Molecular Genetics and Genomics

, Volume 290, Issue 3, pp 1169–1180 | Cite as

Next-generation transcriptome sequencing, SNP discovery and validation in four market classes of peanut, Arachis hypogaea L.

  • Ratan Chopra
  • Gloria Burow
  • Andrew Farmer
  • Joann Mudge
  • Charles E. Simpson
  • Thea A. Wilkins
  • Michael R. Baring
  • Naveen Puppala
  • Kelly D. Chamberlin
  • Mark D. Burow
Original Paper

Abstract

Single-nucleotide polymorphisms, which can be identified in the thousands or millions from comparisons of transcriptome or genome sequences, are ideally suited for making high-resolution genetic maps, investigating population evolutionary history, and discovering marker–trait linkages. Despite significant results from their use in human genetics, progress in identification and use in plants, and particularly polyploid plants, has lagged. As part of a long-term project to identify and use SNPs suitable for these purposes in cultivated peanut, which is tetraploid, we generated transcriptome sequences of four peanut cultivars, namely OLin, New Mexico Valencia C, Tamrun OL07 and Jupiter, which represent the four major market classes of peanut grown in the world, and which are important economically to the US southwest peanut growing region. CopyDNA libraries of each genotype were used to generate 2 × 54 paired-end reads using an Illumina GAIIx sequencer. Raw reads were mapped to a custom reference consisting of Tifrunner 454 sequences plus peanut ESTs in GenBank, compromising 43,108 contigs; 263,840 SNP and indel variants were identified among four genotypes compared to the reference. A subset of 6 variants was assayed across 24 genotypes representing four market types using KASP chemistry to assess the criteria for SNP selection. Results demonstrated that transcriptome sequencing can identify SNPs usable as selectable DNA-based markers in complex polyploid species such as peanut. Criteria for effective use of SNPs as markers are discussed in this context.

Keywords

Peanut Groundnut Arachis Transcriptome SNP KASP 

Abbreviations

SNP

Single-nucleotide polymorphism

EST

Expressed sequence tag

RIN

RNA integrity number

TSA

Transcriptome shotgun assembly

O/L

Oleic/linoleic

RFLP

Restriction fragment length polymorphism

AFLP

Amplified fragment length polymorphism

SSR

Simple sequence repeat

Supplementary material

438_2014_976_MOESM1_ESM.tif (194 kb)
Figure 1: Contig distribution of the custom reference assembly, for contigs with length ≥ 200 bp (TIFF 194 kb)
438_2014_976_MOESM2_ESM.tif (252 kb)
Figure 2: Histogram of the number of SNPs across 36,102 contigs which were aligned to the custom reference (TIFF 251 kb)
438_2014_976_MOESM3_ESM.tif (247 kb)
Figure 3: FAD2 gene sequences, showing similarity of homoeologous copies in the region resulting in functional differences between alleles, and interference from homoeologous copies in the KASP assay. Differences between genes and alleles for each gene in this region are highlighted. KASP primers designed to amplify each gene are shown above and below each gene as the sequence that they are intended to amplify; SNP1_ASP is the pair of allele-specific primer designed to selectively amplify each allele of SNP1; SNP 1_C is the SNP1 common primer; designations are similar for SNP 2. When genomic DNA is amplified, SNP 1 primers will distinguish the two alleles of sequence 1 (FAD2B), and will amplify sequence 2 (FAD2A) giving the sequence 1 reference allele score for sequence 2. Likewise, SNP 2 primers will distinguish the two alleles of sequence 2 (FAD2A), but will also amplify sequence 1 (FAD2B), giving the sequence 2 (FAD2A) reference allele score (TIFF 247 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Ratan Chopra
    • 1
  • Gloria Burow
    • 2
  • Andrew Farmer
    • 3
  • Joann Mudge
    • 3
  • Charles E. Simpson
    • 4
  • Thea A. Wilkins
    • 1
  • Michael R. Baring
    • 5
  • Naveen Puppala
    • 6
  • Kelly D. Chamberlin
    • 7
  • Mark D. Burow
    • 1
    • 8
  1. 1.Department of Plant and Soil SciencesTexas Tech UniversityLubbockUSA
  2. 2.USDA-ARS-CSRLLubbockUSA
  3. 3.National Center for Genome ResourcesSanta FeUSA
  4. 4.Texas A&M AgriLife ResearchStephenvilleUSA
  5. 5.Heep CenterTexas A&M AgriLife ResearchCollege StationUSA
  6. 6.Agricultural Science CenterNew Mexico State UniversityClovisUSA
  7. 7.USDA-ARSStillwaterUSA
  8. 8.Texas A&M AgriLife ResearchLubbockUSA

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