Comparative Genetic Mapping and Discovery of Linkage Disequilibrium Across Linkage Groups in White Clover (Trifolium repens L.)

Abstract

White clover (Trifolium repens L.) is an allotetraploid species (2n = 4X = 32) that is widely distributed in temperate regions and cultivated as a forage legume. In this study, we developed expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers, constructed linkage maps, and performed comparative mapping with other legume species. A total of 7982 ESTs that could be assembled into 5400 contigs and 2582 singletons were generated. Using the EST sequences that were obtained, 1973 primer pairs to amplify EST-derived SSR markers were designed and used for linkage analysis of 188 F(1) progenies, which were generated by a cross between two Japanese plants, '273-7' and 'T17-349,' with previously published SSR markers. An integrated linkage map was constructed by combining parental-specific maps, which consisted of 1743 SSR loci on 16 homeologous linkage groups with a total length of 2511 cM. The primer sequences of the developed EST-SSR markers and their map positions are available on http://clovergarden.jp/. Linkage disequilibrium (LD) was observed on 9 of 16 linkage groups of a parental-specific map. The genome structures were compared among white clover, red clover (T. pratense L.), Medicago truncatula, and Lotus japonicus. Macrosynteny was observed across the four legume species. Surprisingly, the comparative genome structure between white clover and M. truncatula had a higher degree of conservation than that of the two clover species.

Keywords: comparative map; expressed sequence tag–simple sequence repeat; linkage disequilibrium; white clover.

Figure 1

An integrated linkage map of white clover generated from a mapping population of ‘273-7’ × ‘T17-349’. Positions of bi-parental, ‘273-7’-specific, and ‘T17-349’-specific loci are indicated by colors of linkage bars. Those showing normal segregation of bi-parental, ‘273-7’-specific, and ‘T17-349’-specific loci are green, red, and blue, respectively. Those showing distorted segregation of bi-parental, ‘273-7’-specific, and ‘T17-349’-specific loci are yellow-green, pink, and aqua, respectively. Distorted loci are preferentially represented when multiple loci, including the distorted loci, are closely located. Bars attached to linkage groups show numbers of mapped multiple loci per each 5 cM. The orange and yellow colors indicate when all multiple loci generated from a single marker are mapped onto the same or homeologous linkage groups, respectively, whereas purple represents other cases.

Figure 2

Patterns of LD blocks of the parental-specific maps. Upper right and lower left triangles show LD blocks of ‘273-7’-specific and ‘T17-349’-specific maps, respectively. Colors in triangles indicate magnitudes of LD (r²) as listed on the right side.

Figure 3

Graphical view of syntenic blocks between white and red clover. Loci generated common or orthologous markers identified by BLASTX searches with a cutoff E-value ≤ 1e−10. (A) Colors of lines represent white clover homeologous linkage groups of orthologous loci. (B) Colors of lines represent red clover linkage groups of orthologous loci.

Figure 4

Graphical view of syntenic relationship between white clover, red clover, M. truncatula, and L. japonicus. Homologous regions were identified by BLASTX searches with a cutoff E-value ≤ 1e−10. Synteny blocks were defined as the region where three or more conserved homologs were located within a 10-cM region in the white and red clover linkage maps, and a 500-kb DNA stretch in the reference genomes. Syntenic regions between the two clover species and model legumes are connected by colored lines. Line colors represent white clover homeologous linkage groups and red clover linkage groups of orthologous loci. (A) Syntenic relationships between two clover species and M. truncatula. (B) Syntenic relationships between two clover species and L. japonicus.