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Abstract Cotton (Gossypium hirsutumL.) is the key renewable fibre crop worldwide, yet its yield and fibre quality show high variability due to genotype-specific traits and complex interactions among cultivars, management practices and environmental factors. Modern breeding practices may limit future yield gains due to a narrow founding gene pool. Precision breeding and biotechnological approaches offer potential solutions, contingent on accurate cultivar-specific data. Here we address this need by generating high-quality reference genomes for three modern cotton cultivars (‘UGA230’, ‘UA48’ and ‘CSX8308’) and updating the ‘TM-1’ cotton genetic standard reference. Despite hypothesized genetic uniformity, considerable sequence and structural variation was observed among the four genomes, which overlap with ancient and ongoing genomic introgressions from ‘Pima’ cotton, gene regulatory mechanisms and phenotypic trait divergence. Differentially expressed genes across fibre development correlate with fibre production, potentially contributing to the distinctive fibre quality traits observed in modern cotton cultivars. These genomes and comparative analyses provide a valuable foundation for future genetic endeavours to enhance global cotton yield and sustainability. 

Ethyl methanesulfonate (EMS) mutagenesis offers important advantages for improving crops, such as cotton, with limited diversity in elite gene pools. EMS-induced point mutations are less frequently associated with deleterious traits than alleles from wild or exotic germplasm. From 157 mutant lines that have significantly improved fiber properties, we focused on nine mutant lines here. A total of eight populations were developed by crossing mutant lines in different combinations into GA230 (GA2004230) background. Multiple lines in each population were significantly improved for the fiber trait that distinguished the donor parent(s), demonstrating that an elite breeding line (GA230) could be improved for fiber qualities using the mutant lines. Genotypes improved for multiple fiber traits of interest suggesting that allele pyramiding is possible. Compared to midparent values, individual progeny in the population conferred fiber quality improvements of as much as 31.7% (in population O) for micronaire (MIC), 16.1% (in population P) for length, 22.4% (in population K) for strength, 4.1% (in population Q) for uniformity, 45.8% (in population N) for elongation, and 13.9% (in population O) for lint percentage (lint%). While further testing for stability of the phenotype and estimation of yield potential is necessary, mutation breeding shows promise as an approach to reduce the problem of the genetic bottleneck of upland cotton. The populations developed here may also contribute to identifying candidate genes and causal mutations for fiber quality improvement.