Herbert Léo, Vernet Aurore, Frouin Julien, Meunier Anne Cécile, Di Mattia Jeremy, Wang Minghui, Sidhu Gaganpreet K, Mathis Luc, Nicolas Alain, Guiderdoni Emmanuel, Fayos Ian
Meiogenix SA, Paris, France.
Centre de coopération internationale en recherche agronomique pour le développement (CIRAD), Unité mixte de recherche - Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP) Institut, Montpellier, France.
Front Plant Sci. 2025 May 1;16:1580225. doi: 10.3389/fpls.2025.1580225. eCollection 2025.
Meiotic crossovers shuffle the genetic information transmitted by the gametes. However, the potential to recover all the combinations of the parental alleles remains limited in most organisms, including plants, by the occurrence of only few crossovers chromosome and a prominent bias in their spatial distribution. Thus, novel methods for stimulating recombination frequencies and/or modifying their location are highly desired to accelerate plant breeding.
Here, we investigate the use of a dCas9-SPO11-1 fusion and clusters of 11 gRNAs to alter meiotic recombination in two chromosomal regions of a rice hybrid (KalingaIII/Kitaake). To accurately genotype rare recombinants in regions of few kbp, we improved the digital PCR-based pollen-typing method in parallel.
Expression of the dCas9-SPO11-1 fusion protein under the ubiquitous promoter was obtained in leaves/anthers/meiocytes and found to complement the sterility of the mutant line. We observed a 3.27-fold increase over wild-type (p<0.001) of recombinant pollens in a transgenic hybrid line (7a) targeting a chromosome 7 region. In the offspring plant 7a1, a significant 2.05-fold increase (p=0.048) was observed in the central interval (7.2 kb) of the Chr. 7 target region. This stimulation of meiotic recombination is consistent with the expression of the dCas9-SPO11-1 fusion and gRNAs as well as with the ChIP-revealed binding of dCas9-SPO11-1 to the targeted region. In contrast, no stimulation was observed in other transgenic lines deficient in the above pre-requisite features, expressing the dCas9-SPO11-1 fusion but no gRNAs or targeting a Chr.9 region.
These results open new avenues to locally stimulate meiotic recombination in crop genomes and paves the way for a future implementation in plant breeding programs.
减数分裂交叉重组可使配子传递的遗传信息发生重排。然而，在包括植物在内的大多数生物中，由于每条染色体上发生的交叉重组数量很少且其空间分布存在显著偏差，恢复亲本等位基因所有组合的可能性仍然有限。因此，迫切需要新的方法来提高重组频率和/或改变其位置，以加速植物育种进程。
在此，我们研究了使用dCas9-SPO11-1融合蛋白和11个gRNA簇来改变水稻杂交种（KalingaIII/北陆）两个染色体区域的减数分裂重组。为了准确对几千碱基对区域内的罕见重组体进行基因分型，我们同时改进了基于数字PCR的花粉分型方法。
在叶片/花药/减数分裂细胞中获得了在遍在启动子控制下的dCas9-SPO11-1融合蛋白表达，并发现其可弥补突变系的不育性。在靶向7号染色体区域的转基因杂交系（7a）中，我们观察到重组花粉比野生型增加了3.27倍（p<0.001）。在子代植株7a1中，在7号染色体目标区域的中央区间（7.2 kb）观察到显著增加了2.05倍（p = 0.048）。减数分裂重组的这种增强与dCas9-SPO11-1融合蛋白和gRNA的表达以及ChIP揭示的dCas9-SPO11-1与靶向区域结合一致。相比之下，在缺乏上述必备特征的其他转基因系中未观察到增强，这些系表达dCas9-SPO11-1融合蛋白但不表达gRNA或靶向9号染色体区域。
这些结果为在作物基因组中局部刺激减数分裂重组开辟了新途径，并为未来在植物育种计划中的应用铺平了道路。
