Development Of EST-SSR Markers From Malus And Their Transferability To Pyrus

With the development of genomics,the progress of DNA sequencing technology has cut down the cost,which made randomly sequencing of large collections of eDNA libraries a practical technology.Thousands of EST sequences produced during the process have provided an important source for developing SSR marker.As a new kind of molecular marker,EST-SSR has been developed and used in many plant species because it has several advantages over genome-derived SSRs such as low cost,high transferability besides possessing the features of the latter one.In this paper,we gave a brief introduction about principle and characteristics of EST-SSR technique. Moreover,its application in some research fields of pomology,including genetic diversity analysis,genetic linkage map construction,comparative mapping and molecular phylogeny were reviewed.And in this study,147 SSR primer pairs were developed from Malus EST database.After preliminary testing,94 of these 147 primer pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic of which 18 were further used to assess genetic diversity among 20 Malus sp.or cultivars.Besides,in order to evaluate the transferability of the newly exploited markers across genera,primer selecting and analysis of genetic relationships of a collection of Pyrus were also conducted.The results are as follows:(1)Eighteen pairs of SSR primers amplified discrete bands within the expected size range on all of 20 Malus accessions and 126 alleles were detected in total,with an average of 6.94 alleles per locus.Polymorphie information content values per locus ranged from 0.5238 to 0.9127,with a mean PIC value of 0.831 for all loci,displaying high polymorphism.The Genetic Similarity value among these species varied from 0.5238 to 0.9127,averagely 0.6749.UPGMA cluster analysis showed that twenty Malus accessions could be classified into five groups at the similarity level of 0.6800. In first group,6 apple eultivars including 'Liaofu','Jizaohong','Qinguan', 'Xiushuiguoguang','Qiujin' and 'Huaguan' clustered together,and the similarity coefficient between each other was high,verifying close genetic relationship among them.A genetic similarity value of 0.8333 was found between M.domestica subsp. chinensis Li Y.N.and M.sieversii subsp,kirghisorum which is the subspecies of M. sieversii,demonstrating their close relationship.This result is in agreement withY N Li's(1999)viewpoint that M.domestica subsp,chinensis Li Y.N.originated from M. sieversii.What's more,the sequence alignments of 3 loci showed that priming sites were conserved,whereas microsatellite repeats were present in all materials but structurally variable.(2)All of the 94 Malus EST-SSRs mentioned above were used to amplify on four accessions of Pyrus,of which,72 SSRs were functional and 40 SSRs produced polymorphic amplification and then 8 SSR loci were randomly selected to analyze genetic relationships of 24 Pyrus accessions.Eight primer-pairs produced expected bands with the similar size in apples and a total of 59 alleles were generated with an average of 7.375 per locus.The observed heterozygosity of different loci ranged from 0.29(MES₉₆)to 0.83(MES₁₃₈),with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus(Bao et al.2007).From UPGMA clusters analysis,all the accessions were distinctly divided into two groups at the similarity level of 0.6800:Asian pears and occidental pears.Within Asian pears, Chinese sand pear mingled with Chinese white pears suggesting a close relationship between them,which supports the previous viewpoint of Chinese white pears as a variety or an ecotype of Chinese sand pears(P.pyrifolia White Pear Group)(Bao et al. 2008;Teng et al.2002;Teng and Tanabe 2004).In addition,Japanese peals scatted in the group consisting of Chinese sand pears and Chinese white pears,especially some had high affinities to Chinese sand pears from Zhejiang and Fujian province, indicating a close relationship between them.