The continuing increase in size and quality of the “short reads” raw data is a significant help for the quality of the assembly obtained through various bioinformatics tools. However, building a reference genome sequence for most plant species remains a significant challenge due to the large number of repeated sequences which are problematic for a whole-genome quality de novo assembly. Furthermore, for most SNP identification approaches in plant genetics and breeding, only the “Gene-space” regions including the promoter, exon and intron sequences are considered. Results: We developed the iPea protocol to produce a de novo Gene-space assembly by reconstructing, in an iterative way, the non-coding sequence flanking the Unigene cDNA sequence through addition of next-generation DNA-seq data. The approach was elaborated with the large diploid genome of pea (<em>Pisum sativum</em> L.), rich in repetitive sequences. The final Gene-space assembly included 35,400 contigs (97 Mb), covering 88 % of the 40,227 contigs (53.1 Mb) of the PsCam_low-copy Unigen set. Its accuracy was validated by the results of the built GenoPea 13.2 K SNP Array. Conclusion: The iPEA protocol allows the reconstruction of a Gene-space based from RNA-Seq and DNA-seq data with limited computing resources.< Leer menos