COMPROBACIÓN DE LA HIPÓTESIS

The EMS mutagenized SL-lines carry a mutation load of one mutation per 502 kb in the M2 generation (Perry et al., 2009). Backcrossing of an identified mutant plant to its wild-type ecotype background decreases this mutation load by 50% and produces plants that still carry the mutation of interest, but less background mutations that probably could interfere with the original mutant phenotype. Therefore, brush mutants were crossed to L. japonicus Gifu wild-type plants and progenies of these crosses were already determined to segregate the mutant phenotype in a 3:1 ratio and to show the same phenotypes as the original brush mutants (Maekawa-Yoshikawa et al., 2009). Since the phenotyping of the F3 (MG-20 x brush) progenies revealed new phenotypic classes, progenies of the brush backcross populations were analysed in detail. The cosegregation of the root and nodulation phenotype as well as the cosegregation of the mutant phenotypes and the EMS-induced mutation located in CNGC1 was determined. In total, three backcross progenies were checked and segregating populations were phenotyped and genotyped (Figure 33). All the analysed progenies segregated mutant roots and some of these showed white bumps or nodules and also some without nodules could be observed, whereas all plants with wild-type roots also had wild-type nodules (Figure 33).

Figure 33. Phenotypes of brush backcross progenies.

Backcross progenies were grown at 26°C, inoculated with M. loti expressing DsRed and phenotyped 3 wpi. All three F1 plants had the same parent plants (K3790/SL0979 x K6215/Gifu). K6216 and K6222 originated from one F1 pod. *One plant (M0027) was heterozygous at the EMS-induced mutation in CNGC1, but showed a mutant phenotype.

A dCAPS marker was used to genotype the EMS-mutation in CNGC1 in brush backcross progenies. One individual (M0027, F4) showed a mutant phenotype, but was heterozygous for the mutation in CNGC1 (Figure 33), as confirmed by sequencing. All the other mutant backcross plants were homozygous for the mutant allele at the site of the mutation. To confirm this result, more backcross plants were investigated to find further mutant plants that are heterozygous at the mutation in CNGC1. Their number could determine the distance from the mutation in CNGC1

to the putative brush locus. In total, 215 F4 plants were phenotyped and genotyped with the dCAPS marker. Thereof three plants were again heterozygous at the mutation in CNGC1, but showed a mutant root phenotype. This results in a calculated distance of 0,9 cM from the mutation in CNGC1 to the putative mutation causing the root phenotype, under the assumption that the dCAPS scoring was correct.

Progeny of M0027 (brush x Gifu wt, F4) was phenotyped at 18°C and at 26°C to check whether the temperature-sensitive nodulation and root growth phenotype could still be observed and whether they were cosegregating. Therefore, seedlings were grown for six weeks at 18°C on plates before transferring them to pots to prevent a masking effect of the short and thick roots on the nodulation phenotype, because these very short roots almost never exhibited infection structures. Previous temperature-shift experiments already demonstrated that after transferring the plants to 26°C, long, wild type-like roots of brush mutants were still impaired in nodulation (Figure 24).

Figure 34. Root systems of brush backcross mutants resembled the brush root phenotype.

L. japonicus Gifu (A, D), brush mutants (B, E) and brush backcross mutants (BC F5, progeny of M0027; C, F) were grown for six weeks at 18°C on plates before transferring them to pots, where they were cultivated at 18°C (A - C) or 26°C (D - F). After one week plants were inoculated with M. loti MAFF DsRed. Pictures were taken 12 dpi. Arrowheads point to nodules. At 18°C brush backcross mutants (C) exhibited more nodules compared to brush mutants (B). The root systems of brush backcross mutants (C, F) resembled brush mutant roots (B, E) at 18°C and at 26°C. Scale bars = 1 cm. Wild-type plants are shown at lower magnification than the mutants.

M0027 showed a mutant root and nodulation phenotype and therefore should have carried two mutant alleles in case of a recessive mutation, but, as already mentioned, is was heterozygous at the mutation in CNGC1. Measuring the root lengths of wild-type plants, brush mutants and self progeny of M0027 revealed that at 18°C and at 26°C roots of M0027 self-progeny plants were significantly longer than those of brush mutants, but still shorter than wild-type roots (Figure 35C). Importantly, self-progeny did not segregate wild-type plants thus excluding a phenotypic miss- scoring of the M0027 recombinant individual. Furthermore, the two different temperatures did not influence the root lengths (Figure 35C). At 18°C the backcross plants produced more nodules compared to brush mutants by trend, but significantly less than wild-type plants (Figure 34A – C, Figure 35A). At 26°C, neither backcross nor brush mutants showed any infected nodule, instead at both conditions white bumps could be detected (Figure 35A and B). There was a tendency of backcross plants compared to brush mutants to have more white bumps (Figure 35B).

seemed that also the lateral root growth was less impaired in the backcross mutants at 18°C compared to brush mutants (Figure 34B and C). Apparently the reduced mutation load of the backcross mutants led to a reduced phenotypic strength of the characteristic brush root and nodulation phenotype, but still both phenotypes were evident and significantly different from the wild type. This result, together with the F4 data from the brush x MG-20 cross, strongly suggests the presence of multiple (EMS-generated) modifier loci that influence the strength of the temperature response as well as the root length of brush mutants.

Figure 35. Analysis of nodulation and root lengths of brush and brush backcross mutants.

L. japonicus Gifu wild-type plants (white boxplots), brush mutants (grey boxplots) and brush backcross plants (BC F5, progeny of M0027; striped boxplots) had been grown for 6 weeks at 18°C. Subsequently they had been cultivated at 18°C or 26°C for one week, before they were inoculated with M. loti MAFF expressing DsRed. Twelve dpi nodules (A) and white bumps (B) per root system were counted and the length of their root systems was measured in cm (C) (n = 10-13).

(A) Compared to wild-type plants brush backcross mutants formed less nodules at 18°C (Welsh’s t-test, *** p-value < 0.001). Only two brush mutants out of 10 showed 1 nodule. At 26°C neither brush mutants nor brush backcross mutants formed any nodule. (B) At 18°C white bumps could be detected at brush mutants and brush backcross plants. At 26°C only one brush mutant plant out of 11 formed one white bump, whereas 8 out of 12 brush backcross plants exhibited white bumps. (C) Regarding the root lengths, brush mutants and brush backcross mutants had significantly shorter roots compared to the wild-type at 18°C and at 26°C, but brush backcross mutants produced longer