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Abstract
Crape myrtle (Lagerstroemia spp.) is widely utilized in landscape owing to its diverse flower colors, extended blooming period, attractive bark, and ease of maintenance. Targeted breeding has produced elite semi dwarf, disease tolerant cultivars such as ‘Pristine Crystal’, ‘Pristine Lilac’, and ‘Pristine Ruby’. Notably, these cultivars exhibit exceptional clean foliage during the summer season, enhanced disease resistance, and semi-dwarf characteristics that further facilitate their use in landscape settings. Despite decades of breeding work on crape myrtle, fundamental cytogenetic parameters, including genome size and chromosome number, have remained inadequately defined. Using a refined fluorescent staining protocol we confirmed that Lagerstroemia species possess 2n = 48 chromosomes, and flow cytometric analyses showed genome sizes of 0.70–0.79 pg across the genus. Interspecific hybridization proved critical for trait enhancement. From 3,126 controlled crosses among elite cultivars and L. speciosa we obtained 731 fruits; ‘Pristine Lilac’ was the most effective maternal parent, whereas ‘Pristine Crystal’ and ‘Pristine Ruby’ excelled as pollen donors. For hybrids 21H026, 21H040, and 21H043, Woody Plant Medium plus moderate 6 benzylaminopurine (BA), indole 3 butyric acid (IBA), and gibberellic acid (GA₃) produced the longest shoots and strongest roots, while MS based media maximized shoot multiplication. A leaf derived explant flowered in vitro within 60 d, indicating potential for annual, first year blooming cultivars. To expand genetic diversity, we optimized ethyl methanesulfonate (EMS) mutagenesis. EMS concentration was the chief determinant of germination and survival; exposure time showed genotype specific effects, with LD₅₀ values differing significantly between 6 and 12 h treatments, underscoring the need for cultivar tailored protocols. Finally, we generated a high quality, haplotype resolved genome of L. speciosa using PacBio Revio long reads, Hi C scaffolding, and RNA seq support. Although k-mer analysis estimated a 348 Mb genome, flow cytometry converged on ~367 Mb per haploid genome. The two haplotypes span 343.4 Mb and 317.0 Mb with scaffold N50s of 13.95 Mb and 12.75 Mb, respectively, and BUSCO completeness >93%. Repeat annotation showed ~40% repetitive content. This reference genome, together with the cytogenetic, tissue culture, and EMS datasets, provides a robust platform for identifying genes controlling ornamental traits, disease resistance, and stress tolerance, thereby accelerating future crape myrtle breeding.