Molecular Genetics and Genomics

, Volume 279, Issue 5, pp 535–543 | Cite as

Genomic DNA sequence comparison between two inbred soybean cyst nematode biotypes facilitated by massively parallel 454 micro-bead sequencing

  • Sadia Bekal
  • J. P. Craig
  • M. E. Hudson
  • T. L. Niblack
  • L. L. Domier
  • K. N. LambertEmail author
Original Paper


Heterodera glycines, the soybean cyst nematode (SCN), is a damaging agricultural pest that could be effectively managed if critical phenotypes, such as virulence and host range could be understood. While SCN is amenable to genetic analysis, lack of DNA sequence data prevents the use of such methods to study this pathogen. Fortunately, new methods of DNA sequencing that produced large amounts of data and permit whole genome comparative analyses have become available. In this study, 400 million bases of genomic DNA sequence were collected from two inbred biotypes of SCN using 454 micro-bead DNA sequencing. Comparisons to a BAC, sequenced by Sanger sequencing, showed that the micro-bead sequences could identify low and high copy number regions within the BAC. Potential single nucleotide polymorphisms (SNPs) between the two SCN biotypes were identified by comparing the two sets of sequences. Selected resequencing revealed that up to 84% of the SNPs were correct. We conclude that the quality of the micro-bead sequence data was sufficient for de novo SNP identification and should be applicable to organisms with similar genome sizes and complexities. The SNPs identified will be an important starting point in associating phenotypes with specific regions of the SCN genome.


SNP Indel Heterodera glycines DNA Polymorphism Genetic 



This work was supported by grant 2006-35604-16731 from the United States Department of Agriculture National Research Initiative Competitive Grants Program. The CFAR program at the University of Illinois at Urbana-Champaign. The Illinois Soybean Production Operating Board, The North Central Regional Integrated Pest Management, Competitive Grants Program and the United Soybean Board. We also wish to thank Kam Colgrove for his technical support. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. This work has been carried out in compliance with the current laws governing genetic experimentation in the United States of America.


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410PubMedGoogle Scholar
  2. Angly FE, Felts B, Breitbart M, Salamon P, Edwards RA, Carlson C, Chan AM, Haynes M, Kelley S, Liu H, Mahaffy JM, Mueller JE, Nulton J, Olson R, Parsons R, Rayhawk S, Suttle CA, Rohwer F (2006) The marine viromes of four oceanic regions. PLoS Biol 4:e368PubMedCrossRefGoogle Scholar
  3. Atibalentja N, Bekal S, Domier LL, Niblack TL, Noel GR, Lambert KN (2005) A genetic linkage map of the soybean cyst nematode Heterodera glycines. Mol Gen Genomics 273:273–281CrossRefGoogle Scholar
  4. Bekal S, Niblack TL, Lambert KN (2003) A chorismate mutase from the soybean cyst nematode Heterodera glycines shows polymorphisms that correlate with virulence. Mol Plant Microbe Interact 16:439–446PubMedCrossRefGoogle Scholar
  5. Choi I Y, Hyten DL, Matukumalli LK, Song Q, Chaky JM, Quigley CV, Chase K, Lark KG, Reiter RS, Yoon MS, Hwang EY, Yi SI, Young ND, Shoemaker RC, van Tassell CP, Specht JE, Cregan PB (2007) A soybean transcript map: gene distribution, haplotype and SNP analysis. Genetics 176:685–696PubMedCrossRefGoogle Scholar
  6. Dong K, Opperman CH (1997) Genetic analysis of parasitism in the soybean cyst nematode Heterodera glycines. Genetics 146:1311–1318PubMedGoogle Scholar
  7. Edwards RA, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M, Peterson DM, Saar MO, Alexander S, Alexander EC Jr, Rohwer F (2006) Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7:57PubMedCrossRefGoogle Scholar
  8. Girard A, Sachidanandam R, Hannon GJ, Carmell MA (2006) A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 442:199–202PubMedGoogle Scholar
  9. Goldberg SM, Johnson J, Busam D, Feldblyum T, Ferriera S, Friedman R, Halpern A, Khouri H, Kravitz SA, Lauro FM, Li K, Rogers YH, Strausberg R, Sutton G, Tallon L, Thomas T, Venter E, Frazier M, Venter JC (2006) A sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes. Proc Natl Acad Sci USA 103:11240–11245PubMedCrossRefGoogle Scholar
  10. Green RE, Krause J, Ptak SE, Briggs AW, Ronan MT, Simons JF, Du L, Egholm M, Rothberg JM, Paunovic M, Paabo S (2006) Analysis of one million base pairs of Neanderthal DNA. Nature 444:330–336PubMedCrossRefGoogle Scholar
  11. Hudson TJ, Stein LD, Gerety SS, Ma J, Castle AB, Silva J, Slonim DK, Baptista R, Kruglyak L, Xu SH, Hu X, Colbert AM, Rosenberg C, Reeve-Daly MP, Rozen S, Hui L, Wu X, Vestergaard C, Wilson KM, Bae JS, Maitra S, Ganiatsas S, Evans CA, DeAngelis MM, Ingalls KA, Nahf RW, Horton LT Jr, Anderson MO, Collymore AJ, Ye W, Kouyoumjian V, Zemsteva IS, Tam J, Devine R, Courtney DF, Renaud MT, Nguyen H, O’Connor TJ, Fizames C, Faure S, Gyapay G, Dib C, Morissette J, Orlin JB, Birren BW, Goodman N, Weissenbach J, Hawkins TL, Foote S, Page DC, Lander ES (1995) An STS-based map of the human genome. Science 270:1945–1954PubMedCrossRefGoogle Scholar
  12. Huse SM, Huber JA, Morrison HG, Sogin ML, Welch DM (2007) Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biol 8:R143PubMedCrossRefGoogle Scholar
  13. International HapMap Consortium (2005) A haplotype map of the human genome. Nature 437:1299–1320CrossRefGoogle Scholar
  14. Korbel JO, Urban AE, Affourtit JP, Godwin B, Grubert F, Simons JF, Kim PM, Palejev D, Carriero NJ, Du L, Taillon BE, Chen Z, Tanzer A, Saunders AC, Chi J, Yang F, Carter NP, Hurles ME, Weissman SM, Harkins TT, Gerstein MB, Egholm M, Snyder M (2007) Paired-end mapping reveals extensive structural variation in the human genome. Science 318:420–426PubMedCrossRefGoogle Scholar
  15. Korf I, Flicek P, Duan D, Brent MR (2001) Integrating genomic homology into gene structure prediction. Bioinformatics 17:S140–148PubMedGoogle Scholar
  16. Krause L, Diaz NN, Bartels D, Edwards RA, Puhler A, Rohwer F, Meyer F, Stoye J (2006) Finding novel genes in bacterial communities isolated from the environment. Bioinformatics 22:e281–e289PubMedCrossRefGoogle Scholar
  17. Lambert KN, Bekal S, Domier LL, Niblack TL, Noel GR, Smyth CA (2005) Selection of Heterodera glycines chorismate mutase-1 alleles on nematode-resistant soybean. Mol Plant Microbe Interact 18:593–601PubMedCrossRefGoogle Scholar
  18. Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI, Schuster SC, Schleper C (2006) Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature 442:806–809PubMedCrossRefGoogle Scholar
  19. Macas J, Neumann P, Navratilova A (2007) Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula. BMC Genomics 8:427PubMedCrossRefGoogle Scholar
  20. Malhi RS, Sickler B, Lin D, Satkoski J, Tito RY, George D, Kanthaswamy S, Smith DG (2007) MamuSNP: a resource for Rhesus Macaque (Macaca mulatta) genomics. PLoS ONE 2:e438PubMedCrossRefGoogle Scholar
  21. Mardis ER (2006) Anticipating the 1,000 dollar genome. Genome Biol 7:112PubMedCrossRefGoogle Scholar
  22. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF, Rothberg JM (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376–380PubMedGoogle Scholar
  23. Ng P, Tan JJ, Ooi HS, Lee YL, Chiu KP, Fullwood MJ, Srinivasan KG, Perbost C, Du L, Sung WK, Wei CL, Ruan Y (2006) Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes. Nucleic Acids Res 34:e84PubMedCrossRefGoogle Scholar
  24. Niblack TL, Arelli PR, Noel GR, Opperman CH, Orf JH (2002) A revised classification scheme for genetically diverse populations of Heterodera glycines. J Nematology 34:279–288Google Scholar
  25. Niblack TL, Heinz RD, Smith GS, Donald PA (1993) Distribution, density, and diversity of Heterodera glycines populations in Missouri. Ann Appl Nematology 25:880–886Google Scholar
  26. Picoult-Newberg L, Ideker TE, Pohl MG, Taylor SL, Donaldson MA, Nickerson DA, Boyce-Jacino M (1999) Mining SNPs from EST databases. Genome Res 9:167–174PubMedGoogle Scholar
  27. Poinar HN, Schwarz C, Qi J, Shapiro B, Macphee RD, Buigues B, Tikhonov A, Huson DH, Tomsho LP, Auch A, Rampp M, Miller W, Schuster SC (2006) Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Science 311:392–394PubMedCrossRefGoogle Scholar
  28. Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11PubMedCrossRefGoogle Scholar
  29. Rouppe van der Voort JNAM, van Eck HJ, van Zandvoort PM, Overmars H, Helder J, Bakker J (1999) Linkage analysis by genotyping of sibling populations: agenetic map for the potato cyst nematode contructed using “pseudo-F2” mapping strategy. Mol Gen Genet 261:1021–1031CrossRefGoogle Scholar
  30. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386PubMedGoogle Scholar
  31. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  32. Schmitt DP (2004). In: Schmitt DP, Wrather JA, Riggs RD (eds) Biology and management of soybean cyst nematode. Walsworth Publishing Company, Marceline, pp 1–8Google Scholar
  33. Shaffer C (2007) Next-generation sequencing outpaces expectations. Nat Biotechnol 25:149PubMedCrossRefGoogle Scholar
  34. Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc Natl Acad Sci USA 103:12115–12120PubMedCrossRefGoogle Scholar
  35. Swaminathan K, Varala K, Hudson ME (2007) Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey. BMC Genomics 8:132PubMedCrossRefGoogle Scholar
  36. Taillon-Miller P, Gu Z, Li Q, Hillier L, Kwok PY (1998) Overlapping genomic sequences: a treasure trove of single-nucleotide polymorphisms. Genome Res 8:748–754PubMedGoogle Scholar
  37. Thomas RK, Nickerson E, Simons JF, Janne PA, Tengs T, Yuza Y, Garraway LA, LaFramboise T, Lee JC, Shah K, O’Neill K, Sasaki H, Lindeman N, Wong KK, Borras AM, Gutmann EJ, Dragnev KH, DeBiasi R, Chen TH, Glatt KA, Greulich H, Desany B, Lubeski CK, Brockman W, Alvarez P, Hutchison SK, Leamon JH, Ronan MT, Turenchalk GS, Egholm M, Sellers WR, Rothberg JM, Meyerson M (2006) Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nat Med 12:852–855PubMedCrossRefGoogle Scholar
  38. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027–1031PubMedCrossRefGoogle Scholar
  39. Wei C, Lamesch P, Arumugam M, Rosenberg J, Hu P, Vidal M, Brent MR (2005) Closing in on the C. elegans ORFeome by cloning TWINSCAN predictions. Genome Res 15:577–582PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Sadia Bekal
    • 1
  • J. P. Craig
    • 1
  • M. E. Hudson
    • 1
  • T. L. Niblack
    • 1
  • L. L. Domier
    • 2
  • K. N. Lambert
    • 1
    Email author
  1. 1.Crop SciencesUniversity of IllinoisUrbanaUSA
  2. 2.USDA-ARS, Crop SciencesUniversity of IllinoisUrbanaUSA

Personalised recommendations