Integration of the FISH pachytene and genetic maps of Medicago truncatula

A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was const ructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meio tic prophase stage are 20 times longer than at mitotic metaphase, and displ ay a well differentiated pattern of brightly fluorescing heterochromatin se gments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochrom atin patterns, and the positions of three repetitive sequences (5S rDNA, 45 S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybr idization (FISH). We determined the correlation between genetic linkage gro ups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC ) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive se quences on the pachytene chromosomes, and compared the genetic and cytologi cal distances between markers. The mapping resolution was determined in a e uchromatic part of chromosome 5 by comparing the cytological distances betw een FISH signals of clones of a BAC contig with their corresponding physica l distance, and showed that resolution in this region is about 60 kb. The e stablishment of this FISH pachytene karyotype, with a far better mapping re solution and detection sensitivity compared to those in the highly condense d mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.