Next-generation transcriptome sequencing, SNP discovery and validation in four market classes of peanut, Arachis hypogaea L.
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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.
KeywordsPeanut Groundnut Arachis Transcriptome SNP KASP
Expressed sequence tag
RNA integrity number
Transcriptome shotgun assembly
Restriction fragment length polymorphism
Amplified fragment length polymorphism
Simple sequence repeat
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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