# Introgression reshapes recombination distribution in grapevine interspecific hybrids

Marion Delame, Emilce Prado, Sophie Blanc, Guillaume Robert-Siegwald, Christophe Schneider, Pere Mestre, Camille Rustenholz, Didier Merdinoglu

► To cite this version:

Marion Delame, Emilce Prado, Sophie Blanc, Guillaume Robert-Siegwald, Christophe Schneider, et al. Introgression reshapes recombination distribution in grapevine interspecific hybrids. TAG Theoretical and Applied Genetics, 2019, 132 (4), pp.1073-1087. 10.1007/s00122-018-3260-x. hal-02629272

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# Introgression reshapes recombination distribution in grapevine interspecific hybrids

Marion Delame¹,², Emilce Prado¹, Sophie Blanc¹, Guillaume Robert¹, Pere Mestre¹, Camille Rustenholz¹, Didier Merdinoglu¹

¹Université de Strasbourg, INRA, SVQV UMR-A 1131, F-68000 Colmar, France

²AgroParisTech, Direction des Formations Doctorales, 19 avenue du Maine, 75015 Paris, France

* Corresponding author

E-mail: didier.merdinoglu@inra.fr

## Abstract

*Muscadinia rotundifolia*, an American species related to the European grapevine *Vitis vinifera*, has a high level of resistance to several grapevine major diseases. Being particularly interesting for grape breeding, resistance factors from *M. rotundifolia* have been transferred into a cultivated background while eliminating the unwanted cultural traits and off-flavours by backcross-based introgression. However, crosses between both species most often led to sterile hybrids and developmental abnormalities in the offspring. In this context, identify the genetic and genomic features that impede crosses between the two species is essential. To this end, three mapping populations were generated by pseudo-backcrosses using *M. rotundifolia* as the donor parent and several *V. vinifera* cultivars as the recurrent parents. Genotyping-by-sequencing was used to establish high-density genetic linkage maps and to determine the origin of each chromosome segment of the analysed individuals. Several large regions displaying species-related distortions of segregation were identified. A good collinearity of the SNP positions was observed between parental maps, confirming the synteny between both species, except on lower arm of chromosome 7. Interestingly, we observed on partially hybrid regions that recombination events are enhanced in homologous regions whereas nearly none were observed in homeologous ones. Altogether, these results provide new insight to improve the use of *M. rotundifolia* in grape breeding, notably, to optimize the introgression process.

## Introduction

Wild species related to crops have provided plant breeders with a wide range of genetic resources which has incontestably contributed to the emergence of modern agriculture and to the agricultural economy world-wide. All the functional categories of traits, including abiotic stress tolerance, yield, quality, male sterility, benefited the use of wild relatives. But pest and disease resistance gathers over 80% of the beneficial traits conferred by genes derived from wild relatives that were exploited for over a century [1]. Despite the undeniable importance of wild species in crop improvement for resistance, breeders have often faced the challenge of obtaining viable and fertile interspecific hybrids, which has consequently limited the optimal use of wild genetic resources [1-7].
Grapevine, which does not escape this reality, is a crop particularly threatened by numbers of severe pests and diseases. Indeed, from the 19th century, European viticulture has undergone radical changes as a result of the introduction of major diseases from North America: powdery mildew, downy mildew and phylloxera, respectively caused by the fungus *Erysiphe necator*, the oomycete *Plasmopara viticola* and the aphid *Daktulosphaira vitifoliae*. Almost all the European grapevine cultivars (*Vitis vinifera ssp. sativa*) are susceptible to these pathogens. But, fortunately, many sources of resistance were found in Asian and American wild *Vitis* species [8-10]. More, several genetic factors conferring protection against downy mildew, powdery mildew and phylloxera have been recently identified [11-16]. Besides the true *Vitis* species, *Muscadinia rotundifolia* is a North American grapevine related to *V. vinifera* which has the unique feature of displaying a high resistance level to many major grapevine pathogens: *E. necator*, *P. viticola* and *D. vitifoliae* but also *Guignardia bidwellii*, a fungus responsible for black-rot disease, *Xiphinema index*, a nematode vector of grapevine fanleaf virus, and *Xylella fastidiosa*, a bacteria responsible for Pierce's disease [17-20].

*M. rotundifolia* being particularly interesting for grape breeding, crosses with *V. vinifera* were attempted as soon as the end of the 19th century and a first success was obtained by Detjen [21]. In the 1980s, successful crosses produced fertile hybrids and allowed the use of *M. rotundifolia* in French breeding programs [22]. Backcross-based introgressions were attempted to transfer resistance factors from *M. rotundifolia* into cultivated backgrounds while eliminating the unwanted cultural traits and off-flavours. In the meanwhile, genetic factors conferring resistance to powdery and downy mildew were identified and molecular makers allowing marker assisted selection were developed [14, 23-28]. However, the poor vigour and low fertility of the *V. vinifera* × *M. rotundifolia* hybrids and backcrosses hampered a broader and convenient utilization of *M. rotundifolia* in breeding programs. The low rate of successful crosses and the sterility of interspecific hybrids were first attributed to the difference in chromosome numbers between both species, 2n=38 chromosomes for *V. vinifera* vs 2n=40 for *M. rotundifolia*. Cytogenetic studies showed that some mismatches between chromosomes of the same pair occur during meiosis of F1 hybrids and individuals derived from the first pseudo-backcross [29-30]. But the small size of the chromosomes did not allow to determine which ones were really involved in these mismatches. Blanc et al. [27] established a genetic linkage map of *M. rotundifolia* using SSR markers which showed a high level of macrosynteny with *V. vinifera*. The same work also revealed that chromosome 7 of *V. vinifera* matches with chromosomes 7 and 20 of *M. rotundifolia*, then explaining the origin of the difference of chromosome number between both species. Nevertheless, the explanation of low prolificity and fertility of interspecific crosses remains still unknown, except that, both species belonging to two different subgenera (*Vitis* and *Muscadinia*), their divergence is rather high compared to the genetic distance between *Vitis* subgenus species [31].

No efficient tools are thus available to improve the process of introgression of resistance genes from *M. rotundifolia* into *V. vinifera*. That is why we decided to acquire new data about the compatibility between the genomes of both species by studying the recombination rate between homeologous chromosome pairs in *V. vinifera* × *M. rotundifolia* interspecific hybrids. Our study is based upon pseudo-F1 hybrids derived from crosses between *M. rotundifolia* and *V. vinifera* and pseudo-backcross populations derived from successive crosses between pseudo-F1 hybrids and various *V. vinifera* cultivars. The genetic maps previously developed with these populations were mainly built thanks to microsatellite markers and did not offer a density of markers sufficient for a fine analysis of recombination rate along the chromosomes [24, 26]. Genotyping-by-sequencing (GBS) appears to be a simple robust method allowing the discovery of thousands of SNPs for hundreds of individuals rapidly and at low cost [32]. A SNP genetic linkage map has already been established using GBS for an
interspecific cross between *V. vinifera* and *V. rupestris* [33]. However, GBS has never been used to study crosses between *V. vinifera* and *M. rotundifolia*. In this study, we thus used GBS to perform a fine-scale analysis of the recombination rate along the genome in various interspecific contexts represented by three pseudo-backcross populations at different step of introgression.

## Results

### Genetic mapping

All the data put together, 263,034 SNPs were detected by GBS. After screening for SNPs having less than 5% of missing data, 9,006, 9,132 and 8,489 SNPs were obtained for BC1, BC2 and BC4 populations, respectively (Table 1). For each population, respectively 40%, 27% and 53% of these SNPs displayed redundancy and were thus discarded because bringing no new information for genetic mapping. The parental origin of 2.4%, 3.2% and 4.9% of the remaining SNPs, called non-redundant, was undetermined in BC1, BC2 and BC4 populations, respectively, because their genotype was unknown for the parents. They were thus discarded from the following analyses. In addition, in BC1 population, 19% of SNPs were found homozygous in both parents and thus did not segregate. This rate dramatically increased to 37% and 54% for the BC4 and BC2 populations, respectively. Moreover, 855 and 733 SNPs displayed a segregation distortion in BC1 and BC2 populations, respectively, while of only 200 in BC4 population. Finally, after performing all the filtering steps, 3,415, 2,210 and 2,222 SNPs were available for genetic mapping for BC1, BC2 and BC4 populations, respectively.

<table><caption>Table 1. Number of SNP markers conserved after each filtering step for the three populations</caption><thead><tr><th rowspan="2">Filtering steps</th><th colspan="3">Mapping populations</th></tr><tr><th>BC1</th><th>BC2</th><th>BC4</th></tr></thead><tbody><tr><td>Total SNPs detected</td><td colspan="3">263,034</td></tr><tr><td>SNPs ≤ 5% missing data</td><td>9,006</td><td>9,132</td><td>8,489</td></tr><tr><td>Non-redundant SNPs</td><td>5,396</td><td>6,677</td><td>4,068</td></tr><tr><td>SNPs with known segregation</td><td>5,266</td><td>6,460</td><td>3,868</td></tr><tr><td>SNPs with at least one heterozygous parent</td><td>4,270</td><td>2,943</td><td>2,427</td></tr><tr><td>SNPs with segregation distortion</td><td>855</td><td>733</td><td>205</td></tr><tr><td>SNPs available for genetic mapping</td><td>3,415</td><td>2,210</td><td>2,222</td></tr></tbody></table>

We established parental genetic linkage maps for each population. The number of mapped markers depended on the parent types and the backcross levels (Table 2). *V. vinifera* maps consisted of 927, 996 and 1,177 markers, respectively, for BC1, BC2 and BC4 populations whereas hybrid maps included 2,860, 1,516 and 1,521 markers, respectively. Conversely, the genetic distance covered was quite similar between maps, from 1,153.7 cM (1771P) to 1,455.8 cM (Cabernet Sauvignon), whatever the parent and the population and was not correlated to the number of mapped markers. The average physical spacing between two successive markers was rather low, specially for hybrids maps which displayed a density of one marker per 0.17 – 0.32 Mbp. Despite the overall high marker density, some local gaps in markers were observed on genetic maps.
<table><caption>**Table 2. Statistics for parental maps of BC1, BC2 and BC4 populations.** Into brackets, extrema per chromosome.</caption><thead><tr><th rowspan="2">Mapping population</th><th colspan="2">BC1</th><th colspan="2">BC2</th><th colspan="2">BC4</th></tr><tr><th>♂ Cabernet Sauvignon</th><th>♀ 8624</th><th>♂ Syrah</th><th>♀ 22-8-78</th><th>♂ Nebbiolo</th><th>♀ 1771P</th></tr></thead><tbody><tr><td>Parent</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Number of mapped markers</td><td>927</td><td>2,860</td><td>996</td><td>1,516</td><td>1,177</td><td>1,521</td></tr><tr><td>Total length of the map (cM)</td><td>1,455.8</td><td>1,325.3</td><td>1,242.8</td><td>1,272.1</td><td>1,368.5</td><td>1,153.7</td></tr><tr><td>Average spacing between single genetic positions (cM)</td><td>2.0 (0.9-4.9)</td><td>1.6 (0.3-21.2)</td><td>1.8 (0.9-7.5)</td><td>1.1 (0.3-2.8)</td><td>1.2 (0.8-1.8)</td><td>1.0 (0.3-1.7)</td></tr><tr><td>Average spacing between markers (cM/Mbp)</td><td>1.57/0.52</td><td>0.46/0.17</td><td>1.25/0.49</td><td>0.84/0.32</td><td>1.16/0.41</td><td>0.76/0.32</td></tr><tr><td>Extrema of the maximum spacing between markers per chromosome (cM)</td><td>6.2-32</td><td>3.0-43.9</td><td>5.4-18.6</td><td>3.5-57.5</td><td>5.5-41.9</td><td>5.4-47.4</td></tr><tr><td>Coverage rate</td><td>0.93 (0.79-0.98)</td><td>0.96 (0.75-1.00)</td><td>0.88 (0.22-0.99)</td><td>0.89 (0.11-1.00)</td><td>0.95 (0.78-0.99)</td><td>0.94 (0.79-0.99)</td></tr><tr><td>Contraction rate</td><td>1.02 (0.69-1.45)</td><td>0.90 (0.62-1.18)</td><td>0.85 (0.43-1.19)</td><td>0.93 (0.63-1.95)</td><td>0.94 (0.76-1.41)</td><td>0.80 (0.52-1.39)</td></tr></tbody></table>

A coverage rate was calculated for each chromosome as the ratio of physical distance between distal mapped markers on the total physical chromosome length. The coverage rates were high, varying from 88% to 96%, but singularities were identified. Chromosomes 7, 8 and 12 were entirely covered for BC1 hybrid map and chromosome 8 for BC2 hybrid map (S1 Table). Conversely, for BC2 population, chromosomes 8 of *V. vinifera* map and 11 of hybrid map were poorly covered with respectively 22% and 11% coverage. Despite quite high covering rates, some chromosomes harbour badly covered regions: chromosomes 1, 11, 13 and 15 of the hybrid map in BC1 population, and in the BC2 population, chromosomes 6 and 16 of the *V. vinifera* map and chromosomes 1 and 6 of the hybrid map (S1 Fig.). Low coverage rates have two origins: i) a low density of SNPs detected in the corresponding regions, e.g. chromosome 6 of both parental maps of the BC2 population, ii) a segregation distortion which led us to eliminate markers for mapping, especially chromosome 13 in the BC1 population and chromosomes 8 and 11 in the BC2 population (S2 Fig.).

BC1 population, initially composed with 177 individuals, was dramatically reduced through the years due to developmental abnormalities which were heightened after establishment in the vineyard. The segregation distortion before and after establishment in the vineyard was estimated by performing a $\chi^2$-test between observed and expected Mendelian ratios of SSR markers (Table 3). On chromosome 15, segregation distortion was already present before planting and stayed stable afterwards. On chromosome 13, segregation distortion appeared only after planting. On chromosomes 4 and 11, segregation distortion was present before planting and increased afterwards whereas, on chromosome 1, it decreased.
<table><caption>**Table 3.** Evolution over the years of the segregation distortion of eight SSR markers on BC1 hybrid map. Segregation distortion is estimated by a $\chi^2$-test comparing observed and expected allelic Mendelian ratios, before (initial p-value) and after (final p-value) establishment in the vineyard.</caption><thead><tr><th>Chromosome</th><th>SSR marker</th><th>Initial p-value</th><th>Final p-value</th></tr></thead><tbody><tr><td>1</td><td>VVIn61</td><td>0.001</td><td>0.014</td></tr><tr><td rowspan="2">4</td><td>VVIp25b</td><td>0.048</td><td>0.033</td></tr><tr><td>VMC2e10</td><td>0.135</td><td>0.008</td></tr><tr><td>11</td><td>VVIv35a</td><td>0.017</td><td>0.001</td></tr><tr><td rowspan="3">13</td><td>VVIn62</td><td>0.323</td><td>4.10<sup>-4</sup></td></tr><tr><td>VMC3d12</td><td>0.102</td><td>4.10<sup>-7</sup></td></tr><tr><td>VMC3b12</td><td>0.144</td><td>0.002</td></tr><tr><td>15</td><td>VVIm42b</td><td>1.10<sup>-4</sup></td><td>0.003</td></tr></tbody></table>

A contraction rate was calculated for each chromosome as the ratio of genetic distance and physical distance between the two distal markers. A contraction rate below 0.9 means that recombination rate is lower than observed in the *V. vinifera* reference map [34]. Conversely, a contraction rate above 0.9 means that recombination rate is higher than observed in the grapevine reference map. No difference was measured in average contraction rate between *V. vinifera* and hybrid maps, both reaching a value (0.93) very close to the reference linkage map. Forty two percent of the chromosomes displayed a contraction rate close to the reference, between 0.8 and 1.0, but some specific patterns were highlighted. For instance, chromosome 14 was contracted at different level in the majority of parental maps. Chromosome 7 was dilated in all the *V. vinifera* maps, with contraction rates of 1.45, 1.14 and 1.41 for BC1, BC2 and BC4 populations, respectively. On the contrary, chromosome 9 was contracted in the hybrid maps, especially of the BC1 and BC2 populations with contraction rates of 0.69 and 0.63, respectively. Similarly, chromosome 1 was contracted in the BC1 and BC4 hybrid maps with contraction rates of 0.46 and 0.61, respectively. Substantial contraction was also observed in *V. vinifera* maps (chromosomes 12 and 14 in Syrah map (respectively, 0.67 and 0.69)) (S2 Table).

Study of recombination showed as well different behaviours depending on the population. Both maps of the BC1 population showed a high level of collinearity between genetic and physical maps, whatever the chromosome (S1 Fig.). This result is consistent with the high level of macrosynteny observed between *V. vinifera* and *M. rotundifolia* [27]. In this population, recombination rate was similar between both parents with, nevertheless, some exceptions. Chromosome 7 displayed a fall in recombination rate in its lower half (Fig 1A). Unlike the BC1 maps, marker order was not conserved between BC2 maps and high changes in terms of recombination rates were observed depending on chromosomes (Fig 1B). For instance, recombination rates of chromosomes 4 and 8 were similar in both parents. On the contrary, for chromosomes 2, 12 and 14, hybrid map locally revealed a higher recombination rate than *V. vinifera* map, whereas other regions displayed an opposite pattern or even did not recombine at all. The BC4 population showed the same pattern of recombination as observed in the BC2 population but for different chromosomes (Fig 1C). For instance, variations in recombination rate along chromosome 5 were similar between both parents. On the contrary, chromosome 14 showed a lower recombination rate on hybrid map than on *V. vinifera* map at the beginning of the chromosome and a reversal of trend at the end of the chromosome. Finally, both parent maps have a similar total genetic length but with different local recombination rates.
**Fig 1.** Variation of genetic distance depending on physical distance on the hybrid and *V. vinifera* maps. A. Chromosomes 7 and 10 of the BC1 population. B. Chromosomes 4, 8, 2, 12 and 14 of the BC2 population. C. Chromosomes 5 and 14 of the BC4 population. For each cross, *V. vinifera* maps are represented with green triangles, and hybrid maps with red dots. Bars below the graphs show the regions covered by homeologous pairs of chromosomes (*V. vinifera* - *M. rotundifolia*; in red) and homologous regions (*V. vinifera* - *V. vinifera*; in dark grey).

<figure><img src="image_2.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /><figcaption>BC2 - Chromosome 12</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>BC2 - Chromosome 14</figcaption></figure>

<figure><img src="image_6.png" /></figure>

C.

<figure><img src="image_7.png" /><figcaption>BC4 - Chromosome 14</figcaption></figure>

<figure><img src="image_8.png" /></figure>

### Chromosome painting

By comparison to the PN40024 reference genome, 91,865 SNPs were identified in Trayshed and 110,815 SNPs in the seven accessions of *M. rotundifolia* taken altogether. A rate of SNPs coming from *M. rotundifolia*, τᵢ, was calculated as the ratio of the number of SNPs from *M. rotundifolia* detected on those available. When the rate was above the set threshold, the region was identified as homeologous (*V. vinifera* - *M. rotundifolia*). The threshold was set to balance the chance to detect a false
homeologous region and the chance not to detect a true homeologous region. In the F1 hybrid, a threshold set to 0.5 allowed to detect the entire genome as homeologous, as expected. For the BC1 and BC4, a threshold of 0.6 was used. Because of a lack of information on the exact *M. rotundifolia* parent of the BC2, the threshold was set to 0.5.

In 1771P parent of BC4 population, five regions derived from *M. rotundifolia* were identified, on chromosomes 8, 13, 14, 18 and 19 (Fig 2A). Analysis of the transmission of these regions highlights different patterns (Table 4). Chromosomes 8, 18 and 19 bore a large *M. rotundifolia* region covering over 80%. Respectively 15%, 32% and 13% of the individuals showed a recombination event in these homeologous regions. Conversely, chromosomes 13 and 14 bore a smaller *M. rotundifolia* region covering 24.7% and 30.3% of the chromosome length, respectively. Only one individual out of 79 displayed a recombination event in these homeologous regions, i.e. *M. rotundifolia* region was either entirely kept or entirely lost in the quasi-totality of individuals. These results allowed to establish a link between proportion of *M. rotundifolia* remaining region and frequency of recombinant individuals.

<caption>**Fig 2.** Homologous and homeologous regions in 1771P (A) and 22-8-78 (B). Chromosomes are horizontally represented with their physical length on the x-axis. Homeologous regions, where $τ_i$ is above the threshold, are in red; homologous regions, where $τ_i$ is below the threshold, are in dark grey.</caption>

A.

<figure><img src="image_9.png" /></figure>

B.

<figure><img src="image_10.png" /><figcaption>**Table 4.** Relative length and transmission in BC4 population of homeologous regions of 1771P parent. For each chromosome, the proportion of homeologous regions (PHr) is calculated as the ratio between the length of the homeologous region (LHr) and the chromosome length (CL). The number of individuals which either entirely lost the homeologous region (IL), entirely conserved it (IC) or had one recombination event in it (IR1) was recorded.</figcaption></figure>

<table><thead><tr><th>Chromosome</th><th>CL (Mb)</th><th>LHr (Mb)</th><th>PHr (%)</th><th>IL</th><th>IC</th><th>IR1</th></tr></thead><tbody><tr><td>8</td><td>22.55</td><td>18.34</td><td>81.3</td><td>41</td><td>26</td><td>12</td></tr><tr><td>13</td><td>29.08</td><td>7.17</td><td>24.7</td><td>41</td><td>37</td><td>1</td></tr><tr><td>14</td><td>30.27</td><td>7.25</td><td>30.3</td><td>32</td><td>46</td><td>1</td></tr><tr><td>18</td><td>34.57</td><td>33.66</td><td>97.4</td><td>28</td><td>26</td><td>25</td></tr><tr><td>19</td><td>24.70</td><td>22.71</td><td>92.0</td><td>37</td><td>32</td><td>10</td></tr></tbody></table>
In 22-8-78 parent of BC2 population, regions from *M. rotundifolia* were transmitted on almost all chromosomes (Fig 2B). Chromosomes 3, 4, 7, 10, 18 and 19 derived entirely from *V. vinifera* whereas chromosomes 8, 9 and 12 were almost entirely from *M. rotundifolia*. Other chromosomes have smaller regions from *M. rotundifolia*. Interestingly, chromosome 14 carried two distal telomeric regions derived from *M. rotundifolia*, revealing two likely crossovers for the meiosis of the corresponding pseudo-F1 parent. During the F1 meiosis, chromosomes 3, 4, 7, 8, 10, 18 and 19 did not recombine, all except 8 being totally from *V. vinifera*. Other chromosomes displayed one or two recombination events. As in BC4 population, various recombination patterns were observed in BC2 population (Table 5). For chromosomes 8 and 12, with a homeologous region covering over 95% of the chromosome, respectively 47% and 19% of the individuals carried at least one or more recombination events. Conversely, in small homeologous regions, covering less than 25% of the chromosome length, less than 1% of individuals displayed a recombination event. Again, a link between proportion of *M. rotundifolia* remaining region and frequency of recombinant individuals is supported by these results.

<table><caption>**Table 5. Relative length and transmission in BC2 population of homeologous regions of 22-8-78 parent.** For each chromosome, the proportion of homeologous regions (PHr) is calculated as the ratio between the length of the homeologous region (LHr) and the chromosome length (CL). The number of individuals which either entirely lost the homeologous region (IL), entirely conserved it (IC), had one recombination event in it (IR1) or more (IR2) was recorded.</caption><thead><tr><th>Chromosome</th><th>CL (Mb)</th><th>LHr (Mb)</th><th>PHr (%)</th><th>IL</th><th>IC</th><th>IR1</th><th>IR2</th></tr></thead><tbody><tr><td>1</td><td>24.23</td><td>9.42</td><td>30.4</td><td>67</td><td>133</td><td>10</td><td>0</td></tr><tr><td>2</td><td>18.89</td><td>13.67</td><td>54.6</td><td>108</td><td>102</td><td>0</td><td>0</td></tr><tr><td>5</td><td>25.65</td><td>5.29</td><td>24.4</td><td>119</td><td>89</td><td>2</td><td>0</td></tr><tr><td>6</td><td>22.65</td><td>16.73</td><td>66.9</td><td>115</td><td>85</td><td>10</td><td>0</td></tr><tr><td>8</td><td>22.55</td><td>22.55</td><td>100.0</td><td>46</td><td>63</td><td>98</td><td>3</td></tr><tr><td>9a</td><td rowspan="2">23.01</td><td>6.72</td><td>29.2</td><td>86</td><td>74</td><td>50</td><td>0</td></tr><tr><td>9b</td><td>13.15</td><td>57.2</td><td>109</td><td>89</td><td>12</td><td>0</td></tr><tr><td>11</td><td>20.12</td><td>13.77</td><td>68.4</td><td>174</td><td>33</td><td>3</td><td>0</td></tr><tr><td>12</td><td>24.27</td><td>23.07</td><td>95.1</td><td>89</td><td>80</td><td>40</td><td>1</td></tr><tr><td>13</td><td>29.08</td><td>14.30</td><td>4.9</td><td>95</td><td>115</td><td>0</td><td>0</td></tr><tr><td>14a</td><td rowspan="2">30.27</td><td>0.88</td><td>2.9</td><td>104</td><td>105</td><td>1</td><td>0</td></tr><tr><td>14b</td><td>3.78</td><td>12.5</td><td>97</td><td>113</td><td>0</td><td>0</td></tr><tr><td>15</td><td>20.30</td><td>2.14</td><td>10.6</td><td>120</td><td>90</td><td>0</td><td>0</td></tr><tr><td>16</td><td>23.57</td><td>7.22</td><td>30.6</td><td>116</td><td>93</td><td>1</td><td>0</td></tr><tr><td>17</td><td>18.69</td><td>1.58</td><td>8.5</td><td>87</td><td>123</td><td>0</td><td>0</td></tr></tbody></table>

We represented together the recombination fractions and the nature of regions, homologous or homeologous, at the genome scale (Fig 3). We observed as a general rule that low recombination regions matched homeologous regions on the BC2 and BC4 hybrid maps.
<caption>**Fig 3.** Plot of estimated recombination fractions for all pairs of markers of the BC4 (A) and BC3 (B) hybrid genetic maps. Markers are plotted ordered on the x-axis and on the y-axis. Recombination fraction is plotted thanks to a colour scale. Red dot indicates that the two markers, respectively on x- and y-axis, are highly linked (low recombination fraction) and blue dot indicates that markers poorly or not linked (high recombination fraction). Chromosome limits are identified at the left of the heatmap. Homeologous and homologous regions are identified at the top.</caption>

A.

<figure><img src="image_11.png" /></figure>

B.

<figure><img src="image_12.png" /></figure>
In homeologous regions, a transmission rate was calculated for each SNP as the percentage of individuals of each of the three populations carrying the *M. rotundifolia* allele of its hybrid parent (Fig 4). As expected, transmission rates were around 0.5 in the majority of regions. But, as described above, some regions showed an unbalanced transmission. Surprisingly, the segregation distortion was not always in favour of the *V. vinifera* allele. For instance, in BC1 population, alleles from *M. rotundifolia* were transmitted in 62% of the offspring on chromosome 1, whereas, on chromosome 11, they were transmitted in only 39% of the offspring. At the upper part of the chromosome 13, alleles from *M. rotundifolia* were present in near than 80% of the BC1 individuals. In BC2 population, segregation distortion observed on chromosomes 1 and 11 displayed the same pattern as in the BC1 population: alleles from *M. rotundifolia* are conserved in 68% of the individuals on chromosome 1 and in 17% of the individuals on chromosome 11.

<caption>**Fig 4. Transmission rate of M. rotundifolia SNPs in the BC populations.** The transmission rate is calculated for each SNP as the percentage of individuals in a population harbouring the allele from *M. rotundifolia*. A. Chromosomes 8, 1, 11 and 13 of BC1 population. B. Chromosomes 8, 1, 11 and 13 of BC2 population. Orange dots are the positions of SNPs used to calculate transmission rate. Homologous regions are represented with a grey background on the graphs.</caption>

A.

<figure><img src="image_13.png" /></figure>
## B.

<figure><img src="image_14.png" /></figure>

## Discussion

Although *M. rotundifolia* is highly resistant to numerous grapevine diseases, its use in breeding programs has been limited due to the difficulty to obtain fertile progeny in interspecific crosses. In our study, we used three populations of different level of pseudo-backcross to investigate the transmission rate of chromosomic regions of *M. rotundifolia* in a *V. vinifera* background. We also established high-density genetic linkage maps of the parents of the populations to study marker order and recombination rates across the genome and in introgressed regions.

### Synteny and divergence between *V. vinifera* and *M. rotundifolia* genomes

The comparison of marker order in the hybrid map and in the *V. vinifera* linkage map of BC1 population with their physical position confirms the high level of macrosynteny between *V. vinifera* and *M. rotundifolia* genomes already observed by Blanc *et al.* [27]. More, no translocation or chromosomal inversion was identified.

Despite an alignment only possible against the *V. vinifera* reference genome (PN40024 12Xv2), we detected SNPs all over the genome whatever the cross. This confirms that sequences are close enough to allow alignment of *M. rotundifolia* reads against the *V. vinifera* genome. However, the analysis of the number of SNPs detected in each region of the genome revealed some differences. More SNPs were mapped in the hybrid maps than in the *V. vinifera* ones (Table 2); similarly homeologous regions were better covered than homologous ones (S1 Fig.). In addition, the more advanced is the backcross, the lower is the number of detected SNPs and the higher is the homozygosity. All these observations converge to state that homeologous regions display a higher level of polymorphisms than the homologous ones.
Our genetic linkage maps allowed to establish 19 linkage groups. This number is consistent with the 19
pairs of chromosomes of *V. vinifera* and not with the 20 pairs of chromosomes of *M. rotundifolia*.
Cytogenetic studies showed that the F1 hybrid carries 39 chromosomes, 19 inherited from *V. vinifera*
and 20 inherited from *M. rotundifolia* [29]. Blanc *et al.* [27] showed that linkage groups 7 and 20 of *M.
rotundifolia* match respectively the upper and lower arms of chromosome 7 of *V. vinifera*. We showed
that allele segregation of chromosome 7 fitted the expected Mendelian ratios. This signifies that,
during F1 meiosis, chromosomes 7 and 20 from *M. rotundifolia* and chromosome 7 from *V. vinifera* are
equally distributed in the gametes. In addition, recombination rate on the upper part of the
chromosome 7 of BC1 hybrid map was similar to BC1 *V. vinifera* map (Fig 1A). But conversely, in the
lower part of the same chromosome, the recombination rate in hybrid map was extremely low
compared to the *V. vinifera* map. Consequently, we hypothesize that, during meiosis of fertile F1
hybrid, the number of crossovers between *M. rotundifolia* chromosome 7 and the upper part of *V.
vinifera* chromosome 7 is normal while it is extremely low between *M. rotundifolia* chromosome 7 and
the lower part of *V. vinifera* chromosome 7. This observation reveals a high divergence between both
genomes on the lower arm of the chromosome 7 which could partly explain the low level of fertility of
pseudo-F1 hybrids [29-30]. In addition, this mismatch allows us to predict the difficulty to introgress in
*V. vinifera* a genetic factors carried by *M. rotundifolia* chromosome 20.

Distortion of segregation

Many segregation distortions were observed in the hybrid maps of BC1 and BC2 populations but also
in the BC2 *V. vinifera* map (S2 Fig.). This phenomenon is common in interspecific crosses [35-38]. It has
also been observed in crosses between *V. vinifera* cultivars [39-40]. However, some particular patterns
arise from our results. Technical causes, such as multilocus SNPs or scoring errors, generally generate
isolated markers. But the majority of distorted markers that we observed were clustered in large region
covering several Mbp.

Interestingly, whereas BC mapping populations have two origins of *M. rotundifolia* and various origins
of *V. vinifera*, the same clusters of distorted markers are shared between mapping populations.
Markers on chromosomes 1, 11 and 15 were distorted exactly in the same way in BC1 and BC2 hybrid
maps (Fig 4). These clusters are probably linked to genomic specificities of each species rather than to
accession features. Thus, the risk to observe these distortions in a major part of the crosses between
*M. rotundifolia* and *V. vinifera* whatever the accessions used seems rather high. This kind of bias could
have an impact on the efficiency of the introgression by potentially reducing the number of individuals
with interesting traits, when the favourable allele determining a trait is in coupling with the weakly
transmitted region. In addition to species related distortions, our results also show that specific
interactions between accessions may unpredictably arise in an isolated cross and create a strong
distortion, as observed on chromosome 13 of the BC1 hybrid map.

In interspecific backcrosses, alleles from the recurrent parent are expected to be favoured over alleles
from the donor parent [35] But surprisingly, we observed that, in homeologous distorted regions, *M. rotundifolia* alleles were not systematically eliminated in favour of *V. vinifera* alleles (Fig 4). For instance, *M. rotundifolia* alleles were preferentially conserved on chromosomes 1 and 13 whereas they were preferentially eliminated on chromosomes 11 and 15. Conservation of donor alleles in a recurrent background was already observed in other interspecific backcrosses and pseudo-backcrosses [35, 41-42]. Myburg *et al.* [35] explained this phenomenon by “the alleviation of genetic load by donor alleles in the recurrent genetic backgrounds”, i.e. the increase of fitness in hybrids. Rieseberg *et al.* [43] gave an alternative explanation involving the existence of “selfish” genes “that enhance the success of
gametes they inhabit even if they pose a significant fitness cost during the diploid phase of the life cycle”.

BC1 population was dramatically reduced through the years due to developmental abnormalities which increased after establishment in the vineyard. SSR data acquired on seedlings allowed us to identify segregation distortions (Table 3) and to distinguish three different cases: i) segregation distortion was already present at early stage of development, in SSR data acquired on seedlings and stayed stable after planting, as observed on chromosome 15; ii) segregation distortion was not present at early stage of development but appeared after planting, as observed on chromosome 13; iii) segregation distortion was present at early stage of development but evolved by increasing or decreasing after planting, as observed on chromosomes 4 and 11, and 1, respectively. Several causes can be associated to the first case of distortion: issues during gametogenesis, fertilization or seed germination. In the second case, the distortion is caused by developmental and abiotic stress leading to the death of plants after establishment in the vineyard. The third case combine both causes: alleles or genotypes favoured during gametogenesis, fertilization or germination were either favoured again after establishment in the vineyard or eliminated.

In BC2 population, both maps, *V. vinifera* and hybrid, displayed distortion of segregation on the same chromosomes (chromosomes 4, 6, 8 and 18). These clusters were located either in a homeologous region, where *M. rotundifolia* alleles were either eliminated (chromosome 6), or favoured (chromosome 8), or in a homologous one (chromosomes 4 and 18). We assume that post-zygotic lethal interactions occurred between alleles of the two parents, influencing the viability of zygotes, the germination rate of seeds or the seedling survival. This phenomenon, which has already been reported in other plant species [35], is thus not specific to grapevine.

Our results show that a special attention have to be given to regions harbouring unbalanced segregations, particularly to species related distortions. Indeed, by favouring some alleles or genotypes at the expense of others, segregation distortion impact qualitatively and quantitatively the introgression of interesting or unfavourable traits. Some interesting alleles may be lost quickly if they are in a region preferentially eliminated. Moreover, if these alleles are present in few individuals, the probability to have interesting genotypes with a desired combination of traits is low.

## Recombination suppression in homeologous regions

The difference in recombination rate observed between the BC1 hybrid map, derived from the 8624 F1 parent, and the other hybrid maps, derived from 22-8-78 and 1771P BC parents, is the most surprising result of our study (Fig 3). In the BC1 hybrid map, recombination pattern was similar to that of the BC1 *V. vinifera* map, except for chromosome 7, as discussed above. Genetic distances were conserved between both parental maps, even if variations of the ratio genetic distance / physical distance were observed all along the chromosomes. Conversely, in the BC2 and BC4 hybrid maps, recombination patterns were extremely different from those of the *V. vinifera* maps. Recombination is suppressed in homeologous regions and enhanced in homologous regions of recombined chromosomes. The main difference between F1 and BC parents is the proportion of *M. rotundifolia* genome and thus the percentage of homeologous regions. In an F1 parent, all chromosome pairs are homeologous, i.e. associate one chromosome from *V. vinifera* to one chromosome from *M. rotundifolia*. On the contrary, in the BC parents, some parts of the genome are homologous, i.e. both chromosomes of a pair are inherited from *V. vinifera*, while some others remain homeologous.

The similar genetic distance of BC1 hybrid map and *V. vinifera* maps can be explained by two phenomena. Firstly, a failure to form at least one crossing-over between chromosomes of the same
pair during meiosis may lead to sterility by creating unbalanced gametes [44-46]. During F1 meiosis, only cells with at least one crossing-over per chromosome are able to produce viable gametes and then, generate a progeny. Secondly, *V. vinifera* and *M. rotundifolia* have small chromosomes and, according to the mean distance of genetic linkage maps, only 1-2 recombinations occur per chromosome during meiosis. The similar size of the F1 and *V. vinifera* maps obtained in this study is probably the consequence of these two prerequisites: a minimum of one recombination per chromosome and a maximum of two recombinations per chromosome. More, the distances between markers are conserved in both parental maps which tends to indicate that the divergence between both species is stable along the genome, except the lower arm of chromosome 7.

In the 22-8-78 BC1 and 1771P BC3 parents, each pair of chromosome is composed of one chromosome from *V. vinifera* and one chromosome from the hybrid parent (F1 and BC2) introgressed with a variable-length segment from *M. rotundifolia*, according to the individuals (Fig 2). We observed that recombination rate in 22-8-78 BC1 and 1771P BC3 meiosis depended on the size of the introgressed segment. When the chromosome was entirely inherited from *M. rotundifolia*, it behaved as in the 8624 F1 meiosis. The recombination events occurred along the chromosome as expected in intraspecific cross (Fig 3), which led to a linkage map with genetic distances similar to *V. vinifera*. When the pairs of chromosomes are composed of both homologous and homeologous regions, recombination occurs preferentially in homologous regions rather than in homeologous regions. The importance of this effect is correlated to the size of introgressed region: smaller is the homeologous region, higher is the recombination suppression it undergoes. This recombination suppression in homeologous regions in interspecific hybrids has already been observed in tomato [47], lettuce [48], barley [49] and cotton [50]. Plant material used in these studies were introgression lines or substitution lines selected for their unique introgressed region. Here, we first report about the effect of variable-length introgressed regions on several chromosomes. Our results highlight that the behaviour at meiosis in terms of crossovers of each chromosome pair only depends on its percentage of homeologous region and is unrelated to the behaviour of the other chromosome pairs.

Canady *et al.* [47] established that recombination suppression observed in tomato is not due to chromosomal rearrangements and proposed as a cause the lack of sequence identity. As explained above, macrosynteny between *M. rotundifolia* and *V. vinifera* is conserved but sequence divergence between the two genomes is substantial [31]. According to Kauppi *et al.* [51], three reasons can be assumed to explain the distribution of recombination: 1) "the balance between the need for recombination and the need to minimize the breakdown of favourable haplotypes", 2) "the facilitation of the optimal mechanical/biochemical function of chiasmata in chromosome segregation", 3) "the efficiency of the region to repair". Moreover, several studies have shown that DNA mismatch repair system restricted homeologous recombination in model systems, such as yeast [52] and Arabidopsis [53].

However, in our study, even if recombination is suppressed in homeologous regions, total genetic length of each chromosome is conserved between *V. vinifera* maps and hybrids maps whatever the backcross level and the proportion of homeologous region. The higher recombination rate in homologous regions balances the lack of recombination in homeologous regions. Consequently, at a chromosome scale, the presence of a homeologous segment amplifies recombination in the remaining homologous region. Analysis of allotriploids in *Brassicas* led Pele et al [54] to similar conclusions. As observed in the F1 meiosis, the need for at least one recombination on the chromosome to produce viable gamete is probably responsible for this offsetting mechanism.
**Conclusion**

Our results bring new insight to address and overcome the difficulties met in the exploitation of wild
species through interspecific crosses in grape breeding. In particular, phenomena uncovered about the
behaviour of homeologous regions in various genetic contexts open concrete and practical
perspectives to make the introgression process more effective and to enhance the creation of new
allelic combinations. More, the first elements acquired in other plant species suggest the possibility of
generalization of these perspectives.

**Material and methods**

Plant material

Three pseudo-backcross (BC) populations derived from two sources of *M. rotundifolia* were used in this study. A BC1 mapping population of 68 individuals was obtained from the cross between 8624 ((V. vinifera cv. Carignan x V. vinifera cv. Cabernet Sauvignon) x M. rotundifolia cv. Trayshed) as female parent and V. vinifera cv. Cabernet Sauvignon as pollen donor. A BC4 mapping population of 79 individuals, derived from the BC1 by three consecutive pseudo-backcrosses, was then obtained from the cross between 1771P ((BC1 x V. vinifera cv. Barbera) x V. vinifera cv. Tempranillo) and V. vinifera cv. Nebbiolo. A BC2 mapping population of 248 individuals was obtained from a cross between 22-8-78 ([[(V. vinifera cv. Cabernet Sauvignon x V. vinifera cv. Alicante Bouschet) x M. rotundifolia cv. NC184-4] x [(V. vinifera cv. Mourvèdre x V. vinifera cv. Cabernet Sauvignon) x V. vinifera cv. Pinot meunier]]) as female parent and V. vinifera cv. Syrah as pollen donor.

SSR marker and GBS analyses

Genomic DNA was extracted from 80 mg of young expanding leaves using the Qiagen DNeasy® 96 Plant Kit (Qiagen S.A., Courtaboeuf, France) as described by the supplier. Microsatellite analysis was performed as described in Blasi *et al.* [13]. DNA samples following quality prerequisites were sent to the Genomic Diversity Facility at the Institute of Biotechnology of Cornell University (Ithaca, NY, USA) where GBS analysis, sequence alignment to *V. vinifera's* reference genome (PN40024 12Xv2; https://urgi.versailles.inra.fr/jbrowse/gmod_jbrowse/?data=myData%2FVitis%2Fdata_gff&loc=chr1%3A9694432..14541139&tracks=Vitis%20vinifera%20cv.PN40024%20assembly%2012XV2%2CCRIBI_V1%2CREPET_TE%2Cscaffolds%2CSNP_Discovery_Vitis_vinifera&highlight=) and SNP calling were achieved. The reference-based pipeline used for bioinformatics analysis was the 'discovery' pipeline described in TASSEL 3.0 documentation and in Glaubitz *et al.* [55].

Genetic mapping

SNP markers obtained by GBS were filtered to only keep the most informative and reliable markers.
The first step consisted in cleaning the data. For each genotype, SNPs with read depth (DP) lower than
4 or allelic depths (AD) for the reference and alternate alleles lower than 2 were set as missing data.
Then, for each population of backcross, SNP markers were eliminated if: 1) they had more than 5% of
missing data in the population, 2) their segregation in the population was strictly identical to the
segregation of another SNP, 3) the origin of their segregation was unknown because genotypes of both
parents were missing, 4) they did not segregate in the population because both parents were
homozygous. The goodness-of-fit between observed and expected Mendelian ratios was analysed for
each marker locus using a χ2-test. Markers showing segregation distortion (p-value < 0.05) were not
included in the final maps. The same filters were applied to SSR markers.
Parental genetic linkage maps were established for each population. Distribution of markers in linkage groups and phase analysis was performed with JoinMap 3.0 [56]. The threshold value of logarithm of odd (LOD) score was set at 3.0 to claim linkage between markers. Linkage analysis was then performed with the R/qt1 package of the R software (version 3.3.2, © 2016 The R Foundation for Statistical Computing) [57]. Linkage groups were numbered according to the reference genome [58].

A covering rate was estimated for each map using the physical position of distal markers for each chromosome. A contraction rate, allowing to link genetic distance and physical size, was also calculated per chromosome as:

$$C = \frac{Dg}{Dp} \times 300 000$$

where *Dg* is the genetic distance in cM between the two distal markers, *Dp* the physical distance in bp between the same two markers and 300 000 a conversion factor between centimorgans and base pairs. Due to the way of calculation, the contraction rate of the framework reference linkage map published by Doligez et al. [34] is 0.9.

In this paper, we distinguish the maps of the F1, BC1 and BC3 parents, called hybrid maps from the maps of the *V. vinifera* parents, called *V. vinifera* maps.

## Chromosome painting

The aim of chromosome painting is to identify *M. roduntifolia* regions remaining in each BC individual thanks to specific SNPs. Resequencing data were available for seven *M. rotundifolia* cultivars: Trayshed, Fry, Carlos, Dulcet, Regale, Yuga x Carlos and Michaux. Resequencing datasets were deposited under DOI 10.15454/1.5009976984187844E12 and are available through the following web page: https://urgi.versailles.inra.fr/Projects/Achieved-projects/Muscares. They will be made available through the EMBL database for final publication. For BC1 and BC4 populations, both derived from Trayshed, chromosome painting was performed with the Trayshed data. *M. rotundifolia* parent of the BC2 population being not available, resequencing data from all the seven *M. rotundifolia* accessions were used for the analysis.

First, SNPs from *M. rotundifolia* that could be found in the offspring were determined. Illumina reads from the seven accessions of *M. rotundifolia* were aligned against the *V. vinifera* reference genome (PN40024 12Xv2), using the alignment gsnap software [59]. We performed the analysis using the following parameters: --batch=4, --npaths =3, --max-mismatches=12. Reads were then parsed with an Edit Distance of 15, and we retained only the alignments that were paired and that matched with a unique location on the reference genome. SNPs from these reads were called, using the default parameters for SAMtools mpileup [60], followed by bcftools, as described on the SAMtools webpage (http://samtools.sourceforge.net/mpileup.shtml) and with the following settings: --multiallelic-caller, --skip-variants=Indel and --pval-threshold=1. SNP calling for *M. rotundifolia* accessions was performed at the same positions than those provided in the GBS vcf file, and a vcf file was generated for each accession. Data were then cleaned for their quality: QUAL score lower than 30, DP score lower than 4 or higher than 25 were considered as missing data. A pool of *M. rotundifolia* SNPs was established using each position with at least one alternative allele in Trayshed, for BC1 and BC4, or in one of the 7 cultivars, for the BC2.

Second, SNPs from *M. rotundifolia* identified in the previous step were searched in the offspring. After eliminating the positions with missing data, SNPs that were actually present were determined for each individual of each population. These SNPs were heterozygous and had one allele from *M. rotundifolia*. Finally, a rate of the SNPs coming from *M. rotundifolia* was calculated on sliding windows of 20 consecutive SNPs as:
$$ \tau_i = \frac{Nr_i}{Np_i} $$

where $τ_i$ was the rate of SNPs coming from *M. rotundifolia* for the window i, $Nr_i$ the potential number of SNPs from *M. rotundifolia* that could be found in the window i, $Np_i$ the real number of SNPs from *M. rotundifolia* detected in the window i. The window was slid of 1 SNP. A threshold of $τ_i$, beyond which the region was assigned to *M. rotundifolia*, was fitted for each population according to the BC level and the quality of the data.

## Acknowledgments

We gratefully acknowledge the Fondation Jean Poupelain for funding our work through the HealthyGrape2 program. We are grateful to E. Duchêne for useful discussions on R scripts. We thank, V. Dumas and J. Misbach for excellent assistance in plant maintenance and growing.

## Author Contributions

Conceptualization: DM. Formal analysis: MD SB. Funding acquisition: DM. Investigation: MD EP PM. Methodology: DM MD CR. Project administration: DM. Supervision: DM CR MD. Visualization: MD. Writing - original draft: MD DM. Writing - review & editing: MD DM CR EP PM.

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## Supporting information

S1 Fig. Parental maps of the BC1 (a), BC4 (b) and BC2 (c) populations.

S2 Fig. Evolution of the segregation distortion along the chromosomes of the hybrid parents of the BC1 (a) and BC2 (b) populations and of the V. vinifera parent of the BC2 population (c).

S1 Table. Covering rates of the chromosomes of the parental maps of the BC1, BC2 and BC4 populations.

S2 Table. Contraction rates of the chromosomes of the parental maps of the BC1, BC2 and BC4 populations.

## Figure captions

**Fig 1.** Variation of genetic distance depending on physical distance on the hybrid and V. vinifera maps. A. Chromosomes 7 and 10 of the BC1 population. B. Chromosomes 4, 8, 2, 12 and 14 of the BC2 population. C. Chromosomes 5 and 14 of the BC4 population. For each cross, V. vinifera maps are represented with green triangles, and hybrid maps with red dots. Bars below the graphs show the
regions covered by homeologous pairs of chromosomes (V. vinifera - M. rotundifolia; in red) and
homologous regions (V. vinifera - V. vinifera; in dark grey).

**Fig 2. Homologous and homeologous regions in 1771P (A) and 22-8-78 (B).** Chromosomes are horizontally represented with their physical length on the x-axis. Homeologous regions, where $τ_i$ is above the threshold, are in red; homologous regions, where $τ_i$ is below the threshold, are in dark grey.

**Fig 3. Plot of estimated recombination fractions for all pairs of markers of the BC4 (A) and BC3 (B) hybrid genetic maps.** Markers are plotted ordered on the x-axis and on the y-axis. Recombination fraction is plotted thanks to a colour scale. Red dot indicates that the two markers, respectively on x- and y-axis, are highly linked (low recombination fraction) and blue dot indicates that markers poorly or not linked (high recombination fraction). Chromosome limits are identified at the left of the heatmap. Homeologous and homologous regions are identified at the top.

**Fig 4. Transmission rate of M. rotundifolia SNPs in the BC populations.** The transmission rate is calculated for each SNP as the percentage of individuals in a population harbouring the allele from M. rotundifolia. a. Chromosomes 8, 1, 11 and 13 of BC1 population. b. Chromosomes 8, 1, 11 and 13 of BC2 population. Orange dots are the positions of SNPs used to calculate transmission rate. Homologous regions are represented with a grey background on the graphs.