Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives

Abstract

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.

Keywords: BAC; BIBAC; Tripsacum; maize; parviglumis.

Figure 1

DNA analysis of random BAC clones from the Parviglumis BAC library by pulsed-field gel electrophoresis. DNA samples were digested with I-SceI and separated on a 1% agarose CHEF gel. The 6.38-kb common band is the vector pIndigoBac536-S I-SceI fragment. Markers used are MidRange PFG marker I (New England Biolabs).

Figure 2

Insert size distributions of the seven ZMAP BAC and BIBAC libraries. In each histogram, the x-axis represents insert size range (kilobases) and the y-axis represents the number of clones within a particular insert size range. (A) Parviglumis BAC library: the average insert size is 148 kb based on 387 clones. (B) Tripsacum BAC library: the average insert size is 139 kb based on 412 clones. (C) Zheng58 BAC library: the average insert size is144 kb based on 348 clones. (D) Chang7-2 BAC library: the average insert size is 139 kb based on 349 clones. (E) Mo17 BAC library: the average insert size is 122 kb based on 238 clones. (F) B73 BIBAC library: the average insert size is 92 kb based on 282 clones. (G) Sorghum BIBAC library: the average insert size is 111 kb based on 256 clones.

Figure 3

DNA analysis of random BIBAC clones from the sorghum Nengsi-1 BIBAC library by pulsed-field gel electrophoresis. DNA samples were digested with I-SceI and separated on a 1% agarose CHEF gel. The 23.2-kb common band is the vector BIBAC-S I-SceI fragment. Markers used are MidRange PFG marker I (New England Biolabs).

Figure 4

Stability of sorghum Nengsi-1 BIBAC clones in Agrobacterium. BIBAC clones with different insert sizes from 58 to 160 kb were selected randomly from the sorghum Nengsi-1 BIBAC library. Plasmid DNA of each BIBAC clone was transferred into Agrobacterium EHA105 cells. Single Agrobacterium EHA105 transformed colonies were cultured at 28° for 48 and 96 hr (subcultured after 48 hr). BIBAC DNA of each culture was prepared via an indirect method (Shi et al. 2011) and analyzed with I-SceI. The molecular weight marker was the MidRange PFG marker I (NEB).