Integration of cytogenetic and genetic linkage maps unveils the physical architecture of tomato chromosome 2

Abstract

We report the integration of the linkage map of tomato chromosome 2 with a high-density bacterial artificial chromosome fluorescence in situ hybridization (BAC-FISH)-based cytogenetic map. The euchromatic block of chromosome 2 resides between 13 and 142 cM and has a physical length of 48.12 microm, with 1 microm equivalent to 540 kb. BAC-FISH resolved a pair of loci that were 3.7-3.9 Mb apart and were not resolved on the linkage map. Most of the regions had crossover densities close to the mean of approximately 200 kb/cM. Relatively hot and cold spots of recombination were unevenly distributed along the chromosome. The distribution of centimorgan/micrometer values was similar to the previously reported recombination nodule distribution along the pachytene chromosome. FISH-based physical maps will play an important role in advanced genomics research for tomato, including map-based cloning of agronomically important traits and whole-genome sequencing.

Figure 1.—

Cytological architecture of tomato chromosome 2. (A) DAPI-stained pachytene chromosome 2. (B) FISH pattern on pachytene chromosome 2 using probes for both digoxigenin-labeled BAC clone LE_HBa0303I24 and biotin-labeled BAC clone LE_HBa0177F12. DAPI-stained chromosomes and FISH signals were converted to a black-and-white image to enhance the visualization of distribution of euchromatin and heterochromatin on the pachytene chromosome. NOR, nucleolar organizing region; CEN, centromere; PH, pericentromeric heterochromatin; TEL, telomere; EU, euchromatin; HETERO, heterochromatin. Bar, 10 μm.

Figure 2.—

Physical coverage of the genetic linkage map of tomato chromosome 2. (A) The FISH signal from the BAC clone located at the 0-cM position was detected on the nucleolar organizing region (NOR). Digoxigenin-labeled LE_HBa0007F24 (red) anchored by cLEC-7-P21 (0 cM) was observed at the distal ends of the short arm, which is marked by a 45S rDNA locus. (B) Pachytene chromosomes of tomato were hybridized with biotin-labeled SL_MboI0006E22 (green). Arrowheads indicate pachytene chromosome 2. (C) The pachytene chromosomes in B were hybridized with digoxigenin-labeled Arabidopsis pAtT4 containing the telomere-specific sequence. (D) Sequencing results indicate that the telomere-specific repeat sequence containing SL_Mbo0006E22 is 100 kb away from LE_HBa0032J10. Bar, 10 μm.

Figure 3.—

Integration of cytogenetic and genetic maps. (A) Pachytene chromosome hybridized using five biotin- or digoxigenin-labeled BAC clones (LE_HBa0303I24, 13 cM; LE_HBa0072A04, 46 cM; LE-HBa0204D01, 74.5 cM; LE_HBa0172G12, 106 cM; LE_HBa0177F12, 142 cM). G1–G4 represent designated genetic distance blocks of 33, 27, 33, and 36 cM, respectively. (A1–A4) Twenty-eight BAC clones hybridized to pachytene chromosome 2. The green and red dots on each line indicate BAC–FISH results from the left and the right, respectively; the distance is not proportional to the actual distance. The blue dotted circle indicates the physical gap. The red dotted circle indicates the reversed order of loci between the genetic and the cytogenetic maps. The pink dotted circle indicates two separate loci that were colocalized in the genetic map.

Figure 4.—

Comparison of linkage and physical distances of loci. Left: the tomato genetic linkage map 2; map positions are given in centimorgans (yellow). Right: the physical distance in base pairs (red) corresponding to 1 cM. Horizontal bars represent the analyzed loci (yellow, corresponding to the genetic linkage map 2); vertical bars indicate the regions analyzed using BAC–FISH.

Figure 5.—

Determination of the centimorgan/micrometer relationships. (A) Chromosome diagram with genetic (left) and cytological (right) distances between analyzed loci. Red ellipses indicate the analyzed loci. (B) Comparison of the RN distribution and the centimorgan:micrometer ratio along the length of chromosome 2. The y-axis represents the long arm of chromosome 2. The top x-axis is the number of RNs in 0.1-μm intervals along the chromosome. The bottom x-axis is the centimorgan:micrometer ratio along the chromosome. The red line indicates the general trend in the RN distribution, redrawn from Sherman and Stack (1995). The horizontal bars indicate the ratio of the genetic distance between analyzed loci (values to the left in B) to the cytological distance between the analyzed BACs (values to the right in B). The value of each bar is shown at the right of the bar.

Figure 6.—

Confirmation of “next” BAC clones. (A) After “seed” BAC clone sequencing, we performed a BLASTN search of BAC end sequences. The selected next BAC candidates were verified using BAC–FISH. Digoxigenin-labeled seed BAC clones LE_H168N10 (red) and biotin-labeled next BAC candidates LE_M045L06 (green) were hybridized with pachytene chromosome 2. Colocalized red and green signals indicate that the analyzed next BAC clones are located next to the seed BAC clones in the tomato genome. (B) Both clones were then hybridized with extended DNA fiber (DNA from interphase nuclei, which have been extended into individual fibers).