Construction And Application Of SSR Characteristic Fingerprinting Data Table For Pear

Pear belonging to the Pyrus of the subfamily Romoideae in the family Rosaceae,is perennial deciduous trees or shrubs.The origin centers of Pyrus is China,which is abundant in Pyrus germplasm resources deposits.Moreover,the cultivation area and fruit production of pear ranked the third in fruit tree production in china,and ranked first place at the cultivation area and fruit production of pear in the world.Furthermore,thereareagreat variety of pearsinChina.Then,it is very hard for pear germplasm identification on the basis of morphological characteristics.A more reliable and convenient approach is DNA molecular marking technique.SSR molecular marker has the features of high efficiency,good repeatability and high polymorphism.Developing enough SSR molecular marker is very meaningful for the construction of genetic linkage map,genetic diversity analysis and molecular marker-assisted breeding.The purpose of our study is screening out suitable SSR core primers which can accurately and effectively evaluate pear germplasm.It is very meaningful for variety protection,safeguarding the rights of breeders and the healthy development of fruit industry.In our study,1334 SSR primer pairs were initially screened on 12 pear germplasmswith distant genetic relationship.The SSR primer pairs consisted of 800 pairs of SSR primers from the articles about pears and apples,and 534 pairs of SSR primers developed from pear genome by our team.Then 48 pear germplasms with different geographical origins were chosen to screen the initial selected primers.Then the second selected primers were screened by fluorescence SSR markers detection technique with capillary electrophoresis.Finally,a set of SSR core primers were identified,which was suitable for genetic diversity analysis,construction of fingerprint database and identification of pear cultivars.Then,125 pear germplasms were analyzed using the core primers to conduct studies of genetic diversity and cluster analysis.PowerMarker version 3.25 software were used to calculate genetic diversity of pear cultivars.NTSYS-pc2.10 software were used to conduct the cluster analysis.The major results as follows:1.Optimization of SSR-PCR in pear cultivars:The optimized reaction conditions for SSR in thisstudy are as follows:The PCR mixture was performed in a final volume of 20?L containing1×PCR Buffer(Mg²⁺),2.5 mM dNTPs0.2 mmol/L,primer 0.5?mol/L,Taq DNA polymerase 0.025 U/?L,DNAtemplate2.5 ng/?L.2.Screening of SSR Core Primers for pear cultivars:A total of 25 pairs of SSR core primers with high polymorphism and good repeatability were identified by polyacrylamide gel electrophoresis and capillary electrophoresis.Then,the genetic diversity of 48 pear cultivars were analyzed using these 25core markers.The results showed that 387 alleles were amplified and the number of alleles per locus ranged from 9 to 22,with an average value of 15.48.The locus heterozygosity ranged from 0.44 to 0.92,with an average value of 0.76.The polymorphic information content?PIC?ranged from 0.70 to 0.92,with an average value of 0.85.The genetic diversity index ranged from 0.73 to 0.92,with an average value of 0.87.These indexes showed that the 25 pairs of SSR primers with high polymorphism can evaluate 48 pear cultivars genetic diversity effectively.Based on the SSR data,the similarity of 48 pear cultivars investigated in this study could be depicted in a UPGMA phenogram.The dendrogram showed that the results of molecular clustering largely agreed with the pedigree and geographic origin.These results showed that the 25 pairs of SSR primers with high polymorphism were the core primers for pear germplasm identification.3.Construction of SSR characteristic fingerprinting data table and Molecular ID for pear cultivars:124 pear cultivars were genetically identified by the 25 pairs of SSR core primers.Among them,115 pear cultivars could be differentiated successfully except for 9 relative cultivars in group 4,and 21-69 genotype were amplified.According to the number of genotype,the 25 pairs of SSR core primerswere encoded from most to least.Moreover,the pear cultivars were encoded and the data were marked by ground color for distinguishing by excel.Then,SSR characteristic fingerprinting data table for 124 pear cultivars was generated.Assignment the top 10 SSR loci on the SSR characteristic fingerprinting data table used the number from 00 to 99.Finally,Cascading the number of assignment according to the SSR characteristic fingerprinting data table generated the molecular ID for 124 pear cultivars.‘Fuanjianba'and other 99 cultivars could be differentiated using only 1 pair of primers,‘Chech 1'and other 16 varieties could be differentiated using combination primers,and another 10 pear cultivars only generated one characteristic genotype in 25 SSR loci.And on this basis we constructeda molecular ID using the top 10 SSR fingerprint date on the SSR characteristic fingerprinting data table.The 115 cultivars which were differentiated successfully have their unique molecular ID.4.The genetic diversity analysis of 125 pear germplasms by 25 pairs of SSR core primers:453alleles were amplified in 125 pear cultivars by 25 pairs of SSR core primers,and the number of alleles per locus ranged from 9 to 26,with an average value of 18.12.The number of genotypes ranged from 21to 72,With an average of 46.52.The polymorphic information content?PIC?ranged from 0.71 to 0.92,with an average value of 0.85.The UPGMA cluster analysis showed that 125 pear germplasms could be divided into into two major clusters,including the Asian pear and the European pear.Chinese white pears?P.bretschneideri Rehd.?,Chinese sand pears?P.pyrifolia Nakai?,Ussurian pears?P.ussuriensis Maxim.?,Sinkian pears?P.sinkiangensis Yü?and wild Duli were clustered together.Moreover,European pear?P.communis L.?and Sinkian pears?P.sinkiangensis Yü?‘Duxiaxi'were grouped together.