The high-quality genome of diploid strawberry (Fragaria nilgerrensis) provides new insights into anthocyanin accumulation

Abstract

Fragaria nilgerrensis is a wild diploid strawberry species endemic to east and southeast region in Asia and provides a rich source of genetic variations for strawberry improvement. Here, we present a chromosome-scale assembly of F. nilgerrensis using single-molecule real-time (SMRT) Pacific Biosciences sequencing and chromosome conformation capture (Hi-C) genome scaffolding. The genome assembly size was 270.3 Mb, with a contig N50 of ∼8.5 Mb. A total of 28 780 genes and 117.2 Mb of transposable elements were annotated for this genome. Next, detailed comparative genomics with the high-quality F. vesca reference genome was conducted to obtain the difference among transposable elements, SNPs, Indels, and so on. The genome size of F. nilgerrensis was enhanced by around 50 Mb relatively to F. vesca, which is mainly due to expansion of transposable elements. In comparison with the F. vesca genome, we identified 4 561 825 SNPs, 846 301 Indels, 4243 inversions, 35 498 translocations and 10 099 relocations. We also found a marked expansion of genes involved in phenylpropanoid biosynthesis, starch and sucrose metabolism, cyanoamino acid metabolism, plant-pathogen interaction, brassinosteroid biosynthesis and plant hormone signal transduction in F. nilgerrensis, which may account for its specific phenotypes and considerable environmental adaptability. Interestingly, we found sequence variations in the upstream regulatory region of FnMYB10, a core transcriptional activator of anthocyanin biosynthesis, resulted in the low expression level of the FnMYB10 gene, which is likely responsible for white fruit phenotype of F. nilgerrensis. The high-quality F. nilgerrensis genome will be a valuable resource for biological research and comparative genomics research.

Keywords: Fragaria nilgerrensis; MYB10; Hi-C; PacBio SMRT; promoter activity; transposable element.

Figure 1

Gene family and genome evolution of Fragaria nilgerrensis. (a) The estimation of divergence time and expansion (red), contraction (green) of gene families in F. vesca, Malus × domestica, Prunus avium, Prunus persica, Pyrus Communis, Pyrus bretschneideri, Rosa chinensis, Rubus occidentalis, Arabidopsis thaliana, Vitis vinifera, Solanum lycopersicum, Citrus sinensis and Oryza sativa. A phylogenetic tree was performed based on 373 single‐copy orthologous genes using O. sativa as the outgroup. The numerical value beside each node is the estimated divergent time. (b) The distribution of single‐copy, multiple‐copy, unique and other orthologues in F. vesca, M. × domestica, P. avium, P. persica, P. communis, P. bretschneideri, R. chinensis, R. occidentalis, A. thaliana, V. vinifera, S. lycopersicum, C. sinensis and O. sativa.

Figure 2

Comparative analysis and evolution events in the Fragaria nilgerrensis genome. (a) Genome duplication in F. nilgerrensis (Fn), F. vesca (Fv), P. avium (Pa), R. chinensis (Rc) and R. occidentali (Ro) genomes as revealed through 4DTv analyses. The percentages of the orthologous pairs (Fn vs Fv) between F. nilgerrensis (Fn) and F. vesca (Fv), the orthologous pairs (Fn vs Pa) between F. nilgerrensis (Fn) and P. avium (Pa), the orthologous pairs (Fn vs Rc) between F. nilgerrensis (Fn) and R. chinensis (Rc), the orthologous pairs (Fn vs Ro) between F. nilgerrensis (Fn) and R. occidentali (Ro), and paralogous gene pairs within the F. nilgerrensis (Fn vs Fn) genomes are plotted against their calculated 4DTv values; (b) distribution of synonymous substitution rates (Ks) for homologous gene groups. (c) Venn diagram represents the shared and unique gene families among F. nilgerrensis, F. vesca, Prunus avium, Rosa chinensis and Rubus occidentalis.

Figure 3

Functional classifications of expansion, contraction and unique genes in Fragaria nilgerrensis by KEGG. (a) Functional classification of expansion genes in F. nilgerrensis, 1. phenylpropanoid biosynthesis, 2. starch and sucrose metabolism, 3. cyanoamino acid metabolism, 4. ribosome, 5. spliceosome, 6. plant–pathogen interaction, 7. brassinosteroid biosynthesis, 8. galactose metabolism, 9. oxidative phosphorylation, 10. carbon metabolism, 11. plant hormone signal transduction, 12. pentose and glucuronate interconversions, 13. amino sugar and nucleotide sugar metabolism, 14. photosynthesis, 15. DNA replication, 16. nucleotide excision repair, 17. mismatch repair, 18. homologous recombination, 19. cysteine and methionine metabolism, 20. biosynthesis of amino acids, 21. pentose phosphate pathway, 22. glutathione metabolism, 23. sulphur metabolism, 24. RNA transport, 25. endocytosis, 26. purine metabolism, 27. pyrimidine metabolism, 28. inositol phosphate metabolism, 29. porphyrin and chlorophyll metabolism, 30. RNA polymerase, 31. phosphatidylinositol signalling system, 32. protein processing in endoplasmic reticulum, 33. glycine, serine and threonine metabolism, 34. monobactam biosynthesis, 35. lysine biosynthesis, 36. aminoacyl‐tRNA biosynthesis, 37. 2‐Oxocarboxylic acid metabolism, 38. ubiquitin mediated proteolysis；(b) Functional classification of contraction genes in F. nilgerrensis, 1. protein processing in endoplasmic reticulum, 2. spliceosome, 3. endocytosis, 4. pentose and glucuronate interconversions, 5. starch and sucrose metabolism, 6. linoleic acid metabolism, 7. alpha‐linolenic acid metabolism, 8. mRNA surveillance pathway, 9. plant hormone signal transduction, 10. tryptophan metabolism, 11. phenylpropanoid biosynthesis, 12. flavonoid biosynthesis, 13. stilbenoid, diarylheptanoid and gingerol biosynthesis, 14. steroid biosynthesis, 15. tyrosine metabolism, 16. isoquinoline alkaloid biosynthesis, 17. tropane, piperidine and pyridine alkaloid biosynthesis, 18. glycine, serine and threonine metabolism, 19. valine, leucine and isoleucine biosynthesis, 20. arginine and proline metabolism, 21. phenylalanine metabolism, 22. glutathione metabolism, 23. glycerophospholipid metabolism, 24. terpenoid backbone biosynthesis, 25. monoterpenoid biosynthesis, 26. carbon metabolism, 27. biosynthesis of amino acids, 28. ribosome biogenesis in eukaryotes, 29. protein export, 30. plant–pathogen interaction; (c) Functional classification of unique genes in F. nilgerrensis, 1. ribosome, 2. plant–pathogen interaction, 3. purine metabolism, 4. plant hormone signal transduction, 5. carbon metabolism, 6. ubiquitin mediated proteolysis, 7. phagosome, 8. citrate cycle, 9. pyrimidine metabolism, 10. RNA polymerase, 11. DNA replication, 12. glycolysis, 13. RNA transport, 14. SNARE interactions in vesicular transport, 15. endocytosis, 16. pentose and glucuronate interconversions, 17. oxidative phosphorylation, 18. photosynthesis, 19. lysine degradation, 20. tryptophan metabolism, 21. glutathione metabolism, 22. starch and sucrose metabolism, 23. brassinosteroid biosynthesis, 24. triterpenoid biosynthesis, 25. tropane, piperidine and pyridine alkaloid biosynthesis, 26. 2‐Oxocarboxylic acid metabolism, 27. biosynthesis of amino acids, 28. RNA degradation, 29. spliceosome, 30. proteasome, 31. nucleotide excision repair, 32. mismatch repair, 33. homologous recombination, 34. protein processing in endoplasmic reticulum, 35. peroxisome, 36. AGE‐RAGE signalling pathway in diabetic complications, 37. pentose phosphate pathway.

Figure 4

Genome landscape of Fragaria nilgerrensis. Elements are shown in the following scheme (from inner to outer). (i) Syntenic relationships among different chromosomes of F. nilgerrensis; (ii) distribution of repeats (window size, 100 kb); (iii) distribution of Copia elements (window size, 100 kb); (iv) distribution of Gpysy elements (window size, 100 kb); (v) distribution of TIR elements (window size, 100 kb); (vi) distribution of GC content (window size, 100 kb); (vii) gene density (window size, 100 kb).

Figure 5

The comparison of Fragaria nilgerrensis and F. vesca genomes. (a) The distribution of transposable element (TE) in the exon, promoter (2kb up‐ 5' UTR) and downstream (1kb‐down‐3' UTR) regions of genes in the F. nilgerrensis and F. vesca genome. (b) Distribution of insertion ages of LTR retrotransposons. The x‐axis represents the estimated insertion age of the LTR retrotransposons. The y‐axis represents the number of intact LTR retrotransposons. (c) Syntenic blocks share between the F. nilgerrensis and F. vesca genomes. (d) The numbers of SNPs, Indels, inversions, translocations, relocations in F. nilgerrensis genome compared with F. vesca genome. (e) The distribution of structural variation (SV) in the exon, promoter (2kb up‐ 5' UTR) and downstream (1kb‐down‐3' UTR) regions of genes in the F. nilgerrensis genome.

Figure 6

The anthocyanin content and anthocyanin‐related gene expression between Fragaria nilgerrensis (Fn) and F. vesca (Fv). (a) The anthocyanin content and compositions between the mature fruits of F. nilgerrensis (Fn) and F. vesca (Fv). (b) Simplified scheme of anthocyanin biosynthetic and regulatory pathway in plants. Biosynthetic genes are shown in right or left side of arrow and the transcription factors are shown in oval. PAL phenylalanine ammonia lyase, C4H cinnamate‐4‐hydroxylase, 4CL 4‐coumarate CoA ligase, CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone 3‐hydroxylase, F3’H flavonoid 3’‐hydroxylase, DFR dihydroflavonol 4‐reductase, ANS anthocyanidin synthase, LDOX, leucoanthocyanidin dioxygenase, UFGT, UDP‐flavonoid glucosyl transferase. (c) The gene expression heatmap of anthocyanin‐related genes in three developmental stages (green, turn and ripe stages) of white‐fruited F. nilgerrensis (Fn) and red‐fruited F. vesca (Fv). Heml software is used for making gene expression heatmap.

Figure 7

The comparison of sequences and transcription activity of MYB10 promoter between Fragaria nilgerrensis (Fn) and F. vesca (Fv). (a) The comparison of MYB10 promoter and protein between F. nilgerrensis (Fn) and F. vesca (Fv). Promoter and protein are shown as lines and boxes, respectively. Red lines (1–8) represent missing cis‐acting elements in the promoter of F. nilgerrensis compared with F. vesca. Red lines (9, 10) represent missing cis‐acting elements in the promoter of F. vesca compared with F. nilgerrensis. Red lines (11, 12) represent missing amino acids in F. nilgerrensis compared with F. vesca. Amino acids changes between F. nilgerrensis (Fn) and F. vesca (Fv) are shown in boxes. (b) Schematic diagrams of the Luc, ProFnMYB10‐Luc and ProFvMYB10‐Luc reporter constructs used for tobacco transient expression assay. (c) Luciferase activity assay. The Luc, ProFnMYB10‐Luc and ProFvMYB10‐Luc are transformed into Nicotiana benthamiana. (d) The comparison of luciferase activity. The transcriptional activity of these infiltrated tobacco leaves based on the ratio of LUC to REN is investigated by Dual Luciferase Reporter Gene assay kit. Different letters indicate significant differences (P < 0.05, based on Duncan’s multiple range test).