José Cuenca, Yann Froelicher, Pablo Aleza, José Juárez, Luis Navarro, and Patrick Ollitrault

Multilocus half-tetrad analysis and centromere mapping in Citrus: Evidences of SDR mechanism for 2n megagametophyte production and partial chiasma interference in mandarin cv ‘Fortune’

José Cuenca, Yann Froelicher, Pablo Aleza, José Juárez, Luis Navarro, and Patrick Ollitrault

Supplementary information

Validation of MAC-PR estimation of allele dosage

The inference of a diploid gamete structure when the two parents share one allele requires the ability to estimate allelic dosage in triploid progenies. In this paper we have used the MAC-PR method (Esselink et al.,2004) to estimate allelic dosage after verification of its applicability in our populations and markers as described below.

In order to verify that the MAC-PR method can be applied to estimate the allelic dosage of our markers in triploid genotypes, with our PCR conditions, a preliminary trial was performedwith five markers. The DNA of two haploid lines (from Clementine cv ‘Nules’ and Pumello cv ‘Chandler’) displaying allelic variability were mixed at different ratios (1:2, 1:1, 2:1 and 3:1). Ten replications (PCR and run in the genetic fragment analyzer) were done for each ratio and marker. Area ratios among the two observed peaks (alleles) were calculated for all runs and the correlation between DNA ratio and peak area ratio was analyzed. An illustration is given for the mest56 marker (figure SI1), showing a very high correlation among DNA proportion and peak area ratio (r2=0.9902). Similar results were observed for the four other markers.

Figure SI1: Relationship between several DNA proportions of Clementine and Chandler haploids and peak area ratio for three repetition of the mest56 marker.

To avoid misinterpretation of allele dosage associated with PCR allele competition, the triploid hybrid peak area ratios were systematically corrected by ‘Fortune’ genitor peak ratio as:

SD

where SD is the estimated allele dosage ratio in the sample; Si and Fi are the peak area for allele i for the analysed sample and ‘Fortune’ diploid cultivar, respectively (see figure SI2).

Figure SI2: Observed peaks for the CAC15 marker in Fortune, Murcott, the FM60 hybrid and the FM38 hybrid showing respectively ratio 2:1 and 1:2 between alleles 169 and 178. SD=(S1·F2)/(S2·F1)

Ten technical replications (independent PCR) were done for mCrCIR06B05with two triploid genotypes with ratios 1:2 and 2:1to determine the discrimination power of the assignment of observed peak area ratios. Theresults (figure SI3-a) testified the high stability of allele dosage evaluation.

Figure SI3: Scatterplot of peak area ratios for ten PCR repetitions of two genotypes with ratios 1:2 and 2:1 for two alleles of mCrCIR06B05 marker (a) and distribution of peak area ratios for all the genotypes and markers showing 1:2 and 2:1 ratios (b).

The unambiguous differentiation of allele dosage in heterozygous triploids has been confirmed during progeny analysis by the very clear bimodal distribution of the peaks area ratio of the different triploid hybrids for all markers (Figure SI3-b). These results validate the method of area peak ratios used for genotyping the triploid progenies in this study.

Method for Half tetrad analysis

The analysis was made according to Tavoletti et al. (1996) assuming that multiple crossover does not occur between contiguous markers. Under this hypothesis, each crossover between two markers leads to a change from homozygosity to heterozygosity between marker i and i+1 in the case of SDR and a half change from heterozygosity to homozygosity in the case of FDR. Thus, the distance between two adjacent markers (dMiMi+1) can be estimated by the proportion of 2n gamete with changes (homozygosity versus heterozygosity; CMiMi+1) between the two markers. For FDR, dMiMi+1 = CMiMi+1 and for SDR, dMiMi+1= ½ CMiMi+1.

The probability of each observed genotype profile (Gj), considering that the locus is homozygous (Ho) or heterozygous (He), was calculated as:

P(Gj)

where:

• PMiMi+1is the probability of configuration for two adjacent loci whose interval does not include the centromere; being PMiMi+1= CMiMi+1in case of configuration change and PMiMi+1= 1- CMiMi+1 when no change occurs between the adjacent loci.
• PCMaand PCMa+1 are the probability of the observed configuration for the two loci flanking the centromere, PCMa(and identically PCMa+1) values being (1-dCMa), (dCMa), (2dCMa) and (1-2dCMa) respectively for the following situation (He, FDR), (Ho, FDR), (He, SDR), (Ho, SDR); with dCMa being the mapped distance between the centromere and the given flanking locus.

Under FDR or SDR models and different centromere positions, the probability of the observed 2n gamete population (P) is:

P

where n is the number of 2n gametes with the genotype j and C a combinatory coefficient constant for the set of observed data.

Both under FDR and SDR models, the centromere position was virtually moved from before the first considered locus along the linkage group to after the last considered locus (intervals of 0.5 cM) and relative probability (P/C) was estimated for each position. The LOD between best position under SDR and FDR was calculated in order to determine the mode of 2n gamete restitution and the position of the centromere was considered as the one producing the highest relative probability under the identified mode of restitution.