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Abstract: A total of 8375 genic simple sequence repeat (SSR) loci were discovered from a unigene set assembled from 116 282 transcriptomic unigenes in this study. Dinucleotide repeat motifs were the most common with a frequency of 65.11%, followed by trinucleotide (32.81%). A total of 4100 primer pairs were designed from the SSR loci. Of these, 343 primer pairs (repeat length ≥15 bp) were synthesized with an M13 tail and tested for stable amplification and polymorphism in four Pyrus accessions. After the preliminary test, 104 polymorphic genic SSR markers were developed; dinucleotide and trinucleotide repeats represented 97.11% (101) of these. Twenty-eight polymorphic genic SSR markers were selected randomly to further validate genetic diversity among 28 Pyrus accessions. These markers displayed a high level of polymorphism. The number of alleles at these SSR loci ranged from 2 to 17, with a mean of 9.43 alleles per locus, and the polymorphism information content (PIC) values ranged from 0.26 to 0.91. The UPGMA (unweighted pair-group method with arithmetic average) cluster analysis grouped the 28 Pyrus accessions into two groups: Oriental pears and Occidental pears, which are congruent to the traditional taxonomy, demonstrating their effectiveness in analyzing Pyrus phylogenetic relationships, enriching rare Pyrus EST-SSR resources, and confirming the potential value of a pear transcriptome database for the development of new SSR markers.

基于“酥梨”芽转录组的简单序列重复（SSR）标记开发

研究目的：基于转录组数据开发具有扩增率高和跨物种转移性的基因组编码区内的SSR（genic-SSR）标记，为梨属植物的分子系统发育关系和遗传多样性相关研究提供新的方法。
创新要点：首次利用梨属植物的转录组测序（RNA-seq）数据结合M-13荧光尾巴高效率地开发了104个genic-SSR标记，并成功将其应用于梨属植物的系统发育关系研究中。
研究方法：应用生物信息学软件从转录组测序数据中搜索SSR位点和设计相应引物，结合高效的M-13荧光尾巴的方法筛选多态性高的SSR标记。
重要结论：转录组数据能够为梨属植物分子系统发育关系和遗传多样性研究提供新的SSR标记来源。

关键词：简单序列重复（SSR）标记；转录组；遗传多样性；梨属

[1] Aldasoro, J.J., Aedo, C., Garmendia, F.M., 1996. The genus Pyrus L. (Rosaceae) in south-west Europe and North Africa. Bot J Linn Soc, 121(2):143-158. 

[2] Ashrafi, H., Hill, T., Stoffel, K., 2012. De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes. BMC Genomics, 13(1):571

[3] Bao, L., Chen, K., Zhang, D., 2007. Genetic diversity and similarity of pear (Pyrus L.) cultivars native to East Asia revealed by SSR (simple sequence repeat) markers. Genet Resour Crop Ev, 54(5):959-971. 

[4] Barbar, T., Palma-Silva, C., Paggi, G.M., 2007. Cross-species transfer of nuclear microsatellite markers: potential and limitations. Mol Ecol, 16(18):3759-3767. 

[5] Bassil, N., Postman, J.D., 2010. Identification of European and Asian pears using EST-SSRs from Pyrus . Genet Resour Crop Ev, 57(3):357-370. 

[6] Bell, R.L., Zwet, T., 1998. Breeding for host resistance to pear psylla: evaluation of parental germplasm. Acta Hortic (ISHS), 484:471-476. 

[7] Blanca, J., Cañizares, J., Roig, C., 2011. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics, 12(1):104

[8] Bouck, A., Vision, T., 2006. The molecular ecologist’s guide to expressed sequence tags. Mol Ecol, 16(5):907-924. 

[9] Cao, Y., Tian, L., Gao, Y., 2012. Genetic diversity of cultivated and wild Ussurian Pear (Pyrus ussuriensis Maxim.) in China evaluated with M13-tailed SSR markers. Genet Resour Crop Ev, 59(1):9-17. 

[10] Chen, X., Sun, D., Rong, D., 2011. A recessive gene controlling male sterility sensitive to short daylength/low temperature in wheat (Triticum aestivum L.). J Zhejiang Univ-Sci B (Biomed & Biotechnol), 12(11):943-950. 

[11] Cloutier, S., Niu, Z., Datla, R., 2009. Development and analysis of EST-SSRs for flax (Linum usitatissimum L.). Theor Appl Genet, 119(1):53-63. 

[12] Cordeiro, G.M., Casu, R., McIntyre, C.L., 2001. Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum. Plant Sci, 160(6):1115-1123. 

[13] Doyle, J., Doyle, J., 1987. Genomic plant DNA preparation from fresh tissue-CTAB method. Phytochem Bull, 19(11):11-15. 

[14] Dreisigacker, S., Zhang, P., Warburton, M.L., 2004. SSR and pedigree analyses of genetic diversity among CIMMYT wheat lines targeted to different megaenvironments. Crop Sci, 44(2):381-388. 

[15] Dutta, S., Kumawat, G., Singh, B.P., 2011. Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh]. BMC Plant Biol, 11(1):17

[16] Gupta, P., Varshney, R., 2000. The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica, 113(3):163-185. 

[17] Kantety, R.V., La Rota, M., Matthews, D.E., 2002. Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol, 48(5-6):501-510. 

[18] Katayama, H., Adachi, S., Yamamoto, T., 2007. A wide range of genetic diversity in pear (Pyrus ussuriensis var. aromatica) genetic resources from Iwate, Japan revealed by SSR and chloroplast DNA markers. Genet Resour Crop Ev, 54(7):1573-1585. 

[19] Kaur, S., Pembleton, L.W., Cogan, N.O., 2012. Transcriptome sequencing of field pea and faba bean for discovery and validation of SSR genetic markers. BMC Genomics, 13(1):104

[20] Kimura, T., Shi, Y.Z., Shoda, M., 2002. Identification of Asian pear varieties by SSR analysis. Breed Sci, 52(2):115-121. 

[21] Lesser, M.R., Parchman, T., Buerkle, C., 2012. Cross-species transferability of SSR loci developed from transcriptome sequencing in lodgepole pine. Mol Ecol Resour, 12(3):448-455. 

[22] Liu, G., Li, W., Zheng, P., 2012. Transcriptomic analysis of ‘Suli’ pear (Pyrus pyrifolia white pear group) buds during the dormancy by RNA-Seq. BMC Genomics, 13(1):700

[23] Liu, K., Muse, S.V., 2005. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics, 21(9):2128-2129. 

[24] Mardis, E.R., 2008. The impact of next-generation sequencing technology on genetics. Trends Genet, 24(3):133-141. 

[25] Nei, M., 1973. Analysis of gene diversity in subdivided populations. PNAS, 70(12):3321-3323. 

[26] Nishitani, C., Terakami, S., Sawamura, Y., 2009. Development of novel EST-SSR markers derived from Japanese pear (Pyrus pyrifolia). Breed Sci, 59(4):391-400. 

[27] Pavlicek, A., Hrda, S., Flegr, J., 1999. Free-tree—freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD analysis of genus Frenkelia . Folia Biol (Praha), 45(3):97-99. 

[28] Potter, D., Eriksson, T., Evans, R.C., 2007. Phylogeny and classification of Rosaceae. Plant Syst Evol, 266(1-2):5-43. 

[29] Powell, W., Machray, G.C., Provan, J., 1996. Polymorphism revealed by simple sequence repeats. Trends Plant Sci, 1(7):215-222. 

[30] Reilly, J.F., Martinez, S.D., Mickey, G., 2002. A novel role for farnesyl pyrophosphate synthase in fibroblast growth factor-mediated signal transduction. Biochem J, 366(2):501-510. 

[31] Rubtsov, G.A., 1944. Geographical distribution of the genus Pyrus and trends and factors in its evolution. Am Nat, 78(777):358-366. 

[32] Schuelke, M., 2000. An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol, 18(2):233-234. 

[33] Singh, H., Deshmukh, R.K., Singh, A., 2010. Highly variable SSR markers suitable for rice genotyping using agarose gels. Mol Breed, 25(2):359-364. 

[34] Teng, Y., Tanabe, K., 2004. Reconsideration on the origin of cultivated pears native to East Asia. Acta Hortic, 634:175-182. 

[35] Teng, Y., Tanabe, K., Tamura, F., 2002. Genetic relationships of Pyrus species and cultivars native to East Asia revealed by randomly amplified polymorphic DNA markers. J Am Soc Hortic Sci, 127(2):262-270. 

[36] Thiel, T., Michalek, W., Varshney, R., 2003. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet, 106(3):411-422. 

[37] Tth, G., Gspri, Z., Jurka, J., 2000. Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res, 10(7):967-981. 

[38] van Oosterhout, C., Hutchinson, W.F., Wills, D.P., 2004. MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes, 4(3):535-538. 

[39] Varshney, R.K., Graner, A., Sorrells, M.E., 2005. Genic microsatellite markers in plants: features and applications. Trends Biotechnol, 23(1):48-55. 

[40] Varshney, R.K., Grosse, I., Hhnel, U., 2006. Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome. Theor Appl Genet, 113(2):239-250. 

[41] Vendramin, E., Dettori, M.T., Giovinazzi, J., 2006. A set of EST-SSRs isolated from peach fruit transcriptome and their transportability across Prunus species. Mol Ecol Notes, 7(2):307-310. 

[42] Wang, Y.W., Samuels, T.D., Wu, Y.Q., 2011. Development of 1030 genomic SSR markers in switchgrass. Theor Appl Genet, 122(4):677-686. 

[43] Wang, Z., Gerstein, M., Snyder, M., 2009. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet, 10(1):57-63. 

[44] Wu, J., Wang, Z., Shi, Z., 2013. The genome of the pear (Pyrus bretschneideri Rehd.). Genome Res, 23(2):396-408. 

[45] Wnsch, A., Hormaza, J.I., 2007. Characterization of variability and genetic similarity of European pear using microsatellite loci developed in apple. Sci Hortic, 113(1):37-43. 

[46] Yamamoto, T., Kimura, T., Sawamura, Y., 2001. SSRs isolated from apple can identify polymorphism and genetic diversity in pear. Theor Appl Genet, 102(6-7):865-870. 

[47] Yamamoto, T., Kimura, T., Shoda, M., 2002. Genetic linkage maps constructed by using an interspecific cross between Japanese and European pears. Theor Appl Genet, 106(1):9-18. 

[48] Yamamoto, T., Kimura, T., Soejima, J., 2004. Identification of quince varieties using SSR markers developed from pear and apple. Breed Sci, 54(3):239-244. 

[49] Yao, L., Zheng, X., Cai, D., 2010. Exploitation of Malus EST-SSRs and the utility in evaluation of genetic diversity in Malus and Pyrus . Genet Resour Crop Ev, 57(6):841-851. 

[50] Yeh, F., Boyle, T.J.B., 1997. Population genetic analysis of codominant and dominant markers and quantitative traits. Belg J Bot, 129:157

[51] You, F.M., Huo, N., Gu, Y.Q., 2008. BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics, 9(1):253

[52] Zhang, G., Xu, S., Mao, W., 2013. Determination of the genetic diversity of vegetable soybean [Glycine max (L.) Merr.] using EST-SSR markers. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 14(4):279-288. 

[53] Zheng, X., Hu, C., Spooner, D., 2011. Molecular evolution of Adh and LEAFY and the phylogenetic utility of their introns in Pyrus (Rosaceae). BMC Evol Biol, 11(1):255