Analysis of Mitochondrial Sequence Deletion in the <i>atp9</i> 5′UTR Region and Design of Molecular Markers in Cotton (<i>Gossypium</i> spp.)

ABSTRACT Cotton ( Gossypium ) is the most important fibre crop in the world, consisting of 45 diploid and 7 tetraploid cotton species. These cotton varieties are valuable for breeding, but their molecular identification methods still need further study. We identified an insert/deletion site (AATTT) at the atp 9 5′UTR region in the mitochondrial genome of cotton, which could be used to distinguish different cotton species. In this study, the target fragments of 33 cotton species (29 diploid and 4 tetraploid species) were amplified by PCR, and the PCR products potentially containing an Eco R I restriction site were subsequently digested and analysed. The sequencing results revealed that 27 out of 33 cotton species lacked ‘AATTT’ sequences, while six cotton species ( G. longicalyx , G. hirsutum , G. barbadense , G. tomentosum , G. mustillinum and G. darwinii ) were found to possess the sequences. Additionally, 39 SNPs were found in this region, and specific molecular markers for G. stocksii , G. bickii and E‐genome were developed, respectively. The comparative analysis of mitochondrial sequences from diploid and tetraploid cotton species elucidated their genetic diversity and evolutionary relationships, and species‐specific markers were able to discriminate among these species, thereby provided a foundation for more targeted use of wild genetic resources in cotton improvement and efforts to ensure their conservation.