SNP-PHAGE Applications

SNP-PHAGE peline has been used at Soybean Genomics and Improvement Laboratory at the USDA, ARS, Beltsville to analyze more than 3000 STS. Zhu et al. 2003 have used this software to analyze >76 kbp of soybean sequence and identified 281 polymorphisms. DNA sequences amplified from genomic DNA using PCR primers designed to complete genes, cDNAs and random genomic sequence. 28.7 kbp of coding sequence, 37.9 kbp of noncoding perigenic DNA and 9.7kbp of random genomic sequence to identify polymorphisms and thereby estimate sequence variation in different regions of the genome. They calculated the nucleotide diversity expressed as Watterson’s q as 0.00097 with 0.00053 in coding and 0.00111 in non coding perigenic regions. From the haplotype analysis it was observed that 6 genotypes represented 75% of all sequence variations in 25 genotypes and 90% of commonly identified polymorphisms.

Choi et al.,(Choi et al. 2003) used this software and identified approximately 5000 polymorphisms by the random 3’ sequence amplification method. PCR primer success rate was 57%. 44% of the succesfully amplified sequences had at least one intron. Nucleotide diversity (θ) in introns is 1.456 ×10-3, in exons 0.834 ×10-3 57% of the polymorphisms found are in exon regions. These polymorphisms will be very useful in mapping and characterizing the genes for important agronomic traits. Approximately 1000 polymorphisms were identified by targeted sequence amplification method.

Choi et al (Choi et al. 2004) analyzed chromatogram data from several legumes amplified from soybean primers using this software. In order to assist in the development of a legume-wide universal set of PCR-based markers, 1204 of these soybean-derived sequence tagged sites ( STS) were used to attempt amplification in other legume species including common bean (Phaseolus vulgaris), cowpea (Vigna ungiculata) chickpea (Cicer arietinum) pea (Pisum sativum), peanut (Arachis hypogea), barrel medic (Medicago truncatula) and Lotus japonicus. Two commonly used mapping parents of each species were used in SNP discovery. Sequenable products were obtained for 15.6, 14.0., 6.2, 5.6, 2.7, 2.7 and 2.1% respectively. Sequence comparison of the two genotypes from each species indicated that nucleotide diversity (θ) was the lowest in soybean (0.001), cowpea (0.00095) and Lotus (0.0001) while nucleotide diversity of common bean and pea were the highest, θ = 0.0048 and θ = 0.0042, respectively. A total of 23 primer sets were discovered as universal primers in five legume species; soybean, common bean, cowpea, chickpea and barrel medic. The SNP markers discovered from this study can serve as genetic markers for each legume species mapping. Hyten et al is using this software to study linkage disequilibrium (LD) in soybean.

Publications / Poster Presentations

• Matukumalli L. K, 2004.
  Development of bioinformatics applications for prediction and validation of polymorphisms in soybean (Glycine max) genome using EST data.
  Doctoral Thesis submitted to George Mason University, Aug 2004.
• Choi, I.-Y., D.L. Hyten, L. K. Matukumalli, S.-I. Yi, and P.B. Cregan, 2004.
  SNP Discovery in Legume Species Using Primers Derived from Soybean Unigenes
  Beltsville Agriculture Research Center Poster Day, Beltsville, MD
• Hyten D.L, Song Q.J, Cregan P.B, 2003,
  Linkage Disequilibrium in Four Soybean Populations,
  PAG XI The International Conference on the Status of Plant & Animal Genome Research, San Diego, CA
• Ik-young Choi, DL Hyten, Matukumalli LK , P.B.Cregan, 2003.
  Single Nucleotide Polymorphism discovery in 3’ EST sequence of soybean,
  PAG XI The International Conference on the Status of Plant & Animal Genome Research, San Diego, CA
• Lakshmi K. Matukumalli ., John J. Grefenstette., Curtis P. Van Tassell., Ik-Young Choii and Perry B Cregan. 2004
  In silico prediction and validation of polymorphisms in soybean genome using EST data.
  Beltsville Agriculture Research Center Poster Day, Beltsville, MD.
• Zhu Y.L, Song Q.J, Hyten D.L, Van Tassell C.P, Matukumalli L.K, Grimm D.R, Hyatt S.M,
  Fickus E.W, Young N.D, Cregan P.B. 2003.
  Single-nucleotide polymorphisms in soybean. Genetics 163:1123-1134.
• Matukumalli, LK., Grefenstette, JJ., Van Tassell, CP., Choi, I-Y., Cregan, PB.
  Development of Algorithms for prediction and validation of polymorphisms in polyploids (soybean) using EST data.
  TIGR GSAC Conference 2004.
• Jeanne L. Burton1, Sally A. Madsen, Ling-Chu Chang, Patty S.D. Weber, Guilherme J.M. Rosa, Lakshmi K. Matukumalli and Tad S. Sonstegard , 2004.
  Expression Profiles And SNP Analysis Of Genes That Regulate Neutrophil Apoptosis, Endothelial Adhesion, And Extracellular Matrix Remodeling At Parturition In Dairy Cows.
  PAG XIII The International Conference on the Status of Plant & Animal Genome Research, San Diego, CA
• Eun-Young Hwang, Kyujung Van, Moon Young Kim, Lakshmi Kumar, Suk-Ha Lee, Gary Stacey, Perry B Cregan, 2004.
  Single Nucleotide Polymorphisms Using Primers Designed To Methyl-Filtrated Clones In Soybean
  PAG XIII The International Conference on the Status of Plant & Animal Genome Research, San Diego, CA
• Ik-Young Choi , David L. Hyten , Lakshmi K. Matukumalli , Eun-Young Hwang , Perry B. Cregan, 2004.
  SNP Discovery In 3'-EST Sequence Of Soybean
  PAG XIII The International Conference on the Status of Plant & Animal Genome Research, San Diego, CA
• Lakshmi K Matukumalli, John J Grefenstette, Curtis P Van Tassell, Ik-Young Choii, Perry B Cregan, 2004.
  Application of Machine Learning programs towards Accelerating Polymorphisms Discovery
  7th Annual Conference on Computational Genomics