| Genomic library with high-molecular weight inserts is a powerful tool for establishing cytological markers and integrating genetic and physical maps. In this study, the genomic DNA of Brassica juncea was extracted by SDS method.Then the DNA was randomly sheared into36-48kb fragments, which were further connected into pCC2FOS vectors. After that, recombinant vectors were transformed into the E.coli strain EPI300competent cells and10000-30000clones were obtained per transformation. Finally, the Fosmid library of B. juncea, which consisted of60000clones, was established by three times of connection and transformation. Subsequently, twenty clones were selected randomly to identify its quality. The results showed as following:the inserting frequency of the library was100%, the size of inserts ranged from22kb to45kb and32kb in average, the library was1.8genome equivalents coverage by considering that the genomic size of B. juncea was1068Mb. Next, in order to identify the genetic stabilization of the inserts in the E.coli, six clones were selected randomly and cultured about one hundred generations. The restriction results of those clones suggested that there were no recombination, insertion or deletion in the inserts during the subculture, which means stable passage of Fosmid clones. The Fosmid library was the foundation for screening of cytological markers.Molecular markers were pre-selected from different linkage groups of Brassica. According to the sources, the markers could be divided into three classes:(ⅰ) considering the homology of A-and B-genome, twenty SSR markers were selected from different linkage groups of Brassica campestris,(ⅱ) twelve SSR markers were selected from Brassica nigra,(ⅲ) fourteen intron polymorphism (IP) markers were selected from B-genome linkage groups of B. juncea. PCR results verified that seventeen primers from (i), nine primers from (ii) and fourteen primers from (iii)could amplify products from genomic DNA of both B. nigra and B. juncea, so as to be applied in screening of cytological markers.In this study, the genomic library was treated with PCR pooling protocol to improve the screening efficiency. Fifty clones constituted a sub-pool and kept in a hole of96-well plate. A96-well plate was a sub-pool library and thirteen sub-pool libraries were retained. Positive clones were obtained by the two-step PCR protocol. First, the positive sub-pools were obtained by screening the sub-pool libraries, and then, the positive clones corresponding to the sub-pools were amplified by a second step PCR. At present, nineteen markers have obtained their corresponding positive clones in five sub-pool libraries. In other words, twenty-three positive clones were picked out in twenty-four thousand single clones. Both nia-m103a and Ni2-E04could be amplified products using the clone F12-26as template, which indicated that these two markers may be linked in B. juncea.Finally, the plasmids DNA of the positive clones were extracted and labeled with digoxigenin-dUTP (Roche, Indianapolis, IN) via the nick translation labeling method. The metaphase chromosomes of B. nigra were selected and prepared for chromosome fluorescence in situ hybridization (FISH). According to the hybridization signals, twenty-three positive clones could be divided into three types:(i) the signals located in the centromeric regions of all chromosomes,(ii) the signals detected only on one pair of chromosomal arms,(ⅲ) no signal was detected.In conclusion, an unbiased genomic Fosmid library of B. juncea was successfully established and two types of B-genome cytological markers were obtained in this study. It provides useful cytogenetic markers for further genetic and cytogenetic analysis in B. nigra For example evolutionary relationship analysis of different genomes in Brassica, identification of B-genome specific chromosome, monitoring of chromosome behavior in the somatic hybridization generation, for the cytological locating of the excellent traits or even for the construction of B-genome physical map. |
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