Background: In water lily (Nymphaea) hybrid breeding, breeders often encounter non-viable seeds, which make it difficult to transfer desired or targeted genes of different Nymphaea germplasm. We found that pre-fertilization barriers were the main factor in the failure of the hybridization of Nymphaea. The mechanism of low compatibility between the pollen and stigma remains unclear; therefore, we studied the differences of stigma transcripts and proteomes at 0, 2, and 6 hours after pollination (HAP). Moreover, some regulatory genes and functional proteins that may cause low pollen-pistil compatibility in Nymphaea were identified. Results: RNA-seq was performed for three comparisons (2 vs 0 HAP, 6 vs 2 HAP, 6 vs 0 HAP), and the number of differentially expressed genes (DEGs) was 8,789 (4,680 were up-regulated), 6,401 (3,020 were up-regulated), and 11,284 (6,148 were up-regulated), respectively. Using label-free analysis, 75 (2 vs 0 HAP) proteins (43 increased and 32 decreased), nine (6 vs 2 HAP) proteins (three increased and six decreased), and 90 (6 vs 0 HAP) proteins (52 increased and 38 decreased) were defined as differentially expressed proteins (DEPs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs and DEPs were mainly involved in cell wall organization or biogenesis, S-adenosylmethionine (SAM) metabolism , hydrogen peroxide decomposition and metabolism, reactive oxygen species (ROS) metabolism, secondary metabolism, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis. Conclusions: Our transcriptomic and proteomic analysis highlighted specific genes, incuding those in ROS metabolism, biosynthesis of flavonoids, SAM metabolism, cell wall organization or biogenesis and phenylpropanoid biosynthesis that warrant further study in investigations of the pollen-stigma interaction of water lily. This study strengthens our understanding of the mechanism of low pollen-pistil compatibility in Nymphaea at the molecular level, and provides a theoretical basis for overcoming the pre-fertilization barriers in Nymphaea in the future.

Background: Although there has been a long history of cultivation and research on Nymphaea, the taxonomic relationships and evolutionary relationships among Nymphaea species remain controversial. The chloroplast (cp) genome can provide a new method to determine species origin, evolution, and phylogenetic relationships of Nymphaea. However, there are few studies on the cp genomes of Nymphaea, and the data of their genomes are very scarce. The complete cp genomes of seven Nymphaea species were sequenced by high-throughput sequencing technology, and the structural characteristics and phylogenetic relationships of cp genomes were analyzed.Results: A total of 126–129 genes were annotated in seven Nymphaea species, including 81–84 protein coding genes, eight rRNA, and 37 tRNA genes. A comparative cp genomic analysis of seven Nymphaea species showed that the cp gene sequence of Nymphaea was consistent, with no signs of reverse rearrangement. The codons preferentially ended with A/U. The cp genomes of seven Nymphaea species contained 147–168 dispersed repeats with a length of 15–19 bp and 979 simple sequence repeats (SSRs). Using N. colorata as the reference sequence, a total of 8,328 single nucleotide polymorphisms (SNPs) and 1,579 insertions/deletions were obtained. The degree of variation of the cp genome of the seven Nymphaea species in rpoA–rpl20, rbcL–ndhC, ndhD–ndhF, and trnN-GUU–ndhA regions is relatively high; related regions can be used as potential molecular markers for population genetics research. KaKs analysis showed that the ycf2 gene was positively selected. The results of the phylogenetic analysis showed that the genus Nymphaea can be divided into five branches: subgenus Nymphaea, subgenus Hydrocallis, subgenus Lotos, subgenus Anecphya, and subgenus Brachyceras. Conclusions: The cp genome of Nymphaea is very conservative in structure and composition, but it has rich variation in LSC and SSC regions. The phylogenetic analysis showed that Nymphaea could be further divided into five subgenera, and Euryale and Victoria were most closely related to Nymphaea. This study provided insight on the structure and composition of the cp genome of Nymphaea. Moreover, our results have provided more sequence information and genome resources for follow-up studies on Nymphaea.

Background: In water lily (Nymphaea) hybrid breeding, breeders often encounter non-viable seeds, which make it difficult to transfer desired or targeted genes of different Nymphaea germplasm. We found that pre-fertilization barriers were the main factor in the failure of the hybridization of Nymphaea. The mechanism of low compatibility between the pollen and stigma remains unclear; therefore, we studied the differences of stigma transcripts and proteomes at 0, 2, and 6 hours after pollination (HAP). Moreover, some regulatory genes and functional proteins that may cause low pollen-pistil compatibility in Nymphaea were identified. Results: RNA-seq was performed for three comparisons (2 vs 0 HAP, 6 vs 2 HAP, 6 vs 0 HAP), and the number of differentially expressed genes (DEGs) was 8,789 (4,680 were up-regulated), 6,401 (3,020 were up-regulated), and 11,284 (6,148 were up-regulated), respectively. Using label-free analysis, 75 (2 vs 0 HAP) proteins (43 increased and 32 decreased), nine (6 vs 2 HAP) proteins (three increased and six decreased), and 90 (6 vs 0 HAP) proteins (52 increased and 38 decreased) were defined as differentially expressed proteins (DEPs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs and DEPs were mainly involved in cell wall organization or biogenesis, S-adenosylmethionine (SAM) metabolism , hydrogen peroxide decomposition and metabolism, reactive oxygen species (ROS) metabolism, secondary metabolism, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis. Conclusions: Our transcriptomic and proteomic analysis highlighted specific genes, incuding those in ROS metabolism, biosynthesis of flavonoids, SAM metabolism, cell wall organization or biogenesis and phenylpropanoid biosynthesis that warrant further study in investigations of the pollen-stigma interaction of water lily. This study strengthens our understanding of the mechanism of low pollen-pistil compatibility in Nymphaea at the molecular level, and provides a theoretical basis for overcoming the pre-fertilization barriers in Nymphaea in the future.