Genome size and Survey sequencing of tropical viviparous Nymphaea ‘Paul Stetson’

【Objective】 The study aimed to investigate the genome size and related characteristics of the tropical viviparous Nymphaea ’Paul Stetson’ and thereby provide reference for constructing a whole-genome map, mining functional genes for key traits, and accelerating the molecular breeding of tropical viviparous water lilies. 【Method】Young root tips（2-3 cm） and young and unfolded leaves（3-5 cm） of Nymphaea ’Paul Stetson’ were used as experimental materials, with maize—whose genome size was known—serving as an internal reference. Flow cytometry was employed to estimate the genome size of Nymphaea ’Paul Stetson’, and the results were compared with those of the diploid Nymphaea colorata to preliminarily assess its ploidy. In addition, fluorescence in situ hybridization（FISH） was used to analyze the chromosome number and length. The genome size, ploidy, heterozygosity rate and repetition rate of Nymphaea ’Paul Stetson’ were further clarified combined with K-mer analysis on whole-genome survey sequencingbioinformatics and bioinformatics information. 【Result】 Flow cytometry estimated the genome size of Nymphaea ’Paul Stetson’ to be 0.82 Gb, and compared with the genome of diploid Nymphaea ’Paul Stetson’, it could be preliminatively identified as tetraploid. Genomic DAPI fluorescence staining and FISH further confirmed that the genome of Nymphaea ’Paul Stetson’ was tetraploid, comprising 56 chromosomes with lengths ranging from 0.45 to 1.50 μm and a karyotype formula of 2n=4x=56. BGI sequencing platform was used to carry out Survey sequencing analysis of Nymphaea ’Paul Stetson’, and 615552560 raw data were obtained, with a total of 92.33 Gb, in which GC content was 39.45%, Q20 was 97.08%, and Q30 was 91.97%. There were 615552500 clean reads（89.17 Gb）, of which the GC content was 39.00%, Q20 was 97.11% and Q30 was 92.05%. The total sequencing depth of Survey was 106.9X. The genome size of the revised Nymphaea ’Paul Stetson’ was 834.12Mb, the heterozygous rate was 1.95%, and the repetition rate was 68.48%. The results of Smudgeplot analysis also showed that Nymphaea ’Paul Stetson’ was tetraploid, and the frequency of tetraploid AAAB was the highest（0.43）. 【Conclusion】 The genome of Nymphaea ’Paul Stetson’ is a complex tetraploid characterized by high heterozygosity and a high repetition rate. Its structure is hypothesized to be of the AAAB type, comprising 3 sets of homologous haploid genomes, which presents great challenges for genome assembly.