Diversification and independent domestication of Asian and European pears
- Jun Wu†1,
- Yingtao Wang†2,
- Jiabao Xu†3,
- Schuyler S. Korban†4,
- Zhangjun Fei†5, 6,
- Shutian Tao†1,
- Ray Ming4, 7,
- Shuaishuai Tai3,
- Awais M. Khan5,
- Joseph D. Postman8,
- Chao Gu1,
- Hao Yin1,
- Danman Zheng4,
- Kaijie Qi1,
- Yong Li2,
- Runze Wang1,
- Cecilia H. Deng9,
- Satish Kumar9,
- David Chagné9,
- Xiaolong Li1,
- Juyou Wu1,
- Xiaosan Huang1,
- Huping Zhang1,
- Zhihua Xie1,
- Xiao Li2,
- Mingyue Zhang1,
- Yanhong Li3,
- Zhen Yue3,
- Xiaodong Fang3,
- Jiaming Li1,
- Leiting Li1,
- Cong Jin1,
- Mengfan Qin1,
- Jiaying Zhang1,
- Xiao Wu1,
- Yaqi Ke1,
- Jian Wang3, 10,
- Huanmimg Yang3, 10 and
- Shaoling Zhang1Email author
© The Author(s). 2018
Received: 5 February 2018
Accepted: 11 May 2018
Published: 11 June 2018
Pear (Pyrus) is a globally grown fruit, with thousands of cultivars in five domesticated species and dozens of wild species. However, little is known about the evolutionary history of these pear species and what has contributed to the distinct phenotypic traits between Asian pears and European pears.
We report the genome resequencing of 113 pear accessions from worldwide collections, representing both cultivated and wild pear species. Based on 18,302,883 identified SNPs, we conduct phylogenetics, population structure, gene flow, and selective sweep analyses. Furthermore, we propose a model for the divergence, dissemination, and independent domestication of Asian and European pears in which pear, after originating in southwest China and then being disseminated throughout central Asia, has eventually spread to western Asia, and then on to Europe. We find evidence for rapid evolution and balancing selection for S-RNase genes that have contributed to the maintenance of self-incompatibility, thus promoting outcrossing and accounting for pear genome diversity across the Eurasian continent. In addition, separate selective sweep signatures between Asian pears and European pears, combined with co-localized QTLs and differentially expressed genes, underline distinct phenotypic fruit traits, including flesh texture, sugar, acidity, aroma, and stone cells.
This study provides further clarification of the evolutionary history of pear along with independent domestication of Asian and European pears. Furthermore, it provides substantive and valuable genomic resources that will significantly advance pear improvement and molecular breeding efforts.
Pear (Pyrus), one of the most economically important temperate fruit tree species, with an annual worldwide production of ~ 18 million tons (2015, FAOSTAT), belongs to the subtribe Malinae of the Amygdaloideae subfamily within Rosaceae . The genus Pyrus includes at least 22 recognized species , with more than 5000 accessions maintained worldwide. These accessions display wide morphological and physiological variability, as well as broad adaptations to wide agro-ecological ranges. As a self-incompatible flowering plant, pear is an obligate outcrosser. It is important to note that hybridization in pear occurred not only intraspecies but also interspecies, despite its wide geographic distribution. Although many pear groups are deemed as different species, they are in fact rather similar to subpopulations based primarily on their distinguishable phenotypes. Therefore, it is likely that inter-‘species’ hybridizations and genetic admixtures must have occurred among pear groups without reproductive barriers . Nevertheless, they have long been widely recognized and deemed as “species” in pear research studies [4, 5].
The ancient Pyrus lineage probably arose during the Tertiary period, between 65 to 55 million years ago (MYA), in the mountainous regions of southwestern China . Subsequently, it was dispersed across mountainous ranges both eastward and westward. This oriental and occidental geographical distribution of pear led to the respective development of Asian and European pears . The earliest cultivation of Asian pears can be traced back to about 3300 years ago , with commercial orchards known to have existed for more than 2000 years in China . Similarly, European pears have been cultivated for more than 3000 years, with distinct named cultivars recorded as early as 300 B.C. .
Pyrus communis, the predominant cultivated species of European pear, bears typical pear-shaped fruits with soft and smooth flesh, few stone cells, along with strong aroma and flavor. The major cultivated species in Asia, including P. pyrifolia, P. bretschneideri, P. sinkiangensis, and P. ussuriensis, bear round-shaped fruits with crisp flesh, high sugar content, low acid content, minimal aroma, and mild flavor. The genetic variations and domestication processes responsible for these observed phenotypic differences in fruit trait characters between European and Asian pears are not well understood.
Indeed, these wide genetic variations present in pear accessions, belonging to various Pyrus species, have made it quite difficult to identify relationships among pear germplasm collections. Consequently, current available pear DNA sequence data are inadequate to delineate clear population-level relationships among various pear species [10–12]. In the past two decades, whole-genome sequencing tools have revolutionized the field of life sciences as they have provided unprecedented new means and opportunities to explore and understand genetic variation, evolution, and domestication processes of agricultural crops . Owing to its self-incompatibility and long generation cycle, among other factors, genetic and molecular analyses of pear have been rather challenging and slow. However, recent completion of whole-genome sequencing of ‘Dangshansuli’, an Asian pear , and “Bartlett”, a European pear , has yielded new knowledge, including characterization of the genomic structure, chromosome evolution, and patterns of genetic variation related to important agricultural traits.
In this study, we conducted population-level analysis of genetic variation of pears based on the resequencing of genomes of a diverse group of 57 wild and 56 cultivated Pyrus accessions from wide geographical regions. A total of 18,302,883 genome-wide SNPs were identified and used in multiple analyses. Findings were then used to propose a model to explain divergence, dissemination, and domestication of Asian and European pears. Of particular note, analysis of the evolutionary rate and balancing selection of S-locus genes highlights the impact of self-incompatibility on the genetic diversity of pear, which likely had a strong influence on gene flow and observed genetic variations in Pyrus. Furthermore, selective sweeps associated with agriculturally important genes were detected in cultivated Asian and European pears. In addition to evolutionary and functional genomics insights, this study provides an unprecedented amount of genomic data that will almost certainly enable important advances in modern pear improvement and molecular breeding programs.
Results and discussion
Sequencing and mapping of pear accessions
Summary of genetic diversity in different pear groups
Pear accessions—groups (number of samples)
Cultivated Asian (31)
Wild Asian (32)
Cultivated European (25)
Wild European (25)
Among all identified SNPs, a total of 14.1% were located in coding regions: 7.7% were non-synonymous and 6.4% were synonymous, with a non-synonymous/synonymous ratio of 1.2 (Additional file 1: Table S2). These SNPs had potential effects on a total of 13,838 genes (32.3% of pear genes; Additional file 1: Table S3). The proportion of non-synonymous SNPs in coding regions of pear (7.7%) and apple (10.5%)  was higher than that detected in either soybean  (1.9%) or maize  (0.66%), underscoring the presence of higher levels of genetic variation in pear and other fruit trees compared to some annual crops (Additional file 1: Note 2). To validate SNP calling, both PCR amplification and Sanger sequencing were conducted for 510 randomly selected SNPs in 55 pear accessions, and a 97.5% consistency for SNP calling was obtained (Additional file 3).
Nucleotide diversity and linkage disequilibrium in genomes of Asian and European pears
The nucleotide diversity (ϴπ) of pear at the whole-genome level across all pear accessions was 5.5 × 10− 3 (Table 1). This was higher than those reported for other perennial crops such as peach (1.5 × 10− 3) , cassava (2.6 × 10− 3) , and grapevine (5.1 × 10− 3) , but lower than that reported for date palm (9.2 × 10− 3) . Notably, both wild and cultivated Asian pears had higher nucleotide diversity (5.21 × 10− 3 and 4.76 × 10− 3, respectively) than either wild (3.57 × 10− 3) or cultivated (3.53 × 10− 3) European pears. However, in both Asian and European pears, similar levels of nucleotide diversities were detected for wild and cultivated accessions. This is in sharp contrast to findings reported in both soybean  and rice , wherein strong positive selection has contributed to wide differences in nucleotide diversity observed between wild and cultivated populations .
Our findings of similar levels of nucleotide diversity between wild and cultivated pears, short LD distances, and rapid LD decay in cultivated pears all support relatively weak selection during pear domestication. This could be explained by high outcrossing rates that are maintained by self-incompatibility, as well as the short domestication history and long generation time of this perennial fruit crop . Furthermore, in view of the fact that the major method for propagation of pear cultivars is by grafting, this would contribute to low numbers of sexual generations during the domestication history, and would also contribute to the weak selection during pear domestication.
The phylogeny and structure of Asian and European pear populations
On the other hand, the phylogenetic analysis revealed that European cultivated pears formed a clade that was nested within wild European pear accessions (Fig. 2a). However, it was interesting to note that there was little change in the population structure for European pears, with increasing K values from 2 to 7 (Additional file 1: Figure S3a). In view of the high polymorphism and diversity of Asian pears, which might influence the population structure of European pears, the population structure analysis for European pears was conducted independently. It was found that European pears could be classified into two groups (Fig. 2b). European group I included wild accessions from Europe and North Africa. Almost all of the cultivated European pears clustered together into European group II, except for the wild accession Pyw-ni1 belonging to P. nivalis, an atypical pear used in the production of “perry” cider. Among 13 wild accessions in European group I, P. pyraster accessions were those most closely related to European group II accessions. Thus, it seems that P. pyraster, which grows widely throughout Europe , is likely the progenitor species from which cultivated European pears are derived. It is important to note that very few changes in clustering of European pears were observed at different K values (Additional file 1: Figure S3a). This is supported by findings obtained from ϴπ analyses revealing that, relative to sampled Asian pears, there was lower genetic diversity present among sampled European pear accessions. Thus, for subsequent gene flow and identity-by-descent (IBD) analyses, European group I accessions were split into two subgroups based on their geographical sampling locations. European group I subgroup 1 included accessions that were relatively closer to east Asia, while European group I subgroup 2 accessions were relatively farther away from east Asia (Additional file 1: Table S5; Fig. 2a).
Subsequently, gene flow within and between Asian and European pears were explored. First, we used groups from the phylogenetic tree to conduct a gene flow analysis using Treemix within Asian pears. While we detected a relatively strong gene flow from Asian group I to Asian group IV (P. sinkiangensis), we did not detect significant gene flow among the other pairings of Asian pear groups (Fig. 2c). As for European pears, extensive gene flow (P value = 2.2e-308; F_statistic = 0.988) was detected between European group II and European group I subgroup 2 (Fig. 2d). To further investigate gene flow between Asian and European pears, an analysis was conducted using all groups with no significant gene flow between Asian and European groups. A weak gene flow was detected from European pear group accessions to the Asian pear accession of P. sinkiangensis (Fig. 2e). All these findings are consistent with the earlier hypothesis that P. sinkiangensis is derived from a hybridization between Asian and European pears.
An IBD analysis of Asian group IV (P. sinkiangensis) was conducted to verify gene flow into this species from both cultivated Asian and cultivated European pears (Fig. 2f). It was observed that the proportion of genetic background from Asian cultivated pears was 45.3–61.8% in P. sinkiangensis, which was higher than that detected from European group II (17.9–35.3%). IBD analysis of European group I revealed that P. sinkiangensis contained 10.9–23.0 and 11.8–26.7% of the genetic backgrounds of European subgroups 1 and 2, respectively. This was lower than that detected for European group II, thus indicating that cultivated European pears contributed a higher proportion of genetic background to P. sinkiangensis compared with wild European pears. Therefore, this IBD analysis further supported Treemix results noting that P. sinkiangensis was the product of a hybridization that occurred between cultivated Asian and cultivated European pears. These findings are reasonable to expect from a historical perspective, as there was extensive cultural contact along the Silk Road from 207 BCE to 220 CE . Interestingly, there is a historical record from about 2000 years ago of a Han dynasty diplomat, Qian Zhang, bringing over cultivated Asian pear to the Xinjiang region . Given our finding that the admixed P. sinkiangensis species must have resulted from hybridization between cultivated Asian and cultivated European pears, we can speculate that historical and commercial influences may have contributed to the development of this unique species of cultivated pear.
Origin and dissemination of wild pears
Levels of population differentiation, FST, were then estimated across all chromosomes among the three groups of wild accessions (Fig. 3e). The FST between Asian group III and Asian group II was smaller than that between European group I and Asian group II. This suggested that the divergence of Asian and European groups preceded the divergence of Asian group II from Asian group III. Furthermore, when ϴπ analysis was conducted to evaluate levels of genetic diversity of these groups, it was found that Asian group II and Asian group III showed the highest levels of diversity (Fig. 3f) when compared to other groups.
In addition, IBD analysis was conducted to assess the same DNA segments within and across accessions (Fig. 3g). Overall, IBD values were higher for accessions in both Asian group II (0.59) and Asian group III (0.54), followed by those found in European group I, including central Asia (0.47), Western Asia (0.38), and the European mainland (0.439). Results of population structure analyses of wild pear accessions along with their geographical distributions support the hypothesis that pear must have originated in what is now known as the southwest region of China. Subsequently, it was then disseminated throughout central Asia before it was further spread over to western Asia and then to Europe.
A proposed model for the evolutionary pathway of pear
The hypothetical common ancestor of both Asian and European pears seems to have originated in China, subsequently disseminated through central Asia, and then eventually on to western Asia and Europe. Considering the fact that there is no reproductive isolation in the population, and given that pear is a typical self-incompatible species and an obligate outcrosser, it is likely that a “continent-wide species” must have undergone local adaptation followed by independent domestication processes for each of Asian and European pears. Each of these domestication processes must have involved selection for distinct phenotypic traits, including distinctive fruit shape, flavor, and texture traits that are now characteristic of Asian and European pears [32, 33].
This proposed model clarifies present-day complex relationships among the large numbers of so-called pear species. The five currently recognized cultivated pear species have been domesticated from three wild species. This is quite different from other crops which are often domesticated from a single wild species [34, 35]. The only species of European pear, P. communis, is derived from a wild European species, P. pyraster. One of the four species of Asian pear, the cultivated P. ussuriensis, is derived from the wild P. ussuriensis. Two other cultivated species of Asian pear, P. pyrifolia and P. bretschneideri, are derived from a common ancestor, the wild P. pyrifolia. Finally, the admixed species of the fourth cultivated Asian pear, P. sinkiangensis, is derived from hybridization that must have occurred within the last 3000 years between the cultivated European pear (P. communis) and the cultivated Asian pear, either P. pyrifolia or P. bretschneideri. The IBD analysis indicates similarly sized genome contributions from P. pyrifolia and P. bretschneideri to P. sinkiangensis.
Balancing selection along with rapid evolution of S-RNase genes have strengthened self-incompatibility in pear
Pear exhibits typical gametophytic self-incompatibility (GSI), which is controlled by a single multi-allelic locus, the S-locus. The S-locus contains the pistil determinant, S-RNase, and candidate pollen determinant S-locus haplotype F-box genes, SFB genes [14, 36]. It is commonly known that the S-RNase gene exhibits high sequence variability among different pear cultivars [37, 38].
To analyze allelic diversity of the S-RNase gene, cleaned reads of each pear accession were mapped onto the S-RNase locus of the reference pear genome of ‘Dangshansuli’. A total of 92 SNPs were detected among S-RNase alleles of wild accessions and 141 SNPs among S-RNase alleles of cultivated accessions of pear. Mean ϴπ values were 1.70 × 10− 1 for wild accessions and 1.72 × 10− 1 for cultivated accessions (Additional file 1: Table S6). These mean ϴπ values were much higher than the mean diversity of the genes (1.56 × 10− 2) detected in the whole genome. Notably, the high genetic diversity of S-RNase alleles was almost identical in both cultivated and wild accessions (Additional file 4), suggesting that this gene has not experienced strong selection pressure under human intervention. Meanwhile, both cultivated and wild Asian and European pears had positive Tajima’s D values (> 2.0) for the S-RNase gene. This indicated that a balancing selection must have contributed to maintenance of high levels of polymorphisms. This finding was also supported by high π and FST values obtained for both Asian and European pears (Additional file 1: Table S7).
We speculated that a fast evolution might help to account for the wide variability observed in the S-RNase gene. Therefore, the evolution rates of S-RNase and other genes under balancing selection were compared. The evolution rate of the S-RNase gene of pear was estimated to be at least 1.91e− 09 sites/year, whereas those for other genes under balancing selection ranged between 2.31e− 10 and 6.10e− 10 sites/year. Therefore, the evolution rate of the S-RNase gene remains higher than the estimated evolution rates of other balance-selected genes (Additional file 1: Note 3). These findings support the hypothesis that rapid evolution of the S-RNase gene may have led to its high variability, which is consistent with the theory that reproduction-related genes show higher evolution rates [39, 40]. This has likely contributed to strengthening of GSI and promoting outcrossing, thus facilitating genetic recombination among genotypes of different genetic backgrounds of pear.
Independent domestication processes for each of Asian and European pears
The different genes identified in selective sweeps of Asian and European pears were found to be enriched for 47 and 34 biological processes, respectively, including growth, response to cold, meristem and flower development, and single-organism metabolic processes, which could be involved in the distinct domestication pathways that have contributed to different traits of Asian and European pears. For example, in Asian pears, 11 cell wall degradation-related genes were found in selective sweep regions (Additional file 7), while none were found in selective sweep regions of European pears. These domestication-related genes might contribute to the crisp fruit flesh texture observed in Asian pears, compared with the soft and fine flesh texture of European pears. Four genes associated with fruit size, including one YABBY (Pbr003157.1; Additional file 1: Figure S4), two cyclin-like genes (Pbr015160.2 and Pbr028956.1), and one EXP4 (expansin-A4-like, Pbr041772.1), were found in selective sweeps of Asian pears. In contrast, two different fruit size-related genes, Pbr012098.1 and Pbr012099.1, which are homologous to tomato fw2.2 , were found in selective sweeps of European pears. This result indicates that different genome regions were selected for fruit size in Asian and European pears.
Fewer genes involved in organic acid metabolism were identified in selective sweeps. The citric acid-related gene Pbr014969.1 (Additional file 1: Figure S6), a homolog of ACLA-3 that controls the synthesis of citrate , was identified in Asian pears, whereas three malate acid biosynthesis or transport-related genes, Pbr013232.1, Pbr013272.1, and Pbr030186.1, were identified in European pears. These findings further support an earlier observation regarding the presence of different dominant acid components in Asian and European pears .
Stone cells specifically accumulate in pear flesh and can detract from eating quality. The stone cell content shows a close relationship with biosynthesis, transfer, and deposition of lignin in cell walls. Six genes related to lignin biosynthesis in the selective sweep regions (Additional file 1: Figure S7), including two peroxidases (POD; Pbr000146.1 and Pbr15965.1), two hydroxycinnamoyl transferases (HCT; Pbr006408.1 and Pbr012356.2), one ferulate-5-hydroxylase (F5H; Pbr031416.1), and one cinnamoyl-CoA reductase (CCR; Pbr039962.1), were found in Asian pears, while only two CCR genes (Pbr013290.1 and Pbr039962.1) were detected in European pears. This finding may help to explain the higher concentration of stone cells in Asian pears than European pears.
As for volatile compounds in pear fruit, a total of 12 and three genes annotated in fatty acid metabolism pathways were identified in selective sweeps of Asian and European pears, respectively. Four alcohol dehydrogenase genes (Pbr003230.1, Pbr027590.1, Pbr027591.1, and Pbr034873.1) related to aroma biosynthesis were detected in Asian pears, while only two (Pbr013212.1 and Pbr028181.1) were found in European pears, indicating that aroma in these two groups of pears is regulated by different genes in the metabolic pathway.
It is interesting to note that the 47 genes in selective sweeps of both Asian and European pears include one POD gene (Pbr013214.1) related to stone cell formation and one cyclin-like gene (Pbr035650.1) related to fruit size development, suggesting a certain degree of convergent domestication of fruit quality in Asian and European pears.
Since the selective sweep signatures could colocalize with many important agronomic traits, potentially some of which have been implicated in quantitative trait loci (QTL) studies. We looked for enrichment in selective sweep signatures and candidate QTL regions. The selected regions included previously reported QTL with accurate chromosome information from hybridized segregating populations of pear [45–47] and a newly constructed pear population (“Niikata” × “Hongxiangsu”; Additional file 8). We found 208 and 14 selective sweeps from Asian and European pears that overlapped, respectively, with QTL regions (permutation test P values of 1E-5 and 0.35, respectively), indicating significant colocalization signals in Asian pears. The weak colocalization signals in European pears might be due to the fact that the QTL used here were mostly identified from Asian pear, while few fruit related QTL have been reported in European pears (Fig. 5e). In the overlapping regions of QTL and selective sweeps, a total of 151 and 17 genes were identified in selective sweeps of Asian pears and European pears, respectively (Additional file 9). Among them, 94 genes were mapped to sugar-related QTL, 20 to fruit size, 14 to acidity, ten to firmness, 18 to fruit shape, and three to stone cell content. These results strongly support that genes with selective sweep signatures in QTL regions might play important roles to regulate the distinct phenotypic traits selected in Asian versus European pears.
In this study, we report on genome variation mapping of 113 wild and cultivated pear accessions, collected from worldwide germplasm material. Our findings provide insights that informed our proposed model for the divergence, dissemination, and independent domestication of Asian and European pears. A rapid LD decay was identified in pear, thus revealing a characteristic weak domestication process for this perennial fruit tree. Separate selective sweep signatures identified between Asian and European pears underlined the distinct phenotypic traits observed in pear, including fruit acidity, sugar, and stone cell content, among others. Population structure analysis provided new evidence to support the admixed genetic background of some pear species, which was likely driven by self-incompatibility. Furthermore, analysis of the nucleotide diversity of the S-RNase gene controlling self-incompatibility suggested that a potential mechanism which promoted outcrossing must have accounted for the extensive genome diversity observed in pear.
Finally, it bears repeating that this study offers an unprecedentedly large amount of genomic resources for wild and cultivated pears. This, alongside our identification of candidate genes in selective sweep regions and colocalized QTLs, will significantly contribute to efforts for genetic improvement and molecular breeding of pear. Further, these findings raise intriguing questions that will almost certainly set the stage for the next phase of global pear and perennial tree fruit research.
Sampling information and sequencing
In this study, a total of 113 accessions, belonging to 33 Pyrus species from 26 countries and spanning a wide geographic distribution, were collected and sequenced. This collection covers accessions from all five of the major cultivated species of pear and from most recognized wild species (Additional file 2 and Additional file 1: Figure S1).
Genomic DNA was extracted from leaves using the CTAB method. Paired-end DNA libraries with short inserts (~ 500 bp) were constructed according to the manufacturer’s instructions and sequenced using the HiSeq™ 2000 or Hiseq™ 4000 platforms (Illumina, USA). To retain reads of high quality, reads with fewer than 5% N (missing) bases and with fewer than 50% of bases of base quality < 5 were deemed as cleaned reads. All other reads were discarded.
Reference genome selection and SNP calling
First, to facilitate the selection of an appropriate reference genome, we performed comparison of two published pear genomes: the Asian pear genome ‘Dangshansuli’ and the European pear genome Bartlett [14, 15]. For the details of our assembly quality assessment based on the contig N50, the scaffold N50, and the scaffold size values, as well scaffold to chromosome anchoring ratios, see Additional file 1: Table S1. Second, to clarify differences between these two potential reference genomes, we conducted synteny analysis of both genomes and also used all-versus-all BLASTP (E-value less than 1e-5) analysis to identify orthologous genes of the two genomes. Here, an orthologous gene was defined as a positive reciprocal BLASTP hit between the two genomes. MCScanX  was used to analyze synteny blocks. Third, for SNP calling, we used ‘Dangshansuli’ as the reference genome and the following protocol: 1) SOAPaligner (version 2.22 beta)  was used to map cleaned reads to the pear reference genome ; 2) based on genome coordinates and following removal of potential PCR duplicates, alignments were used to build a consensus sequence for each accession using SOAPsnp (version 1.04) ; 3) further filtration was conducted to obtain an accurate genotype for each site in each accession using the following criteria: (a) the quality value should be more than 20, (b) the number of unique reads for a confirmed genotype should be higher than 2, and (c) the copy number for each site had to be less than 1.5; and 4) the confirmed credible genotype from all accessions for each site and biallelic SNPs with missing rates of less than 0.5 were deemed as SNP variants in the population. Further, to ensure that the variant mapping rates of divergent samples would not deleteriously affect the analysis of θπ, θw, and Tajima’s D and so on, SNPs that were present in the syntenic blocks and that had a missing rate of < 10% among the accessions for both Asian pears and European pears were selected to validate the findings, yielding results that were consistent with mapping to the ‘Dangshansuli’ Asian pear genome (Additional file 1: Note 1). This four-step process led us to ultimately select ‘Dangshansuli’ as the reference genome. A total of 510 SNP loci were randomly selected, and Primer 3 was used to design primers for PCR-based sequence verification. Following PCR amplification, fragments were Sanger sequenced by Invitrogen Inc. (USA).
Population genetics analysis
Genetic distances determined in analyses of 113 accessions and 57 wild accessions were calculated by sampling with replacement SNPs (200 times) using the p-distance method , and neighbor-joining trees were constructed using the neighbor program in the EMBOSS toolbox . Trees were then merged, and Figtree (http://tree.bio.ed.ac.uk/software/figtree/) was used to adjust the neighbor-joining tree. A principal component analysis (PCA) was conducted using the eigen function in R base to obtain an eigenvector. The top four eigenvectors of samples were plotted using the ggplot2 R package . FRAPPE  was used to infer the population structure among samples, wherein the maximum iteration time was set to 10,000, and the number of population groups (K) was varied from 2 to 5. To determine the most appropriate population structure’s classification for all 113 accessions, FRAPPE analysis  was performed 20 times on 1000 randomly selected SNPs at 4dTv (four-fold degenerate site) for each K value from 1 to 10 according to Evanno G et al. .
To estimate the gene flow between Asian and European pears, both wild and cultivated pears, 4.7 M SNPs were selected with the following criteria: the missing rate was < 0.9 in both Asian and European pears. Based on these SNPs, we used Treemix version 1.13 to investigate the gene flow between groups/subgroups, with the settings: “-se -bootstrap -k 500 -m”, wherein the number (−m) varied from 1 to 5.
Nucleotide diversity analyses were conducted, including the average pairwise divergence within a population (θπ) , the Watterson’s estimator (θw) , and Tajima’s D . A sliding window of 10 kb, along with a step of 5 kb, was used to estimate the θπ, θw, and Tajima’s D values. For each window, these values were calculated using an in-house Perl script with the Bio::PopGen package. Pairwise FST values  were computed in the same windows to measure the population differentiation between groups. We also calculated the nucleotide diversity for various types of genomic regions (mRNA, CDS, introns, UTRs, and intergenic regions).
LD and LD blocks
Correlation coefficients (r 2 ) of alleles were calculated using Haploview  to measure LD values in each of the four pear populations (i.e., Asian cultivated, Asian wild, European cultivated, and European wild). The parameters were set as follows: -maxdistance 200, -dprime, -minGeno 0.6, -minMAF 0.05, and -hwcutoff 0.01. LD decays were then plotted using a custom R script for each of the four pear populations. The parameter “-blockoutput GAB” was added to the Haploview program to detect LD blocks for each of the four pear populations.
Identification of identical-by-descent segments between Asian and European pear
Using pairwise accessions, identical-by-descent (IBD) regions were identified in contiguous 10-kb windows with no overlaps. The number of SNPs in each window should be more than 10. Similarity scores were calculated using the p-distance method  in each of the windows. Windows with percent similarity scores higher than 95% were deemed as IBD windows. The percentage of IBD windows along the entire genome was calculated for every pair of accessions.
where n corresponds to the number of accessions for a geographical group, m corresponds to the number of all Asian wild pear accessions, P ij is the percentage of IBDs in the genome for a pair of accessions (each from a different groups), and k is the count of P ij between a geographical group and all Asian wild pear accessions.
Self-incompatibility gene analysis
By mapping all pear sequencing reads to identified S-RNase alleles using bwa version 0.7.12-r1039) , cleaned reads specific for S-RNase genes were identified and used to call SNPs using the GATK package Haplotype Caller . Based on these called SNPs, θw and θπ, which indicate the nucleotide diversity, of S-RNase genes were calculated for different pear groups. The evolution rate of S-RNase and other genes under balancing selection were calculated using the d/2 T formula, where d is the nucleotide diversity and T is the duration of the time since divergence from the most recent common ancestor.
Divergence time of Asian and European pears
Various plant species, including Vitis vinifera (common European grape) , Carica papaya (papaya) , Fragaria vesca (woodland strawberry) , Prunus persica (peach) , Malus x domestica (cultivated apple) , Arabidopsis thaliana , and Populus trichocarpa (black cottonwood) , were used to estimate the divergence time of cultivated Chinese pears (P. bretschneideri)  from cultivated European pears (Pyrus communis) . A total of 420 single-copy gene families in all nine species were identified. Based on 4dTv (four-fold degenerate sites) in these 420 single-copy gene families, a phylogenetic tree was constructed using PhyML (v3.0) . Based on this phylogenetic tree and known divergence time range between Populus trichocarpa and Arabidopsis thaliana (100–120 MYA), we used MCMCTREE (PAML version 4.l4) to estimate the divergence time between cultivated Asian and cultivated European pears [68, 69].
Selective signals in pear
First, we removed the admixed genotypes (Additional file 1: Table S8) based on population structure analysis (Fig. 2a). Then, for Asian pears, cultivated accessions of P. pyrifolia and P. bretschneideri were used to detect selective sweeps. This strategy was used due to the admixture nature of P. sinkiangensis and availability of a limited number of P. ussuriensis accessions (Additional file 1: Table S8). Finally, 89 pear accessions including 19 wild Asian, 22 cultivated Asian, 24 wild European, and 24 cultivated European accessions (Additional file 10) were used for selective sweep analysis.
SNPs with missing rates of less than 0.5 in both Asian and European pears were deemed as common SNPs for selection sweep analysis. To identify regions with signals for selective sweeps in cultivated pears, θπ, Tajima’s D, reduction of diversity (ROD = 1 − θπcul/θπwild), and FST parameters were calculated in non-overlapping windows of 10 kb along the entire pear genome, based on common SNPs. Regions (10-kb window) with signals for selective sweeps were identified using the following criteria: among the top 5% of FST, ROD > 0.5, and bottom 10% of Tajima’s D distribution. Regions with balancing selection were identified using the bottom 5% of FST, and top 5% of Tajima’s D, and the top 10% of θπ.
RNA sequencing and sequence mapping
RNA was extracted from fruit flesh for a total of 24 samples (eight species × three stages). RNA sequencing libraries were constructed using the Illumina standard mRNA-Seq Prep Kit (TruSeq RNA and DNA Sample Preparation Kits version 2). Single end RNA-Seq data were generated with length of 49 bp. Reads were filtered and trimmed and then mapped onto ‘Dangshansuli’ (Pyrus bretschneideri) coding sequences using SOAPaligner .
Sugar content measurements
High performance liquid chromatography (HPLC) was used to measure pear fruit sugars, including sucrose, glucose, fructose, and sorbitol. Sugars were extracted from pear flesh by grinding, and then were dissolved and filtered through a SEP-C18 cartridge (Waters, WAT021515) and Sep-Pak filter. Sugars were processed using a Waters 1525 system (Waters, Shanghai, China); the column was 6.5 mm × 300 mm, inner diameter, 10 μm particle size (Waters), with a Sugar-pak 1 Guard-Pak Holder and Insert (Waters) cartridge for the guard column. Column temperature was set to 85 °C, and 35 °C was the reference cell temperature.
Association of selective sweep regions with QTLs
Selective sweep regions were associated with QTLs identified in pears, including previously published QTLs and new QTLs related to fruit quality that we identified in the course of the present study. Two F1 populations were used for these new QTL mapping analyses, including an F1 population containing 102 individuals derived from crossing ‘Bayuehong’ × ‘Dangshansuli’ (phenotyping of different fruit traits, including sugar content, acid content, stone cell content, and fruit size, were conducted in 2014 and 2015) and an F1 pear population of 176 individuals from a cross between ‘Niikata’ × ‘Hongxiangsu’ using an 8× re-sequencing strategy (phenotyping of fruit-related traits such as sugar content, acidity content, stone cell content), which were investigated in 2015 and 2016.
MapQTL6.0 (https://www.kyazma.nl/index.php/MapQTL/) was used for linkage map construction using a regression mapping algorithm and the Kosambi function. MapQTL6.0 was also used to perform interval mapping and to conduct MQM and Kruskal-Wallis tests to evaluate candidate QTLs. Finally, markers with p < 0.005 and interval mapping LOD values higher than 3.5 were identified as QTLs. In comparison with the overlap of selective sweep regions and QTLs, we used enrichment tests with a sliding window size of 10 kb with 100,000 repetitions throughout the genome to find the overlap regions with candidate QTL regions.
This work was funded by the National Natural Science Foundation of China (31725024, 31471839, 31672111, and 31772276), the Earmarked Fund for China Agriculture Research System (CARS-28), the Science Foundation for Distinguished Young Scholars in Jiangsu Province (BK20150025), and the “333 High Level Talents Project” of Jiangsu Province (BRA2016367), and the US National Science Foundation (IOS-1539831).
Availability of data and materials
All of the raw reads generated in this study have been deposited in the NCBI database under BioProject accession PRJNA381668 . The variation datasets are available through the pear genome database website  or are freely available upon request. Supplementary information and source data files are available with the online version of this article. European wild pear accessions used in this study have been kindly provided to us by the USDA-ARS National Clonal Repository in Corvallis, Oregon following our request, and Asian wild pear accessions were collected by Nanjing Agricultural University, all those are publically available.
JW and SLZ conceived and designed the experiments. JW, SLZ, YTW, SSK, JDP, YL, XL, and CJ contributed samples. STT, KJQ, JYW, XSH, HPZ, and ZHX contributed to phenotyping. JW, LTL, and MFQ performed QTL analysis of F1 populations. SK and CHD provided the GBS (genotype by sequencing) QTL data for pear germplasm. JBX, JW, SST, ZJF, XLL, MYZ, RZW, YHL, ZY, XDF, JW, and HMY analyzed the data and performed statistical analyses. HY, DMZ, JML, JYZ, XW, and YQK performed the experiments. JW, CG, JBX, and SLZ wrote the manuscript with help from SSK, ZJF, RM, MAK, and DC. All authors read and approved the final manuscript.
The authors declare no competing financial interests.
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