De novo assembly and characterization of the carrot transcriptome reveals novel genes, new markers, and genetic diversity

Massimo Iorizzo,Douglas A Senalik,Marta Matvienko,Megan Bowman,Allen Van Deynze,Dariusz Grzebelus,Hamid Ashrafi,Philipp W Simon,Pablo F Cavagnaro

doi:10.1186/1471-2164-12-389

Abstract

BackgroundAmong next generation sequence technologies, platforms such as Illumina and SOLiD produce short reads but with higher coverage and lower cost per sequenced nucleotide than 454 or Sanger. A challenge now is to develop efficient strategies to use short-read length platforms for de novo assembly and marker development. The scope of this study was to develop a de novo assembly of carrot ESTs from multiple genotypes using the Illumina platform, and to identify polymorphisms.ResultsA de novo assembly of transcriptome sequence from four genetic backgrounds produced 58,751 contigs and singletons. Over 50% of these assembled sequences were annotated allowing detection of transposable elements and new carrot anthocyanin genes. Presence of multiple genetic backgrounds in our assembly allowed the identification of 114 computationally polymorphic SSRs, and 20,058 SNPs at a depth of coverage of 20× or more. Polymorphisms were predominantly between inbred lines except for the cultivated x wild RIL pool which had high intra-sample polymorphism. About 90% and 88% of tested SSR and SNP primers amplified a product, of which 70% and 46%, respectively, were of the expected size. Out of verified SSR and SNP markers 84% and 82% were polymorphic. About 25% of SNPs genotyped were polymorphic in two diverse mapping populations.ConclusionsThis study confirmed the potential of short read platforms for de novo EST assembly and identification of genetic polymorphisms in carrot. In addition we produced the first large-scale transcriptome of carrot, a species lacking genomic resources.

Highlights

Among generation sequence technologies, platforms such as Illumina and SOLiD produce short reads but with higher coverage and lower cost per sequenced nucleotide than 454 or Sanger
Sequencing and assembly To develop an overview of the carrot transcriptome and obtain an initial comparison of cultivated and wild carrot transcripts, normalized cDNA libraries were constructed from four sources: two orange unrelated inbred lines of European origin, B493 and B6274 with Imperator and Nantes root shapes, respectively, [18]; a purple/ yellow inbred line B7262 derived from an intercross between purple Turkish and orange Danvers (European) carrots [19]; and a pool of F4 RILs derived from a cross between B493 and QAL, a wild carrot from North America
At most two haplotypes are represented among these transcripts

Summary

Introduction

Among generation sequence technologies, platforms such as Illumina and SOLiD produce short reads but with higher coverage and lower cost per sequenced nucleotide than 454 or Sanger. A challenge now is to develop efficient strategies to use short-read length platforms for de novo assembly and marker development. The scope of this study was to develop a de novo assembly of carrot ESTs from multiple genotypes using the Illumina platform, and to identify polymorphisms. The most widely cultivated member of the Apiaceae, carrot Sativus L.) is a diploid (2n = 2x = 18) with a relatively small genome of 480 Mb. The most widely cultivated member of the Apiaceae, carrot Sativus L.) is a diploid (2n = 2x = 18) with a relatively small genome of 480 Mb It has a history of cultivation as a root crop that dates back about 1100 years to Afghanistan [1]. The carrot plastome has been sequenced [11], a 17-fold bacterial artificial chromosome library (BAC) has been developed and characterized [12], and microsatellite markers are being developed (unpublished data, Cavagnaro et al.), only relatively modest genomic resources have been developed for carrot [13]

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Discussion

Conclusion