Contrasting genetic variation and positive selection followed the divergence of NBS-encoding genes in Asian and European pears

Abstract

Background: The NBS disease-related gene family coordinates the inherent immune system in plants in response to pathogen infections. Previous studies have identified NBS-encoding genes in Pyrus bretschneideri ('Dangshansuli', an Asian pear) and Pyrus communis ('Bartlett', a European pear) genomes, but the patterns of genetic variation and selection pressure on these genes during pear domestication have remained unsolved.

Results: In this study, 338 and 412 NBS-encoding genes were identified from Asian and European pear genomes. This difference between the two pear species was the result of proximal duplications. About 15.79% orthologous gene pairs had Ka/Ks ratio more than one, indicating two pear species undergo strong positive selection after the divergence of Asian and European pear. We identified 21 and 15 NBS-encoding genes under fire blight and black spot disease-related QTL, respectively, suggesting their importance in disease resistance. Domestication caused decreased nucleotide diversity across NBS genes in Asian cultivars (cultivated 6.23E-03; wild 6.47E-03), but opposite trend (cultivated 6.48E-03; wild 5.91E-03) appeared in European pears. Many NBS-encoding coding regions showed Ka/Ks ratio of greater than 1, indicating the role of positive selection in shaping diversity of NBS-encoding genes in pear. Furthermore, we detected 295 and 122 significantly different SNPs between wild and domesticated accessions in Asian and European pear populations. Two NBS genes (Pbr025269.1 and Pbr019876.1) with significantly different SNPs showed >5x upregulation between wild and cultivated pear accessions, and > 2x upregulation in Pyrus calleryana after inoculation with Alternaria alternata. We propose that positively selected and significantly different SNPs of an NBS-encoding gene (Pbr025269.1) regulate gene expression differences in the wild and cultivated groups, which may affect resistance in pear against A. alternata.

Conclusion: Proximal duplication mainly led to the different number of NBS-encoding genes in P. bretschneideri and P. communis genomes. The patterns of genetic diversity and positive selection pressure differed between Asian and European pear populations, most likely due to their independent domestication events. This analysis helps us understand the evolution, diversity, and selection pressure in the NBS-encoding gene family in Asian and European populations, and provides opportunities to study mechanisms of disease resistance in pear.

Keywords: Expansion; NBS; Nucleotide diversity; Pear; Positive selection.

Fig. 1

The maximum likelihood (ML) Phylogenetic analysis of NBS-encoding genes from P. bretschneideri and P. communis. The phylogenetic tree was obtained using the IQ-TREE software on the basis of amino-acid sequences of NBS-encoding genes with ultrafast bootstrap 1000. ① Two subfamilies are shown. Red represents non-TIR group and Blue represents TIR group. ② The six classes of NBS-encoding genes are marked by different colors. Green means CC-NBS-LRR type, light yellow means TIR-NBS-LRR type, yellow means NBS type, light blue means TIR-NBS type, orange means CC-NBS type and purple means NBS-LRR type. ③ Domains of NB-ARC, LRR and TIR are displayed on the tree (CC domain was not shown), yellow means TIR domain, red means NB-ARC domain and blue means LRR domain

Fig. 2

Chromosome location of NBS-encoding genes in P. bretschneideri (a) and P. communis (b). Different colors represent different types of NBS-encoding genes. Blue (NBS-LRR), purple (TIR-NBS-LRR), red (CC-NBS-LRR), green (NBS), cyan (TIR-NBS), black (CC-NBS). The x-axis represents the chromosome number, and the y-axis represents chromosome length

Fig. 3

Distribution of Ks values of NBS genes pairs. Orange lines at the x-axis and the gray dotted box represented the Ks value region from 0.15 to 0.30. a Distribution of Ks values in P. bretschneideri and P. communis, respectively (b) Distribution of Ks values of Orthologous gene pairs between two pear species. Letter ‘Pb’ means P. bretschneideri, and letter ‘Pc’ means P. communis. Ks represents synonymous substitutions per site

Fig. 4

Synteny analysis of the NBS-encoding genes between P. bretschneideri and P. communis. Letter ‘ch’ means the chromosome of P. bretschneideri and letter ‘eu’ means the chromosome of P. communis. Brown lines means collinearity relationships among genes between P. bretschneideri and P. communis

Fig. 5

a Distribution of nucleotide diversities (π) in different pear groups. Pink (all pear accessions), green (Asian and European), orange (Cultivated and wild), blue (Asian cultivated and Asian wild), purple (European cultivated and European wild). b Distribution of F_ST values across the whole NBS-encoding gene family of Asian pears. Red plot is the top 5% F_ST value (> 0.14) in Asian pears. c Distribution of F_ST values across the whole NBS-encoding gene family of European pears Blue plot is the top 5% F_ST value (> 0.13) in European pears

Fig. 6

Expression profiles of 17 NBS-encoding genes in four pear accessions at enlarged stage by RT-qPCR analysis and RNA-seq data. The relative expression levels of each individual NBS-encoding genes were normalized by the Pyrus GAPDH gene. The expression level of PyW14 sample was used as a reference (relative expression level = 1). The letter “r” represents the correlation between RNA-seq data and RT-qPCR data. Bars represent mean relative expression level of genes (rose red: PyW14, purple: PyW13, blue: PyL2, cyan: PyI1); lines represent value of RPKM. Mean relative expression value and corresponding standard deviation were calculated by the three replicates of relative expression value. Four pears included Asian wild (PyW13,14) and cultivated (PyL2,PyI1) accessions. Left y-axis represents the relative expression level and right y-axis represents RPKM values

Fig. 7

Divergence of Pbr025269.1 between Asian cultivated and wild groups. a One significantly different non-synonymous SNP located on NBS domain. b Genotyping distribution of SNP1 in Asian cultivated and wild groups (chi-square test, p-value = 1.42E-08). c Ka/Ks ratio and nucleotide diversity of Pbr025269.1 in Asian wild and cultivated groups. Bars mean nucleotide diversities and spots mean Ka/Ks ratios. d Expression profiles of Pbr025269.1 in ten Asian cultivated (PyL2, PyL7, PyL8, PyL9, PyL10, PyL11, PyI1, PyI9, PyI11, PyI14) and seven Asian wild (PyW5, PyW6, PyW7, PyW9, PyW12, PyW13, PyW14) pear accessions at enlarged stage by RT-qPCR analysis. The relative expression levels of each individual NBS-encoding genes were normalized by the Pyrus GAPDH gene. The expression level of PyW14 sample was used as a reference (relative expression level = 1). Each point means relative expression level of one pear accession. ‘**’ means significant difference (T-test, p-value< 0.05) between wild and cultivated groups. (e) RPKM value of Pbr025269.1 in P. calleryana and ‘hongfen’ pear (an Asian cultivate pear), ‘Treat’ means inoculated A. alternata groups and ‘CK’ means control groups; red represents P. calleryana, blue represents ‘hongfen’(P. pyrifolia)