The fresh leaves of 286 parents were collected in the field, and the genomic DNA was extracted by CTAB method [35]. The concentration and purity of DNA were determined by Nano Drop2000 spectrophotometer, and the quality was determined by 1% agarose gel electrophoresis. A total of 198 polymorphic SSR markers were utilized from previous studies and listed in Table S3 [36].

The DNA concentration of qualified samples were adjusted to 100 ng/μL for restriction-site associated DNA sequencing (RAD-Seq) by Huada Gene Co., Ltd. (Shenzhen, China). The steps were as follows: (1) DNA digestion; (2) add bar-coded adapters; (3) DNA fragmentation; (4) DNA recovery and purification; (5) DNA amplification; (6) DNA recovery and purification; (7) sequenced on Illumina Hiseq 2000 system. The raw reads were aligned with G.hirsutum L. TM-1 reference genome v 1.1 ( by BWA software and the parameters were set to mem-t8. SNP genotypic data were obtained by SNP Calling, with GATK and SAMTools packages [37, 38]. The probability of the fragments mapped to the reference genome was 93.4–99.6%, the coverage on the genome was 0.07–7%, and the average sequencing depth was 1.48. The sequencing data had been deposited to NCBI under the accession number: PRJNA353524.

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