CONSTRUCTION AND CHARECTERIZATION OF CARA CARA NAVEL ORANGE(Citrus sinensis Osbeck)BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY AND.pdf

1、Citrus species constitute one of the major fruit crops of thesubtropical regions with great economic importance. In the presentresearch, we reported the construction and characterization ofdeep-coverage bacterial artific

2、ial chromosome (BAC) library of C.sinensis Osbeck cv. Cara Cara, its preliminary chromosome analysis andGenomic In situ hybridization (GISH) analysis to confirm the putativehybrid origin of Gsinensis. Good high-molecular

3、-weight DNA wasproduced well by nuclei preparation from young leaves, partially digestedby HindIII and two cycles of size selection by pulsed field gelelectrophoresis (PFGE). The library consists of 94 microtiter plates

4、 in a384-well format. From the analysis of 205 randomly selected clones fromthe library, approximately 98％ of the Cara Cara HindⅢ library clonescontain nuclear DNA inserts with an average insert size of 126 kb,providing

5、coverage of 11.8 haploid genome equivalents. This provides a99％ probability of finding any specific sequence from the library. Thelibrary was further evaluated for contamination with high-copy vector,empty pIndigoBac536

6、vector, and organellar DNA sequences. To estimatethe possibility of isolating specific clone, Southern hybridization andPCR-based screening were performed with the intent to isolate gene ofPhytoene synthase, an important

7、 enzyme catalyzing the first committedstep of carotenoid biosynthetic pathway. Eight positive clones harboringthe gene encoding phytoene synthase [Psy (EC 2.5.1.32)] were identifiedfrom this library using filter hybridiz

8、ation procedure. To study thechromosomes of this species,we performed4',6-diamidino-2-phenylindole (DAPI) staining, which clearly revealed 18metaphasic chromosomes in C. sinensis, with size ranges from 2-4.4μm.Genomic in

9、 situ hybridization (GISH) analysis showed that the C.reticulata probe binds nearly all the regions of C. sinensis chromosomes,which showed the inability of this technique, as it was not found suitablefor the identificat

10、ion of putative nature of h?rid origin of C. sinensis.Our work on the construction and characterization of BAC library ofsweet orange reveals the robust protocol for the BAC library constructionof tree species. These pro

11、cedures can be applied for the other woodyspecies BAC library construction as well. In plants, genomic scienceholds promise of improvements and in the citrus genera, the developmentof genomic tools will be crucial for fu

12、rther improvement of citrus species.Highly representative BAC library and further identification of gene andgenomic region of interest from the most economically important speciesof citrus will help us to understand the