蚕豆EST-SSR分子标记的建立及其应用
摘要
蚕豆是世界上重要的豆科植物之一,是人类饮食中淀粉和蛋白质的重要来源,中国和欧洲是蚕豆主要的生产地。由于其部分交叉授粉,蚕豆具有高度异质性,因此品种间以及地方品种间包含了广泛的遗传变异,使保护种质资源变得更加昂贵和困难。尽管其种植历史悠久并且有很高的经济价值,但是对蚕豆分子标记仍然稀少。因此,在发展蚕豆更可靠更有效的分子标记将在多样性分析和资源保护中起重要作用。
EST-SSR分子标记在植物遗传分析和育种研究中已成为一个有力的工具。由于它从转录本推导,这些标记基因的SSR标记比传统的分子标记占有优势: EST-SSR分子标记的开发更简捷更廉价,相关物种具有较高的通用性,并更紧密地连接已知功能基因。
我们下载了NCBI数据库中公布的蚕豆5031条EST序列,通过对5031条EST序列进行拼接,得到Unigene1148条,通过SSR搜索软件搜索到107个SSR位点,SSRs的出现频率为9.32%。其中三核苷酸在107个SSR重复序列中是最为丰富的重复类型,占44.86%。而在三核苷酸中AAG是最丰富的重复基元,占有12.5%。AG和TC是二核苷酸中最丰富的,各占33%。其它SSR重复类型所占比例相差不大。我们首次分离和鉴定了11个蚕豆EST- SSR分子标记其等位基因变异数为每个位点2-3,各多态性引物的PIC值变化范围为0.06到0.43,平均PIC值为0.2919。在此基础上,我们利用已开发的11个EST-SSR分子标记对来自中国和欧洲的29个品种进行主成分分析和聚类分析,结果表明,这些品种可明显分为两个类群,我国蚕豆品种的遗传多样性较低,需要引入外源种质以拓展遗传基础。因此表明,利用EST-SSR标记对蚕豆种质资源评价是有效的。
进一步对这11个蚕豆EST-SSR分子标记在豌豆上的通用性进行检测发现72.73%在豌豆上具有通用性,其中75.00%通用性引物具有多态性。可见,新开发标记具有对豌豆进行进一步遗传研究的潜力。
Faba bean (Vicia faba L.) is one of the most important legumes in the world. It provides an important source of protein and starch for human diets in many countries.The main faba bean producers of the world in China and Europe. Due to its partial cross-pollination, faba bean is highly heterogeneous and consequently contains broad genetic variation within varieties and landraces, which make the conservation of germplasm resources more expensive and difficult. However, despite its long history of cultivation and economic importance, the number of molecular markers available for faba bean is still scarce. Therefore, developing more reliable and efficient molecular markers would be valuable in diversity analysis and resource conservation for faba bean.
EST-derived microsatellite markers (also known as EST-SSR markers) have become a powerful tool in plant genetic analysis and breeding research. Due to their derivation from transcripts, these markers have some intrinsic benefits over genomic SSR markers: they are easier and less expensive to develop, possess higher transferability to related species, and are more closely connected with known function genes.
5031 ESTs from Genbank database in NCBI, 1148 were obtained from 5031 Broad bean ESTs. We found 107 ESTs by software, accounting for 9.32% of ESTs were mined out. Trinucleotide repeats were the most abundant repeat class, and accounted for 44.86% of all found SSRs. And AAG are the most frequent motifs in trinucleotide, accounting for 12.5%.AG and TC are the most frequent motifs in dinucleotide, accounting for 33% each other. However,there were no significant differences among other types. In this research, we report for the first time the isolation and characterization of EST-SSRs in faba bean, including their potential for diversity analysis in different individuals and their transferability to a related pea species (Pisum sativum L.). A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe with two to three alleles per locus. The polymorphism information content value ranged from 0.06 to 0.43 with an average of 0.2919.Genetic similarity estimates from 11 SSRs showed two main cluster that comprised all Chinese genotypes and European genotypes, respectively. An average genetic similarity of 73.52% among the Chinese individuals. The results of this study proved that the EST-SSR marker is very effective in evaluation of faba bean germplasm.
Furthermore, transferable analysis revealed that 72.73% amplified in Pisum sativum L., 75.00% detected polymorphism.These results indicate the potential of these newly developed markers for furt-her genetic investigations of pea.
EST-SSR分子标记在植物遗传分析和育种研究中已成为一个有力的工具。由于它从转录本推导,这些标记基因的SSR标记比传统的分子标记占有优势: EST-SSR分子标记的开发更简捷更廉价,相关物种具有较高的通用性,并更紧密地连接已知功能基因。
我们下载了NCBI数据库中公布的蚕豆5031条EST序列,通过对5031条EST序列进行拼接,得到Unigene1148条,通过SSR搜索软件搜索到107个SSR位点,SSRs的出现频率为9.32%。其中三核苷酸在107个SSR重复序列中是最为丰富的重复类型,占44.86%。而在三核苷酸中AAG是最丰富的重复基元,占有12.5%。AG和TC是二核苷酸中最丰富的,各占33%。其它SSR重复类型所占比例相差不大。我们首次分离和鉴定了11个蚕豆EST- SSR分子标记其等位基因变异数为每个位点2-3,各多态性引物的PIC值变化范围为0.06到0.43,平均PIC值为0.2919。在此基础上,我们利用已开发的11个EST-SSR分子标记对来自中国和欧洲的29个品种进行主成分分析和聚类分析,结果表明,这些品种可明显分为两个类群,我国蚕豆品种的遗传多样性较低,需要引入外源种质以拓展遗传基础。因此表明,利用EST-SSR标记对蚕豆种质资源评价是有效的。
进一步对这11个蚕豆EST-SSR分子标记在豌豆上的通用性进行检测发现72.73%在豌豆上具有通用性,其中75.00%通用性引物具有多态性。可见,新开发标记具有对豌豆进行进一步遗传研究的潜力。
Faba bean (Vicia faba L.) is one of the most important legumes in the world. It provides an important source of protein and starch for human diets in many countries.The main faba bean producers of the world in China and Europe. Due to its partial cross-pollination, faba bean is highly heterogeneous and consequently contains broad genetic variation within varieties and landraces, which make the conservation of germplasm resources more expensive and difficult. However, despite its long history of cultivation and economic importance, the number of molecular markers available for faba bean is still scarce. Therefore, developing more reliable and efficient molecular markers would be valuable in diversity analysis and resource conservation for faba bean.
EST-derived microsatellite markers (also known as EST-SSR markers) have become a powerful tool in plant genetic analysis and breeding research. Due to their derivation from transcripts, these markers have some intrinsic benefits over genomic SSR markers: they are easier and less expensive to develop, possess higher transferability to related species, and are more closely connected with known function genes.
5031 ESTs from Genbank database in NCBI, 1148 were obtained from 5031 Broad bean ESTs. We found 107 ESTs by software, accounting for 9.32% of ESTs were mined out. Trinucleotide repeats were the most abundant repeat class, and accounted for 44.86% of all found SSRs. And AAG are the most frequent motifs in trinucleotide, accounting for 12.5%.AG and TC are the most frequent motifs in dinucleotide, accounting for 33% each other. However,there were no significant differences among other types. In this research, we report for the first time the isolation and characterization of EST-SSRs in faba bean, including their potential for diversity analysis in different individuals and their transferability to a related pea species (Pisum sativum L.). A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe with two to three alleles per locus. The polymorphism information content value ranged from 0.06 to 0.43 with an average of 0.2919.Genetic similarity estimates from 11 SSRs showed two main cluster that comprised all Chinese genotypes and European genotypes, respectively. An average genetic similarity of 73.52% among the Chinese individuals. The results of this study proved that the EST-SSR marker is very effective in evaluation of faba bean germplasm.
Furthermore, transferable analysis revealed that 72.73% amplified in Pisum sativum L., 75.00% detected polymorphism.These results indicate the potential of these newly developed markers for furt-her genetic investigations of pea.
引文
[1] Chen, C.X., et al., Verification of Mandarin and Pummelo Somatic Hybrids by Expressed Sequence Tag-Simple Sequence Repeat Marker Analysis. Journal of the American Society for Horticultural Science, 2008. 133(6): p. 794-800.
[2] Gershon, D., Bioinformatics in a post-genomics age. Nature, 1997. 389(6649): p. 417-418.
[3] Adams, M.D., et al., Complementary-DNA Sequencing - Expressed Sequence Tags and Human Genome Project. Science, 1991. 252(5013): p. 1651-1656.
[4] Kunkel, L., et al., A Large Insert X-Chromosome Specific Library for the Identification of Rflp Haplotypes. American Journal of Human Genetics, 1983. 35(6): p. A176-A176.
[5] Nakai, R., Y. Shoyama, and S. Shiraishi, Genetic characterization of epimedium species using random amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (RFLP) diagnosis. Biological & Pharmaceutical Bulletin, 1996. 19(1): p. 67-70.
[6] Li, X.Y., et al., Analysis of expressed sequence tags from Prunus mume flower and fruit and development of simple sequence repeat markers. Bmc Genetics, 2010. 11: p. 66.
[7] Xie, W.G., et al., Genetic diversity analysis and transferability of cereal EST-SSR markers to orchardgrass (Dactylis glomerata L). Biochemical Systematics and Ecology, 2010. 38(4): p. 740-749.
[8] Hu, J.B., X.Y. Zhou, and J.W. Li, Development of novel EST-SSR markers for cucumber (Cucumis sativus) and their transferability to related species. Scientia Horticulturae, 2010. 125(3): p. 534-538.
[9] Siju, S., et al., Development, Characterization and Cross Species Amplification of Polymorphic Microsatellite Markers from Expressed Sequence Tags of Turmeric (Curcuma longa L.). Molecular Biotechnology, 2010. 44(2): p. 140-147.
[10] Sax, K., The Association of Size Differences with Seed-Coat Pattern and Pigmentation in PHASEOLUS VULGARIS. Genetics, 1923. 8(6): p. 552-560.
[11] Bennett, M.D. and J.B. Smith, Nuclear dna amounts in angiosperms. Philos Trans R Soc Lond B Biol Sci, 1976. 274(933): p. 227-274.
[12] Johnston, J.S., et al., Reference standards for determination of DNA content of plant nuclei. Am J Bot, 1999. 86(5): p. 609.
[13] Walker, D.R., et al., A QTL that enhances and broadens Bt insect resistance in soybean. Theor Appl Genet, 2004. 109(5): p. 1051-1057.
[14] Shoemaker, R.C., et al., Genome duplication in soybean (Glycine subgenus soja). Genetics, 1996. 144(1): p. 329-338.
[15] Avila, C.M., et al., Identification of RAPD markers linked to the Uvf-1 gene conferring hypersensitive resistance against rust (Uromyces viciae-fabae) in Vicia faba L. Theor Appl Genet, 2003. 107(2): p. 353-358.
[16] Sillero, J.C., M.T. Moreno, and D. Rubiales, Characterization of new sources of resistance to Uromyces viciae-fabae in a germplasm collection of Vicia faba. Plant Pathol, 2000. 49.
[17] Tuberosa, R. and S. Salvi, Genomics-based approaches to improve drought tolerance of crops. Trends Plant Sci, 2006. 11(8): p. 405-412.
[18] Buhariwalla, H.K., et al., Development of ESTs from chickpea roots and their use in diversity analysis of the Cicer genus. BMC Plant Biol, 2005. 5: p. 16-23.
[19] Song, Q.J., et al., A new integrated genetic linkage map of the soybean. Theor Appl Genet, 2004. 109(1): p. 122-8.
[20] Zeng, S.H., et al., Development of a EST dataset and characterization of EST-SSRs in a traditional Chinese medicinal plant, Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Bmc Genomics, 2010. 11: p. 94-97.
[21] Varshney, R.K., et al., In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal species. Cellular & Molecular Biology Letters, 2002. 7(2A): p. 537-546.
[22] Zhang, R., et al., Development of Juglans Regia SSR Markers by Data Mining of the EST Database. Plant Molecular Biology Reporter, 2010. 28(4): p. 646-653.
[23] Kantety, R.V., et al., Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Molecular Biology, 2002. 48(5): p. 501-510.
[24] Cardle, L., et al., Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genetics, 2000. 156(2): p. 847-854.
[25] Chen, C.X., et al., Mining and characterizing microsatellites from citrus ESTs. Theoretical and Applied Genetics, 2006. 112(7): p. 1248-1257.
[26] Thiel, T., et al., Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theoretical and Applied Genetics, 2003. 106(3): p. 411-422.
[27] Kota, R., et al., Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.). Genome, 2001. 44(4): p. 523-528.
[28]龚亚明, et al., EST-SSR荧光标记毛细管电泳检测法在豌豆上的应用及评价.浙江农业学报, 2009. 21(6): p. 540-543.
[29] Gong, Y.M., et al., Generation and Characterization of 11 Novel Est Derived Microsatellites from Vicia Faba (Fabaceae). American Journal of Botany, 2010. 97(7): p. E69-E71.
[30] Leigh, F., et al., Assessment of EST- and genomic microsatellite markers for variety discrimination and genetic diversity studies in wheat. Euphytica, 2003. 133(3): p. 359-366.
[31] Gong, Y.M., et al., Developing new SSR markers from ESTs of pea (Pisum sativum L.). Journal of Zhejiang University-Science B, 2010. 11(9): p. 702-707.
[32] Mulato, B.M., et al., Genetic diversity in soybean germplasm identified by SSR and EST-SSR markers. Pesquisa Agropecuaria Brasileira, 2010. 45(3): p. 276-283.
[33] Li, W., et al., Use of chromosome walking in discovery of single-nucleotide polymorphism in noncoding regions of a candidate actin gene in Pinus radiata. J Appl Genet, 2010. 51(3): p. 275-281.
[34] Yeh F C , Y.R. C, and B. T., POPGENE (Version 1.31): Microsoft Window-bases freeware for population genetic analysis, University of Alberta and the Centre for International Forestry Research. 1999.
[35] Yu, J.K., et al., Development and mapping of EST-derived simple sequence repeat markers for hexaploid wheat. Genome, 2004. 47(5): p. 805-818.
[36] Lu, Y.D., et al., Mining, characterization, and exploitation of EST-derived microsatellites in Gossypium barbadense. Chinese Science Bulletin, 2010. 55(18): p. 1889-1893.
[37]魏利斌,张海洋, and郑永战等,芝麻EST-SSR标记的开发和初步研究.作物学报, 2008. 29(1):.: p. 20-35.
[38] Yasodha, R., et al., Eucalyptus microsatellites mined in silico: survey and evaluation. Journal of Genetics, 2008. 87(1): p. 21-25.
[39] Yan, Q.L., et al., Identification of microsatellites in cattle unigenes. Journal of Genetics and Genomics, 2008. 35(5): p. 261-266.
[40] Varshney, R.K., A. Graner, and M.E. Sorrells, Genic microsatellite markers in plants: features and applications. Trends in Biotechnology, 2005. 23(1): p. 48-55.
[41] Poncet, V., et al., SSR mining in coffee tree EST databases: potential use of EST-SSRs as markers for the Coffea genus. Molecular Genetics and Genomics, 2006. 276(5): p. 436-449.
[42] Gupta, P.K., et al., Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat. Molecular Genetics and Genomics, 2003. 270(4): p. 315-323.
[43] Metzgar, D., J. Bytof, and C. Wills, Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome Research, 2000. 10(1): p. 72-80.
[44] Hisano, H., et al., Characterization of the soybean genome using EST-derived microsatellite markers. DNA Res, 2007. 14(6): p. 271-281.
[45] Choudhary, S., et al., Development of chickpea EST-SSR markers and analysis of allelic variation across related species. Theoretical and Applied Genetics, 2009. 118(3): p. 591-608.
[46] Hanai, L.R., et al., Development, characterization, and comparative analysis of polymorphism at common bean SSR loci isolated from genic and genomic sources. Genome, 2007. 50(3): p. 266-277.
[47] Ince, A.G., M. Karaca, and A.N. Onus, Polymorphic Microsatellite Markers Transferable Across Capsicum Species. Plant Molecular Biology Reporter, 2010. 28(2): p. 285-291.
[48] Gupta, S.K. and T. Gopalakrishna, Development of unigene-derived SSR markers in cowpea (Vigna unguiculata) and their transferability to other Vigna species. Genome, 2010. 53(7): p. 508-523.
[49] Eujayl, I., et al., Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat. Theoretical and Applied Genetics, 2002. 104(2-3): p. 399-407.
[50] Saha, M.C., et al., Tall fescue genomic SSR markers: development and transferability across multiple grass species. Theoretical and Applied Genetics, 2006. 113(8): p. 1449-1458.
[51] Cho, Y.G., et al., Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.). Theoretical and Applied Genetics, 2000. 100(5): p. 713-722.
[52] Scott, K.D., et al., Analysis of SSRs derived from grape ESTs. Theoretical and Applied Genetics, 2000. 100(5): p. 723-726.
[53] Zeid, M., et al., Simple sequence repeats (SSRs) in faba bean: new loci from Orobanche-resistant cultivar 'Giza 402'. Plant Breeding, 2009. 128(2): p. 149-155.
[54] Wolfe, M.S., Crop strength through diversity. Nature, 2000. 406(6797): p. 681-682.
[2] Gershon, D., Bioinformatics in a post-genomics age. Nature, 1997. 389(6649): p. 417-418.
[3] Adams, M.D., et al., Complementary-DNA Sequencing - Expressed Sequence Tags and Human Genome Project. Science, 1991. 252(5013): p. 1651-1656.
[4] Kunkel, L., et al., A Large Insert X-Chromosome Specific Library for the Identification of Rflp Haplotypes. American Journal of Human Genetics, 1983. 35(6): p. A176-A176.
[5] Nakai, R., Y. Shoyama, and S. Shiraishi, Genetic characterization of epimedium species using random amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (RFLP) diagnosis. Biological & Pharmaceutical Bulletin, 1996. 19(1): p. 67-70.
[6] Li, X.Y., et al., Analysis of expressed sequence tags from Prunus mume flower and fruit and development of simple sequence repeat markers. Bmc Genetics, 2010. 11: p. 66.
[7] Xie, W.G., et al., Genetic diversity analysis and transferability of cereal EST-SSR markers to orchardgrass (Dactylis glomerata L). Biochemical Systematics and Ecology, 2010. 38(4): p. 740-749.
[8] Hu, J.B., X.Y. Zhou, and J.W. Li, Development of novel EST-SSR markers for cucumber (Cucumis sativus) and their transferability to related species. Scientia Horticulturae, 2010. 125(3): p. 534-538.
[9] Siju, S., et al., Development, Characterization and Cross Species Amplification of Polymorphic Microsatellite Markers from Expressed Sequence Tags of Turmeric (Curcuma longa L.). Molecular Biotechnology, 2010. 44(2): p. 140-147.
[10] Sax, K., The Association of Size Differences with Seed-Coat Pattern and Pigmentation in PHASEOLUS VULGARIS. Genetics, 1923. 8(6): p. 552-560.
[11] Bennett, M.D. and J.B. Smith, Nuclear dna amounts in angiosperms. Philos Trans R Soc Lond B Biol Sci, 1976. 274(933): p. 227-274.
[12] Johnston, J.S., et al., Reference standards for determination of DNA content of plant nuclei. Am J Bot, 1999. 86(5): p. 609.
[13] Walker, D.R., et al., A QTL that enhances and broadens Bt insect resistance in soybean. Theor Appl Genet, 2004. 109(5): p. 1051-1057.
[14] Shoemaker, R.C., et al., Genome duplication in soybean (Glycine subgenus soja). Genetics, 1996. 144(1): p. 329-338.
[15] Avila, C.M., et al., Identification of RAPD markers linked to the Uvf-1 gene conferring hypersensitive resistance against rust (Uromyces viciae-fabae) in Vicia faba L. Theor Appl Genet, 2003. 107(2): p. 353-358.
[16] Sillero, J.C., M.T. Moreno, and D. Rubiales, Characterization of new sources of resistance to Uromyces viciae-fabae in a germplasm collection of Vicia faba. Plant Pathol, 2000. 49.
[17] Tuberosa, R. and S. Salvi, Genomics-based approaches to improve drought tolerance of crops. Trends Plant Sci, 2006. 11(8): p. 405-412.
[18] Buhariwalla, H.K., et al., Development of ESTs from chickpea roots and their use in diversity analysis of the Cicer genus. BMC Plant Biol, 2005. 5: p. 16-23.
[19] Song, Q.J., et al., A new integrated genetic linkage map of the soybean. Theor Appl Genet, 2004. 109(1): p. 122-8.
[20] Zeng, S.H., et al., Development of a EST dataset and characterization of EST-SSRs in a traditional Chinese medicinal plant, Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Bmc Genomics, 2010. 11: p. 94-97.
[21] Varshney, R.K., et al., In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal species. Cellular & Molecular Biology Letters, 2002. 7(2A): p. 537-546.
[22] Zhang, R., et al., Development of Juglans Regia SSR Markers by Data Mining of the EST Database. Plant Molecular Biology Reporter, 2010. 28(4): p. 646-653.
[23] Kantety, R.V., et al., Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Molecular Biology, 2002. 48(5): p. 501-510.
[24] Cardle, L., et al., Computational and experimental characterization of physically clustered simple sequence repeats in plants. Genetics, 2000. 156(2): p. 847-854.
[25] Chen, C.X., et al., Mining and characterizing microsatellites from citrus ESTs. Theoretical and Applied Genetics, 2006. 112(7): p. 1248-1257.
[26] Thiel, T., et al., Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theoretical and Applied Genetics, 2003. 106(3): p. 411-422.
[27] Kota, R., et al., Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.). Genome, 2001. 44(4): p. 523-528.
[28]龚亚明, et al., EST-SSR荧光标记毛细管电泳检测法在豌豆上的应用及评价.浙江农业学报, 2009. 21(6): p. 540-543.
[29] Gong, Y.M., et al., Generation and Characterization of 11 Novel Est Derived Microsatellites from Vicia Faba (Fabaceae). American Journal of Botany, 2010. 97(7): p. E69-E71.
[30] Leigh, F., et al., Assessment of EST- and genomic microsatellite markers for variety discrimination and genetic diversity studies in wheat. Euphytica, 2003. 133(3): p. 359-366.
[31] Gong, Y.M., et al., Developing new SSR markers from ESTs of pea (Pisum sativum L.). Journal of Zhejiang University-Science B, 2010. 11(9): p. 702-707.
[32] Mulato, B.M., et al., Genetic diversity in soybean germplasm identified by SSR and EST-SSR markers. Pesquisa Agropecuaria Brasileira, 2010. 45(3): p. 276-283.
[33] Li, W., et al., Use of chromosome walking in discovery of single-nucleotide polymorphism in noncoding regions of a candidate actin gene in Pinus radiata. J Appl Genet, 2010. 51(3): p. 275-281.
[34] Yeh F C , Y.R. C, and B. T., POPGENE (Version 1.31): Microsoft Window-bases freeware for population genetic analysis, University of Alberta and the Centre for International Forestry Research. 1999.
[35] Yu, J.K., et al., Development and mapping of EST-derived simple sequence repeat markers for hexaploid wheat. Genome, 2004. 47(5): p. 805-818.
[36] Lu, Y.D., et al., Mining, characterization, and exploitation of EST-derived microsatellites in Gossypium barbadense. Chinese Science Bulletin, 2010. 55(18): p. 1889-1893.
[37]魏利斌,张海洋, and郑永战等,芝麻EST-SSR标记的开发和初步研究.作物学报, 2008. 29(1):.: p. 20-35.
[38] Yasodha, R., et al., Eucalyptus microsatellites mined in silico: survey and evaluation. Journal of Genetics, 2008. 87(1): p. 21-25.
[39] Yan, Q.L., et al., Identification of microsatellites in cattle unigenes. Journal of Genetics and Genomics, 2008. 35(5): p. 261-266.
[40] Varshney, R.K., A. Graner, and M.E. Sorrells, Genic microsatellite markers in plants: features and applications. Trends in Biotechnology, 2005. 23(1): p. 48-55.
[41] Poncet, V., et al., SSR mining in coffee tree EST databases: potential use of EST-SSRs as markers for the Coffea genus. Molecular Genetics and Genomics, 2006. 276(5): p. 436-449.
[42] Gupta, P.K., et al., Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat. Molecular Genetics and Genomics, 2003. 270(4): p. 315-323.
[43] Metzgar, D., J. Bytof, and C. Wills, Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome Research, 2000. 10(1): p. 72-80.
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