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Gossypium australe F. Muell (2n = 2x = 26) is a wild perennial species possessing agronomic useful traits that would be interesting to introgress into G. hirsutum L. (2n = 4x = 52), the main cultivated cotton species. To isolate monosomic alien addition lines (MAALs) of G. australe in G. hirsutum, the [2(G. hirsutum · G. australe) · G. hirsutum] pentaploid (2n = 5x = 65) was backcrossed as male parent to G. hirsutum. Analysis of 42 BC1 plants and seven alien addition lines, already available, with 150 SSR markers developed from G. hirsutum revealed a cross-species amplification rate of 100% and a polymorphism rate of 56%. Eighty polymorphic SSR markers generated 87 G. australe-specific loci that have been assigned by a hierarchical cluster analysis to 13 linkage groups corresponding to the 13 chromosomes of G. australe. Analysis by SSR markers and genomic in situ hybridization of the self-progeny of disomic alien addition lines, backcross progeny of the pentaploid, allowed the isolation of five new MAALs. (Resume d'auteur)

All protocols for Next Generation Sequencers (NGS) start with the preparation of a DNA library from extracted DNA. Quality, number, and length of the sequences produced by NGS all depend on the quality of the library that was prepared, itself dependent on the DNA quality. Generally, when using NGS, one would wish to obtain the genomic information contained in the nucleus without an excessive proportion of cytoplasmic DNA. Multiple copies of cytoplasmic genomes are present in every cell (11 to 70 copies, depending on the developmental stage and/or physiological status) ( Tymms et al., 1983 ). The chloroplast DNA of plants, therefore, represents 17 - 23% of their total DNA ( Boffey and Leech, 1982 ). In this study, we developed a protocol that strongly reduces the amount of cytoplasmic DNA. The improvement in quality makes the method perfectly adapted to library construction for NGS. We applied this extraction method to the grapevine ( Vitis vinifera L.) and coffee tree ( Coffea canephora Pierre ex A. Froehner). These species are known to contain many secondary metabolites, which can generate diffi culties for DNA extraction ( Peterson et al., 1997 ; Mattivi et al., 2006 ). To establish the protocol, we were partially inspired by a classic protocol for nuclear DNA plant extraction destined for the construction of BAC (Bacterial Artifi cial Chromosome) libraries ( Peterson et al., 1997 ). The two stages of the protocol are: (i) isolation of nuclei and (ii) nuclear DNA extraction. Nuclear DNA obtained with this protocol was then sequenced using 454/GS-FLX technology (Roche, Basel, Switzerland) with the Titanium kit. All protocols for Next Generation Sequencers (NGS) start with the preparation of a DNA library from extracted DNA. Quality, number, and length of the sequences produced by NGS all depend on the quality of the library that was prepared, itself dependent on the DNA quality. Generally, when using NGS, one would wish to obtain the genomic information contained in the nucleus without an excessive proportion of cytoplasmic DNA. Multiple copies of cytoplasmic genomes are present in every cell (11 to 70 copies, depending on the developmental stage and/or physiological status) ( Tymms et al., 1983 ). The chloroplast DNA of plants, therefore, represents 17 - 23% of their total DNA ( Boffey and Leech, 1982 ). In this study, we developed a protocol that strongly reduces the amount of cytoplasmic DNA. The improvement in quality makes the method perfectly adapted to library construction for NGS. We applied this extraction method to the grapevine ( Vitis vinifera L.) and coffee tree ( Coffea canephora Pierre ex A. Froehner). These species are known to contain many secondary metabolites, which can generate diffi culties for DNA extraction ( Peterson et al., 1997 ; Mattivi et al., 2006 ). To establish the protocol, we were partially inspired by a classic protocol for nuclear DNA plant extraction destined for the construction of BAC (Bacterial Artifi cial Chromosome) libraries ( Peterson et al., 1997 ). The two stages of the protocol are: (i) isolation of nuclei and (ii) nuclear DNA extraction. Nuclear DNA obtained with this protocol was then sequenced using 454/GS-FLX technology (Roche, Basel, Switzerland) with the Titanium kit.