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DNA Research Advance Access originally published online on January 11, 2006
DNA Research 2005 12(5):301-364; doi:10.1093/dnares/dsi018
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© The Author 2006. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

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

Shusei Sato1 {dagger}, Sachiko Isobe2 {dagger}, Erika Asamizu1, Nobuko Ohmido3, Ryohei Kataoka3, Yasukazu Nakamura1, Takakazu Kaneko1, Nozomi Sakurai1, Kenji Okumura2, Irina Klimenko4, Shigemi Sasamoto1, Tsuyuko Wada1, Akiko Watanabe1, Mitsuyo Kohara1, Tsunakazu Fujishiro1 and Satoshi Tabata1,*

1Kazusa DNA Research Institute 2-6-7 Kazusa-kamatari, Kisarazu Chiba 292-0818, Japan
2National Agricultural Research Center for Hokkaido Region (NARCH) Hitsujigaoka 1, Toyohira, Sapporo, 062-8555, Hokkaido, Japan
3Faculty of Human Development, Kobe University Tsurukabuto 3-11, Nada, Kobe 657-8501, Japan
4All-Russian Williams Fodder Crop Research Institute 141055 Lugovaya, Moscow Region, Russia

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 ~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 F1 progeny by agarose gel electrophoresis, followed by genotyping for the primer pairs showing polymorphisms using 188 F1 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.

Key words: red clover; EST; microsatellite marker; genetic linkage map

*To whom correspondence should be addressed. Tel. +81-438-52-3933, Fax. +81-438-52-3934, E-mail: tabata{at}kazusa.or.jp

{dagger}These authors contributed equally to the work

Communicated by Masahiro Yano


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