A deep-coverage tomato BAC library and prospects toward development of an STC framework for genome sequencing

Abstract

Recently a new strategy using BAC end sequences as sequence-tagged connectors (STCs) was proposed for whole-genome sequencing projects. In this study, we present the construction and detailed characterization of a 15.0 haploid genome equivalent BAC library for the cultivated tomato, Lycopersicon esculentum cv. Heinz 1706. The library contains 129,024 clones with an average insert size of 117.5 kb and a chloroplast content of 1.11%. BAC end sequences from 1490 ends were generated and analyzed as a preliminary evaluation for using this library to develop an STC framework to sequence the tomato genome. A total of 1205 BAC end sequences (80.9%) were obtained, with an average length of 360 high-quality bases, and were searched against the GenBank database. Using a cutoff expectation value of <10(-6), and combining the results from BLASTN, BLASTX, and TBLASTX searches, 24.3% of the BAC end sequences were similar to known sequences, of which almost half (48.7%) share sequence similarities to retrotransposons and 7% to known genes. Some of the transposable element sequences were the first reported in tomato, such as sequences similar to maize transposon Activator (Ac) ORF and tobacco pararetrovirus-like sequences. Interestingly, there were no BAC end sequences similar to the highly repeated TGRI and TGRII elements. However, the majority (70.3%) of STCs did not share significant sequence similarities to any sequences in GenBank at either the DNA or predicted protein levels, indicating that a large portion of the tomato genome is still unknown. Our data demonstrate that this BAC library is suitable for developing an STC database to sequence the tomato genome. The advantages of developing an STC framework for whole-genome sequencing of tomato are discussed.

Figure 1

Sizing of tomato BAC inserts. An ethidium bromide-stained gel of plasmid DNA digested with NotI enzyme to release the tomato genomic inserts from the 7.4-kb pBeloBAC11 vector. Lanes 1 and 30 contain λ mid-range PFGE size markers (New England Biolabs).

Figure 2

Insert size distribution of the tomato BAC library based on the sizing of 498 random clones by PFGE. Of the clones in the library 78% contained inserts >100 kb.

Figure 3

The distribution of 306 STCs originated from tomato genomic DNA based on putative identification with an E < 10⁻⁶.