ISBP and SSR markers derived from the wheat chromosome 4D survey sequencing

VANZETTI L ; ROMERO J; CLAVIJO B; TABBITA F; GARBUS I ; LOMBARDO L; BONAFEDE M, ; CATIVELLI M, ; LOMBARDO L, ; RONCALLO P, ; TRANQUILLI G, ; PANIEGO N, ; HELGUERA M, ; ECHENIQUE V.

San Diego

Conferencia; Plant and Animal Conference; 2012

schergo

Flow sorting of individual chromosomes followed by shotgun sequencing using next generation sequencing (NGS) technologies have been applied to 4D chromosome arms from Chinese Spring (CS). Sequence data equivalent to a 3.6x chromosome coverage was produced by shotgun sequencing with a Roche 454 NGS platform. Sequences were assembled by the Newbler program producing contigs for each arm. This assembly was used to identify molecular markers like insertion site-based polymorphisms (ISBP) markers and microsatellites (SSRs) with potential use in germplasm characterization, genome saturation and marker assisted selection in wheat. ISBP markers were designed with the IsbpFinder program. A total of 79,891 and 65,433 ISBP markers were developed for chromosomes 4DS and 4DL, respectively. These markers were mostly designed on sites involving low copy DNA and CACTA and Gypsy transposable elements. About 23% of them showed a high confidence level. From this group a set of 30 markers for each chromosome arm was selected and tested with CS cytogenetic stocks (nullitetrasomic and ditelosomic lines) to validate their chromosome specificity. Validated markers were tested against a set of CS 4D-deletion lines, to asses their physical location. On the other hand from a total of 38,673 SSRs detected using the Sputnik algorithm 17,570 (45.43%) were located on 4DL and 21,103 on 4DS (54.56 %). The predominant motifs were trinucleotides (39%), followed by dinucleotides (29%), pentanucleotides (17%) and tetranucleotides (15%). These results show the potentiality of survey sequencing to deliver useful resources for applied genetics, and also revealed in some extent the reliability of the DRAFT assemblyproduced.