Development Of Apple SSR And SNP Markers And Application In Genetic Map Construction And Cultivar Identification

Apple?Malus domestica Borkh.?is one of the most important fruit crops widely cultivated in temperate regions of the world.It plays an important role in fruit industry in China.The development of apple commercial planting mode puts forward higher requirements for the new cultivars.At present,the main approaches of breeding new apple varieties includes conventional hybridization,bud selection,seedling selection and mutation breeding.Among them,conventional cross breeding is expected to obtain excellent variation types through gene recombination,and considered to be principle pathway to achieve innovation and utilization of apple germplasm.However,apple is a perennial crop with highly heterozygous genotype.The ong growth cycle and juveniale phase of seedling bring many difficulties to apple breeding.With the development of bioinformatics and molecular biology,molecular marker-assisted selection can effectively accelerate apple breeding process,but developping valuable molecular markers is one of preconditions of molecular breeding for apple.In this study,the apple genome sequences publicly published were used for analyzing the types and distribution features of apple SSR markers by the bioinformatics methods,and the PCR reaction system was established and optimized.Moreover SNP markers were developed through whole genome re-sequencing technology,and single nucleotide variations also were recognized.The genetic map of apple was constructed using SNP and SSR markers,and finally many apple varieties were identified on the basis of simple sequence repeat markers.This study aimed to lay the foundation for the mapping of genes related agronomic characters and molecular markers assisted breeding in apple.The main results were as follows:?1?SSRs from the apple genome were screened using MISA software,and the SSR primers were designed by primer3_core program based on the Perl language platform.There were 163,426 microsatellite repeat sequences in the apple genome,with an average of 1 SSR per 3,219.79 bp,one to six nucleotides motifs types could be detected and they were distributed unequally on the 17 chromosomes of apple.Orthogonal design was applied to optimize SSR-PCR amplification system of apple in five factors such as primers,Taq polymerase,Mg²⁺,dNTP and DNA template.An optimal SSR-PCR reaction system of apple was established,and reaction mixtures in 20?L contained primers 1.0?mol·L^-1,Taq polymerase,0.01875 U·?L^-1,Mg²⁺,1.50?mol·L^-1,dNTP,0.15?mol·L^-1,and DNA template5.0 ng·?L^-1.?2?A whole genome re-sequencing project on'Fuji'and'Golden Delicious'was carried out.Pair-end sequencing strategy was used on Illumina Hiseq 2000 platform.The read length was 100 bp with an average of 500 bp insertion fragment.Throurh mapping with reference sequnence,3,183,680 SNPs were detected in'Fuji'and'Golden delicious'in all,and there was one polymorphic SNP marker every 270 bp in apple genome.Anylysis results showed that their variable frequencies were significant difference between transitions?A/G,G/A?and transversions?A/C,A/T,G/C,G/T?,such as 66.98%and 33.02%,and the ratio between them was 2.03.?3?122 individuals in F1 population derived from‘Fuji'x‘Golden Delicious'and the two parents were used to construct genetic linkage map with SNP markers developed via molecular biological techniques,combined with SSR markers.When constructing map with Joinmap 4.0,the threshold value of LOD as 3.0 was set to distinguish linkage groups,and genetic distance based on SNP markers were estimated by Kosambi's function and regression algorithm.Finally,301 markers were mapped on 17 linkage groups.The results showed that the genetic map spanned 3,410.76 cM,and the distance between adjacent markers was 11.33 cM.In additions,linkage group sizes ranged from 88.65 to 326.63 cM among groups,and average length was 200.63 cM.?4?After analysing SSR characteristic of the whole apple genome,8 pair SSR primers with good polymorphism were selected and identified apple varieties by new method named MCID.60 apple varieties were successfully distinguished from each other based on analyzing the SSR-PCR fingerprints,and an apple identification diagram was established finally.