De Novo Characterization of the Root Transcriptome and Development of EST-SSR Markers in Paris polyphylla Smith var. yunnanensis, an Endangered Medical Plant

Authors
L. Wang 1
Y. Yang 2
Y. Zhao 2
S. Yang 1
S. Udikeri 3
T. Liu 1
1 Yunnan Research Center on Good Agricultural Practice for Dominant Chinese Medicinal Materials, Yunnan Agricultural University, Kunming, 650201, People’s Republic of China.
2 China Tobacco Yunnan Industrial Co., Ltd, Kunming 650201. People’s Republic of China.
3 University of Agricultural Sciences, Dharwad, Pin-580005, Karnataka, India.
Abstract
Paris polyphylla Smith var. yunnanensis (Liliaceae) is an important traditional medicinal plant of the Yunnan Province in China. However, the genomic information regarding this plant is limited. To further understand its molecular background, we conducted Illumina HiSeq 2000 second-generation sequencing of this plant species. Approximately 30,198,679 reads with an average length of 202 bases were obtained from its root cells. These reads were assembled into 56,095 unique sequences and approximately 49.7% of the unique sequences were annotated by Basic Local Alignment Search Tool (BLAST) similarity searches against public sequence databases. Most of these unigenes were mapped to carbohydrate metabolism, energy metabolism, and secondary metabolite biosynthetic pathways. Additionally, 3,853 EST-SSRs were identified as potential molecular markers in our unigenes. Of these, 9 nuclear SSR markers were employed to assess genetic diversity and structure of 11 geographically disjunct populations. The present study revealed a moderate genetic diversity (He= 0.527) and low genetic differentiation (Fst= 0.103), which may be ascribed to an earlier period of more pronounced gene flow when the species had a more continuous distribution. The 11 studied populations were divided into two clusters based on the UPGMA dendrogram, which were not congruent with their geographical distributions. Overall, the root transcriptome sequences generated in this study reveal novel gene expression profiles and offer important clues for further study of the molecular mechanism of Paris’ root secondary metabolite synthesis and population genetics. The EST-SSR markers identified will also facilitate marker-assisted selection in Paris breeding.

Keywords

1. Abbasi, Z., Arzani, A. and Majidi, M. M. 2014. Evaluation of Genetic Diversity of Sugar Beet (Beta vulgaris L.) Crossing Parents Using Agro-morphological Traits and Molecular Markers. J. Agr. Sci. Tech., 16(6): 1397-1411
2. Berardini, T. Z., Mundodi, S., Reiser, L., Huala, E., Garcia-Hernandez, M., Zhang, P., Mueller, L. A., Yoon, J., Doyle, A. and Lander, G. 2004. Functional Annotation of the Arabidopsis Genome Using Controlled Vocabularies. Plant. Physiol., 135: 745-755.
3. Botstein, D., White, R. L., Skolnick, M. and Davis, R. W. 1980. Construction of a Genetic Linkage Map in Man Using Restriction Fragment Length Polymorphisms. Am. J. Hum. Genet. 32: 314–331
4. Chen, S., Luo, H., Li, Y., Sun, Y., Wu, Q., Niu, Y., Song, J., Lv, A., Zhu, Y., Sun, C., Steinmetz, A. and Qian, Z. 2011. 454 EST Analyses Detects Genes Putatively Involved in Ginsenoside Biosynthesis in Panax ginseng. Plant. Cell. Report., 30: 1593-1601.
5. Chi, K. R. 2008. The Year of Sequencing. Nat. Method., 5: 11–14.
6. Corey, E. J., Matsuda, S. P. T. and Bartel, B. 1993. Isolation of an Arabidopsis thaliana Gene Encoding Cycloartenol Synthase by Functional Expression in a Yeast Mutant Lacking Lanosterol Synthase by the Use of a Chromatographic Screen. Proc. Natl. Acad. Sci. USA, 90: 11628–11632.
7. Doyle, J. J. and Doyle, J. L. 1987. A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochem. Bull., 19: 11-15.
8. Frank, M. Y., Naxin, H., Yong, Q. G., Ming, C. L., Ma, Y. Q., Hane, D., Lazo, G. R., Dvorak, J. and Anderson, O. D. 2008. BatchPrimer3: A High Throughput Web Application for PCR and Sequencing Primer Design. BMC Bioinforma., 9: 253.
9. Ghaedrahmati, M., Mardi, M., Naghavi, M.R., Majidi Heravan, E., Nakhoda, B., Azadi, A. and Kazemi, M. 2014. Mapping QTLs Associated with Salt Tolerance Related Traits in Wheat (Triticum aestivum L.). J. Agr. Sci. Tech., 16(6): 1413-1428.
10. Guo, C. K., Zhang, S., Kong, W. J. and Li, Q. T. 2006. Clinical Study of Bi Yan Qing Du Granule and Bi Yan Shu Oral Liquid in Treatment Patients with Nasopharyngeal Carcinoma after Radiotherapy. Chin. Cancer, 15: 113–115.
11. Guo, S. Y., Zheng, J., Joung, G., Liu, S., Zhang, Z., Crasta, O. R., Sobral, B. W., Xu, Y., Huang, S. and Fei, Z. 2010. Transcriptome Sequencing and Comparative Analysis of Cucumber Flowers with Different Sex Types. BMC Genom., 11: 384
12. Hamrick, J. L. and Godt, M. J. W. 1996. Effects of Life History Traits on Genetic Diversity in Plant Species. Phil. Trans. R. Soc. B, 351: 1291–1298.
13. Haralampidis, K., Trojanowska, M., Osbourn, A. E. 2002. Biosynthesis of Triterpenoid Saponins in Plants. Adv. Biochem. Eng. Biotechnol. 75: 31-49.
14. He, J., Yang, B. Y., Chen, S. F., Gao, L. M. and Wang, H. 2007. Assessment of genetic diversity of Paris ployphylla (Trilliaceae) by ISSR markers. Acta Botanica Yunnanica, 29: 388-392.
15. Herrera, J. B., Bartel, B., Wilson, W. K. and Matsuda, S. P. 1998. Cloning and Characterization of the Arabidopsis thaliana Lupeol Synthase Gene. Phytochem. ,49: 1905–11.
16. Iseli, C., Jongeneel, C. V. and Bucher, P. 1999. ESTScan: A Program for Detecting, Evaluating, and Reconstructing Potential Coding Regions. Proc. Int. Conf. Intell. Syst. Mol. Biol., 4: 138–148.
17. Ji, Y. H., PETER, W. F., Li, H., Xiao, T. J. and Zhou, Z. K. 2006. Phylogeny and Classification of Paris (Melanthiaceae) Inferred from DNA Sequence Data. Ann. Bot., 98: 245–256.
18. Kalinowska, M., Zimowski, J., Paczkowski, C. and Wojciechowski, Z. A. 2005. The Formation of Sugar Chains in Triterpenoid Saponins and Glycoalkaloids. Phytochem. Rev., 4: 237–257.
19. Kanehisa, M. and Goto, S. 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic. Acids. Res., 28: 27-30.
20. Kumar, S., Kalra, S., Kumar, S., Kaur, J. and Singh, K. 2012. Differentially Expressed Transcripts from Leaf and Root Tissue of Chlorophytum borivilianum: A Plant with High Medicinal Value. Gene., 511: 79–87.
21. Lee, M. H., Jeong, J. H., Seo, J. W., Shin, C. G.., Kim, Y. S., In, J. G., Yang, D. C., Yi, J. S. and Choi, Y. E. 2004. Enhanced Triterpene and Phytosterol Biosynthesis in Panax ginseng Overexpressing Squalene Synthase Gene. Plant Cell. Physiol., 45: 976–984.
22. Lee, R. K. Y., Ong, R. C. Y., Cheung, J. Y. N., Li, Y. C., Chan, J. Y. W. and Lee, M. M. S. 2009. Polyphyllin D: A Potential Anti-cancer Agent to Kill Hepatocarcinoma Cells with Multi-drug Resistance. Curr. Chem. Biol., 3: 89–99.
23. Li, H. 1998. The Phylogeny of the Genus Paris L. In: “The Genus Paris (Trilliaceae)”, (Ed.): Li, H.. Science Press, Beijing, PP. 8–65.
24. Liu, H., Zhang, T., Chen, X. Q., Huang, Y. and Wang, Q. 2006. Steroidal Saponins of Paris polyphylla Smith var. yunnanensis. Chin. J. Nat. Med., 4: 265–7.
25. Liu, K. and Muse, S. V. 2005. PowerMarker: Integrated Analysis Environment for Genetic Marker Data. Bioinforma., 21: 2128-2129.
26. Luo, H. M., Sun, C. and Sun, Y. Z. 2011. Analysis of the Transcriptome of Panax notoginseng Root Uncovers Putative Triterpene Saponin-biosynthetic Genes and Genetic Markers. BMC Genom., 12(Suppl. 5) S5.
27. Mardis, E. R. 2008. The Impact of Next-generation Sequencing Technology on Genetics. TIG, 24: 133–141.
28. Margulies, M., Egholm, M., Altman, W. E., Attiya, S. and Bader, J. S. 2005. Genome Sequencing in Microfabricated High-density Picolitre Reactors. Nature, 37: 376–380.
29. Matsuda, H., Pongpiriyadacha, Y., Morikawa, T., Kishi, A., Kataoka, S. and Yoshikawa, M. 2003. Protective Effects of Steroid Saponins from Paris polyphylla var. yunnanensis on Ethanol- or Indomethacin-induced Gastric Mucosal Lesions in Rats: Structural Requirement for Activity and Mode of Action. Bioorg. Med. Chem. Lett., 13: 1101–1106.
30. Miller, M. P. 1997. Tools for Population Genetic Analysis, Version 1.3. Department of Biological Sciences, Northern Arizona University, Flagstaff AZ.
31. Morozova, O. and Marra, M. 2008. Applications of Next-generation Sequencing Technologies in Functional Genomics. Genom., 2: 255–264.
32. Nybom, H. 2000. Effects of Life History Traits and Sampling Strategies on Genetic Diversity Estimates Obtained with RAPD Markers in Plants. Perspect. Plant. Ecol., 3: 93–114.
33. Nybom, H. 2004. Comparison of Different Nuclear DNA Markers for Estimating Intraspecific Genetic Diversity in Plants. Mol. Ecol., 13: 1143–1155.
34. Parchman, T. L., Geist. K. S., Grahnen, J. A., Benkman, C. W. and Buerkle, C. A. 2010. Transcriptome Sequencing in an Ecologically Important Tree Species: Assembly, Annotation, and Marker Discovery. BMC Genom., 11: 180.
35. Phillips, D. R., Rasbery, J. M., Bartel, B. and Matsuda, S. P. T. 2006. Biosynthetic Diversity in Plant Triterpene Cyclization. Curr. Opin. Plant Biol., 9: 305–314.
36. Qin, B., Eagles, J., Mellon, F. A., Mylona, P., Pena-Rodriguez, L. and Osbourn, A. E. 2010. High throughput Screening of Mutants of Oat that Are Defective in Triterpene Synthesis. Phytochem., 71: 1245–1252.
37. Rohmer, M. 2003. Mevalonate-independent Methylerythritol Phosphate Pathway for Isoprenoid Biosynthesis. Elucidation and Distribution. Pure. Appl. Chem., 75: 375–387.
38. Selkoe, K. A. and Roonen, R. J. 2006. Microsatellites for Ecologists: A Practical Guide to Using and Evaluating Microsatellite Markers. Ecol. Lett., 9: 615–629.
39. Sharma, R. K., Bhardwaj, P., Negi, R., Mohapatra, T. and Ahuja, P. S. 2009. Identification, Characterization and Utilization of Unigene Derived Microsatellite Markers in Tea (Camellia sinensis L.). BMC Plant. Biol., 9: 53.
40. Temnykh, S., DeClerck, G., Lukashova, A., Lipovich, L., Cartinhour, S. and McCouch, S. 2001. Computational and Experimental Qnalysis of Microsatellites in Rice (Oryza sativa L.): Frequency, Length Variation, Transposon Associations, and Genetic Marker Potential. Genom. Res., 11: 1441-1452.
41. Thiel, T., Michalek, W., Varshney, R. K. and Graner, A. 2003. Exploiting EST Databases for the Development and Characterization of Gene-derived SSR-markers in Barley (Hordeum vulgare L.). Theor. Appl. Genet., 106: 411–422.
42. Tiwari, J. K., Ballabha, R. and Tiwari, P. 2010. Ethnopaediatrics in Garhwal Himalaya Uttarkhand India (Psychomedicine and Medicine). New York Sci. J., 3: 123-126.
43. Varshney, R. K., Graner, A., Sorrells, M. E. 2005. Genic Microsatellite Markers in Plants: Features and Applications. Trend. Biotechnol., 23: 48–55.
44. Vincken, J. P., Heng, L., deGroot, A. and Gruppen, H. 2007. Saponins: Classification and Occurence in the Plant Kingdom. Phytochem., 68: 275–297.
45. Wang, L., Li, P. and Brutnell, T. P. 2010. Exploring Plant Transcriptomes Using Ultra High-throughput Sequencing. Brief Funct. Genom., 9: 118–128.
46. Wang, W., Wang, Y., Zhang, Q., Qi, Y. and Guo, D. 2009. Global Characterization of Artemisia annua Glandular Trichome Transcriptome Using 454 Pyrosequencing. BMC Genom., 10: 465.
47. Weber, A. P., Weber, K. L., Carr, K., Wilkerson, C. and Ohlrogge, J. B. 2007. Sampling the Arabidopsis Transcriptome with Massively Parallel Pyrosequencing. Plant. Physiol., 144: 32–42.
48. Wu, X., Wang, L., Wang, G.. C., Wang, H., Dai, Y., Yang, X. X., Ye, W. C. and Li, Y. L. 2013. Triterpenoid Saponins from Rhizomes of Paris polyphylla var. yunnanensis. Carbohydr. Res., 4: 3681–3687.
49. Xu, T. H., Ma, X. X., Xu, Y. J., Xie, S. X., Zhao, H. F., Si, Y. S., Han, D., Li, Y., Niu, J. Z. and Xu, D. M. 2007. New Steroidal Saponin from Paris polyphylla Sm. var. yunnanensis (France). Hand-Mazz. Chem. J. Chin. U, 28: 2303–6.
50. Yeh, F. C., Yang, R. C. and Boyle, T. 1999. POPGENE 32, Version 1.31. Population Genetics Software, Edmonton University of Alberta.
51. Zalapa, J. E., Cuevas, H., Zhu, H., Steffan, S., Senalik, D., Zeldin, E. and McCown, B. 2012. Using Nest-generation Sequencing Approaches to Isolate Simple Sequence Repeat (SSR) Loci in the Plant Sciences. Am. J. Bot., 99: 193-208.
52. Zhang, C. B., Peng, B., Zhang, W. L., Wang, S. M., Sun, H., Dong, Y. S. and Zhao, L. M. 2014. Application of SSR Markers for Purity Testing of Commercial Hybrid Soybean (Glycine max L.). J. Agr. Sci. Tech., 16(6): 1389-1396.
53. Zhang, S. P. 2007. Research Progress on Chemical Constituents and Pharmacological Effect of Genus Paris. Strait. Pharm. J., 19: 4–7.
54. Zhao, J. L., Mou, Y., Shan, T. J., Li, Y., Zhou, L. G., Wang, M. G.. and Wang, J. G. 2010 .Antimicrobial Metabolites from the Endophytic Fungus Pichia guilliermondii Isolated from Paris polyphylla var. yunnanensis. Mol., 15: 7961–7970.
55. Zhou, X., Su, Z., Sammons, R. D., Peng, Y. H., Tranel, P. J., Stewart, C. N. and Yuan, J. S. 2009. Novel Software Package for Cross-Platform Transcriptome Analysis (CPTRA). BMC Bioinforma., 10: S16.

1.       bbasi, Z., Arzani, A. and Majidi, M. M. 2014. Evaluation of Genetic Diversity of Sugar Beet (Beta vulgaris L.) Crossing Parents Using Agro-morphological Traits and Molecular Markers. J. Agr. Sci. Tech., 16(6): 1397-1411
2.       Berardini, T. Z., Mundodi, S., Reiser, L., Huala, E., Garcia-Hernandez, M., Zhang, P., Mueller, L. A., Yoon, J., Doyle, A. and Lander, G. 2004. Functional Annotation of the Arabidopsis Genome Using Controlled Vocabularies. Plant. Physiol., 135: 745-755.
3.       Botstein, D., White, R. L., Skolnick, M. and Davis, R. W. 1980. Construction of a Genetic Linkage Map in Man Using Restriction Fragment Length Polymorphisms. Am. J. Hum. Genet. 32: 314–331
4.       Chen, S., Luo, H., Li, Y., Sun, Y., Wu, Q., Niu, Y., Song, J., Lv, A., Zhu, Y., Sun, C., Steinmetz, A. and Qian, Z. 2011. 454 EST Analyses Detects Genes Putatively Involved in Ginsenoside Biosynthesis in Panax ginseng. Plant. Cell. Report., 30: 1593-1601.
5.       Chi, K. R. 2008. The Year of Sequencing. Nat. Method., 5: 11–14.
6.       Corey, E. J., Matsuda, S. P. T. and Bartel, B. 1993. Isolation of an Arabidopsis thaliana Gene Encoding Cycloartenol Synthase by Functional Expression in a Yeast Mutant Lacking Lanosterol Synthase by the Use of a Chromatographic Screen. Proc. Natl. Acad. Sci. USA, 90: 11628–11632.
7.       Doyle, J. J. and Doyle, J. L. 1987. A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochem. Bull., 19: 11-15.
8.       Frank, M. Y., Naxin, H., Yong, Q. G., Ming, C. L., Ma, Y. Q., Hane, D., Lazo, G. R., Dvorak, J. and Anderson,O. D. 2008. BatchPrimer3: A High Throughput Web Application for PCR and Sequencing Primer Design. BMC Bioinforma., 9: 253.
9.       Ghaedrahmati, M., Mardi, M., Naghavi, M.R., Majidi Heravan, E., Nakhoda, B., Azadi, A. and Kazemi, M. 2014. Mapping QTLs Associated with Salt Tolerance Related Traits in Wheat (Triticum aestivum L.). J. Agr. Sci. Tech., 16(6): 1413-1428.
10.    Guo, C. K., Zhang, S., Kong, W. J. and Li, Q. T. 2006. Clinical Study of Bi Yan Qing Du Granule and Bi Yan Shu Oral Liquid in Treatment Patients with Nasopharyngeal Carcinoma after Radiotherapy. Chin. Cancer, 15: 113–115.
11.    Guo, S. Y., Zheng, J., Joung, G., Liu, S., Zhang, Z., Crasta, O. R., Sobral, B. W., Xu, Y., Huang, S. and Fei, Z. 2010. Transcriptome Sequencing and Comparative Analysis of Cucumber Flowers with Different Sex Types. BMC Genom., 11: 384
12.    Hamrick, J. L. and Godt, M. J. W. 1996. Effects of Life History Traits on Genetic Diversity in Plant Species. Phil. Trans. R. Soc. B, 351: 1291–1298.
13.    Haralampidis, K., Trojanowska, M., Osbourn, A. E. 2002. Biosynthesis of Triterpenoid Saponins in Plants. Adv. Biochem. Eng. Biotechnol. 75: 31-49.
14.    He, J., Yang, B. Y., Chen, S. F., Gao, L. M. and Wang, H. 2007. Assessment of genetic diversity of Paris ployphylla (Trilliaceae) by ISSR markers. Acta Botanica Yunnanica, 29: 388-392.
15.    Herrera, J. B., Bartel, B., Wilson, W. K. and Matsuda, S. P. 1998. Cloning and Characterization of the Arabidopsis thaliana Lupeol Synthase Gene. Phytochem. ,49: 1905–11.
16.    Iseli, C., Jongeneel, C. V. and Bucher, P. 1999. ESTScan: A Program for Detecting, Evaluating, and Reconstructing Potential Coding Regions. Proc. Int. Conf. Intell. Syst. Mol. Biol., 4: 138–148.
17.    Ji, Y. H., PETER, W. F., Li, H., Xiao, T. J. and Zhou, Z. K. 2006. Phylogeny and Classification of Paris (Melanthiaceae) Inferred from DNA Sequence Data. Ann. Bot., 98: 245–256.
18.    Kalinowska, M., Zimowski, J., Paczkowski, C. and Wojciechowski, Z. A. 2005. The Formation of Sugar Chains in Triterpenoid Saponins and Glycoalkaloids. Phytochem. Rev., 4: 237–257.
19.    Kanehisa, M. and Goto, S. 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic. Acids. Res., 28: 27-30.
20.    Kumar, S., Kalra, S., Kumar, S., Kaur, J. and Singh, K. 2012. Differentially Expressed Transcripts from Leaf and Root Tissue of Chlorophytum borivilianum: A Plant with High Medicinal Value. Gene., 511: 79–87.
21.    Lee, M. H., Jeong, J. H., Seo, J. W., Shin, C. G.., Kim, Y. S., In, J. G., Yang, D. C., Yi, J. S. and Choi, Y. E. 2004. Enhanced Triterpene and Phytosterol Biosynthesis in Panax ginseng Overexpressing Squalene Synthase Gene. Plant Cell. Physiol., 45: 976–984.
22.    Lee, R. K. Y., Ong, R. C. Y., Cheung, J. Y. N., Li, Y. C., Chan, J. Y. W. and Lee, M. M. S. 2009. Polyphyllin D: A Potential Anti-cancer Agent to Kill Hepatocarcinoma Cells with Multi-drug Resistance. Curr. Chem. Biol., 3: 89–99.
23.    Li, H. 1998. The Phylogeny of the Genus Paris L. In: “The Genus Paris (Trilliaceae)”, (Ed.): Li, H.. Science Press, Beijing, PP. 8–65.
24.    Liu, H., Zhang, T., Chen, X. Q., Huang, Y. and Wang, Q. 2006. Steroidal Saponins of Paris polyphylla Smith var. yunnanensis. Chin. J. Nat. Med., 4: 265–7.
25.    Liu, K. and Muse, S. V. 2005. PowerMarker: Integrated Analysis Environment for Genetic Marker Data. Bioinforma., 21: 2128-2129.
26.    Luo, H. M., Sun, C. and Sun, Y. Z. 2011. Analysis of the Transcriptome of Panax notoginseng Root Uncovers Putative Triterpene Saponin-biosynthetic Genes and Genetic Markers. BMC Genom., 12(Suppl. 5) S5.
27.    Mardis, E. R. 2008. The Impact of Next-generation Sequencing Technology on Genetics. TIG, 24: 133–141.
28.    Margulies, M., Egholm, M., Altman, W. E., Attiya, S. and Bader, J. S. 2005. Genome Sequencing in Microfabricated High-density Picolitre Reactors. Nature, 37: 376–380.
29.    Matsuda, H., Pongpiriyadacha, Y., Morikawa, T., Kishi, A., Kataoka, S. and Yoshikawa, M. 2003. Protective Effects of Steroid Saponins from Paris polyphylla var. yunnanensis on Ethanol- or Indomethacin-induced Gastric Mucosal Lesions in Rats: Structural Requirement for Activity and Mode of Action. Bioorg. Med. Chem. Lett., 13: 1101–1106.
30.    Miller, M. P. 1997. Tools for Population Genetic Analysis, Version 1.3. Department of Biological Sciences, Northern Arizona University, Flagstaff AZ.
31.    Morozova, O. and Marra, M. 2008. Applications of Next-generation Sequencing Technologies in Functional Genomics. Genom., 2: 255–264.
32.    Nybom, H. 2000. Effects of Life History Traits and Sampling Strategies on Genetic Diversity Estimates Obtained with RAPD Markers in Plants. Perspect. Plant. Ecol., 3: 93–114.
33.    Nybom, H. 2004. Comparison of Different Nuclear DNA Markers for Estimating Intraspecific Genetic Diversity in Plants. Mol. Ecol., 13: 1143–1155.
34.    Parchman, T. L., Geist. K. S., Grahnen, J. A., Benkman, C. W. and Buerkle, C. A. 2010. Transcriptome Sequencing in an Ecologically Important Tree Species: Assembly, Annotation, and Marker Discovery. BMC Genom., 11: 180.
35.    Phillips, D. R., Rasbery, J. M., Bartel, B. and Matsuda, S. P. T. 2006. Biosynthetic Diversity in Plant Triterpene Cyclization. Curr. Opin. Plant Biol., 9: 305–314.
36.    Qin, B., Eagles, J., Mellon, F. A., Mylona, P., Pena-Rodriguez, L. and Osbourn, A. E. 2010. High throughput Screening of Mutants of Oat that Are Defective in Triterpene Synthesis. Phytochem., 71: 1245–1252.
37.    Rohmer, M. 2003. Mevalonate-independent Methylerythritol Phosphate Pathway for Isoprenoid Biosynthesis. Elucidation and Distribution. Pure. Appl. Chem., 75: 375–387.
38.    Selkoe, K. A. and Roonen, R. J. 2006. Microsatellites for Ecologists: A Practical Guide to Using and Evaluating Microsatellite Markers. Ecol. Lett., 9: 615–629.
39.    Sharma, R. K., Bhardwaj, P., Negi, R., Mohapatra, T. and Ahuja, P. S. 2009. Identification, Characterization and Utilization of Unigene Derived Microsatellite Markers in Tea (Camellia sinensis L.). BMC Plant. Biol., 9: 53.
40.    Temnykh, S., DeClerck, G., Lukashova, A., Lipovich, L., Cartinhour, S. and McCouch, S. 2001. Computational and Experimental Qnalysis of Microsatellites in Rice (Oryza sativa L.): Frequency, Length Variation, Transposon Associations, and Genetic Marker Potential. Genom. Res., 11: 1441-1452.
41.    Thiel, T., Michalek, W., Varshney, R. K. and Graner, A. 2003. Exploiting EST Databases for the Development and Characterization of Gene-derived SSR-markers in Barley (Hordeum vulgare L.). Theor. Appl. Genet., 106: 411–422.
42.    Tiwari, J. K., Ballabha, R. and Tiwari, P. 2010. Ethnopaediatrics in Garhwal Himalaya Uttarkhand India (Psychomedicine and Medicine). New York Sci. J., 3: 123-126.
43.    Varshney, R. K., Graner, A., Sorrells, M. E. 2005. Genic Microsatellite Markers in Plants: Features and Applications. Trend. Biotechnol., 23: 48–55.
44.    Vincken, J. P., Heng, L., deGroot, A. and Gruppen, H. 2007. Saponins: Classification and Occurence in the Plant Kingdom. Phytochem., 68: 275–297.
45.    Wang, L., Li, P.and Brutnell, T. P. 2010. Exploring Plant Transcriptomes Using Ultra High-throughput Sequencing. Brief Funct. Genom., 9: 118–128.
46.    Wang, W., Wang, Y., Zhang, Q., Qi, Y. and Guo, D. 2009. Global Characterization of Artemisia annua Glandular Trichome Transcriptome Using 454 Pyrosequencing. BMC Genom., 10: 465.
47.    Weber, A. P., Weber, K. L., Carr, K., Wilkerson, C. and Ohlrogge, J. B. 2007. Sampling the Arabidopsis Transcriptome with Massively Parallel Pyrosequencing. Plant. Physiol., 144: 32–42.
48.    Wu, X., Wang, L., Wang, G.. C., Wang, H., Dai, Y., Yang, X. X., Ye, W. C. and Li, Y. L. 2013. Triterpenoid Saponins from Rhizomes of Paris polyphylla var. yunnanensis. Carbohydr. Res., 4: 3681–3687.
49.    Xu, T. H., Ma, X. X., Xu, Y. J., Xie, S. X., Zhao, H. F., Si, Y. S., Han, D., Li, Y., Niu, J. Z. and Xu, D. M. 2007. New Steroidal Saponin from Paris polyphylla Sm. var. yunnanensis (France). Hand-Mazz. Chem. J. Chin. U, 28: 2303–6.
50.    Yeh, F. C., Yang, R. C. and Boyle, T. 1999. POPGENE 32, Version 1.31. Population Genetics Software, Edmonton University of Alberta.
51.    Zalapa, J. E., Cuevas, H., Zhu, H., Steffan, S., Senalik, D., Zeldin, E. and McCown, B. 2012. Using Nest-generation Sequencing Approaches to Isolate Simple Sequence Repeat (SSR) Loci in the Plant Sciences. Am. J. Bot., 99: 193-208.
52.    Zhang, C. B., Peng, B., Zhang, W. L., Wang, S. M., Sun, H., Dong, Y. S. and Zhao, L. M. 2014. Application of SSR Markers for Purity Testing of Commercial Hybrid Soybean (Glycine max L.). J. Agr. Sci. Tech., 16(6): 1389-1396.
53.    Zhang, S. P. 2007. Research Progress on Chemical Constituents and Pharmacological Effect of Genus Paris. Strait. Pharm. J., 19: 4–7.
54.    Zhao, J. L., Mou, Y., Shan, T. J., Li, Y., Zhou, L. G., Wang, M. G.. and Wang, J. G. 2010 .Antimicrobial Metabolites from the Endophytic Fungus Pichia guilliermondii Isolated from Paris polyphylla var. yunnanensis. Mol., 15: 7961–7970.
55.    Zhou, X., Su, Z., Sammons, R. D., Peng, Y. H., Tranel, P. J., Stewart, C. N. and Yuan, J. S. 2009. Novel Software Package for Cross-Platform Transcriptome Analysis (CPTRA). BMC Bioinforma., 10: S16.
Journal of Agricultural Science and Technology
Volume 18, Issue 2
March and April 2016
Pages 437-452