Diploid aposporous sunflower forms triploid BIII progeny displaying increased apospory levels and non-random genetic mutations

Apomixis, i.e. asexual reproduction via seeds, could represent a revolution for sunflower breeding. In previous work, we identified a diploid sunflower inbred line (Rf975) naturally displaying aposporous-like embryo sacs (AES-like). Here, we explored the nature (reduced vs. unreduced) and the viability of these AES-like gametophytes by studying the formation of triploid (3x) BIII hybrids (2n + n) in the progeny of Rf975. Flow cytometry of immature seeds revealed that a considerable proportion (42.8 %) of the self-pollination Rf975 progeny were triploids. However, only 36.6% of these triploids reached maturity. Reproductive characterization revealed that 100% of the analyzed triploids displayed AES, and that the average proportion of ovules with AES was 61.9%, a value significantly higher than that detected in line Rf975 (4.6%). Abnormal pollen grains and scarce viable seeds were also detected. A segregant F2 progeny (including diploid and triploid individuals) was obtained by crossing Rf975 and HA89 (a genetically divergent sexual diploid inbred line). Methylation content sensitive enzyme ddRAD (MCSeEd) analysis of the diploid F2 did not reveal individuals with the F1 genetic constitution, proving that aposporous Rf975 is not capable of parthenogenesis. Moreover, both genetic and epigenetic non-random changes were detected in the F2 triploids, in specific genomic loci. Our results indicate that the AES-like gametophytes detected in Rf975 are supernumerary non-reduced embryo sacs unable to carry on parthenogenesis. Moreover, we discovered non-random (epi)genetic changes occurring in sunflower during triploidization events. This research will contribute to the development of essential knowledge and tools to enable the future implementation of apomixis-based breeding programs for this crop.

无融合生殖（apomixis），即通过种子实现的无性繁殖，有望为向日葵育种带来革命性变革。在前期研究中，我们鉴定出一个天然呈现类无孢子生殖胚囊（aposporous-like embryo sacs，AES-like）的二倍体向日葵自交系Rf975。本研究通过分析Rf975后代中三倍体（triploid，3x）BIII杂种（2n + n）的形成情况，解析了这类AES-like配子体（gametophytes）的本质（减数分裂型与非减数分裂型）及其活力。对未成熟种子的流式细胞术（flow cytometry）分析显示，自交的Rf975后代中有相当比例（42.8%）为三倍体。然而其中仅36.6%的三倍体能够发育至成熟。生殖特性鉴定结果显示，所有被分析的三倍体均呈现无孢子生殖胚囊（aposporous embryo sacs，AES），且携带AES的胚囊平均占比为61.9%，这一数值显著高于Rf975品系中的检测值（4.6%）。此外还检测到异常花粉粒以及可育种子稀缺的现象。通过将Rf975与遗传背景差异显著的有性生殖二倍体自交系HA89杂交，获得了包含二倍体与三倍体个体的分离F2后代群体。对二倍体F2群体开展的甲基化含量敏感酶ddRAD（Methylation content sensitive enzyme ddRAD，MCSeEd）分析未发现携带F1遗传组成的个体，由此证明无孢子生殖的Rf975无法进行孤雌生殖（parthenogenesis）。此外，在F2三倍体的特定基因组位点中检测到了非随机的遗传与表观遗传改变。本研究结果表明，在Rf975中检测到的AES-like配子体为额外产生的非减数分裂胚囊，无法完成孤雌生殖过程。此外，本研究还发现向日葵在三倍化（triploidization）过程中会发生非随机的（表观）遗传改变。本研究将为该作物基于无融合生殖的育种项目的未来落地提供关键的理论基础与技术工具。