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

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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.

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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

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Acknowledgments

The authors wish to thank Jennifer Chagoya at Texas A&M AgriLife Research, and Halee Hughes and Nancy Layland at the USDA-ARS, Lubbock for technical support. This work was funded by grants from the Texas Peanut Producers Board award CY2008-Burow-TTU-Development to MDB and CES, and 2009-TTU-Burow-Genotyping to MDB, National Peanut Board grant #332/TX-99/1139 to MDB, and #332/TX-99/1213 to MDB and CES, Peanut Foundation grant 04-810-08 to MDB, Ogallala Aquifer Initiative award IPM12.06 to MDB, and United States Department of Agriculture/National Institute of Food and Agriculture Hatch Act award TEX08835 to MDB.

Conflict of interest

The authors declare that they have no conflict of interest. All experiments were performed in accordance with current biomedical research ethical standards in the US.

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Authors and Affiliations

  1. Department of Plant and Soil Sciences, Texas Tech University, Lubbock, TX, 79409, USA

    Ratan Chopra, Thea A. Wilkins & Mark D. Burow

  2. USDA-ARS-CSRL, 3810 4th Street, Lubbock, TX, 79415, USA

    Gloria Burow

  3. National Center for Genome Resources, 2935 Rodeo Park Drive East, Santa Fe, NM, 87505, USA

    Andrew Farmer & Joann Mudge

  4. Texas A&M AgriLife Research, 1229 N. US Highway 281, Stephenville, TX, 76401, USA

    Charles E. Simpson

  5. Heep Center, Texas A&M AgriLife Research, College Station, TX, 77843, USA

    Michael R. Baring

  6. Agricultural Science Center, New Mexico State University, Clovis, NM, 88001, USA

    Naveen Puppala

  7. USDA-ARS, 1301 N. Western, Stillwater, OK, 74075, USA

    Kelly D. Chamberlin

  8. Texas A&M AgriLife Research, 1102 East FM 1294, Lubbock, TX, 79403, USA

    Mark D. Burow

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  1. Ratan Chopra
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  2. Gloria Burow
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  3. Andrew Farmer
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  5. Charles E. Simpson
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Corresponding author

Correspondence to Mark D. Burow.

Additional information

Communicated by S. Hohmann.

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Figure 1: Contig distribution of the custom reference assembly, for contigs with length ≥ 200 bp (TIFF 194 kb)

438_2014_976_MOESM2_ESM.tif

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

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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Chopra, R., Burow, G., Farmer, A. et al. Next-generation transcriptome sequencing, SNP discovery and validation in four market classes of peanut, Arachis hypogaea L.. Mol Genet Genomics 290, 1169–1180 (2015). https://doi.org/10.1007/s00438-014-0976-4

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