High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9

Abstract

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.

Figure 1.

Pachytene Chromosome Identification Based on the Distribution of Repetitive DNA Sequences in Maize Inbred Line KYS Using FISH. In (A) and (B), chromosomes were prepared by the squash procedure, and in (C) and (D), meiocytes were mounted in an acrylamide pad to preserve the 3-D chromosome structure. The 4′,6-diamidino-2-phenylindole (DAPI)–stained chromosomes were converted to black-and-white images and overlaid with signal images. Six repetitive sequences labeled with different fluorochrome combinations show distinct colors on pachytene chromosomes as follows: centromeric CentC repeat (red), telomere sequences (dark blue), 180-bp repeat (green), TR-1 element (pink), and 5S (light blue) and 45S (yellow) rRNA genes. In (A), (B), and (D), all signals are indicated by arrowheads of the same color corresponding with signals except for centromeres and telomeres. TR-1 elements coexisting with 180-bp repeats in the terminal knob on 6S and the tip of 6L are white and are indicated by white arrowheads. The small knob on 4S is indicated by a small white arrow. Bars = 10 μm. (A) Pachytene chromosomes derived from squash preparations were probed with the marker cocktail. The presence of two sites of CentC signals on chromosome 10 is indicated by a red arrow, and the isolated centromere 10 is shown in the inset. (B) Computationally straightened chromosomes were taken from the cell shown in (A). Large white arrow indicates centromeres. Note that small lines almost perpendicular to the straightened chromosome are traces of other chromosomes that cross the straightened chromosome. The inset shows only the telomere signal on 9S. (C) A projection after deconvolution of a 3-D pachytene meiocyte shows signals of the marker cocktail probes. (D) Computationally straightened chromosomes were taken from the cell shown in (C). Some small loci were not detected in 3-D preparations. White arrow indicates centromeres.

Figure 2.

Mapping of bz1, d3, and wx1 Genes on Pachytene Chromosomes Using HR Gene FISH. (A) to (C) Pachytene chromosomes of maize inbred line KYS were probed with 13.3-kb bz1 fragment (red) and FITC-labeled CentC repeats and telomere sequences (green). Note the presence of two CentC signals on chromosome 10 indicated by a white arrow. (B) shows a magnified image of chromosome 9, and the computationally straightened chromosome is shown in (C). (D) and (E) Detection of 3.4-kb d3 fragment (red) on pachytene chromosome 9. The computationally straightened chromosome 9 taken from (D) is shown in (E). (F) and (G) Detection of a 3.1-kb wx1 fragment (red) on pachytene chromosome 9. The computationally straightened chromosome 9 taken from (F) is shown in (G). The three straightened chromosomes in (C), (E), and (G) are shown at the same magnification. The asterisks indicate the terminal knob on 9S. Bars = 10 μm.

Figure 3.

Mapping of Two Nearby Unique Sequences on Pachytene Chromosomes to Demonstrate the Resolution of HR Gene FISH. (A) to (C) Pachytene chromosomes of KYS were hybridized with FITC-labeled sh1 fragment (green) and Cy3-labeled bz1 fragment (red), which are separated by ∼250 kb in B73. (B) shows the magnified image of signals, and the computationally straightened chromosome 9 is shown in (C). (D) Diagrams of the organization of the genomic sh1-bz1 region in B73 and McC based on the results from Fu and Dooner (2002). (E) to (G) Pachytene chromosomes of KYS were hybridized with FITC-labeled uce2 and tac7077 fragments (green) and Cy3-labeled bz1 fragment (red). (F) shows the magnified image of signals, and the computationally straightened chromosome 9 is shown in (G). The straightened chromosomes in (C) and (G) are shown at the same magnification. The asterisks indicate the terminal knob on 9S. Bars = 5 μm in (B) and (F) and 10 μm in all other FISH images.

Figure 4.

Simultaneous Mapping of Nine Loci as well as Centromere and Telomere Markers on Pachytene Chromosome 9 Using HR Gene FISH. (A) The HR gene FISH image of nine loci labeled alternately with FITC or Cy3 on pachytene chromosome 9. Signals are indicated by alternate red and green arrowheads. The asterisk indicates the terminal knob on 9S. C, centromere. (B) Three computationally straightened chromosomes 9 from three independent cells. The left chromosome is straightened from the image shown in (A). Bars = 10 μm.

Figure 5.

Mapping of Genes on the End of the Genetic Map by HR Gene FISH to Demonstrate the Physical Limitation of the Genetic Map. (A) to (C) The HR gene FISH image of FITC-labeled rs1 fragment (green) and Cy3-labeled centromeres and telomeres (red) on pachytene chromosome 7. (B) shows the magnified image of signals, and the computationally straightened chromosome 7 is shown in (C). (D) to (F) The HR gene FISH image of FITC-labeled afd1 fragment (green) and Cy3-labeled centromeres and telomeres (red) on pachytene chromosome 6. (E) shows the magnified image of signals, and the computationally straightened chromosome 6 is shown in (F). (G) to (I) The HR gene FISH image of FITC-labeled kcbp1 fragment (green) and Cy3-labeled 5S rDNA, centromeres, and telomeres (red) on pachytene chromosome 2. (H) shows the magnified image of signals, and the computationally straightened chromosome 2 is shown in (I). Bars = 10 μm.

Figure 6.

Detection of Homologs of the MADS Box Gene Family Using the zmm1 Gene as Probe. (A) Pachytene chromosomes of maize inbred line KYS were hybridized with Cy3-labeled zmm1 genomic fragment (red) and FITC-labeled CentC repeats (green). (B) The computationally straightened chromosomes 4, 5, 7, 9, and 10 taken from the cell shown in (A). Bars = 10 μm.

Figure 7.

An Integrated Cytogenetic Map of Maize Chromosome 9 Showing Comprehensive Correlation between Gene Positions, Chromosome Structure, Genetic Maps, the BAC Contigs, and the RN Map. (A) The high-resolution genetic IBM2 map is shown in part (Maize GDB; http://www.maizegdb.org/). (B) The genetic coverage of 24 BAC contigs anchored on linkage group 9 is shown along the IBM2 map in order (http://www.genome.arizona.edu/fpc/WebAGCoL/maize/index.html; new version released on July 19, 2005). The length of the contig corresponds to the position of its corresponding genetic locus on the IBM2 map. Six contigs are anchored to chromosome 9 by nine FISH probes in silico, and the serial numbers of these contigs are listed at left. (C) Ideogram of pachytene chromosome 9 of maize inbred line KYS based on HR gene FISH mapping results in this study. Heterochromatic and knob regions with bright DAPI staining are shown in dark blue. Green and red circles represent FITC and Cy3 signals, respectively, of FISH generated by the nine-gene probe cocktail. The positions of genes on the chromosome are drawn based on the data in Table 3. The breakpoints of several cytogenetic stocks are indicated by horizontal lines crossing the chromosome. The chromosomal positions of genes and breakpoints are given at right as fractions of chromosome arm length. (D) UMC98 genetic map redrawn in part from Davis et al. (1999). Highlighted boxes are core markers. (E) The straightened chromosome 9 from the left image shown in Figure 4B. DAPI staining was converted to black-and-white images and overlaid with signal images. The genetic distances between genes, reported in centimorgans, based on UMC98 map are listed at the left of the chromosome, and the cytological distances between genes, reported in micrometers, are shown at the right of the chromosome. (F) A comparison of RN distribution along the length of chromosome 9 and the values of centimorgans/micrometers between genes. The chromosome is represented on the y axis with the short arm on top. The top x axis is the numbers of RN in 0.2-μm intervals along the chromosome, and the red line shows the general trend of the RN distribution redrawn from Anderson et al. (2003). The horizontal bars represent the ratios of the genetic distance between genes (centimorgans; the value listed at the left of the chromosome in [E]) to the cytological distance between genes (micrometers; the value listed at the right of the chromosome in [E]). The value for each bar is shown at the right of the bar.