De novo assembly of a wild pear (Pyrus betuleafolia) genome

Abstract

China is the origin and evolutionary centre of Oriental pears. Pyrus betuleafolia is a wild species native to China and distributed in the northern region, and it is widely used as rootstock. Here, we report the de novo assembly of the genome of P. betuleafolia-Shanxi Duli using an integrated strategy that combines PacBio sequencing, BioNano mapping and chromosome conformation capture (Hi-C) sequencing. The genome assembly size was 532.7 Mb, with a contig N50 of 1.57 Mb. A total of 59 552 protein-coding genes and 247.4 Mb of repetitive sequences were annotated for this genome. The expansion genes in P. betuleafolia were significantly enriched in secondary metabolism, which may account for the organism's considerable environmental adaptability. An alignment analysis of orthologous genes showed that fruit size, sugar metabolism and transport, and photosynthetic efficiency were positively selected in Oriental pear during domestication. A total of 573 nucleotide-binding site (NBS)-type resistance gene analogues (RGAs) were identified in the P. betuleafolia genome, 150 of which are TIR-NBS-LRR (TNL)-type genes, which represented the greatest number of TNL-type genes among the published Rosaceae genomes and explained the strong disease resistance of this wild species. The study of flavour metabolism-related genes showed that the anthocyanidin reductase (ANR) metabolic pathway affected the astringency of pear fruit and that sorbitol transporter (SOT) transmembrane transport may be the main factor affecting the accumulation of soluble organic matter. This high-quality P. betuleafolia genome provides a valuable resource for the utilization of wild pear in fundamental pear studies and breeding.

Keywords: Pyrus betuleafolia; BioNano optical mapping; De novo assembly; Hi-C; PacBio SMRT.

Figure 1

Pyrus betuleafolia‐Shanxi Duli de novo genome assembly. (A) P. betuleafolia‐Shanxi Duli used in this study. (B) Summary of the de novo genome assembly and sequencing analysis of P. betuleafolia‐Shanxi Duli. A, Chromosome number; B, heat map view of genes; C, NBS‐type resistance gene analogues (RGAs); D, repeat density in 200‐kb windows (red, average +1 SD; blue, average −1 SD; yellow, gene and repeat density between red and blue); and E paralogous relationships between P. betuleafolia chromosomes.

Figure 2

Gene family evolution analysis. (A) Venn diagram showing the shared and unique gene families among three pear species. Each number in parentheses represents the number of genes within corresponding families (without parentheses). (B) Expansion and contraction of gene families in six Rosaceae species and tomato. A phylogenetic tree was constructed based on all single‐copy orthologous genes using tomato (Solanum lycopersicum) as the outgroup. Pie diagrams on each branch of the tree represent the proportion of genes undergoing gain (red) or loss (green) events. The numerical value beside each node shows the estimated divergent time.

Figure 3

Comparative analysis and evolution events in the Pbe‐ SD genome. (A) Syntenic blocks shared between the Pbe‐ SD and GDDH13 genomes. Grey lines connect matched gene pairs, with one set highlighted in red. (B) Ks distribution for paralogous and orthologous genes in comparisons of the Pbe‐ SD, DSHS and Bartlett genomes and Ka/Ks distribution in comparisons of the Pbe‐ SD and DSHS genomes.

Figure 4

Distribution of RGAs in chromosomes. (A) Distribution of RGAs along the Pbe‐ SD chromosomes. The bottom graph shows the absolute number of genes homologous to nucleotide‐binding site–leucine‐rich repeat (NBS‐LRR‐encoding) proteins, receptor‐like protein kinases (RLKs), receptor‐like proteins (RLPs) and transmembrane coiled‐coil (TMCC) proteins along each of the 17 chromosomes. The top four graphs show the ratio of the number of genes in each RGA class to the total number of genes in a sliding window of 10 Mb wide. (B) Collinearity comparison of TNL‐type genes on chromosome 2 of the GDDH13 and Pbe‐ SD genomes.

Figure 5

Differential expression of genes involved in sugar/acid metabolism in fruits from DSHS and Pbe‐SD. (A) Sugar/acid synthesis pathway and related structural genes in pear fruit. SUSY: sucrose synthase; SPS: sucrose phosphate synthase; SPP: sucrose‐phosphatase; HK: hexokinase; FK: fructokinase; PK: pyruvate kinase; MDH: malate dehydrogenase; ACO: aconitate hydratase; IDH: isocitrate dehydrogenase; CS: citrate synthesis; NINV: neutral invertase; CWINV: cell wall invertase; vAINV: vacuolar acid invertase; PFK: 6‐phosphofructokinase; SOT: sorbitol transporter; and HT: hexose transporter. (B) Differentially expressed genes in the proanthocyanidin metabolism pathway. Red colour represents higher than log₁₀ (FPKM) data of genes; green colour represents lower than log₁₀ (FPKM) data of genes; and black colour represents log₁₀ (FPKM) = 0. S1, S2 and S3 indicate the three biological replications of Pbe‐SD. D1, D2 and D3 indicate the three biological replications of DSHS.

Figure 6

Differential expression of genes involved in proanthocyanidin metabolism in fruits from DSHS and Pbe‐ SD. (A) Proanthocyanidin synthesis pathway and related structural genes in pear fruit. PAL, phenylalanine ammonia lyase; C4H, cinnamate‐4‐hydroxymate; 4CL, 4‐coumarate:coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3‐hydroxylase, F3'H, flavanone 3’‐hydroxylase; DFR, dihydroflavonol 4‐reductase; ANS, anthocyanidin synthase; UFGT, UDP‐glucose flavonoid 3‐O‐glucosyl transferase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; GT, glycosyltransferases; and FLS, flavonol synthase. (B) Differentially expressed genes in the proanthocyanidin metabolism pathway. Red colour represents higher than log₁₀ (FPKM) data of genes; green colour represents lower than log₁₀ (FPKM) data of genes; and black colour represents log₁₀ (FPKM) = 0. S1, S2 and S3 indicate the three biological replications of Pbe‐ SD. D1, D2 and D3 indicate the three biological replications of DSHS.