Construction And Characterization Of A BAC Library For Triticum Boeoticum And Isolation Of Centromeric Repetitive Sequences

The genomics research has come into post-genomics era, in another word, functional genomics era. However, it is still in changing from structural genomics era to functional genomics era in our country. We will focus on construction of the platforms for a long time, such as construction of BAC library, physical mapping based on BAC and so on, though the whole genome of rice(Oryza sativa L. Ssp. Indica)has been sequenced in China (Yu et al, 2002). Common wheat (Triticum aestivum L. 2n=42, AABBDD) is one of the most important crops in the world. Because of the very large size and complexity of genome, wheat genomics has progressed very slowly and lagged behind other crops with smaller genomes such as Arabidopsis thalina and Oryza sativa (Lijavetzly et al, 1999). We have constructed BAC library for Triticum boeoticum boiss (2n=14, AbAb), one diploid progenitor of common wheat in order to overcome the difficulties. It will be a useful platform in gene clone and genomic research of common wheat. In construction of BAC library, preparation of vector and large DNA fragments were two critical steps. We used recovered DNA for ligation and transformation. The library consists of about 200 000 clones. 15 360 clones were picked individually and formed ordered library. About 190 000 clones were picked and pooled in six 384-well plates. A random sampling analysis of 200 BAC clones indicated the average insert size was 104 kb. Based on the genome size of T. boeoticum, the library is about 3 times equal to T. boeoticum haploid genome (5 600 Mb). Screening the BAC library with cpDNA sequence psbA gene and mtDNA sequence atp6 gene as probe showed that contamination of the library with chloroplast and mitochondrial clones was less than 1%. In order to study the structure and clone the sequence of the centromere of T. boeoticum, 3072 clones of the BAC library were screened by RCS1 as probes. Thirty-two positive clones were obtained. Then ten positive clones picked at random from the thirty-two positive clones wre probed by RCS1 and Tcs250 respectively again. Six and five clones showed unambiguous positive hybridization respectively. Then three pairs of primers to RCS1 were designed using DNA star software. PCR amplification was carried out using positive BAC clones as templates by the three pairs of primers respectively. We obtained a 600 bp (named TBRCS1) sequence by primer 3. Sequencing analysis revealed that TBRCS1 had 83% homology to RCS1 and 92% to a Ty3/gypsy class of retrotransposon sequence in barley and 96% to a integrase-like gene reported in Aegilops tauschii It indicated that TBRCS1 was part of centromere of T. boeoticum. From these experiments we also illustrated: (1) Isolation and purification of vector using Qiagen Plasmid Purification Kit is a fast, inexpensive, and reliable method. (2) Self-ligation and recovery of vector could improve the quality of vector significantly. (3) Frozen and refrozen vector and ligation solution should be avoided, so aliquots would have to be made. similarly refrozen bacterial culture and glycerol stocks of BAC pools also should be avoided. (4) Pre-electrophoresis could help remove away small DNA fragments mixed in the large DNA fragments. (5) The larger the DNA fragments was, the smaller the actually average inserts and the lower efficiency of transformation was. DNA fragments ranging from 100-200 kb are desirable for ligation and transformation. DNA fragments larger than 300 kb are very poor for ligation and transformation. (6) The quality of large fragments obtained by two steps of electrophoresis was better than that by three steps of electrophoresis because of low percentage of small DNA fragments trapped in large DNA fragments and purification of large DNA fragments but the loss of DNA fragments was very seriously in the two steps of electrophoresis. Enough DNA fragments could be obtained using three steps of electrophoresis, however, overloading the pulsed field gel toincrease the amounts of partially digested DNA can result in an undesirable quality of the large fragments trapped with small size DNA. (7) A molar ratio of vector : insert of about 3-5 : 1 was tested well in our experiment. (8) The highest transformation efficiency was reached by concentrating and desalting the ligation mixture, under optimal conditions, 2 500 ~5 000 clones can be obtained per microlitre of dialyzed ligation mixture. (9) Primary experiments indicated that the pooled BAC clones were stable after 12 h culture. Establishment of T. boeoticum BAC library would be a useful platform in gene clone, physical mapping, the research on centromeres and telomere of wheat and its relatives. This library will be a long-term tool for wheat genomic research.