Molecular Breeding 5: 511–520, 1999. 511 © 1999 Kluwer Academic Publishers. Printed in the Netherlands. A Sorghum propinquum BAC library, suitable for cloning genes associated with loss-of-function mutations during crop domestication Yann-Rong Lin1, Linghua Zhu1, Shuxin Ren1, Jinshui Yang2, Keith F. Schertz3 & Andrew H. Paterson1;4;∗ 1Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843-2474, USA; 2Institute of Genetics, Fudan University, Shanghai, 200433, P.R. China; 3USDA-ARS, College Station, TX 77843-2474, USA; 4Department of Crop and Soil Science, University of Georgia, Athens GA 30602, USA (∗author for correspondence, send correspondence to address #4; fax 002-1-706-583-0162; e-mail [email protected]) Received 23 July 1998; accepted in revised form 23 March 1999 Key words: BAC, positional cloning, Sorghum propinquum Abstract AlargeinsertSorghum propinquum BAC library has been constructed to analyze the physical organization of the sorghum genome and to facilitate positional cloning of sorghum genes and QTLs associated with the early stages of grain crop domestication. This library was established from 12 different ligations using high-molecular-weight DNA generated from either one cycle or two cycles of size selection. This library consists of 38 016 BAC clones with an estimated average insert size of 126 kb and coverage of 6.6 genome equivalents. The 6.6 genome-equivalent BAC library of S. propinquum provides a 99.7% probability of finding one or more BACs that contain genes of interest. Twenty mapped DNA probes, ten homologous and ten heterologous, were used to screen the library, and 121 positive clones were identified, 6.05 per locus or 6.37 per probe. Abbreviations: BAC, bacterial artificial chromosome; QTL, quantitative trait locus Introduction Dominant and additive alleles have advantages over recessive alleles for positional cloning. Test- Genetic mapping of crosses between cultivated plants ing of candidate DNA sequences for mutant com- and their wild relatives has shown that at many loci, plementation requires that the candidate sequence be crop domestication has been associated with selec- genetically dominant or additive to the native se- tion for recessive mutant alleles in the cultivated type. quence. Further, some loss-of function mutations may For example, studies of fruit mass (size) and Brix be deletions that substantially truncate or eliminate (soluble solids concentration) of tomato (Lycopersi- transcripts. This would preclude the use of many meth- con esculentum,2n D 24) have shown that the ods for identifying candidate transcripts. Thus, BAC majority of QTLs with either dominant and/or ad- libraries of wild species that harbor dominant and ditive effects were contributed by wild species [55]. additive alleles for many traits offer unique advan- Six traits related to domestication were analyzed in tages for map-based cloning (positional cloning). This the F2 of Sorghum bicolor BTx623(a cultivar) × S. propinquum BAC library provides a valuable com- Sorghum propinquum (a wild relative of S. bicolor), plement to an established S. bicolor BAC library [52] and S. propinquum possessed most of the dominant for cloning of genes associated with domestication and alleles (Table 1) [39] at five traits (grain shattering, many other traits. plant height, flowering time, tiller number, rhizoma- Sorghum is a suitable species for positional cloning tousness). because of its relatively small genome size and high- density RFLP map. The haploid genome size of 512 Table 1. Mode of gene action of S. propinquum alleles for six recombination (S. Choi and R. Wing, unpublished traits related to domestication. data); and linearized BAC DNA using the Cre-lox sys- Trait Number of Mode of gene actiona tem has been used successfully to generate transgenic genes/QTLs D A R O animals [35]. BAC clones are also suitable for FISH (fluorescence in situ hybridization) analysis which en- Shattering 1 1 0 0 0 ables direct gene localization on chromosomes [29, Plant height 6 4 1 1 1 45, 52]. Flowering 3 2 0 0 0 Tiller number 4 1 2 0 0 We describe the construction and characterization Rhizomatousness 8 2 4 1 1 of a BAC library of S. propinquum. S. propinquum Seed size 9 2 1 0 0 has been utilized in RFLP linkage mapping and QTL Sum 31 12 8 2 2 analysis of many important traits related to sorghum domestication and productivity [16, 31, 38, 40]. Fur- aD, dominant; A, additive; R, recessive; O, overdominant. ther, S. propinquum appears to have been the ancestor that conferred many ‘weediness’ traits to johnsongrass (S. halepense), so offers opportunities to pursue new D sorghum is ca. 760 Mb (1C 0.8 pg) [4], second dimensions in agricultural research [40]. A 2.7× BAC smallest only to rice among major crops in the Poaceae library has already been made for the S. bicolor family, and the chromosome number of both Sorghum mapping parent that was crossed to S. propinquum D bicolor and S. propinquum is 2n 20 [24]. At least for RFLP [16] and QTL mapping [31, 38, 40]. A six sorghum linkage maps have been established by S. propinquum library is a valuable complement. RFLP and other DNA markers [8, 9, 16, 28, 41, 42, 50, 53]. More than 2100 RFLP loci have been mapped × on the interspecific F2 population of S. bicolor Materials and methods S. propinquum [ 16, Paterson et al., unpublished data). This high density of DNA markers, about 0.7 cM or an S. propinquum BAC library construction average of 350 kb between DNA markers, is suitable for fine mapping of gene(s) and QTLs, and physical Megabase DNA isolation for ligation mapping. Vegetative clones of the S. propinquum mapping par- Positional cloning has been successfully used to ent (unnamed accession) were grown in the green- clone several important genes in plants [3, 13, 33, 46]. house. Young leaves were cut into small pieces, One essential factor for positional cloning is a large wrapped in foil, and stored at −80 ◦C. To isolate insert DNA library to expedite ‘chromosome walk- megabase DNA, the nuclei preparation methods of ing’. Although YAC vectors [10] can carry inserts over Zhao et al. [58] and Zhang et al. [57) were adopted 900 kb [18, 20, 26], they are associated with problems with modifications. Young leaf tissue was put in liq- such as chimerism, insert rearrangement, clonal in- uid nitrogen and ground into powder. The powder stability, and difficulty of manipulation. BAC vectors was homogenized with 1× HB buffer (10 mM Tris, [25, 45], which are Escherichia coli F factor-based 80 mM KCl, 10 mM EDTA, 1 mM spermine, 1 mM systems, are easier to manipulate and do not have spermidine; 0.5 M sucrose, pH 9.4–9.5), 0.15% 2- the chimerism problem because they have only one mercaptoethanol and 0.5% Triton. The isolated nuclei or two copies per cell. Xa21, a rice disease resistance were embedded in agarose plugs with SCE buffer gene, is the first example of positional cloning using a (1 M sorbitol, 0.1 M sodium citrate, 60 mM EDTA, BAC library [46]. By anchoring ‘BAC islands’ to high- pH 7.0) and lysed with lysis buffer (1% sodium lauryl density RFLP maps, and aligning BACs using high sarcosine, 0.5 M EDTA pH 9.3–9.4, 1 mg/ml Pro- throughput fingerprinting [32], sequence-ready contig teinase K), at 50 ◦C for 48 h. Lysis buffer was changed maps might be generated for the complex genomes of once during lysis. Before restriction enzyme digestion, many well-mapped crops. Modified BAC vectors are the agarose plugs were washed with 0.1 mM PMSF suitable for mutant complementation testing and gene (phenylmethylsulfonyl fluoridein, Sigma, USA) for expression. For example, binary-BAC (BIBAC) vec- 1 h on ice to inactivate Proteinase K. The PMSF so- tors can be used directly for plant transformation via lution was then replaced with fresh T10E10 (10 mM Agrobacterium tumefaciens [27]; pBACwich vectors Tris-HCl, 10 mM EDTA pH 8.0) by adding fresh can be used for plant transformation by site-specific T10E10 every 20 min for 1 h. DNA agarose plugs were 513 equilibrated with restriction enzyme buffer by adding Ligation and transformation fresh buffer every 30 min for 1 h. Size-selected HindIII DNA fragments were ligated About 60 mg of S. propinquum nuclei-containing into dephosphorylated pBeloBAC11 vector at a ratio agarose plugs, which contained about 1.2 µgofDNA of 15 ng vector to 60 ng insert, in a total volume of in each plug, were sliced into small pieces and equili- 60 µl. Ligations were performed in 1× ligase buffer brated with three different amounts of HindIII (18, 24, with 200 units of T4 DNA ligase (NEB, USA) and or 30 units) plus digestion buffer (New England Bio- incubated at 16 ◦C for at least 16 h. To remove salt labs, USA) on ice for 5 h, to ensure that HindIII could from ligation buffer, ligation mix was placed on a drop diffuse into sliced plugs to achieve an even digestion. dialysis filter (Catalog number VSWP 02500, Milli- ◦ Partial digestions were incubated at 37 Cfor5min. pore, USA) and floated on 0.5× TE for 30 min to 1 h. Partially digested DNA was separated by PFGE in a Ligation mix (3 µl) was used to transform 25 µlof 1% SeaPlaque agarose gel (FMC, USA) in 1× TAE E. coli ElectroMAX DH10B cells (Gibco-BRL, USA) ◦ buffer at 14 C using HEXCHEF 6000 and the MJ by electroporation using the Gene Pulser II (BioRad, Research Programmable Power Inverter (USA). Lig- USA) at the following optimized conditions: capaci- ations 1–9 were prepared from DNA subjected to one tance 25 µF, resistance 100 , and a voltage gradient cycle of size selection with either 5 s or 8 s pulse time.