Construction And Characterization Of Cara Cara Navel Orange (Citrus Sinensis Osbeck) Bacterial Artificial Chromosome Library And Identification Of Clones Containing Phytoene Synthase Gene

Citrus species constitute one of the major fruit crops of the subtropical regions with great economic importance. In the present research, we reported the construction and characterization of deep-coverage bacterial artificial chromosome (BAC) library of C. sinensis Osbeck cv. Cara Cara, its preliminary chromosome analysis and Genomic In situ hybridization (GISH) analysis to confirm the putative hybrid origin of C.sinensis. Good high-molecular-weight DNA was produced well by nuclei preparation from young leaves, partially digested by HindIII and two cycles of size selection by pulsed field gel electrophoresis (PFGE). The library consists of 94 microtiter plates in a 384-well format. From the analysis of 205 randomly selected clones from the library, approximately 98% of the Cara Cara HindIII library clones contain nuclear DNA inserts with an average insert size of 126 kb, providing coverage of 11.8 haploid genome equivalents. This provides a 99% probability of finding any specific sequence from the library. The library was further evaluated for contamination with high-copy vector, empty pIndigoBac536 vector, and organellar DNA sequences. To estimate the possibility of isolating specific clone, Southern hybridization and PCR-based screening were performed with the intent to isolate gene of Phytoene synthase, an important enzyme catalyzing the first committed step of carotenoid biosynthetic pathway. Eight positive clones harboring the gene encoding phytoene synthase [Psy (EC 2.5.1.32)] were identified from this library using filter hybridization procedure. To study the chromosomes of this species, we performed4',6-diamidino-2-phenylindole (DAPI) staining, which clearly revealed 18 metaphasic chromosomes in C. sinensis, with size ranges from 2-4.4μm. Genomic in 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 suitable for the identification of putative nature of hybrid origin of C. sinensis. Our work on the construction and characterization of BAC library of sweet orange reveals the robust protocol for the BAC library construction of tree species. These procedures can be applied for the other woody species BAC library construction as well. In plants, genomic science holds promise of improvements and in the citrus genera, the development of genomic tools will be crucial for further improvement of citrus species. Highly representative BAC library and further identification of gene and genomic region of interest from the most economically important species of citrus will help us to understand the genetic elements that contribute to key aspects of citrus biology, which will make an impact for the future improvements in this crop.