The heterozygous pineapple genome demonstrates the importance of haplotype-resolved plant genomes

Abstract Collapsed (haploid) genome assemblies omit large portions of genetic information, especially in heterozygous, clonally propagated crops such as pineapple. Here, we assembled a telomere-to-telomere, haplotype-resolved genome for a key pre-Colombian cultivar of pineapple (Ananas comosus) ‘Smooth Cayenne’ (F180) using PacBio Hi-Fi and Hi-C data. The two 25-chromosome haplotypes span 858 Mb (N50 ≈ 16.8 Mb) and are >99% complete, each resolving all centromeres and 22 of 25 telomeres. Comparison of the phased chromosomes reveals 1.5 million single nucleotide polymorphisms (SNPs) and 1953 large structural variants (74 inversions, 750 translocations, and 1129 segmental duplications). This assembly reveals that inversions have profoundly impacted the ‘Smooth Cayenne’ genome, reshaping ~3–4% of the total sequence. Structural context dictates the genetic impact of these large inversions, as shown in recombination landscape analysis of 374 F1 seedlings, wherein a 1.3 Mb paracentric inversion on chromosome 20 forms a strict recombination coldspot, whereas a 6 Mb pericentric inversion on chromosome 24 still permits gene flow likely via short double crossovers, albeit at lower rates than the rest of the chromosome. Re-anchoring the 11 879 DArTseq markers from the F1 seedlings to the phased reference assembly, removes the dense network of spurious inter-chromosomal linkage seen in the collapsed F153 ‘Smooth Cayenne’ genome, likely providing markedly cleaner baselines for genome-wide association studies (GWAS) and genomic prediction. These results establish the new F180 assembly as a very high-quality reference, illustrate how undetected inversions can silently constrain genetic gain, and demonstrate the broader value of phased genomes for dissecting heterozygosity, structural variation and meiotic behaviour in perennial crops.