A set of Lotus japonicus Gifu x Lotus burttii recombinant inbred lines facilitates map-based cloning and QTL mapping

Abstract

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype 'Gifu' was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation.

Figure 1.

Marker linkage and recombination landscapes for Gifu × MG-20 and Gifu × L. burttii RIL populations. (A and B) Recombination fractions (rf) and LOD scores for all marker pairs. Recombination fractions are in the upper left triangle and the LOD scores are in the lower right triangle. The LOD scores are for a test of r = 0.5. Red corresponds to a large LOD or a small recombination fraction, indicating a strong marker linkage, while blue is the reverse. Missing values appear in light grey. All plotted values are listed in Supplementary Tables S3 and S4. (A) Gifu × MG-20 RILs. (B) Gifu × L. burttii RILs. (C) The physical distance (in kb) corresponding to a recombination fraction of 1% is plotted at each marker position. A high value indicates a low rate of recombination. (D) The fraction of L. japonicus Gifu genotypes at each marker position is plotted. A fraction of 0.5 (black line) is expected in the absence of segregation distortion.

Figure 2.

sym10 mapping resolution with crosses to L. japonicus MG-20 and L. burttii. The number of informative recombinants is shown in the cross(es) where the marker is polymorphic. The delimited size of the sym10 region is shown for the two crosses individually and with the combined mapping information.

Figure 3.

S. fredii infection phenotypes for L. burttii and Gifu. Six-week-old plate-grown plants inoculated with S. fredii. (A) L. burttii. (B) L. japonicus Gifu. Arrow points to red root nodule.

Figure 4.

QTL analysis of S. fredii nodulation. The trait analysed is the average number of nodules on Gifu × L. burttii RILs inoculated with S. fredii. (A) QTL analysis without cofactors. (B) QTL analysis including markers TM0002, TM0617, and TM0494, corresponding to the three significant peaks in (A), as cofactors. Five and 10% false discovery rate levels are indicated by the upper and lower dashed lines, respectively. Light grey area delimits the results of 1000 QTL analyses on permuted data, which were used to determine the significance thresholds. Dashed vertical grey lines indicate chromosome ends.