Construction of Two Sunflower (Helianthus annuus L.) BAC and BIBAC Libraries and Their Application to Fluorescence In Situ Hybridization (FISH) Jiuhuan Feng1, C. C. Jan2, Brady A. Vick2, Hong-Bin Zhang3 1 North Dakota State University, Department of Plant Sciences, Fargo, ND 58105, USA 2 U. S. Department of Agriculture, Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND 58105-5677, USA 3 Department of Soil and Crop Sciences and Institute for Plant Genomics and Biotechnology, 2123 TAMUS, Texas A&M University, College Station, Texas 77843- 2123, USA Abstract Large-insert BAC and BIBAC libraries have played an essential role in modern genomics research. To facilitate genomic research in sunflower, two complementary BAC and BIBAC libraries were constructed from nuclear DNA of an inbred line, HA89, by using different restriction enzyme and vector systems. The BAC library, constructed with BamH1 in the pECBAC1 vector, contains 107,136 clones with an average insert size of about 140 kb, equivalent to 5.0 times the sunflower haploid genome (3000 Mb/1C). The BIBAC library, constructed with HindIII in the pCLD04541 vector, contains 84,864 clones, with an average insert size of about 137 kb and equivalent to 3.9 times the sunflower haploid genome. Both libraries provide a greater than 99% probability of obtaining a particular DNA sequence. Together, the combined libraries represent about 8.9 equivalents of the sunflower haploid genome. Using a random clone from the BIBAC library as a probe, a fluorescence in situ hybridization (FISH) was conducted in cultivated sunflower. This BIBAC clone produced six strong signals on three pairs of satellited chromosomes, and is likely related to a nucleolus organizing region (NOR). These libraries provide a comprehensive resource for future sunflower genomics and genetics research, including physical mapping and gene cloning, and make it possible to order the molecular markers and BAC and BIBAC clones on the chromosomes of sunflower via FISH technique. Our findings suggest that FISH of BAC clones is an efficient technique for identifying chromosome and for developing a physical map of the sunflower genome. Introduction The molecular biology and cytogenetics of sunflower (Helianthus annuus L.), an economically important oil crop, has lagged behind many other major crop species. Sunflower has small and similar mitotic metaphase chromosomes, making it difficult to distinguish chromosomes by karyotypic analysis and chromosome banding. FISH provides a powerful and efficient means of identifying chromosomes in situ. It has been utilized in many animals and plants, but its application to sunflower remains to be investigated. Screening a BAC library with mapped molecular markers (Jan et al., 1998) would help identify chromosome-specific BAC clones. Chromosome-specific BAC clones can be used as FISH probes to identify chromosome and assign them to specific linkage groups. Moreover, BAC libraries will make physical mapping of the genome possible and facilitate map-based cloning of important genes. Here, we report the construction and characterization of a BAC library and a plant-transformation-competent BIBAC library of sunflower, and the preliminary localization of a random BIBAC clone JF497A2 (140 kb) on sunflower chromosomes by FISH. Material and Methods Plant material HA89, a widely used sunflower inbred line, was used for BAC and BIBAC library construction and chromosome preparations. Construction and analysis of BAC and BIBAC libraries The BAC and BIBAC libraries were constructed from megabase-size DNA isolated from nuclei preparations according to the procedures described by in H-B Zhang (Zhang et al., 1995; Zhang, 2000; http://hbz.tamu.edu). The megabase-size DNA was partially digested with HindIII or BamHI, size-selected on pulsed-field gels and then ligated into either HindIII-digested BIBAC vector pCLD04541 or BamHI-digested vector pECBAC1, respectively. Ligated DNA was transformed into E. coli strain ElectroMAX DH10B competent cells by electroporation using a Cell Porator (Life Technologies, Gibco BRL). White colonies were manually arrayed into 384-well microtiter plates. To estimate the insert sizes of the BAC and BIBAC clones, clones were randomly selected from the libraries. BAC and BIBAC plasmid DNA was isolated by the alkaline lysis method, completely digested with NotI and run on CHEF (contour-clamped homogeneous electric field) gels. Chromosome preparation and FISH Mitotic chromosome spreads from fixed root tips were prepared. A random clone, JF497A2, from the BIBAC library was selected as a probe. The BIBAC plasmid DNA was labeled with digoxigenin-11-dUTP using the nick translation protocol. The procedures for hybridization and signal detection were as described by Feng et al. (2002). The signals were detected with anti-sheep IgG FITC conjugate and the whole chromosomes were counterstained with DAPI. Photographs were taken under a Zeiss Axioplan 2 imaging microscope. Images for the same cell were captured with a different filter for FITC and DAPI, respectively, and by finally creating a composite overlay of the cell. Results and Discussion Characterization of BAC and BIBAC libraries Using the nuclear DNA isolated from fresh young leaves of the inbred line HA89, we constructed a BAC library and a plant-transformation-competent BIBAC library for sunflower. The BAC library contains 107,136 clones constructed with BamHI in the BAC vector pECBAC1. In analysis of 225 randomly selected clones from the library, almost all contained inserts (empty clones <1%) with an average insert size of 140 kb. The BIBAC library consists of 84,864 clones constructed with HindIII in the plant- transformation-competent BIBAC vector pCLD04541. An analysis of 196 clones randomly selected from the BIBAC library indicated that more than 99% of the clones contained inserts with an average insert size of 137 kb. Given that the genome size of sunflower is about 3000 Mb/1C, the BAC and BIBAC libraries are equivalent to 5.0 x and 3.9 x of the sunflower haploid genome, respectively, and either library provides a probability of greater than 99% of containing a particular clone. The two libraries together contain 192,000 clones, representing about 8.9 equivalents of the sunflower haploid genome. Because they were constructed with two restriction enzymes complementary in the restriction site sequences in two different plasmid (F and P2) vector systems, the two libraries would be complementary, thus providing improved genome coverage, compared to either of the libraries alone. Furthermore, because techniques for large DNA fragment transformation have been developed in several plant species, the plant transformability of the BIBAC library will facilitate its role in positional gene cloning, gene characterization and molecular breeding in sunflower. M kb BIBACs BACs 194 145.5 97 48.5 M BACs BIBACs Fig. 1. A CHEF gel image of sunflower BAC and BIBAC clones stained with ethidium bromide. BAC and BIBAC DNA were isolated and digested with NotI. Digested DNA was separated on a 1% gel (6v/cm, 5 s initial pulse, 15 s final pulse, 12.5oC, 16 h). M: Lambda ladder marker. FISH using a random BIBAC clone as a probe When the BIBAC clone JF497A2 (140 kb) was hybridized to the mitotic chromosomes of HA89, six distinct green signals were detected on the termini of three pairs of chromosomes. According to our observation, these three pairs of chromosomes are likely to be satellited chromosomes, and the BIBAC DNA is likely to be related to the NOR DNA sequence. This result is consistent with Raicu’s report (1976) that three pairs of chromosomes have secondary constructions in cultivated sunflower. In addition, Ji et al. (1997) reported differentially bright fluorescence of NORs in somatic and meiotic chromatin after staining with DAPI and PI allowing for detection of major NORs. In our study, the distal regions on the three pairs of chromosomes appear lighter blue than other chromatin under DAPI staining. A B C Fig. 2. FISH images of BIBAC JF497A2. A. DAPI counterstained metaphase chromosomes (2n = 34) of HA89, where there are three pairs of chromosomes having the terminal regions lighter (arrows) than other parts. B. FITC-detected signals after hybridization with BIBAC JF497A2. C. A composite overlay image of the same cell as shown in images A and B, the FISH green signals showing three pairs of major NOR loci. In summary, these BAC and BIBAC libraries provide comprehensive resources for sunflower genomics and genetics research, including physical mapping, gene cloning and molecular breeding in sunflower. The preliminary analysis of FISH strongly suggests that BAC and BIBAC clones can be successfully used as FISH probes in sunflower, making it possible to directly order the BAC and BIBAC clones on chromosomes. Use of the libraries to identify a set of sunflower trisomics by FISH is in progress. References Feng, J.H., and S.Y. Zee. 2002. Chromosome spatial distribution in wheat root tip cells during mitosis. Advances in Chromosome Science: Chromosome Sciences in the New Millennium, vol.1, China Agricultural Sciences and Technology Press, Beijing, pp:160-166. Jan, C.C., B. A. Vick, J. F. Miller, A. L. Kahler, and E.T. Butler III. 1998. Construction of an RFLP linkage map for cultivated sunflower. Theor. Appl. Genet. 96:15-22. Ji, Y., D.A. Raska, T. D. McKnight, M.N. Islam-Faridi, C. F. Crane, M. S. Zwick, R.E. Hanson, H., J. Price, and D. M. Stelly. 1997. Use of meiosis FISH for identification of a new monosome in Gossypium hirsutum L. Genome 40:34-40. Raicu, R., V. Vranceanu, A. Mihailescu, C. Popescu, and M. Kirillova. 1976. Research of the chromosome complement in Helianthus L. genus. Caryologia 29:307-316. Zhang, H. B., X. P. Zhao, X. L. Ding, A. H. Paterson, and R. A. Wing. 1995. Preparation of megabase-size DNA from plant nuclei. Plant J. 7:175-184. Zhang, H. B. 2000. Construction and Manipulation of Large-insert Bacterial Clone Libraries-Manual. Texas A&M University, Texas, USA(http://hbz.tamu.edu) Acknowledgments We thank Wei Sun, Leonard W. Cook and Lisa A. Brown for their assistances in developing the BAC and BIBAC libraries, and Terrance S. Peterson for his assistance in preparing the figures. This work was supported by USDA program grant 5442-21000- 027-02.
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