SUMMARY: As a major crop species and leading natural fiber crop in the world, cotton has a potentially broad genetic base, reflected in the collections of Gossypium species. Cotton breeders therefore have an opportunity to exploit and manipulate many thousand genes in the Gossypium reservoir. To positionally isolate genes in cotton and to physically map the cotton genome, high-quality libraries are needed that are comprised of bacterial artificial chromosome (BAC) clones containing large cotton DNA inserts. Recently, we have constructed three complementary BAC libraries (BamHI, EcoRI, and HindIII) from a Gl₂^e-BC₆ nearly isogenic line (NIL) of G. hirsutum acc. TM-1, a genetic standard of Upland cottons. The cloning vector of pECBAC1 was used in all three libraries (about equal genome coverage). The total number of BAC clones is 115,584, covering 7.2X haploid AD genome equivalents. The average size of DNA inserts is 143 Kb, with a range of 90 Kb to 210 Kb. DNA markers flanking Gl₂^e, the glandless cotton gene, have been identified via linkage analysis of an F₂ population derived from a cross between the TM-1 NILs (with and without Gl₂^e). They are currently being used to screen the BAC library for the isolation of the Gl₂^e gene to engineer the glandless cottonseed, and additional DNA markers are being developed from the positive BAC clones. Other genes, such as those for resistance to the diseases and nematodes in cotton, are also taking advantage of these BAC libraries for positional cloning. The newly constructed BAC libraries provide a rich resource for detailed cotton genomics research.

MATERIALS AND METHODS: ESP is a nearly isogenic line (NIL) to TM-1, a genetic standard of Gossypium hirsutum L. ESP has a major glandless gene (Gl₂^e) that was introduced from an Egyptian cotton "Bahtim 110", a (G. barbadense). Megabase DNA was extracted from ESP leaves and embedded in the LMP agarose micro-beads, and it was partially digested with BamHI, HindHIII, and EcoRI, individually, and size-selected on a pulsed-field gel (PFGE). Ligates were made of the cloning vector pECBAC1 with partially digested DNA fragments that were size-selected for 150 Kb up to 500 Kb. Electrophoration was performed to transform the ligates into E. coli strain DH10B cells. Transformation was achieved for the size range of 150 Kb to 350 Kb. Transformed white colonies were picked up individually into 384-deep well microtiter dishes containing the culture medium for -70^oC storage and for production of working copies of high-density filters. Insert sizes of BAC clones was determined in PFGE, and the blot was probed with total ESP genomic DNA. Fingerprints of ESP BAC clones were made with the Fpase kit that was patented by Zhang and Tao (Invention #. TAMUS 1228). Briefly, Fpase-digested BAC insert DNA was subjected to 4% denaturing polyacryamide gel electrophoresis, and dried gel was autoradiographed to a X-ray film. Linkage analysis of DNA markers with the Gl₂^e gene was performed by use of an F₂ population derived from a cross between TM-1 x ESP, a pair of NILs. The genetic distances were estimated by use of MapMaker 3.0/Exp.

Table 1: Cotton ESP BAC Libraries
Cloning Site	Vector	Number of Clones	Average Insert Size	Genome equivalents	Reference
HindIII	pECBAC1	38,400	148Kb[view]	2.5X	Yu et al. 2000
BanHI	pECBAC1	38,784	138Kb[view]	2.3X	Yu et al. 2000
EcoRI	pECBAC1	38,400	142Kb[view]	2.4X	Yu et al. 2000
Total:		115,584	143Kb	7.2X

Note: ESP is an isogenic line of TM-1, a genetic standard of Gossypium hirsutum L.
The size range of cotton DNA inserts in ESP BAC clones is 90Kb to 240Kb.

Image 1:Transformants of cotton BAC clones




Image 2:Insert DNA sizes of HindIII BAC Library




Image 3:Inserts of Cotton BAC clones probed with Total ESP DNA




Image 4:Fingerprints of cotton BAC clones




Image 5:Detection of Cotton SSR Loci from BAC clones

References:

Yu, J., R.J. Kohel, H.-B. Zhang, J.-M. Dong, L.I. Decanini. 2000. Construction of a cotton BAC library and its applications to gene isolation. Proc. of 8^th International Conference on the Status of Plant and Animal Genome Researches. January 9-13, 2000, San Diego, CA

Yu, J., R. J. Kohel, H.-B. Zhang, J. Dong, Q. Tao, L.I. Decanini, C. Wu, J.L. Lewis. 2000. Construction, characterization, and applications of high-quality bacterial artificial chromosome (BAC) libraries in cotton (Gossypium spp.). (in preparation)

Texas A&M BAC Center

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