Limits...
New insights into the wheat chromosome 4D structure and virtual gene order, revealed by survey pyrosequencing.

Helguera M, Rivarola M, Clavijo B, Martis MM, Vanzetti LS, González S, Garbus I, Leroy P, Šimková H, Valárik M, Caccamo M, Doležel J, Mayer KF, Feuillet C, Tranquilli G, Paniego N, Echenique V - Plant Sci. (2014)

Bottom Line: The virtual order showed a higher collinearity with homeologous 4B compared to 4A.Additionally, a virtual map was constructed and ∼5700 genes (∼2200 on 4DS and ∼3500 on 4DL) predicted.The sequence and virtual order obtained here using the 454 platform were compared with the Illumina one used by the IWGSC, giving complementary information.

View Article: PubMed Central - PubMed

Affiliation: Estación Experimental Agropecuaria Marcos Juárez, Instituto Nacional de Tecnología Agropecuaria (INTA), Marcos Juárez, Córdoba, Argentina. Electronic address: helguera.marcelo@inta.gob.ar.

Show MeSH
Histogram of relative DAPI fluorescence (flow karyotype). Histogram obtained after flow cytometric analysis of mitotic metaphase chromosomes of double ditelosomic line 4D of bread wheat cv. Chinese Spring. Peaks corresponding to telosomes 4DS and 4DL are well discriminated, which facilitated their flow sorting. Sorted chromosome arms were identified after FISH with probes for Afa repeat (yellow-green), which results in characteristic banding pattern (inset). X-axis: relative DAPI fluorescence intensity; Y-axis: number of particles.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4352925&req=5

fig0060: Histogram of relative DAPI fluorescence (flow karyotype). Histogram obtained after flow cytometric analysis of mitotic metaphase chromosomes of double ditelosomic line 4D of bread wheat cv. Chinese Spring. Peaks corresponding to telosomes 4DS and 4DL are well discriminated, which facilitated their flow sorting. Sorted chromosome arms were identified after FISH with probes for Afa repeat (yellow-green), which results in characteristic banding pattern (inset). X-axis: relative DAPI fluorescence intensity; Y-axis: number of particles.

Mentions: Flow cytometric analysis of DAPI-stained chromosomes prepared from double ditelosomic line 4D of cv. Chinese Spring resulted in a histogram of fluorescence intensity (flow karyotype) on which the 4DS and 4DL telosomes formed distinct peaks (Fig. 1). This enabled sorting 4DS and 4DL arms with the average purities of 91.42% and 86.63%, respectively. The sorted fractions were contaminated by a random mixture of fragments of various chromosomes and chromatids without prevalence of a specific chromosome. A total of 106,000 and 59,000 of 4DS and 4DL arms, respectively were sorted and used for DNA amplification. Two separate DNA bulks from flow-sorted arms were prepared using multiple displacement amplification (MDA). To limit representation bias, each DNA bulk was pooled from several independent MDA reactions. The 4DS bulk was combined from three amplifications and yielded 9.63 μg DNA. Similarly, the 4DL bulk yielded 5.44 μg DNA and contained DNA of four MDA reactions. MDA-amplified DNA yielded short fragments, suitable for the construction of a 3 kb-LMP library.


New insights into the wheat chromosome 4D structure and virtual gene order, revealed by survey pyrosequencing.

Helguera M, Rivarola M, Clavijo B, Martis MM, Vanzetti LS, González S, Garbus I, Leroy P, Šimková H, Valárik M, Caccamo M, Doležel J, Mayer KF, Feuillet C, Tranquilli G, Paniego N, Echenique V - Plant Sci. (2014)

Histogram of relative DAPI fluorescence (flow karyotype). Histogram obtained after flow cytometric analysis of mitotic metaphase chromosomes of double ditelosomic line 4D of bread wheat cv. Chinese Spring. Peaks corresponding to telosomes 4DS and 4DL are well discriminated, which facilitated their flow sorting. Sorted chromosome arms were identified after FISH with probes for Afa repeat (yellow-green), which results in characteristic banding pattern (inset). X-axis: relative DAPI fluorescence intensity; Y-axis: number of particles.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352925&req=5

fig0060: Histogram of relative DAPI fluorescence (flow karyotype). Histogram obtained after flow cytometric analysis of mitotic metaphase chromosomes of double ditelosomic line 4D of bread wheat cv. Chinese Spring. Peaks corresponding to telosomes 4DS and 4DL are well discriminated, which facilitated their flow sorting. Sorted chromosome arms were identified after FISH with probes for Afa repeat (yellow-green), which results in characteristic banding pattern (inset). X-axis: relative DAPI fluorescence intensity; Y-axis: number of particles.
Mentions: Flow cytometric analysis of DAPI-stained chromosomes prepared from double ditelosomic line 4D of cv. Chinese Spring resulted in a histogram of fluorescence intensity (flow karyotype) on which the 4DS and 4DL telosomes formed distinct peaks (Fig. 1). This enabled sorting 4DS and 4DL arms with the average purities of 91.42% and 86.63%, respectively. The sorted fractions were contaminated by a random mixture of fragments of various chromosomes and chromatids without prevalence of a specific chromosome. A total of 106,000 and 59,000 of 4DS and 4DL arms, respectively were sorted and used for DNA amplification. Two separate DNA bulks from flow-sorted arms were prepared using multiple displacement amplification (MDA). To limit representation bias, each DNA bulk was pooled from several independent MDA reactions. The 4DS bulk was combined from three amplifications and yielded 9.63 μg DNA. Similarly, the 4DL bulk yielded 5.44 μg DNA and contained DNA of four MDA reactions. MDA-amplified DNA yielded short fragments, suitable for the construction of a 3 kb-LMP library.

Bottom Line: The virtual order showed a higher collinearity with homeologous 4B compared to 4A.Additionally, a virtual map was constructed and ∼5700 genes (∼2200 on 4DS and ∼3500 on 4DL) predicted.The sequence and virtual order obtained here using the 454 platform were compared with the Illumina one used by the IWGSC, giving complementary information.

View Article: PubMed Central - PubMed

Affiliation: Estación Experimental Agropecuaria Marcos Juárez, Instituto Nacional de Tecnología Agropecuaria (INTA), Marcos Juárez, Córdoba, Argentina. Electronic address: helguera.marcelo@inta.gob.ar.

Show MeSH