Development of InDel markers for the restorer gene <Emphasis Type="Italic">Rf1</Emphasis> and assessment of their utility for marker-assisted selection in cotton

The cytoplasmic male sterility (CMS) system is ideal for exploiting heterosis in crops such as cotton. However, CMS-D2, which is based on Gossypium harknessii cytoplasm, is still not widely used for cotton production. In this study, we developed an efficient marker-assisted selection method based on insertion/deletion (InDel) markers that can identify restorer lines carrying Rf1. Whole-genome resequencing was first completed for restorer N(Rf1Rf1)] and maintainer N(rf1rf1)] lines with normal fertile cytoplasm (N). Comparisons with the TM-1 reference genome sequence resulted in the identification of 292,065 and 183,657 InDels for the restorer and maintainer lines, respectively. Most of the InDels in the restorer line were distributed on Chromosome_D05, which carries Rf1. Of the 12 InDel markers near the Rf1 target region that were further validated, four co-dominant markers (i.e., InDel-1891, InDel-3434, InDel-7525, and InDel-9356) co-segregated with Rf1, as verified by a segregation analysis in an F₂ population. We subsequently used InDel-1891 to determine the allele status at the Rf1 locus in a backcross scheme for transferring Rf1. In this study, we developed new markers to increase the marker density in the Rf1 target region, which will be useful for the fine mapping of Rf1. The development of convenient and inexpensive co-segregating InDel markers will facilitate the marker-assisted selection of restorer lines carrying Rf1.