*4.1. Plant Material and DNA Extraction*

We sampled a total of 132 accessions belonging to different species, including five *G. klotzschianum* (D3), two *G. hirsutum* (AD1), 20 *G. barbadense* (AD2), 32 *G. tomentosum* (AD3), 59 *G. darwinii* (AD5), 10 *G. ekmanianum* (AD6), and four *G. stephensii* (AD7), from six islands and the Wild Cotton Germplasm Nursery of China. Among the 132 accessions, 32, 25, 16, 32, 10, 4, and 12 were obtained from Santa Cruz, Isabella, San Cristobal, a Hawaiian island, the Dominican Republic, Wake Atoll (NPGS, USA), and the Wild Cotton Germplasm Nursery of China, respectively (Supplementary Table S1). Seedlings of these accessions were grown at the wild cotton germplasm nursery of China, Sanya Hainan during October 2015, 2016, and 2017, respectively. When the plants were about 30–35 days old, fresh leaves were sampled and immediately frozen at −80 ◦C for later DNA extraction. Total genomic DNA was extracted from the frozen leaves by the cetyltrimethylamonium bromide (CTAB) method as described

by Zhang and Stewart [74] with slight modifications. DNA was quantified using Nanodrop at a 260/280 nm absorbance ratio and the quality was checked by 1% (*w*/*v*) agarose gel electrophoresis.
