Cultivating and Processing Cotton Leaf Tissue for BAC Library Construction

The G. hirsutum genetic standard line Texas Marker-1 (TM-1) seeds were obtained from co-author David M. Stelly (seeds can be requested by email: stelly@tamu.edu) and were propagated in greenhouse conditions for this study. Prior to tissue harvesting, the seedlings were dark-treated for 24 hours to reduce carbohydrate synthesis and photosynthetic byproducts. Approximately 100 grams of young, expanding leaf tissue was harvested, rinsed two times in ddH₂0, blotted dry, and immediately flash frozen in liquid nitrogen. The restriction-derived BAC libraries were constructed by preparing intact nuclei according to previously published methods⁵⁷ with the following modifications: addition of 1% (w/v) soluble PVP-40 (Sigma-Aldrich), 0.1% (w/v) L-ascorbic acid (Sigma-Aldrich), 0.13% (w/v) sodium diethyldithiocarbamate trihydrate (DIECA, Sigma-Aldrich), and 0.4% beta-mercaptoethanol to the nuclei isolation buffer (NIB) right before use. Post nuclei isolation and plug washing, the nuclei plugs were subject to pre-electrophoresis as a first step to remove small DNA (<80 Kb) and positively charged elements that may contribute as enzymatic or cloning inhibitors, following the methods of Osoegawa et al.^{58 (link)} with the following modifications: Plugs were run at 1 s:4 s switch time for 2.5 hours at 4 V/cm and soaked in 10 mM Tris-HCl overnight, changing the buffer at least 3 times. To prepare high molecular weight BAC inserts, the plugs were macerated and partially digested (separately) with, HindIII and BstYI using standard methods. BAC insert size selections, ligations, and transformations were carried out according to the methods of Lou and Wing⁵⁷ .