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Evaluation of the Bacterial Artificial Chromosome Cloning System for Crop Plants

Rod A. Wing, Texas A&M University, Soil & Crop Sciences Department, Texas A&M BAC Center, College Station, TX 77843-2123.
Email: rodwing@tam2000.tamu.edu

Most plant and animal genes are known only by their phenotype (e.g. bacterial disease resistance in plants or cystic fibrosis in humans). A technology, termed "map-based gene cloning", has been developed to isolate such genes based on the position of a target on a genetic map. Since 1986, map-based cloning has been used successfully to isolate over 35 human genetic disease genes and over 20 plants genes. A crucial element of map-based cloning requires the availability of large insert DNA libraries (YACs or BACs) containing DNA inserts from 150 kb to 1000 kb.

Over the past two years our laboratory has explored the use of the BAC cloning system for plants and animals. Based on our studies, the BAC system is emerging as the system of choice for construction of plant genomic libraries with average insert sizes of 150 kb. The BAC vector, pBeloBAC11 (courtesy of M.Simon, Cal Tech), is derived from the endogenous E.coli F-factor plasmid which contains genes for strict copy number control and unidirectional origin of DNA replication. Additionally, pBeloBAC11 has three unique restriction enzyme sites (HindIII, BamH1 & SphI) located within the LacZ gene which can be used as cloning sites for megabase-size plant DNA. BAC libraries are generated by ligating size-selected restriction digested DNA with pBeloBAC11 followed by electroporation into E. coli. BAC library construction and characterization is extremely efficient when compared to YAC library construction and analysis. At present we have constructed representative BAC libraries for Sorghum bicolor (3), Oryza sativa ssp. japonica and indica (4) , Arabidopsis thaliana (2) and bovine (1). We are currently constructing libraries for cotton, sugarcane, tomato, wheat, sorghum propinquum, and corn. Our strategy has been to construct libraries from the parents of widely used mapping populations. We anticipate that by the summer of 1996 we should have the largest collection of plant and animal BAC libraries in the world.

A long term objective our BAC research is to establish a "BAC Center" at Texas A&M University (http://http.tamu.edu:8000/~creel/TAMBAC.html) which will serve as a genetic resource for the plant and animal genome communities world wide. The BAC Center will provide several functions:

  • The construction and maintenance of high quality BAC libraries for plant and animal genomes.
  • Robotic screening of BAC libraries with probes linked to important genes.
  • BAC clone distribution.
  • Integrated physical mapping - FISH (metaphase, interphase and meiotic)
For more informaion concerning BAC work please see our World Wide Web Page at http://http.tamu.edu:8000/~creel/TAMBAC.html. This page describes our Arabidopsis BAC library, The Texas A&M BAC Center, and contains a manual for constructing plant BAC libraries

References and Abstracts

Cai, L., J.F. Taylor, R.A. Wing, D.S. Gallagher, S.S. Woo, and S.K. Davis. 1995. Construction and characterization of a bovine bacterial artificial chromosome library (Genomics 29:413-425).

A bacterial artificial chromosome (BAC) library has been constructed for use in bovine genome mapping using the pBeloBAC11 vector. Currently, the library consists of 23,040 clones, which achieves a 70% probability (P = 0.70) of the library containing a specific unique DNA sequence. An average insert size of 146 kb was estimated from the analysis of 77 randomly selected BAC clones produced by one or two rounds of size selection. The bovine DNA inserts proved to be very stable for at least 100 cell generations. No chimeric clones were detected among 11 large, size-selected BAC clones using fluorescence in situ hybridization (FISH) on metaphase bovine chromosomes. The polymerase chain reaction (PCR) was used to screen the library for single-copy nuclear sequences. Thirty-three of 46 (72%) sequences were present in the library in at least one copy, which is consistent with the estimated 70% probability of this library containing a unique DNA sequence. A BAC clone containing the 3-beta-hydroxy-5-ene steroid dehydrogenase (HSD3B) gene was physically mapped to bovine chromosome 3 by FISH. Two new microsatellite markers were isolated from the HSD3B-positive BAC clone as sequence-tagged sites for genetic mapping. These markers cosegregated, and no recombinants were detected in 193 informative meioses. Plasmid end rescue and the inverse polymerase chain reaction methods were used to rescue both ends of this BAC clone, and chromosome walking was performed using PCR primers designed within the end region sequences. Based on our experimental results, the BAC system provides a very useful tool for complex genome analysis.

Choi, S.D., R. Creelman, J. Mullet, and R.A. Wing. 1995. Construction and characterization of a bacterial artificial chromosome library from Arabidopsis thaliana Weeds World. 2: 17-20.

We constructed an ordered 3,948 clone Arabidopsis BAC library. The library has a combined average insert size of 100 kb (n=54). Assuming a haploid genome size of 100,000 kb, the BAC library contains *3.95 haploid genome equivalents with a 98% probability of isolating a specific genomic region. The library was screened with five Arabidopsis cDNA probes and one tomato probe and all probes hybridized to at least one (in most cases three) BAC clones in the library.

*Note: This library is now 12X and is maintained in the Texas A&M BAC Center and Arabidopsis Biological Resource Center (ABRC), Ohio State University.

Woo S.-S., J. Jiang, B. S. Gill, A. H. Paterson, and R. A. Wing. 1994. Construction and characterization of a bacterial artificial chromosome library for Sorghum bicolor. Nucleic Acids. Res. 22:4922-4931.

The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.

Zhang, H.B., S.D. Choi, S.S. Woo, Z.K. Li, and R.A. Wing. 1996. Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population. (Molecular Breeding, in press).

Rice is a leading grain crop and the staple food for over half the world population. Rice is also an ideal species for genetic and biological studies for cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent recombinant inbred mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14,208 clones with an average insert size of 130 kb, which represents 4.4 rice haploid genome equivalents. Three single copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.

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