Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1-4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

Summary: Cotton fibre is a unicellular seed trichome, and lint fibre initials per seed as a factor determines fibre yield. However, the mechanisms controlling fibre initiation from ovule epidermis are not understood well enough. Here, with single‐cell RNA sequencing (scRNA‐seq), a total of 14 535 cells were identified from cotton ovule outer integument of Xu142_LF line at four developmental stages (1.5, 1, 0.5 days before anthesis and the day of anthesis). Three major cell types, fibre, non‐fibre epidermis and outer pigment layer were identified and then verified by RNA in situ hybridization. A comparative analysis on scRNA‐seq data between Xu142 and its fibreless mutant Xu142 fl further confirmed fibre cluster definition. The developmental trajectory of fibre cell was reconstructed, and fibre cell was identified differentiated at 1 day before anthesis. Gene regulatory networks at four stages revealed the spatiotemporal pattern of core transcription factors, and MYB25‐like and HOX3 were demonstrated played key roles as commanders in fibre differentiation and tip‐biased diffuse growth respectively. A model for early development of a single fibre cell was proposed here, which sheds light on further deciphering mechanism of plant trichome and the improvement of cotton fibre yield.