PCR-based screening of BAC clones of different chromosomes in Chinese cabbage

Abstract

In this paper, taking SSR and functional gene sequence as the primers and the plasmid of first- and second-level pools of bacterial artificial chromosome (BAC) library as templates, the PCR method was used for specific clones of different chromosomes in Chinese cabbage. The results showed that the number of positive clones was 1–11 per primer and the average number of clone was 3.9 by screening 19200 clones of BAC library using 12 pairs of SSR primers from 10 linkage groups individually, which were nearly consistent with about 3.4 times of genome coverage. Positive clones were acquired in chromosome Nos. 2 to 5 and 8 to 10 without screening with the positive clones in chromosome Nos. 1, 6, and 7. In addition, the primer of FLC1 functional gene of chromosome No. 10 was used for PCR screening, and two BAC clones containing FLC1 gene were acquired. Therefore, different specific BAC clones of chromosomes were taken by using SSR primer and functional gene primer. Specific clone screening of chromosomes could provide a probe for identifying the chromosome accurately. Meanwhile, the BAC library screening method was optimized, serving as an effective technical means for quick BAC clone screening.

Keywords

Chinese cabbage / chromosome / SSR primer / mixing pool / PCR screening

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No. 30871713), the Institution Doctoral Program Special Research Foundation, China (No. 20101302110001), and the Natural Sciences Foundation of Hebei Province, China (Nos. C2010000738 and C2010000677).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg