Gossypium klotzschianum (D3-k) genome CRI_v1

To delve into the speciation history of Gossypium genus, the authors introduced four novel assemblies – G. harknessii (Ghar), G. Gossypioides (Ggos), G. trilobum (Gtri), and G. klotzschianum (Gklo).

All samples for DNA-seq and RNA-seq were collected from adult plants in the National Wild Cotton Nursery at the Institute of Cotton Research Institute (ICR), Chinese Academy of Agricultural Sciences (CAAS), in Sanya, China. Briefly, the leaves, flowers, stems, apex, bolls, and flower buds were collected, immediately frozen in liquid nitrogen, and stored at −80 °C. The leaves of 15-day-old seedlings were also collected for a high-throughput chromosome conformation capture experiment.

High molecular weight genomic DNA (gDNA) was extracted with a standard CTAB protocol from the leaves of four cotton species: G. harknessii (D2-2), Ggos (D6), Gtri (D8), and Gklo. PacBio SMRTbell long-read sequencing libraries were prepared from Gklo DNA by fragmenting extracted DNA with a Covaris® g-TUBE® Shearing Device. Illumina (San Diego, CA, USA) paired-end sequencing libraries (with an insert size of 350 bp) were generated from the same extracted Gklo gDNA following the manufacturer’s protocol. Short-insert (220 bp and 500 bp insert size) paired-end and large-insert (3, 4, 5 kb) mate pair libraries were prepared from Ggos, Ghar, and Gtri gDNA. Illumina Hi-C was conducted following a previously published protocol (Berkum et al., 2010). Briefly, fresh leaves from Gklo seedlings were fixed in a 1% formaldehyde solution. The nuclei and chromatin were extracted, then digested with DpnII. The overhangs resulting from DpnII digestion were filled in with biotin-14-dCTP (Invitrogen) and Klenow (New England Biolabs [NEB]). After chromatin dilution and re-ligation, gDNA was extracted and purified; purified DNA was sheared to 300-500 bp with a Bioruptor (Diagenode). Finally, the Hi- C library was prepared as previously described (Servant et al., 2015). Total RNAs were extracted from six different tissues using a TRIzol® Reagent RNA isolation kit (Invitrogen). RNA-seq libraries were prepared using the standard Illumina mRNA-seq library preparation kit. 

The PacBio SMRTbell long-read sequencing library was sequenced on the PacBio Sequel Ⅰ platform. The Hi-C library was sequenced on the Illumina HiSeq X Ten platform. PE150 sequencing was conducted on the Illumina HiSeq X Ten platform for the Gklo library. PE125 sequencing was conducted on the Illumina HiSeq X Ten platform for the prepared Ghar, Ggos,Journal Pre-proof and Gtri libraries. Paired-end 150 bp reads were generated on the Illumina NovaSeq platform for the RNA libraries.

Publication

Xu, et al.,Widespread Incomplete Lineage Sorting and Introgression Shaped Adaptive Radiation in the Gossypium genus. PLANT COMMUNICATIONS  2 October 2023.