Core primers screening for SSR markers and cluster analysis in theplants of Pyrus spp.

 Abstract:【Objective】China is one of the origin centers of Pyrus plants with abundant deposits of Pyrusgermplasm resources. To date, experts have carried out a lot of research on pear cultivars by SSR markers,such as genetic diversity, population structure analysis and construction of fingerprint database, etc.Screenings of primers were necessary for these researches. However, the screening of SSR core primers ongenetic diversity analysis, construction of fingerprint database and identification of pear cultivars had rare⁃ly reported in the literature yet. SSR core primers should have the features of high efficiency, good repeat⁃ability and high polymorphism. The purpose of our study is to screen out suitable SSR core primers, whichcan be accurately used for evaluation of germplasm genetic diversity and genetic relationship of pear cultivars.【Methods】1 334 pairs of SSR primers were initially screened using 12 pear（Pyrus. L）germplasmresources with distant genetic relationship. 1 334 pairs of SSR primers that consisted of 800 pairs of SSRprimers for apples and pears searched from the articles and 534 pairs of SSR primers developed from peartranscriptome were screened. The markers with clear PCR amplified results, unique amplified loci, goodstability and repeatability were Chosen. Then 48 pear germplasm resources with different geographical origins were chosen to screen the initial selected primers. Polyacrylamide gel electrophoresis and ABI3730XL DNA analyzer were used to describe the amplified results, and the secondly selected primerswere screened by fluorescence SSR markers detection technique with capillary electrophoresis. Finally, aset of SSR core primers were identified, which could be suitable for genetic diversity analysis, construction of fingerprint database and identification of pear materials. Moreover, Genetic diversity and clusteranalysis of 48 pear materials were performed to verify the effectiveness of these core primers. The geneticdiversity of 47 pear cultivars and 1 wild germplasm resource were analyzed by Power Marker version 3.25.The cluster analysis were displayed by NTSYS-pc2.10.【Results】A total of 131 pairs of SSR primers wereinitially identified from 1 334 pairs of SSR primers by polyacrylamide gel electrophoresis. A total of 25pairs of SSR core primers with high polymorphism and good repeatability were identified through secondscreening in 48 germplasm resources by polyacrylamide gel electrophoresis and capillary electrophoresis.The primers were located in linkage group 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 15, 17, respectively, with an averageof 1.85 core marker per linkage group. Only 1 SSR marker was located in 1, 6, 7, 8, 9, 11 linkage group;Linkage group 5 harbored the largest number of markers, which contained 5 SSR loci. Then, the geneticdiversity of 48 pear germplasm resources were analyzed using these 25 core markers. The results showedthat 387 alleles were amplified and the number of alleles per locus ranged from 9 at ZFRIt480 to 22 atZFRIt175, with an average value of 15.48. And the average number of genotype per locus was 27.32. Thelocus heterozygosity ranged from 0.44 at ZFRIt045 to 0.92 at ZFRIt175 and ZFRIt260, with an averagevalue of 0.76. The polymorphic information content (PIC) ranged from 0.70 at ZFRIt480 to 0.92 atZFRIt175, with an average value of 0.85. The genetic diversity index ranged from 0.73 at ZFRIt480 to0.92 at ZFRIt175, with an average value of 0.87. These results showed that the 25 pairs of SSR primerswith high polymorphism could evaluate the genetic diversity of the 48 pear materials effectively. Based onthe SSR data, the similarity of the 48 pear materials investigated in this study could be depicted in a UPG⁃MA phenogram. The dendrogram showed that the results of molecular clustering largely agreed with thepedigree and geographic origin. The 48 pear materials were obviously divided into two major clusters, including the Asian pear and the European pear. The Asian pear included cultivated species and wild spe⁃cies Pyrus belulaefolia Bge which were further separated as two major sub-clusters. In the sub-clusters ofcultivated species, pears cultivars belonging to P. ussuriensis were clustered together, such as‘Xiaoxi⁃angshui’‘Xiehuatian’‘Pingxiangli’and‘Qiuxiang’, etc. Moreover, pears cultivars belonging to P.bretschneideri and P. pyrifolia were clustered together, which weren’t completely separated. In the 26 P.bretschneideri cultivars and P. pyrifolia cultivars, Chinese P. pyrifolia cultivars‘Huobali’‘Wanshuhuobali’‘Xiangyunhuobali’and‘Miduxiaohongli’, etc which were originated from Yunnan were gathered together, and separated from the other Chinese P. pyrifolia cultivars. 9 Chinese white pear cultivars including‘Dangshansuli’‘Taihuangli’were gathered together. Two Japan and Korea P. pyrifolia cultivars‘Shinseiki’and‘Whangkeumbae’were gathered together, Pear cultivars from America, Belgium and‘Duxiaxi’from China were clustered to the European pear group. The results were similar to previousstudy.【Conclusion】The core primers could produce clear amplification results with good repeatabilityand high polymorphism, and could be applied to pear germplasm identification, construction of geneticlinkage map, genetic diversity analyze, molecular marker-assisted breeding and DUS (Distinctness、Uniformity and Stability) testing for new cultivars. These results also would provide technical support for newcultivars protection in pear.