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Genome-wide association study and transcriptome of olecranon-type traits in peach (Prunus persica L.) germplasm
Jianliang Liu1,2,3 na1,
Yao Bao1,4 na1,
Yuming Zhong5,
Qin Wang1,4 &
Huifan Liu ORCID: orcid.org/0000-0002-0306-45231,4
The top of the olecranon honey peach (Prunus persica L.) fruit appears similar to an eagle's beak. In this study, a single olecranon honey peach with a round-type fruit was observed in our fruit orchard. To explore the genetic mechanism of olecranon formation, we performed full-length transcriptome sequencing analysis of olecranon and round peaches as well as a genome-wide association study of the association of olecranon-type trait loci.
The gene locus was 26,924,482 base pairs in NC_034014.1. Transcriptome sequencing showed that the clean sequencing data of each sample reached 7.10GB, with 14,360 genes and 23,167 transcripts expressed in both the olecranon honey peach and round peach. Among the 11 differentially expressed genes selected as candidate genes, six were highly expressed in olecranon peach and named as LOC18775282, LOC18772209, LOC18773929, LOC18772013, LOC18773401, and ONT.13798.5. Five genes were highly expressed in round peach and named as LOC18773079, LOC18773525, LOC18773067, LOC18775244, and LOC18772236. Notably, ONT.13798.5 was not previously identified. The genes were within 1 Mb up- or down-stream of the main genome-wide association study locus for olecranon-type traits.
This study revealed loci associated with olecranon and provides useful information for analysis and breeding of olecranon honey peach.
Peach (Prunus persica L.) is a genetic and genomic reference species for the genus Prunus because of its small genome and relatively short juvenile period [1]. Cultivated peach has a diploid (2n = 2x = 16) and relatively small genome (265 Mb) (www.rosaceae.org). Peach shape is an important external characteristic used by consumers to select preferred cultivars [2]. The shape of peach can be classified as round or flat; flat peaches are more popular than round peaches [2, 3]. Most studies of peach shape have focused on flat and round peaches. However, few studies have examined the olecranon honey peach, which was named for its unique surface features, as the top resembles the beak of an eagle. It is considered as one of the best peach varieties and is very popular in China because it is sweet, juicy, and has a crisp texture [4].
Genome-wide association studies (GWAS) were used to identify flat- and round-related loci or genes, indicating that PpCAD1 (ppa003772m, designated as PpCAD1) and 25,060,196 base pairs (bp) of scaffold Pp06 play important roles in the fruit shape [5]. The flat shape is determined by a single gene, S/s, in the distal part of chromosome 6, and the flat phenotype is caused by the partial dominant allele in heterozygosis, from which the dominant homozygote is aborted [5, 6]. Moreover, at 26,924,482 bp of scaffold Pp06, all accessions with round-shaped fruit were found to have an A/A genotype, whereas those with A/T or T/T genotypes formed flat-shaped fruits [3]. In addition, differences in the fruit cell number, cell size, and gene expression patterns between the round and flat fruit varieties and the oblate shape of peach fruits are mainly determined by regulating the production of cells in the vertical direction during early fruit development [2]. However, the molecular features affecting the shape of olecranon honey peach have not been evaluated in genetic or transcriptome studies.
In this study, a GWAS of olecranon traits in 456 peach accessions from across China was conducted to identify gene loci related to fruit shape. The Oxford Nanopore Technologies (ONT, Oxford, UK) sequencing method was used to study differences in the transcription of genes affecting the olecranon trait to narrow the range of related genes and provide a basis for further studies of these genes.
Resequencing 456 accessions of peach
We generated 2814.05Gbp clean reads, and the Q30 reached 92.43 % in whole-genome resequencing of 456 peach accessions from China (Fig. 1 A, Supplemental Table 1) with an average depth of 20X and coverage of 98.76 % for each accession (Supplemental Table 2). The average number of clean reads of the samples was 41,190,056, and the average mapping rate between the samples and reference gene was 97.38 % (Supplemental Table 2). The reads of each accession were mapped against the peach reference genome (release version NCBIv2), and a final set of 2,923,674 high-quality single-nucleotide polymorphisms (SNPs) and 627,795 insertions-deletions (InDels) was identified using SnpEff software. Averages of 231,407 and 121,336 SNPs were the transition type and transversion type, respectively, and the average SNP ratio of the heterozygous types was 63.39 % (Supplemental Table 3). For each accession, we analyzed the distribution of polymorphisms in each genomic region and found that averages of 96,783, 51,340, 85,430, and 63,805 SNPs were within intergenic regions, introns, upstream regions (within 5 kb upstream of transcription start sites), and downstream regions (within 5 kb downstream of transcription stop sites), respectively (Supplemental Table 4). In coding regions, averages of 16,305 nonsynonymous, 25 start-lost, 290 stop-gain, and 158 stop-lost SNPs were annotated (Supplemental Table 4), which led to amino acid changes, splicing variants, longer transcripts, or premature stop codons. Moreover, averages of 14,712, 11,377, 17,855, and 13,285 InDels were within intergenic regions, introns, upstream regions, and downstream regions, respectively (Supplemental Table 5).
Geographic distribution, linkage disequilibrium (LD) decay of 456 peach accessions and genome-wide association scan for olecranon-type traits. (A) Geographic distribution of 456 peach accessions. (B) LD decays of each chromosome. (C) Manhattan plots for FaST-LMM. Red horizontal lines indicate genome-wide significance. (D) Quantile–quantile plot for FaST-LMM. The horizontal axis shows − log10-transformed expected P-values and vertical axis shows − log10-transformed observed P-values
Patterns of LD across peach genome
Linkage disequilibrium (LD) analysis indicated that the physical distance between SNPs (reported as half of the maximum value) occurred at ~ 53.76 kb (r2 = 0.33) for Amygdalus persica L. (Table 1), which was slightly higher than that of wild peach (~ 35 kb) [7] and consistent with the narrow domestication bottleneck. The magnitude of LD decay varied among different chromosomes (Fig. 1D; Table 1). The maximum physical distance between SNPs was observed at ~ 292.11 kb (r2 = 0.35) for NC_034012.1, followed by NC_034013.1 (~ 132.42 kb). The minimum physical distance between SNPs occurred at ~ 20.84 kb (r2 = 0.34).
Table 1 LD blocks were defined using PLINK software
GWAS of olecranon-type traits and prediction of candidate genes
In this study, we counted 296 types of peaches out of the 456 germplasm resources, among which 155 had olecranon and 141 had no olecranon. The peaches could also be divided into 82 nectarines and 225 peaches. The weights of these peaches ranged from 7.08 to 520 g, and the fruit shape index ranged from 0.60 to 1.33 (Supplemental Table 1). To explore the potential of GWAS to identify causal genes of olecranon-type traits, we used the FaST-LMM model to conduct an association study of olecranon-type traits. The locus defined by the SNP at 26,924,482 bp was strongly associated with olecranon-type traits (Fig. 1 C and 1D) and positioned on NC_034014.1, and the minor allele frequency was 0.07, which is greater than 0.05. The SNP locus 26,924,482 bp is homozygous in olecranon peach and heterozygous in round peach.
We selected 38 genes within 1 Mb up- or down-stream of the main GWAS locus for olecranon-type traits (Supplemental Table 6). Through Swiss-PROt database annotation, we found that 20 genes are also expressed in the Arabidopsis genome. Five genes were related to serine/threonine-protein kinase and six were related to non-specific lipid-transfer protein. This variation was highly associated with an A/T polymorphism detected in the fifth intron of LOC18772384 on NC_034014.1. LOC18772384 was annotated as MACPF domain-containing protein CAD1-like (Supplemental Table 6).
Transcriptome material analysis
A single olecranon honey peach tree with round-type fruits was observed in our fruit orchard. A branch of this tree was found to bear only round-type fruits (Fig. 2B), and was found to have an alteration related to fruit-shape in the bud sport. Moreover, other branches bore olecranon-type fruits (Fig. 2 C). To further distinguish fruits with round and olecranon shapes, ripe fruits were characterized (Table 2). The results showed that the olecranon-type fruit weighed 218.50 ± 4.36 g, and had a longitudinal diameter of 69.16 ± 0.49 cm and transverse diameter of 72.87 ± 1.10 cm; the round-type peach weighed 113.92 ± 1.72 g, and had a longitudinal diameter of 62.86 ± 0.63 cm and transverse diameter of 58.71 ± 0.94 cm. According to the t-test, the quality indicators of the two types of peaches differed significantly (P < 0.05). Notably, both the weight and vertical and horizontal diameters of olecranon-type fruit were larger than those of round-type fruit. However, the olecranon-type fruit shape index was smaller than that of round-type fruit; these values were 0.95 ± 0.01 and 1.07 ± 0.00, respectively.
Phenotypic characterization of olecranon honey peach and round peach. (A) Sampled peach trees, (B) round peach, and (C) olecranon honey peach
Table 2 Comparison of quality indices of the two peach fruits
Overview of RNA-Seq data
The full-length transcriptome of six samples was sequenced, and the clean data of each sequence reached 7.10GB. After quality filtering, the read number ranged from 5,687,141 to 7,035,534 bp with an N50 ranging from 1263 to 1423 bp; the mean length of reads ranged from 1,145 to 1,259 bp, with a maximum length of 21,540 among all reads, and the mean q-score was Q9 in each library (Supplemental Table 7). After removing ribosomal RNA, the numbers of full-length reads in the six libraries were 5,195,328 (77.57 %), 481,897 (77.81 %), 4,795,588 (77.20 %), 5,346,222 (77.86 %), 4,312,262 (77.68 %), and 4,847,747 (76.97 %), respectively (Supplemental Table 8). The minimum number of reads per library was 5.55 million for B-2 and the maximum number was 6.87 million for B-1. Approximate 99.75 % of reads were mapped to the peach reference genome V2 (Supplemental Table 8). We analyzed the correlation between expression levels in different samples by Pearson correlation coefficient. The results for B-3 sample were abnormal (Supplementary Figure S1) and thus excluded from subsequent analysis.
DEG and DET analysis
We found 14,360 genes and 23,167 transcripts expressed in both olecranon and round peach (Fig. 3 A, 3B). There were 5782 differentially expressed genes (DEGs) in the two groups, including 3293 up-regulated genes and 2489 down-regulated genes with P-values < 0.05 (Fig. 3 C). There were 11,239 differentially expressed transcripts (DETs) in the two groups, including 6294 up-regulated transcripts and 4945 down-regulated transcripts with P-values < 0.05 (Fig. 3D).
Expression of genes and transcriptome. (A) Venn diagram for genes in round peach and olecranon peach. (B) Venn diagram for transcriptome. (C) Volcano map of the differentially expressed genes (DEGs). Each point in the differential expression volcano chart represents a gene, and the abscissa represents the logarithm of the multiple of the difference in expression of a certain gene in the two samples; the ordinate represents the negative logarithm of statistical significance of the change in gene expression. A larger absolute value of the abscissa indicates a greater fold-difference in expression between the two samples; a larger ordinate value indicates more significant differential expression and greater reliability of the DEGs obtained by screening. The green dots in the figure represent down-regulated DEGs, red dots represent up-regulated DEGs, and black dots represent non-DEGs. (D) Volcano map of DEGs
Functional annotation of DEGs and DETs
To identify genes important for olecranon-type traits, we annotated the DEGs and DETs using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Eukaryotic Orthologous Groups (KOG), Clusters of Orthologous Groups (COG), and eggNOG databases. Based on GO annotation, 3793 DEGs and 7200 DETs were classified into three main categories, including biological process, cellular component, and molecular function (Supplemental Tables 9, Figs. 4 and 5), with each category possessing 20, 16, and 14 terms, respectively. In the biological process classification, genes and transcripts related to metabolic process, cellular, and process were highly enriched. In the cellular component category, the main subcategories were cell and cell part. Catalytic activity and binding were the major subcategories in the biological process classification. A total of 1073 DEGs and 2293 DETs were enriched according to the KEGG database (Supplemental Table 9). The DEGs and DETs were divided into 5 categories, including cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems (Supplementary Figure S2 and S3). Among the DEGs, the most common term was plant hormone signal transduction related to environmental information processing. Among the DETs, the most common term was biosynthesis of amino acids related to metabolism. There were 3154 DEGs and 6222 DETs annotated in the KOG database (Supplementary Table 9). DEGs and DETs were most enriched in the eggNOG database, showing numbers of 5299 and 10,140, respectively (Supplementary Table 9).
Statistical classification of Gene Ontology (GO) annotation for the differentially expressed genes. The abscissa represents the GO classification, left of the ordinate represents the percentage of the number of genes, and right represents the number of genes
Statistical classification of Gene Ontology annotation for the differentially expressed transcripts
Selection of candidate genes for olecranon-type traits
First, we selected 38 genes within 1 Mb up- or down-stream of the main GWAS locus for olecranon-type traits (Supplemental Table 6). The expression of 38 genes in round-type fruits and olecranon-type fruits is shown in Supplemental Tables 10, among which 8 were differentially expressed (false discovery rate (FDR) < 0.01). Eight DEGs were selected as candidates affecting olecranon-type traits, including 4 highly expressed genes in olecranon honey peach (LOC18775282, LOC18772209, LOC18773929, LOC18773401) and 4 highly expressed genes in round peach (LOC18773079, LOC18773525, LOC18773067, LOC18775244) according to the expression pattern shown in the heatmap (Fig. 6 A).
Expression level heatmap of selected differentially expressed genes (DEGs) and differentially expressed transcripts (DETs). (A) DEGs within 1 Mb up- or down-stream of 26,924,482 bp. (B) DETs within 1 Mb up- or down-stream of 26,924,482 bp. A-1; A-2; A-3: Olecranon honey peach; B-1; B-2; B-3: round peach
We selected 80 transcripts with 1 Mb up- or down-stream of the main GWAS locus for olecranon-type traits. Among the 80 transcripts, 39 were not expressed or showed very low expression in the round-type fruits and olecranon-type fruits and 11 transcripts (FDR < 0.01) were differentially expressed (Supplemental Table 11). According to expression pattern in the heatmap (Fig. 6B), 7 genes were highly expressed in olecranon honey peach and 4 genes were highly expressed in round peach. According to sequence comparison, ONT.13798.5 showed no corresponding gene (Table 3). Thus, we selected 10 genes and one new gene (ONT.13798.5) as candidates affecting olecranon-type traits. Eight genes were identical to the candidate genes in Fig. 6 A, namely LOC18775282, LOC18772209, LOC18773929, LOC18773079, LOC18773525, LOC18773067, LOC18775244, LOC18773401, and LOC18785015. The other 2 genes were LOC18772013 and LOC18772236. ONT.13798.5, a new gene, was located on scaffold_6, and its nucleic acid sequence is shown in Supplemental Table 12.
Table 3 Basic information on corresponding genes in the differential transcriptome
We next selected 11 DEGs that may affect olecranon-type traits, which are shown in Table 3. ONT.13798.5 had the shortest length of 513 bp, and LOC18772236 showed the longest length of 4500 bp (Table 3).
Validation of expression patterns of candidate genes
To confirm the accuracy of the ONT full-length transcriptome sequencing results and validate the expression patterns of the candidate genes, RT-PCR of the 11 selected genes was performed (Table 3). The sequences of the gene-specific primers are shown in Table 4. The relative expression levels of all 11 genes were consistent with the results of ONT full-length transcriptome sequencing analysis (Fig. 7). Complete correlations between RNA-Seq data and RT-PCR data were also identified in most of the tested genes (R = 1). Therefore, the qRT-PCR data confirmed the reliability of the transcriptome data.
Table 4 Primers used for quantitative real-time RT-PCR
RT-PCR validation of expression levels of 11 differentially expressed genes identified by RNA-sEq. Olecranon honey peach denoted by A. Round peach denoted by B
Bud mutations (bud sports) are common in many plant species including fruit trees [3], and somatic genetic variation can lead to phenotypic alterations in plants [8]. There are obvious differences in the shape of round peach and olecranon honey peach (Fig. 2). Based on quality indicators, the round peach remarkably differs from olecranon honey peach, particularly in the fruit type (Table 2). These differences indicate that although these peaches are grown on a similar tree, they do not belong to the same variety. In this study, we evaluated fruit type-related indicators, including weight, horizontal and vertical diameters, fruit type index, and the part of the fruit that resembles an eagle's beak. The bud mutant variant of round peach was typically smaller than that of olecranon honey peach based on these indicators. The olecranon honey peach is a new variety with high consumer value in China [4]. However, not all bud sports have a regulatory effect on the fruit type, such as the Beni Shogun apple mutation. This mutation reportedly affects traits such as skin coloration, fruit softening, and starch hydrolysis, whereas the loss of acidity, sugar accumulation, and weight appear to be unaffected by the mutation [9].
GWAS can be used to identify the genes or quantitative trait loci underlying complex traits, such as in melon [5], cotton [10], rapeseed [11], grape [12], and peach [5, 7, 13]. In previous studies of peach fruit shape, some candidate genes for forming the flat peach have been identified, such as ppa003772m [5] and ppa025511m [6], but olecranon-type traits have not been evaluated. In the current study, the olecranon-type trait was located close to the candidate genes identified by linkage analysis. ppa003772m and ppa025511m were not included within 1 Mb up- or down-stream of the main GWAS locus for olecranon-type traits (Supplemental Table 6). Thus, these genes related to eagle beak traits are not related to ppa003772m and ppa025511m. However, a recently reported candidate gene (PpCAD1) for flat-shaped peach at 26,924,482 bp (Supplemental Table 6) is annotated by constitutively activated cell death in GENES1 [5]. PpCAD1 did not differ in the round peach and olecranon honey peach according to ONT full-length sequencing.
Furthermore, to better understand the formation of olecranon-type traits in peach at the molecular level, combined ONT full-length sequencing was performed to comprehensively analyze the transcriptome profile in the olecranon honey peach and round peach. This method overcomes technical limitations by accurately predicting full-length splicing subtypes in second-generation sequencing short-read data [14]. ONT full-length sequencing technology has been used to sequence plants and animals, such as polar bear [15], Hiptage benghalensis [16], zebrafish [17], and Susscrofa [18]. Compared with previous sequencing results [19, 20], the ONT full-length sequencing produced not only longer reads, but also a higher comparison ratio of reads.
Fruit ripening is a developmental process, and fruit shape depends on events that occur early in fruit development [15, 21, 22]. In flat peach, as the typical peach evaluated in fruit shape research, 28 genes related to fruit shape have been identified [2]. Based on their functional annotations and coding sequences, 3 genes were identical among the 11 identified candidate genes, indicating similar mechanisms in regulation of the olecranon-type trait and flat-type trait. Two of three genes are annotated as non-specific lipid-transfer protein (LOC18773079, LOC18773525) and the other is annotated as leucine-rich repeat receptor-like protein (LOC18772236). As shown in Fig. 7, high expression of LOC18773079, LOC18773525, and LOC18772236 inhibits the formation of olecranon. In tomato, four fruit shape genes have been identified [22], named as SUN, OVATE, FAS, and LC. In eggplant, SUN and OVATE exert conserved functions in controlling the fruit shape [23, 24]. FAS expression leads to a flat or oxheart shape, and LC expression controls the meristem size and locule number. No genes belonged to the FAS and LC types among the 11 candidate genes. SUN and OVATE are related to fruit elongation. According to GO annotation, LOC18775244 is related to translational elongation, and its low expression is favorable for the formation of olecranon (Fig. 7). These results contrast those determined by Jian [2] for the flat-type peach, who found that the mechanisms regulating fruit shape between tomato and peach are similar, and those regulating the shape of olecranon and flat peach may differ.
In addition, according to NR annotation, LOC18775282 is related to CBL-interacting protein kinases and highly expressed in olecranon peach. In a study of cotton fiber elongation [25], CBL-interacting protein kinases were also linked to fiber length. LOC18772013 is related to B3 domain-containing protein. As a member of the B3 DNA binding superfamily, auxin-responsive factors can bind to auxin-responsive elements of induced genes and further regulate plant metabolism [26]. Auxin plays an important role in plant development at the cellular level, such as cell extension, division, and differentiation [27]. LOC18773067 was annotated as RING-H2 finger protein ATL74. RING-H2 finger protein plays key roles in regulating growth/developmental processes, hormone signaling, and responses to biotic and abiotic stresses in plants [28]. U-box domain-containing protein and serine/threonine-protein also involved in olecranon formation.
The shape of fruit is an important characteristic affecting consumer decisions. The shape of the top of olecranon honey peach fruit resembles the beak of an eagle, which is popular among consumers. No studies have been performed to examine the olecranon-type trait. In this study, loci associated with the olecranon-type trait were examined by GWAS. The gene loci related to the olecranon-type trait was 26,924,482 bp in NC_034014.1. To further identify olecranon-type trait genes, we performed ONT full-length transcriptome sequencing of young fruits of 3 olecranon honey peaches and 3 round peaches. The sequencing data of each sample reached up to 7.10GB, with 14,360 genes and 23,167 transcripts expressed in both olecranon and round peach. Eleven DEGs were selected as candidate genes, six of which were highly expressed in olecranon peach and named as LOC18775282, LOC18772209, LOC18773929, LOC18772013, LOC18773401, and ONT.13798.5. Five of these were highly expressed in round peach and named as LOC18773079, LOC18773525, LOC18773067, LOC18775244, and LOC18772236. The genes were within 1 Mb up- or down-stream of the main GWAS locus for olecranon-type traits. Notably, ONT.13798.5 is a new gene, and LOC18775244 is related to translational elongation. The formation of olecranon traits is a complex process, which has been shown to be related to cellular signal transduction and plant hormones. These results provide a genetic basis for the olecranon-type trait and can be used to further evaluate the eagle beak shape of olecranon honey peach.
Plant materials and sample collection
A total of 456 peach accessions were collected in China for GWAS and are distributed in the North (315), Northwest (26), Qinghai-Tibet Region (3), South (83), Yangtze River Basin (22), and Yunnan-Guizhou Plateau (7) regions in China (Fig. 1 A). Detailed information on these samples is shown in Supplementary Table 1. The collected peach leaves were immediately frozen at − 80 ℃ for DNA extraction in liquid nitrogen.
In addition, bud mutation was found on a tree of olecranon honey peach in Lianping in northern Guangdong province, China, which was used for full-length transcriptome studies. One branch of this tree produces normal round-shaped fruit, whereas other branches produce olecranon-shaped fruit (Fig. 2). The tree was maintained in the same manner as other peach trees in the orchard. The bud sport was stable for at least 3 years. The round-type fruit was obtained by selecting the branch with the bud sport. To avoiding sampling of chimeras, we selected olecranon honey peaches produced far from the mutated branch. After the olecranon traits had formed, the tops of young fruits (young fruit development) were immediately frozen in liquid nitrogen and stored at − 80 ℃ for full-length transcriptome sequencing and RT-PCR.
Ripening fruits were selected by experienced growers (fruit hanging time of approximately 75 days) and quickly transported to a well-ventilated, sterile laboratory to count the olecranon type traits. The olecranon trait was defined as the tip of a peach shaped like an eagle's beak.
Genomic DNA was extracted from fresh young leaves using the cetyltrimethylammnonium bromide method [29]. At least 5 µg of genomic DNA was used for each accession to construct sequencing libraries according to the manufacturer's instructions (Illumina, San Diego, CA, USA). The libraries were sequenced on an Illumina HiSeq X Ten or NovaSeq 6000 platform, generating 150-bp paired-end reads. Raw sequencing reads were trimmed using Btrim software to filter out low-quality bases and sequences using the following parameters: remove adapter reads; pair-end reads with > 10 % "N" bases; average base-quality of less than 10; quality score of 3′ bases < 50.
SNP and InDel calling
High-quality reads were compared with the peach "NCBIv2" genome using Burrows-Wheeler Aligner (version 0.7.10-r789) with the following parameters: bwa mem 4 –k 19 -M. Next, the alignment bam/sam files were subjected to a series of processing and filtering steps, including position sorting, repeated read marks and removal of local realignments, and basic quality recalibration using Picard (https://sourceforge.net/projects/picard/) and GATK software (v3.8) [30]. SNPs and InDels were filtered by using the HaplotyPecalller tool in GATK software package. SNPs and InDels were annotated based on the Prunus persica (assembly Prunus_persica_NCBIv2) genome using snpEff software [31], and SNPs were categorized into intergenic regions, upstream or downstream regions, and exons or introns.
LD analysis and GWAS
LD was calculated between each pair of SNPs on the same chromosome using PLINK2 software [32]. The values of the squared correlation coefficient (r2) and significance of any LD detected between polymorphic sites were analyzed in all chromosomes with a 1000-kb window. LD blocks were defined based on the following parameters [33]: minor allele frequency > 0.05, r2, ldwindow 999,999, ld-window- r2 0. GWAS of the leaves was based on high-confidence SNPs (minor allele frequency > 0.05, INT > 0.8) from 456 accessions. The Q-value was calculated with ADMIXTURE [34] software, which represents the population structure. The K-value was calculated using SPAGeDi [35] software, which represents the genetic relationship between samples. FaST-LMM [36] was used to test the associations between samples. Both the Q- and K-value were used in TASSEL. The P-value of each SNP was calculated, and –log10P > 7 was defined as the suggestive threshold and genome-wide control threshold.
RNA extraction and transcriptome sequencing
RNA was extracted from six peach samples representing three biological replicates of two peach cultivars. RNA extraction was performed using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA concentration and purity were analyzed with a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Three biological replicates were evaluated for each sample. For ONT RNA sequencing, individual cDNA libraries were constructed from total RNA samples with the SQK-LSK109 RNA Library Prep Kit for Oxford Nanopore (ONT) according to the manufacturer's instruction. The cDNA libraries were sequenced using a PromethlON sequencer (ONT). Primer annealing, reversing transcription into cDNA and plus switch oligo, synthesis of complementary strands, DNA damage and end repair, and magnetic bead purification were performed. Finally, a sequencing linker was added.
Analysis of ONT sequencing data
Raw ONT sequencing reads were filtered to remove low-quality reads (Qscore ≤ 7 and minimum read length ≤ 500 bp). Full-length non-chimeric transcripts were obtained. Clusters of full-length non-chimeric transcripts were obtained after mapping to the reference genome with mimimap2, and consensus isoforms were obtained by polishing within each cluster by pinfish. Redundant consensus sequences were removed by mapping the consensus sequences to the reference genome using minimap2 [37]. Mapped reads were further collapsed by using cDNA Cupcake package with coverage ≤ 0.85, identity ≤ 0.90, and merge sequences showing differences only in the 5′ exons.
For comprehensive functional annotation, the full-length transcripts were analyzed by BLAST against public protein databases, including the NR (NCBI non-redundant protein sequences) database, Pfam (Protein family) database, KEGG database [38], KOG/COG of proteins database [39, 40], Swiss-Prot database [41], and GO database [42].
Quantification of gene/transcript expression levels and differential expression analysis
Full-length reads were mapped to the reference transcriptome sequence. Reads with a match quality > 5 were further quantified. Expression levels were estimated by reads per gene/transcript per 10,000 mapped reads. Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted using the edgeR program package with the one scaling-normalized factor. Differential expression analysis of two samples was performed using the EBSeq R package. The resulting FDR was adjusted using the posterior probability of being differentially expressed. FDR < 0.01 and fold-change ≥ 2 were used as thresholds for significantly different expression. The identified DEGs and DETs were analyzed for GO and KEGG enrichment.
Validation of gene by quantitative real-time RT-PCR
RNAs were isolated from the top of the olecranon honey peach and round peach. Two biological replicates were included. For quantitative real-time RT-PCR analysis, reverse transcription was performed using the TUREscript 1st Stand cDNA SYNTHESIS Kit with cDNA Eraser (Aidlab, Beijing, China) according to the manufacturer's instructions. Gene- or transcript-specific primers were designed with Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA). qPCR was performed on an Applied Biosystems 7500 RealTime PCR System using 2×SYBR® Green qPCR Master Mix (Qiagen, Hilden, Germany). LOC18778082 was selected as a reference gene. The relative expression level was calculated using the following formula: fold-change = 2−△△CT.
Data are expressed as the mean ± standard deviation of three replicates. Significant differences between the means of different parameters were calculated by Duncan's multiple-range test using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to indicate statistically significant results.
The original datasets analyzed in the current study are available in the NCBI Sequence Read Archive (SRA). The date of Olecranon peach Nanopore transcriptome sequencing link to obtain data is https://www.ncbi.nlm.nih.gov/bioproject/PRJNA754788. The date of GWAS link to obtain data is https://www.ncbi.nlm.nih.gov/bioproject/PRJNA758752 .
genome-wide association study.
NR:
NCBI non-redundant protein sequences.
Protein family.
Kyoto Encyclopedia of Genes and Genomes.
Cluster of Orthologous Groups.
Gene Ontology.
DEGs/DETs:
Differentially expressed genes/transcripts.
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We would like to thank guangdong deciduous fruit industry technology innovation team for their technical support and technical advice.
The sample collection was supported by Guangzhou Key Laboratory for Research and Development of Crop Germplasm Resources and Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology. Funding for study design, data analysis, interpretation, and manuscript writing was supported by the Key-Area Research and Development Program of Guangdong Province (2019B020238003), the Guangzhou Key Laboratory for Research and Development of Crop Germplasm Resources (202002010010), and the Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology (2021B1212040013) and the Guangdong Provincial Agricultural Science and Technology Special Commissioner Precision Poverty Alleviation Rural Rejuvenation Project (KA1901003).
Jianliang Liu and Yao Bao contributed equally to this work.
College of Light Industry and Food, Zhongkai University of Agriculture and Engineering, 510225, Guangzhou, Guangdong, China
Jianliang Liu, Yao Bao, Qin Wang & Huifan Liu
Guangzhou Key Laboratory for Research and Development of Crop Germplasm Resources, Zhongkai University of Agriculture and Engineering, 510225, Guangzhou, China
Jianliang Liu
Modern Agriculture Research Center, Zhongkai University of Agriculture and Engineering, 510225, Guangzhou, Guangdong, China
Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology, Guangzhou, Guangdong, China
Yao Bao, Qin Wang & Huifan Liu
College of Environmental Science and Engineering, Zhongkai University of Agriculture and Engineering, 510225, Guangzhou, Guangdong, China
Yuming Zhong
Yao Bao
Qin Wang
Huifan Liu
J.-L.L., H.-F.L. conceived the project and supervised the study. Y.B. performed the experiments and analyzed the data. Y.-M.Z. and Q.W. contributed reagents/materials/ analysis tools. Y.B. and H.-F.L. wrote the article. All authors discussed the results and commented on the manuscript. The author(s) read and approved the final manuscript.
Correspondence to Huifan Liu.
The sampling shall comply with the regulations of the people's Republic of China on wildlife protection, and the samples were collected on private land with permission from the landowner. all the voucher specimens were stored in Guangzhou Key Laboratory for Research and Development of Crop Germplasm Resources in China.
Additional file 1: Supplemental Table S1.
List of 456 peach accessions analyzed in this study. The code, name, scientific name, area, and plantation for 456 re-sequenced peach accessions. Olecranon indicates whether the olecranon-type trait is present; 1 means yes, 0 means no, NA means no statistics. Supplemental Table S2. Sequence information on the genomes of 456 peach accessions. N (%) indicates the percentage of failed sequencing nucleotides. Q30 (%) indicates the percentage of 99.9% well-sequenced nucleotides. Ave_depth indicates average covering depth of sample. Cov_ratio_*(%) indicates the percentage of bases at or above a given depth of coverage as a percentage of the total base number of the reference genome. Properly mapped (%) indicates the percentage of the sequenced data mapped to the reference genomes. Supplemental Table S3. Single-nucleotide polymorphism statistics detected between sample and reference genome. Supplemental Table S4. Statistics of single-nucleotide polymorphism annotation results of samples Supplemental Table S5. Statistics of insertion-deletion annotation results of samples. Supplemental Table S6. Statistical table of genes within 1Mb upstream and downstream of main genome-wide association study sites of olecranon type traits. Supplementary table S7. Clean data statistical table. After the original fastq data were further filtered for short reads and low-quality reads, the total clean data were obtained. A-1; A-2; A-3: Olecranon honey peach; B-1; B-2; B-3: Round peach. Supplementary table S8. Full-length sequence and comparison with reference transcriptome results. Supplementary table S9. Statistical table of the number of annotated genes and transcripts. Supplementary table S10. Statistics of gene expression in round peach and olecranon peach.Supplementary table S11. Statistics of transcripts expression in the round peach and the olecranon peach. The nucleic acid sequence of the ONT gene is as follows.
Additional file 2: Supplemental Figure S1.
Heat map of expression correlation between two samples. Clean data statistical table. After the original fastq data were further filtered for short reads and low-quality reads, the total clean data were obtained. A-1; A-2; A-3: Olecranon honey peach; B-1; B-2; B-3: Round peach.
KEGG classification map of differentially expressed genes. The ordinate is the name of the KEGG metabolic pathway and abscess is the number of genes annotated to the pathway and its proportion to the total number of genes annotated.
KEGG classification map of differentially expressed transcripts.
Liu, J., Bao, Y., Zhong, Y. et al. Genome-wide association study and transcriptome of olecranon-type traits in peach (Prunus persica L.) germplasm. BMC Genomics 22, 702 (2021). https://doi.org/10.1186/s12864-021-08017-y
Olecranon honey peach
Full-length transcriptome sequencing


text:
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redpajama_set_name: RedPajamaGithub


text:
ACCEPTED

#### According to
The Catalogue of Life, 3rd January 2011

#### Published in
in Agerer & Bougher, Aust. Syst. Bot. 14(4): 608 (2001)

#### Original name
Tomentella subamyloidea Agerer, 2001

### Remarks
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text:
Hitlist - Titles w/author : Lunar Science Institute New Keyword search Help
Hitlist - Titles w/author : Lunar Science Institute
1. Abstracts of papers submitted to the fifth Lunar Science Conference : Part I (1974)
Lunar Science Conference; United States. National Aeronautics and Space Administration; Lunar Science Institute
Copies: 1 Lunar Science Conference. Houston,Tx, (5th : 1974) (Conference) 523.33
2. High pressure physics and planetary interiors : proceedings of the conference held at the Lunar Science Institute, Houston, Texas, March 1-3, 1972 (1972)
Runcorn, S.K.; Lunar Science Institute
Copies: 1 Runcorn, S.K. (Stanley Keith), 1922-1995 (Author) 523.031
3. Impact and explosion cratering : planetary and terrestrial implications : proceedings of the Symposium on Planetary Cratering Mechanics, Flagstaff, Arizona, September 13-17, 1976 (1st ed) (1977)
Symposium on Planetary Cratering Mechanics; Roddy, D.J.; Pepin, R.O.; Merrill, R.B.; Lunar Science Institute; Geological Survey (U.S.). Branch of Astrogeologic Studies
Copies: 1 Symposium on Planetary Cratering Mechanics. Flagstaff, Ariz, (1976) (Conference) 523.4-834.4
4. Index to the proceedings of the Lunar and Planetary Science Conferences, Houston, Texas, 1970-1978 (1979)
Masterson, A.R.; Lunar Science Institute; Lunar and Planetary Science Conference
Copies: 2 Masterson, A.R. (Author) 00163/1
5. Proceedings of the eighth Lunar Science Conference. Vol.1: The Moon and the inner solar system (1977)
Lunar Science Conference; Merrill, R.B.; Lunar Science Institute
Copies: 1 Lunar Science Conference. Houston,Tx, (8th : 1977) (Conference) 00163/1
6. Proceedings of the eighth Lunar Science Conference. Vol.2: Petrogenetic studies of mare and highland rocks (1977)
7. Proceedings of the eighth Lunar Science Conference. Vol.3: Planetary and lunar surfaces (1977)
8. Proceedings of the eleventh Lunar and Planetary Science Conference. Vol.1: Igneous processes and remote sensing (1980)
Lunar and Planetary Science Conference; Merrill, R.B.; Lunar Science Institute
Copies: 1 Lunar and Planetary Science Conference. Houston,Tx, (11th : 1980) (Conference) 00163/1
9. Proceedings of the eleventh Lunar and Planetary Science Conference. Vol.2: Meteorite and regolith studies (1980)
10. Proceedings of the eleventh Lunar and Planetary Science Conference. Vol.3: Physical processes (1980)
11. Proceedings of the fifth Lunar Science Conference. Vol.1: Mineralogy and petrology (1974)
Lunar Science Conference; Lunar Science Institute; Lyndon B. Johnson Space Center
12. Proceedings of the fifth Lunar Science Conference. Vol.2: Chemical and isotope analyses (1974)
13. Proceedings of the fifth Lunar Science Conference. Vol.3: Physical properties (1974)
14. Proceedings of the fourth Lunar Science Conference. Vol.1: Mineralogy and petrology (1973)
15. Proceedings of the fourth Lunar Science Conference. Vol.2: Chemical and isotope analyses, organic chemistry (1973)
16. Proceedings of the fourth Lunar Science Conference. Vol.3: Physical properties (1973)
17. Proceedings of the ninth Lunar and Planetary Science Conference. Vol.1: Petrogenetic studies: the Moon and meteorites (1978)
Copies: 1 Lunar and Planetary Science Conference. Houston,Tx, (9th : 1978) (Conference) 00163/1
18. Proceedings of the ninth Lunar and Planetary Science Conference. Vol.2: Lunar and planetary surfaces (1978)
19. Proceedings of the ninth Lunar and Planetary Science Conference. Vol.3: The Moon and the inner solar system (1978)
20. Proceedings of the second Lunar Science Conference. Vol.1: Mineralogy and petrology (1971)
Lunar Science Conference; Levinson, A.A.; Lunar Science Institute
Copies: 2 Lunar Science Conference. Houston,Tx, (2nd : 1971) (Conference) 00163/1

redpajama_set_name: RedPajamaCommonCrawl


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> perfectly well with the 19990607 version.
server will still be active on its previous port.
Next message: Yves Lafon: "Re: remote jigadmin client?"
Previous message: Qiang Lv: "remote jigadmin client?"
In reply to: Anthony B. Coates, ESL +61 2 9373 7972: "Re: Changing to port 80 under NT"


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Two books of Ernst F. Schumacher have been published in Hungarian (Small is Beautiful and Good Work) They provide a foundation to waht is called alternative economics and present a new approach to humanity, rationality and economy. Related Articles No related articles.


text:
Union To Play New York Red Bulls In Open Cup Quarterfinals On July 21
July 8, 20156:16PM EDT
Philadelphia Union will play New York Red Bulls on Tuesday, July 21, at Red Bull Arena in the quarterfinals of the 2015 Lamar Hunt U.S. Open Cup, U.S. Soccer announced Wednesday. The match is set to kick-off at 4 p.m. due to the International Champions Cup taking place at Red Bull Arena later that night.
"We are happy that it's been decided to play this match on the originally designated U.S. Soccer date of July 21, but are very disappointed about the 4 p.m. kick off for our fans that were planning to travel," Union CEO and Operating Partner Nick Sakiewicz said. "We have worked cooperatively with U.S. Soccer to obtain a block of tickets and will be providing round-trip bus transportation to any Union fans that wish to attend the game. There is a limited supply of 500 tickets that will be made available on a first-come, first-serve basis."
The Union reached the quarterfinals of the U.S. Open Cup for the third in time in four years by defeating Rochester Rhinos 0-0 (3-1) in the Fourth Round, and D.C. United, 2-1, in the Round of 16. It's the first time the Red Bulls have reached the quarterfinals since 2011 and they did so by defeating a pair of NASL teams, Atlanta Silverbacks 3-0 in the Fourth Round, and New York Cosmos 4-1 in the Round of 16.
U.S. Soccer also announced the three other quarterfinal fixtures last week. Chicago Fire will host Orlando City SC on Wednesday, July 22. The winner of that game will travel to play the winner of the Union-Red Bulls match in the semifinals. On the other side of the bracket, Real Salt Lake will host L.A. Galaxy on Tuesday, July 14 and Sporting Kansas City will host Houston Dynamo on Tuesday, July 21.
The semifinals are then slated for Aug. 11-12, with the final coming on either Sept. 29 or 30.
The Union will be providing bus transportation for the first 500 fans interested in traveling to Harrison, N.J. for the match. The road trip, which includes round trip bus transportation, a game ticket and light food and beverage will be $10 and can be purchased through www.philadelphiaunion.com/promo. The promo code will be available to Season Ticket Members, who will receive an email, or can contact Union fan services to purchase. Ticketing for the general public will go on sale Friday, July 10 at 10 a.m.


text:
Q: Removing children entries from parent causes error on SaveChanges I am getting the following error:

The association between entity types 'Docket' and 'DocketLine' has been severed but the foreign key for this relationship cannot be set to null. If the dependent entity should be deleted, then setup the relationship to use cascade deletes.

The issue comes about because I have a Docket (header) than has multiple children (DocketLines), and I am doing an update where I am adding new Lines to the docket header, and I am just adding those new DocketLines to the Docket.DocketLines collection (which works fine). But when I attempt to remove a DocketLine from the same collection using Docket.DocketLines.Remove(deletedLine), then this generates the error message above. Any idea why?
I had to change my code to remove the Lines directly from the _context.DocketLines.Remove/RemoveRange(...) collection in the end, and this works, but it seems odd that I would add new items to a child collection to insert new DocketLines, but couldn't remove items from that same collection to remove DocketLines.

A: Inside the DbContext/OnModelCreating method, comment the OnDelete behaviour of the entity that is giving you that problem:

That will allow you to avoid that problem.

redpajama_set_name: RedPajamaStackExchange


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Key things you might want to know!
SkoolBot is a bot that you converse with using normal, natural questions and commands (e.g., "Do I have any math homework this week?"). Skoolbot then connects with your Google Classroom and gets you immediate answers that you can use to plan your day. It focused on questions about homework, grades, and upcoming tests and quizzes.
It also makes communicating with teachers and classmates really simple, without having to remember people's email addresses (e.g., "email my chem teacher", and "ask my history class did anyone see my blue backpack?").
Anyone with a current Google Classroom account from their school can use SkoolBot.
SkoolBot is FREE - how awesome is that?
How do I access SkoolBot?
You can access Skoolbot via Facebook Messenger or kik. Just find Skoolbot in the bot store or marketplace, add it to your buddy list, and start talking to it!
We are in beta testing right now, but you can request to be part of the beta testers by signing up in the form below. If we still have room, we'll be happy send you an invite code!
Sign Up To Request An Invite Code!
Have A Question Or A Problem? Just Reach Out To Us!


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barcelona.cat shortcuts
BCN guide
BCN map
Opting for bicycles
Type of route
The bicycle lane network
Public bicycle scheme
Travelling by bicycle
Respecting other road users
Respecting the right of way
Watching where you park
Cycling with others
Bicycles on public transport
Fostering the use of bicycles
The pick of what's on
Bicing
Bicing is a public bike-sharing scheme that makes 6,000 mechanical and 300 electric bikes available to the general public for journeys around the city. This individual public-transport system makes it easier for the general public to make regular journeys by bicycle, in a sustainable, healthy and economical way.
Bicing is fully integrated into the city's transport system and is easy to use in combination with other means of public transport. This is because it has been designed to ensure public transport access close to bicycle pick-up and drop-off points, based on intermodal criteria for connections between bicycles and other means of transport, as well as proximity to other areas that generate and attract travelling.
The system operates on the basis of a personal, non-transferable, subscription card bearing the user's name. This card identifies the user in the system and must be used at the pick-up and drop-off points, to take out and return bicycles. Bicing users must be over the age of 16.
Bicing website
Ecology, Urban Planning and Mobility Area


I need to configure Chiliproject with LDAP user authentication, that already existing users in LDAP could login to the chili. As I realized from documentation, after users will login with their LDAP auth data corresponding users will be registered (on fly created) in Chili database so that administrators could manage them later.
Could you please describe how I can reach it. I've tried the steps that have been described in documentation and no success.
The Account and Password are the LDAP account ChiliProject will use to connect to your LDAP server. The login field is the LDAP field that ChiliProject will try to match to find the LDAP entry for your user. The first- and surname and e-mail fields are the names of the LDAP fields ChiliProject will use to auto-fill those fields in the ChiliProject user.
This is the page you need.


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What is a Content Studio Leader? [Interview]
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"ReachMobi is far from a typical workplace. To get a better understanding of just how different of a workplace culture the company has, we sat down with Morgan and Mark, two of our Content Studio Managers, to get a feel for what they do and how it differs from other companies.
The Content Studio
To start, we ask about what the "content studio" meant to them. Morgan, a Content Studio Manager, answers, "It's kind of funny, the way you're putting it makes it sound so distant and different. It's really just the way we work. It's a concept, it's the way we divide our business."
Mark, a Content Studio Manager for ReachMobi, adds, "There's the products we offer, like apps and launchers, and each of them has an umbrella, if you will, which is the content studio. Each studio manages its own product, and almost acts like its own little company within the company."
"It's got a lot to do with entrepreneurial spirit"
– Morgan, Content Studio Manager
Morgan tells us. "It's about developing long-term strategy naturally, and helping our people naturally fill out leadership roles. It's a shot at a real-life crucible to prove yourself and your product."
We ask Mark and Morgan what they think the most important fields of expertise for someone to become a content studio head are.
Morgan is quick to tell us, "Anyone who wants to lead needs to be a fast learner. Having knowledge of a wide variety of subjects, of pretty much every part of the business, is required. I think it's most important that any candidate is analytical and has a great grasp on data."
"I think that's all true, and I think being direct, being aggressive in how you market your product is also important," Mark says. "It's worth noting that this position is largely about developing our people to be the best they can be. We're all about building our employees up, and the content studio is a huge part of that."
Finally, we ask Mark and Morgan what they find most rewarding about their positions as content studio heads.
Mark begins, "For me, it's about the competition. It's not a secret that I'm competitive: I want our products to be the best. I want my team's products to be the best. The satisfying thing, for me, is celebrating the big win when we crush our competitors."
Morgan tells us that, for her, "Growing the business is the biggest reward. The success alone is what drives me. I love to think ahead of the data, pull the relevant information from it, and use that to get us ahead and grow our business."
"We're all about building our employees up, and the content studio is a huge part of that."
– Mark, Content Studio Manager
3 Great Reasons to Consider a Job in Tech
Careers & Company
Working in the App World
Searching for Android Developers!


3/18/2005 12:00:00 AM - Advanced Technologies | Although silicone technology has been around for over 5 decades, this unique material continues to find usage in new applications. Its various functionalities allow its usage as fuel resistant gaskets, inert implantable devices in the body, coatings for Atomic Oxygen protection in space, and for interocular lenses for cataract disease. This paper will examine various silicones as effective encapsulants and lenses for High Brightness LEDs (HB LEDs). Physical and optical characteristics will be tested to determine which materials may prove to be the best.


Meet the Prince Royal! Location and luxury are what we think of when we see this gorgeous houseboat, located on the famous Prinsengracht, across from the Anne Frank house. Video. And a second video from guests with a passion!
It's a stylish and comfortable floating residence in the center of the Jordaan area. Decorated with taste and attention by a professional interior designer. The Prince Royal is the place to be for an unique nautical experience in downtown Amsterdam. You have a direct view on the Prinsengracht and the landmark Westerchurch.
The living area is bright and shining, spacious, comfortable and light. The characteristic wooden floor in combination with design furniture gives this area a luxury own identity. There are 2 characteristic bedrooms and 2 full bathrooms in the back and front of the ship. The bedrooms are a little lower but that is part of the history and charm of this houseboat. Both the bedrooms have two big double beds with two separate mattresses next to each other (160cm and 180 cm X 200cm), both with a flat screen TV. Romantic and comfortable!
Both bathrooms have a towel radiator and under the floor heating. The bright and shining bathrooms offers both a modern sink and shower. Bedlinen and towels are provided.
At the outside point of this former freight ship is a nice terrace with a unique view along the canal. You will find a lovely large terrace, with chairs and plants. White swans will pass by together with the canal boats. At the other side of the canal you see the Anne Frank house and the famous Westerchurch. A video has been made from the surroundings last year: You are in the middle of historic Amsterdam but because of the water and the impressive historic canal houses around you the atmosphere is relaxed and peaceful.
The wheelhouse however offers you the best and most exclusive possibility to enjoy the spectacular surrounding of this houseboat. The wheelhouse is reachable from the inside and is higher than the rest of the boat. In this former Captain's area is a luxury place to sit and enjoy for instance a glass of the free wine from the bar with wine fridge!
Our group of four has stayed on stylish and spacious house boats in Amsterdam before, but the Prince Royal is not only a house on the water, it's an actual boat, extremely charming and very original. Beware that you cannot stand upright in both cabins, but you soon don't care, because it feels adventurous and romantic. We loved the terrace to watch the tourists and boats around us. At night the place is surprisingly quiet and we slept like in heaven.
Nous avons été bien accueilli par Rachel qui nous a donné toutes les indications nécessaires.
La péniche est très confortable, bien chauffée, propre. Nous avons apprécié les moments de repos pendant le thé dans la cabine de pilotage !
La péniche est très bien située : les principaux points d'intérêt sont tous à moins de 30 minutes de marche à pied.
Cela donne envie d'y retourner !!
Such an amazing experience. The perfect place to stay in Amsterdam! We can't recommend it enough. Thank you!
Smoking only on the terrace.