| Summary: | Cotton (<i>Gossypium</i> spp.) is the most important natural fiber source in the world. The genetic potential of cotton can be successfully and efficiently exploited by identifying and solving the complex fundamental problems of systematics, evolution, and phylogeny, based on interspecific hybridization of cotton. This study describes the results of interspecific hybridization of <i>G. herbaceum</i> L. (A<sub>1</sub>-genome) and <i>G. mustelinum</i> Miers ex Watt (AD<sub>4</sub>-genome) species, obtaining fertile hybrids through synthetic polyploidization of otherwise sterile triploid forms with colchicine (C<sub>22</sub>H<sub>25</sub>NO<sub>6</sub>) treatment. The fertile F<sub>1</sub>C hybrids were produced from five different cross combinations: (1) <i>G. herbaceum</i> subsp. <i>frutescens</i> × <i>G. mustelinum</i>; (2) <i>G. herbaceum</i> subsp. <i>pseudoarboreum</i> × <i>G. mustelinum</i>; (3) <i>G. herbaceum</i> subsp. <i>pseudoarboreum</i> f. <i>harga</i> × <i>G. mustelinum</i>; (4) <i>G. herbaceum</i> subsp. <i>africanum</i> × <i>G. mustelinum</i>; (5) <i>G. herbaceum</i> subsp. <i>euherbaceum</i> (variety A-833) <i>× G. mustelinum</i>. Cytogenetic analysis discovered normal conjugation of bivalent chromosomes in addition to univalent, open, and closed ring-shaped quadrivalent chromosomes at the stage of metaphase I in the F<sub>1</sub>C and F<sub>2</sub>C hybrids. The setting of hybrid bolls obtained as a result of these crosses ranged from 13.8–92.2%, the fertility of seeds in hybrid bolls from 9.7–16.3%, and the pollen viability rates from 36.6–63.8%. Two transgressive plants with long fiber of 35.1–37.0 mm and one plant with extra-long fiber of 39.1–41.0 mm were identified in the F<sub>2</sub>C progeny of <i>G. herbaceum</i> subsp. <i>frutescens</i> × <i>G. mustelinum</i> cross. Phylogenetic analysis with 72 SSR markers that detect genomic changes showed that tetraploid hybrids derived from the <i>G. herbaceum</i> × <i>G. mustelinum</i> were closer to the species <i>G. mustelinum</i>. The <i>G. herbaceum</i> subsp. <i>frutescens</i> was closer to the cultivated form, and its subsp. <i>africanum</i> was closer to the wild form. New knowledge of the interspecific hybridization and synthetic polyploidization was developed for understanding the genetic mechanisms of the evolution of tetraploid cotton during speciation. The synthetic polyploids of cotton obtained in this study would provide beneficial genes for developing new cotton varieties of the <i>G. hirsutum</i> species, with high-quality cotton fiber and strong tolerance to biotic or abiotic stress. In particular, the introduction of these polyploids to conventional and molecular breeding can serve as a bridge of transferring valuable genes related to high-quality fiber and stress tolerance from different cotton species to the new cultivars.
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