ISBP and SSR Markers Derived from the Wheat Chromosome 4D Survey Sequencing

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 develop 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. A total of 79,891 and 65,433 ISBP markers (created with IsbpFinder software) were obtained from 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 sub-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 also tested against a set of CS 4D-deletion lines, to assess 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 dominant motifs were trinucleotides (39%), followed by dinucleotides (29%), pentanucleotides (17%) and tetranucleotides (15%). These results show the high potentiality of survey sequencing to deliver useful resources for applied genetics, and also revealed in some extent the reliability of the draft assembly
produced.