A large proportion of Earth's biodiversity constitutes organisms that cannot be cultured, have cryptic life-cycles and/or live submerged within their substrates[1][1]–[4][2]. Genomic data are key to unravel both their identity and function[5][3]. The development of metagenomic methods[6][4],[7][5] and the advent of single cell sequencing[8][6]–[10][7] have revolutionized the study of life and function of cryptic organisms by upending the need for large and pure biological material, and allowing generation of genomic data from complex or limited environmental samples. Genome assemblies from metagenomic data have so far been restricted to organisms with small genomes, such as bacteria[11][8], archaea[12][9] and certain eukaryotes[13][10]. On the other hand, single cell technologies have allowed the targeting of unicellular organisms, attaining a better resolution than metagenomics[8][6],[9][11],[14][12]–[16][13], moreover, it has allowed the genomic study of cells from complex organisms one cell at a time[17][14],[18][15]. However, single cell genomics are not easily applied to multicellular organisms formed by consortia of diverse taxa, and the generation of specific workflows for sequencing and data analysis is needed to expand genomic research to the entire tree of life, including sponges[19][16], lichens[3][17],[20][18], intracellular parasites[21][19],[22][20], and plant endophytes[23][21],[24][22]. Among the most important plant endophytes are the obligate mutualistic symbionts, arbuscular mycorrhizal (AM) fungi, that pose an additional challenge with their multinucleate coenocytic mycelia[25][23]. Here, the development of a novel single nuclei sequencing and assembly workflow is reported. This workflow allows, for the first time, the generation of reference genome assemblies from large scale, unbiased sorted, and sequenced AM fungal nuclei circumventing tedious, and often impossible, culturing efforts. This method opens infinite possibilities for studies of evolution and adaptation in these important plant symbionts and demonstrates that reference genomes can be generated from complex non-model organisms by isolating only a handful of their nuclei. [1]: #ref-1 [2]: #ref-4 [3]: #ref-5 [4]: #ref-6 [5]: #ref-7 [6]: #ref-8 [7]: #ref-10 [8]: #ref-11 [9]: #ref-12 [10]: #ref-13 [11]: #ref-9 [12]: #ref-14 [13]: #ref-16 [14]: #ref-17 [15]: #ref-18 [16]: #ref-19 [17]: #ref-3 [18]: #ref-20 [19]: #ref-21 [20]: #ref-22 [21]: #ref-23 [22]: #ref-24 [23]: #ref-25

From single nuclei to whole genome assembly