Comprehensive Structural Analysis of the Genome of Red Clover (Trifolium pratense L.)

With the aim of establishing the basic knowledge and resourcesneeded for applied genetics, we investigated the genome structureof red clover Trifolium pratense L. by a combination of cytological,genomic and genetic approaches. The deduced genome size was440 Mb, as estimated by measuring the nuclear DNA content byflow cytometry. Seven chromosomes could be distinguished bymicroscopic observation of DAPI stained prometaphase chromosomesand fluorescence in situ hybridization using 28S and 5S rDNAprobes and bacterial artificial chromosome probes containingmicrosatellite markers with known positions on a genetic linkagemap. The average GC content of the genomes of chloroplast, mitochondrionand nucleus were shown to be 33.8, 42.9 and 34.2%, respectively,by the analysis of 1.4 Mb of random genomic sequences. A totalof 26 356 expressed sequence tags (ESTs) that were grouped into9339 non-redundant sequences were collected, and 78% of theESTs showed sequence similarity to registered genes, mainlyof Arabidopsis thaliana and rice. To facilitate basic and appliedgenetics in red clover, we generated a high-density geneticlinkage map with gene-associated microsatellite markers. A totalof 7159 primer pairs were designed to amplify simple sequencerepeats (SSRs) identified in four different types of libraries.Based on sequence similarity, 82% of the SSRs were likely tobe associated with genes. Polymorphism was examined using twoparent plants, HR and R130, and 10 F₁ progeny by agarose gelelectrophoresis, followed by genotyping for the primer pairsshowing polymorphisms using 188 F₁ plants from the mapping population.The selected 1305 microsatellite markers as well as the previouslydeveloped 167 restriction fragment length polymorphism markerswere subjected to linkage analysis. A total of 1434 loci detectedby 1399 markers were successfully mapped onto seven linkagegroups totaling 868.7 cM in length; 405 loci (28%) were bi-parental,611 (43%) were specific to HR and 418 (29%) were specific toR130. Each genetic linkage group was linked to a correspondingchromosome by FISH analysis using seven microsatellite markersspecific to each of the linkage groups as probes. Transferabilityof the developed microsatellite markers to other germplasmswas confirmed by testing 268 selected markers on 88 red clovergermplasms. Macrosynteny at the segmental level was observedbetween the genomes of red clover and two model legumes, Lotusjaponicus and Medicago truncatula, strongly suggesting thatthe genome information for the model legumes is transferableto red clover for genetic investigations and experimental breeding.