Chromosome-level assembly, genetic and physical mapping of Phalaenopsis aphrodite genome provides new insights into species adaptation and resources for orchid breeding

Abstract

The Orchidaceae is a diverse and ecologically important plant family. Approximately 69% of all orchid species are epiphytes, which provide diverse microhabitats for many small animals and fungi in the canopy of tropical rainforests. Moreover, many orchids are of economic importance as food flavourings or ornamental plants. Phalaenopsis aphrodite, an epiphytic orchid, is a major breeding parent of many commercial orchid hybrids. We provide a high-quality chromosome-scale assembly of the P. aphrodite genome. The total length of all scaffolds is 1025.1 Mb, with N50 scaffold size of 19.7 Mb. A total of 28 902 protein-coding genes were identified. We constructed an orchid genetic linkage map, and then anchored and ordered the genomic scaffolds along the linkage groups. We also established a high-resolution pachytene karyotype of P. aphrodite and completed the assignment of linkage groups to the 19 chromosomes using fluorescence in situ hybridization. We identified an expansion in the epiphytic orchid lineage of FRS5-like subclade associated with adaptations to the life in the canopy. Phylogenetic analysis further provides new insights into the orchid lineage-specific duplications of MADS-box genes, which might have contributed to the variation in labellum and pollinium morphology and its accessory structure. To our knowledge, this is the first orchid genome to be integrated with a SNP-based genetic linkage map and validated by physical mapping. The genome and genetic map not only offer unprecedented resources for increasing breeding efficiency in horticultural orchids but also provide an important foundation for future studies in adaptation genomics of epiphytes.

Keywords: de novo assembly; fluorescence in situ hybridization; genetic mapping; orchid; plant genome; restriction site-associated DNA sequencing.

Figure 1

The integration of genetic map, genome assembly and FISH mapping. (a) Linkage group 19 with 95 RAD markers spanning 117.8 cM (not all markers are shown on the map). A total of 30 scaffolds were anchored and oriented within the linkage groups. Both purple and orange rectangles represent the anchored scaffolds. Triangles indicate the physical positions of the linkage group 19 specific FISH probes. (b), (c) FISH mapping reveals where DL16‐S96, DL16‐S328 and DL16‐S20 are located on the same chromosome. The chromosomes were stained with DAPI, and images were converted to black and white. Scale bar = 10 μm. (d) An ideogram of straightened pachytene chromosomes with FISH signals corresponding to DL16‐S96, CHS124207, DL16‐S328 and DL16‐S20. Blue and light blue blocks represent heterochromatin and euchromatin, respectively. (e) The locations of recombination hotspots and genetic markers (black triangles) on chromosome 19. The cumulative genetic distance from the end of the short arm to the end of long arm is shown. The locations of the FISH markers are indicated with triangles. The recombination hotspots (>55 cM/Mb, 10 times higher than the average) are indicated with red dashed lines, and the corresponding recombination rates (cM/Mb).

Figure 2

Chromosomal distribution of linkage group‐specific FISH probes. Ideogram showing the location of FISH signals and 90 genetically mapped and unmapped DNA markers on a high‐resolution map of pachytene chromosomes. Dark grey blocks indicate heterochromatin, light grey blocks indicate euchromatin, and the pink line indicates the centromere.

Figure 3

Genomic composition of P. aphrodite. (a) Genomic features of 19 P. aphrodite chromosomes. Track I: density of ESTs (No. of EST/Mb) from 17 orchid species belonging to 12 genera in five subfamilies of Orchidaceae are plotted for nonoverlapping 1‐Mb windows. Track II: chromosomal distribution of noncoding RNA. Track III: base coverage of TE‐related gene models and pseudogenes for nonoverlapping 1‐Mb windows. Track IV: gene density (no. of protein‐coding gene/Mb) for nonoverlapping 1‐Mb windows. Track V: positions of genes associated with important floral traits. Blue dots indicate genes involved in flower pigment biosynthesis and floral scent. Brown dots indicate genes related to flower development. Red dots indicate genes that control flowering. Track VI: yellow lines indicate chromosome regions that contain at least eight paralogous genes. (b) Gene families unique to P. aphrodite and those shared with four other species. The digits under each species name indicate the number of gene families, the number of genes per family and the total gene number (separated by slashes). (c) Comparison of the size of transcription factor families in P. aphrodite, rice and tomato. The colour of each dot indicates the number of genes in each family in P. aphrodite. Families with more than 20 members in P. aphrodite are labelled.

Figure 4

Orchid lineage‐specific duplication of the FAR1/ FRS gene family. (a) Maximum‐likelihood phylogenetic tree of 202 full‐length FAR1/FRS proteins (sequences with length > 400 aa) from 10 species. (b) Phylogeny of the FRS6 subfamily, including 45 amino acid sequences from 11 species. Arabidopsis FRS7 and FRS12 were used as outgroups. (c) Phylogeny of the FRS5‐like subfamily, including 134 amino acid sequences from 10 species. Arabidopsis FRS11 was used as outgroup. In (b) and (c), the purple branches indicate the orchid clade, and red branches indicate the clade of epiphytic orchids.

Figure 5

Phylogenetic tree of AGL6 and expression patterns of P. aphrodite AGL6 and AP3. (a) Red clades indicate orchid family members, and green and blue clades indicate proteins from other monocots and dicots, respectively. The phylogenetic tree was generated from 41 amino acid sequences from 21 species. The nine orchid species belong to five Orchidaceae subfamilies: P. aphrodite, P. bellina, P. lueddemanniana, Gastrodia elata and Oncidium gower ramsey (subfamily Epidendroideae), Apostasia wallichii (subfamily Apostasioideae), Cypripedium formosanum (subfamily Cypripedioideae), Orchis italica (subfamily Orchidoideae) and Vanilla planifolia (subfamily Vanilloideae). (b) RNA‐Seq‐based gene expression heatmap (TPM on log2 scale) of the P. aphrodite AGL6 and AP3 genes. PETAL, the lateral petals; LIP, the labellum; SS, short stalk; LS, long stalk; PO, pollinia. SEED01, protocorm formation; SEED02, protocorm development; SEED03, seedling formation.

Figure 6

Orchid lineage‐specific duplications of MADS‐box genes involved in pollinium development. (a) Maximum‐likelihood phylogenetic tree of MIKC* type MADS‐box proteins from P. aphrodite and 12 other orchid species and eight plants with sequenced genomes. The pollinium‐specific P. aphrodite genes are indicated by pink star next to the sequence ID. (b) Maximum‐likelihood phylogenetic tree of M‐gamma‐type MADS‐box proteins from P. aphrodite and nine other species. (c) Gene expression heatmap of pollinium‐specific MADS‐box genes in P. aphrodite.