP density(129 SNPs in 1000 bp),which might allow discriminating the 360 peach accessions using a single PCR.We believe that a single PCR for the amplification of this region would allow the differentiation of all the peach accessions in this study.