EAGER: Clonal propagation of tomato through seeds

在过去的一个世纪里，杂交种子的大规模使用促成了一场农业革命。杂交作物产量高，但由于遗传分离，其后代产量不稳定，而且往往产量低。这意味着杂交种子必须在每个季节重新生产，使用人工授粉等劳动密集型方法，导致成本大幅提高。因此，杂交作物在许多作物中没有得到充分利用，尤其是发展中国家的农民。最近，一种被称为合成无融合的技术被开发出来，使杂交水稻能够产生与亲本植物基因克隆的种子，这样后代就能产生与亲本植物同样高的产量。该方法模拟了在许多无性植物物种中自然发生的无融合过程，并利用基因组编辑和基因表达操纵来绕过遗传分离和受精。本项目将对水稻单株杂交种子的无性系繁殖方法进行改良，并在双子叶植物番茄上进行试验。如果在番茄上取得成功，该方法可以推广到其他主要的薯类作物，如大豆和棉花，对世界农业产生相应的巨大影响。通过对三个减数分裂基因进行CRISPR-Cas9基因组编辑，消除了减数分裂，随后通过卵细胞表达BABY BOOM家族的胚胎发生转录因子产生孤雌生殖，最近在水稻中实现了有性繁殖植物无性系种子的首次成功繁殖。所得到的种子是无性的，并保持了亲本植物的全基因组杂合性。类似的方法将在本项目中用于双科植物番茄，使杂交番茄植株通过种子无性繁殖。高度保守的减数分裂基因REC8、PAIR1和OSD1的番茄同源基因将通过CRISPR-Cas9进行基因组编辑，以消除减数分裂并形成未减数配子。水稻和拟南芥胚胎因子同源性鉴定的候选胚胎发生因子（包括BABY BOOM转录因子家族）的异位表达可诱导未还原卵细胞的孤雌生殖。假定的克隆后代将通过其染色体倍性进行鉴定，并通过全基因组测序验证，以确认其基因组中亲本杂合性的维持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The large-scale use of hybrid seeds over the past century has contributed to a revolution in agriculture. Hybrid crop plants produce high yields, but due to genetic segregation, their progeny have variable and frequently low yields. This means that hybrid seeds have to be generated afresh for every season, using labor-intensive methods such as hand pollination, resulting in substantially higher costs. Consequently, hybrids are underutilized for many crops, especially by farmers in developing countries. Recently a technology called synthetic apomixis has been developed to enable hybrid rice plants to produce seeds that are genetic clones of the parent plant, such that the progeny can produce the same high yields as the parent plant. The method mimics a process called apomixis that occurs naturally in many asexual plant species, and utilizes genome editing together with manipulation of gene expression to bypass both genetic segregation and fertilization. In this project, the method for clonal propagation of hybrid seeds developed for the monocot plant rice will be modified and tested for use in tomato, which is a dicot species. If successful in tomato, the method can be extended to other major crops that are dicots, such as soybean and cotton, with correspondingly large impacts on world agriculture.The first successful propagation of clonal seeds by a sexually reproducing plant has been recently achieved in rice, through the elimination of meiosis by CRISPR-Cas9 genome editing of three meiotic genes, followed by parthenogenesis arising from egg cell expression of an embryogenic transcription factor of the BABY BOOM family. The resulting seeds were clonal and maintained the genome wide heterozygosity of the parent plant. A similar approach will be used in this project on the dicot plant tomato, to enable the propagation of hybrid tomato plants clonally through seeds. Tomato orthologs of the highly conserved meiosis genes REC8, PAIR1 and OSD1 will be genome edited by CRISPR-Cas9 to eliminate meiosis and form unreduced gametes. Parthenogenesis of the unreduced egg cell will be induced by ectopic expression of candidate embryogenic factors identified by orthology to embryonic factors in rice and Arabidopsis, including those of the BABY BOOM transcription factor family. Putative clonal progeny will be identified by their chromosomal ploidy, and validated by whole genome sequencing to confirm maintenance of parental heterozygosity in their genomes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.