Assignment of genetic linkage maps to diploid Solanum tuberosum pachytene chromosomes by BAC-FISH technology

Abstract

A cytogenetic map has been developed for diploid potato (Solanum tuberosum), in which the arms of the 12 potato bivalents can be identified in pachytene complements using multicolor fluorescence in situ hybridization (FISH) with a set of 60 genetically anchored bacterial artificial chromosome (BAC) clones from the RHPOTKEY BAC library. This diagnostic set of selected BACs (five per chromosome) hybridizes to euchromatic regions and corresponds to well-defined loci in the ultradense genetic map, and with these probes a new detailed and reliable pachytene karyotype could be established. Chromosome size has been estimated both from microscopic length measurements and from 4',6-diamidino-2-phenylindole fluorescence-based DNA content measurements. In both approaches, chromosome 1 is the largest (100-115 Mb) and chromosome 11 the smallest (49-53 Mb). Detailed measurements of mega-base-pair to micrometer ratios have been obtained for chromosome 5, with average values of 1.07 Mb/mum for euchromatin and 3.67 Mb/mum for heterochromatin. In addition, our FISH results helped to solve two discrepancies in the potato genetic map related to chromosomes 8 and 12. Finally, we discuss the significance of the potato cytogenetic map for extended FISH studies in potato and related Solanaceae, which will be especially beneficial for the potato genome-sequencing project.

Fig. 1

Potato cytogenetic map and its alignment to the UHD genetic map of genotype RH. For each chromosome (white numbers), five BAC clones were selected (see Table 1) as cytogenetic markers and were hybridized to DAPI-stained pachytene chromosomes by five-color FISH. The FISH images (right) were straightened and stretched to align with the corresponding genetic maps (left) for relative distance comparison. Each linkage group of the RH genetic map is divided into bin segments (small rectangles) of 0.77-cM length, in which the AFLP marker density is coded by a grayscale value. The black and dark gray bins have the highest marker densities. The centromere locations on the pachytene chromosomes are indicated by asterisks. The corresponding genetic positions of the centromeres are marked according to Park et al. (2007), except for RH linkage group 3 (see text)

Fig. 2

Karyotyping of pachytene chromosomes of Solanum tuberosum cultivar G254. a The complement of pachytene chromosomes. The DAPI-stained chromosomes displayed brightly fluorescent pericentromere heterochromatin around the centromeres (primary constriction within the heterochromatic region). Five-color FISH with ten chromosome-specific BACs allowed the identification of all 12 bivalents in one FISH experiment. b Bivalents were digitally separated from a. Chromosomes were ordered and numbered according to corresponding linkage groups and indicated by arrowheads at centromere position

Fig. 3

a Placement of unassigned linkage group RHU at the end of chromosome 12, by which a new RH12 map was developed. (a) FISH mapping of two BAC clones (RH057E14, green; RH084C24, red) anchored by RHU markers and one BAC clone (RH043B17, yellow) from RH12 bin 39 on the straightened chromosome 12. (b) FISH mapping of BAC clone (RH057E14, red), anchored by an RHU marker, and BAC clone (RH195L21, green), which was mapped to bin 67 of the SH12 map, to the distal part of straightened chromosome 12. b, c One of the most distal BAC clones (RH106H24, green) from chromosome 1 was shown to partially overlap (indicated by yellow color) with telomeric repeats (pAtT4, red). d FISH mapping of two BAC clones from the euchromatic portion of chromosome 9 of which the BAC clone (RH061A13, green signal) bordered the pericentromere heterochromatin. e FISH mapping of three chromosome 8 BAC clones (RH168E16, green; RH048O11, yellow; RH127J02, blue) showed a large physical gap between RH168E16 and RH048O11

Fig. 4

BAC-FISH mapping resolution for chromosome 5. a FISH mapping of 12 BAC clones specific on chromosome 5 showed four gaps along the short arm of chromosome 5. b FISH mapping of BACs located at pericentromere heterochromatin region of chromosome 5. (a) FISH mapping of one BAC clone (RH132D05, green) anchored by a marker from putative centromere bin046. With excess amount of Cot100, it was hybridized at north heterochromatin very proximal to the centromere. Two BAC clones (RH076O08 and RH044A21, red signals) helped us to identify chromosome 5. (b) FISH mapping of two BAC clones (RH094I11, pink; RH048J07, red) anchored by markers from putative centromere bin046. BAC clone (RH075N11, blue) helped us to identify chromosome 5. (c) FISH mapping of three BAC clones (RH130M21, yellow; RH096H07, red; RH071D16, green) anchored by markers from putative centromere bin046. BAC clone (RH075N11, blue) helped us to identify chromosome 5