With the aim of establishing the basic knowledge and resources needed for applied genetics, we investigated the genome structure of red clover Trifolium pratense L. by a combination of cytological, genomic and genetic approaches. The deduced genome size was approximately 440 Mb, as estimated by measuring the nuclear DNA content by flow cytometry. Seven chromosomes could be distinguished by microscopic observation of DAPI stained prometaphase chromosomes and fluorescence in situ hybridization using 28S and 5S rDNA probes and bacterial artificial chromosome probes containing microsatellite markers with known positions on a genetic linkage map. The average GC content of the genomes of chloroplast, mitochondrion and 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 total of 26,356 expressed sequence tags (ESTs) that were grouped into 9339 non-redundant sequences were collected, and 78% of the ESTs showed sequence similarity to registered genes, mainly of Arabidopsis thaliana and rice. To facilitate basic and applied genetics in red clover, we generated a high-density genetic linkage map with gene-associated microsatellite markers. A total of 7159 primer pairs were designed to amplify simple sequence repeats (SSRs) identified in four different types of libraries. Based on sequence similarity, 82% of the SSRs were likely to be associated with genes. Polymorphism was examined using two parent plants, HR and R130, and 10 F(1) progeny by agarose gel electrophoresis, followed by genotyping for the primer pairs showing polymorphisms using 188 F(1) plants from the mapping population. The selected 1305 microsatellite markers as well as the previously developed 167 restriction fragment length polymorphism markers were subjected to linkage analysis. A total of 1434 loci detected by 1399 markers were successfully mapped onto seven linkage groups totaling 868.7 cM in length; 405 loci (28%) were bi-parental, 611 (43%) were specific to HR and 418 (29%) were specific to R130. Each genetic linkage group was linked to a corresponding chromosome by FISH analysis using seven microsatellite markers specific to each of the linkage groups as probes. Transferability of the developed microsatellite markers to other germplasms was confirmed by testing 268 selected markers on 88 red clover germplasms. Macrosynteny at the segmental level was observed between the genomes of red clover and two model legumes, Lotus japonicus and Medicago truncatula, strongly suggesting that the genome information for the model legumes is transferable to red clover for genetic investigations and experimental breeding.

Background: Red clover (Trifolium pratense L.) is a major forage legume that has a strong selfincompatibility system and exhibits high genetic diversity within populations. For several crop species, integrated consensus linkage maps that combine information from multiple mapping populations have been developed. For red clover, three genetic linkage maps have been published, but the information in these existing maps has not been integrated.

The first quantitative trait locus (QTL) analysis of multiple agronomic traits in the model legume Lotus japonicus was performed with a population of recombinant inbred lines derived from Miyakojima MG-20 x Gifu B-129. Thirteen agronomic traits were evaluated in 2004 and 2005: traits of vegetative parts (plant height, stem thickness, leaf length, leaf width, plant regrowth, plant shape, and stem color), flowering traits (flowering time and degree), and pod and seed traits (pod length, pod width, seeds per pod, and seed mass). A total of 40 QTLs were detected that explained 5%-69% of total variation. The QTL that explained the most variation was that for stem color, which was detected in the same region of chromosome 2 in both years. Some QTLs were colocated, especially those for pod and seed traits. Seed mass QTLs were located at 5 locations that mapped to the corresponding genomic positions of equivalent QTLs in soybean, pea, chickpea, and mung bean. This study provides fundamental information for breeding of agronomically important legume crops.