Untangling structural factors and evolutionary drivers in nascent polyploids

Allopolyploids have globally higher fitness than their diploid progenitors however, by comparison, most resynthesized allopolyploids have poor fertility and highly unstable genome. Elucidating the evolutionary processes promoting genome stabilization and fertility is thus essential to comprehend allopolyploid success. Using the Brassica model, we mimicked the speciation process of a nascent allopolyploid species by resynthesizing allotetraploid B. napus and systematically selecting for euploid individuals over eight generations in four independent allopolyploidization events with contrasted genetic backgrounds, cytoplasmic donors and polyploid formation type. We evaluated the evolution of meiotic behavior, fertility and identified rearrangements in S1 to S9 lineages, to explore the positive consequences of euploid selection on B. napus genome stability. Recurrent selection of euploid plants for eight generations drastically reduced the percentage of aneuploid progenies as early as the fourth generation, concomitantly with a quasi disappearance of newly fixed homoeologous rearrangements. The consequences of homoeologous rearrangements on meiotic behavior and seed number strongly depended on the genetic background and cytoplasm donor. The combined use of both self-fertilisation and outcrossing as well as recurrent euploid selection, allowed identification of genomic regions associated with fertility and meiotic behavior, providing complementary evidence to explain B. napus speciation success.

(1) Allopolyploids have globally higher fitness than their diploid progenitors however, by 37 comparison, most resynthesized allopolyploids have poor fertility and highly unstable genome.

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Elucidating the evolutionary processes promoting genome stabilization and fertility is thus 39 essential to comprehend allopolyploid success.

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(2) Using the Brassica model, we mimicked the speciation process of a nascent allopolyploid       lines were selfed (hand pollination of floral buds before anthesis), producing the 'RCC-S1' and 'CRC-S1' 152 progenies ( Fig. 1). Thereafter 11 'RCC-S1' and 10 'CRC-S1' were selfed. These lineages were advanced 153 by SSD until the eighth generation. At each generation, only one plant with 38 chromosomes (euploid) 154 was randomly chosen from the set of euploid offspring. In some cases, lines did not produce any 155 progeny, leading to the extinction of such lineage. In addition to 'RCC' and 'CRC', we produced 156 resynthesized B. napus lines by firstly crossing the B. oleracea 'HDEM' line with the B. rapa 'Z1' line.

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In the first step, we tested if the two groups of plants, with and without deletion, differed for their 267 phenotypic measures using classical Mann-Whitney rank tests. The advantage of such non-parametric 268 test is to be less likely to find false significant differences than using parametric test, and thus

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The dataset presented in this study comprises phenotypic measures of SDD individuals over eight 291 generations for four independent nascent lineages of allopolyploid B. napus. The impact of repetitive 292 euploid selection was visible as soon as the fourth generation and forward, as only 0 to 2.94% of 293 aneuploid individuals were found in the resynthesized S4 to S8 generations compared to 11.46-11.27% 294 in S1-S3 (Supporting Information Fig. S1). In total, we assessed the meiotic behavior and seed yield of  Table S1). This valuable dataset was then analysed in regards to a genotyping 299 dataset performed on the four lines at generation S1, S3, S6 and S8. Each resynthesized B. napus   Fig. S2). In 'RCC', fertility significantly decreased from the 1 st 316 generation to the 4 th (t-test, p<0.01) and again from the 5 th to the 8 th generation (t-test, p<0.01) with 317 a high fertility (mean=572.2) observed in the 5 th generation (comparable to the yield observed in S1) 318 (Fig. 2). In 'CRC', fertility increased in the 2nd generation compared to the first generation (162.5 to 319 395.8 seeds per flower, p<0.05) to drastically decrease until the 8 th generation (t-test, p<0.05) (Fig. 2).

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In 'EMZ', compared to S0 all following generations presented a lower number of seeds. Similarly, 'UG 321 EMZ' individuals exhibited equal or lower number of seeds in the generations following 322 allopolyploidization, only a slight significant decrease was observable from generation S1-S2 to S3 (t-323 test, p<0.05) (Fig. 2) generations. Percentage of cells with 19 bivalents was consistent across generations S1 to S8 in 'RCC' 336 whereas significant differences between generations were visible in the crosses 'CRC', 'EMZ' and 'UG 337 EMZ' (Fig. 2). In 'CRC', percentage of cells with 19 bivalents tended to increase in S3 compared to S1 338 11 (from 21.1% to 44.8%, t-test, p<0.05, Fig. 2) and stabilized in subsequent generations. In 'EMZ', 339 percentage of cells with 19 bivalents decreased drastically from S1 to S2 (t-test, p<0.05, Fig. 2) followed 340 by a slight increase from S2 to S7. In 'UG EMZ', after the drop from S0 to S1, percentage of cells with 341 19 bivalents was found constant with a slight increase in S8 (Fig. 2).

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Finally, we assessed the percentage of cells exhibiting multivalents in the nascent allopolyploids. A 343 similar percentage of cells with multivalents was observed among all lines (27.3% in 'RCC', 28.8% in 344 'CRC', 29.7% in 'EMZ' and 29.1% in 'UG EMZ', Supporting Information Fig. S2). This trend was also found 345 consistent across generations for all resynthesized lines except for 'UG EMZ' where a slight increase in 346 the percentage of multivalents was observed from S1 to S4-S5 (23.4% to 34.7%, t-test, p<0.05, Fig. 2).

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We observed a limited number of homoeologous rearrangements in 'RCC' and 'CRC' compared to 367 other crosses, with the exception of one individual in 'CRC' having a larger number of rearrangements 368 in S6 and S8 (Fig. 3). By contrast, number of homoeologous rearrangements is higher as soon as the S1

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After the first meiosis and its 'genome blender', we focus on the drastic subsequent increase of 428 homoeologous rearrangements from S1 to S4, rapidly followed by a quasi total disappearance of newly