Genomic Analysis of the Glutathione S-Transferase Family in Pear (Pyrus communis) and Functional Identification of PcGST57 in Anthocyanin Accumulation.

Anthocyanin accumulation in vacuoles results in red coloration in pear peels. Glutathione S-transferase (GST) proteins have emerged as important regulators of anthocyanin accumulation. Here, a total of 57 PcGST genes were identified in the European pear 'Bartlett' (Pyrus communis) through comprehensive genomic analysis. Phylogenetic analysis showed that PcGST genes were divided into 10 subfamilies. The gene structure, chromosomal localization, collinearity relationship, cis-elements in the promoter region, and conserved motifs of PcGST genes were analyzed. Further research indicated that glutamic acid (Glu) can significantly improve anthocyanin accumulation in pear peels. RNA sequencing (RNA-seq) analysis showed that Glu induced the expression of most PcGST genes, among which PcGST57 was most significantly induced. Further phylogenetic analysis indicated that PcGST57 was closely related to GST genes identified in other species, which were involved in anthocyanin accumulation. Transcript analysis indicated that PcGST57 was expressed in various tissues, other than flesh, and associated with peel coloration at different developmental stages. Silencing of PcGST57 by virus-induced gene silencing (VIGS) inhibited the expression of PcGST57 and reduced the anthocyanin content in pear fruit. In contrast, overexpression of PcGST57 improved anthocyanin accumulation. Collectively, our results demonstrated that PcGST57 was involved in anthocyanin accumulation in pear and provided candidate genes for red pear breeding.

Genome-wide characterization of simple sequence repeats in Pyrus bretschneideri and their application in an analysis of genetic diversity in pear.

BACKGROUND: Pear (Pyrus spp.) is an economically important temperate fruit tree worldwide. In the past decade, significant progress has been made in pear molecular genetics based on DNA research, but the number of molecular markers is still quite limited, which hardly satisfies the increasing needs of geneticists and breeders. RESULTS: In this study, a total of 156,396 simple sequence repeat (SSR) loci were identified from a genome sequence of Pyrus bretschneideri 'Dangshansuli'. A total of 101,694 pairs of SSR primers were designed from the SSR loci, and 80,415 of the SSR loci were successfully located on 17 linkage groups (LGs). A total of 534 primer pairs were synthesized and preliminarily screened in four pear cultivars, and of these, 332 primer pairs were selected as clear, stable, and polymorphic SSR markers. Eighteen polymorphic SSR markers were randomly selected from the 332 polymorphic SSR markers in order to perform a further analysis of the genetic diversity among 44 pear cultivars. The 14 European pears and their hybrid materials were clustered into one group (European pear group); 29 Asian pear cultivars were clustered into one group (Asian pear group); and the Zangli pear cultivar 'Deqinli' from Yunnan Province, China, was grouped in an independent group, which suggested that the cultivar 'Deqinli' is a distinct and valuable germplasm resource. The population structure analysis partitioned the 44 cultivars into two populations, Pop 1 and Pop 2. Pop 2 was further divided into two subpopulations. Results from the population structure analysis were generally consistent with the results from the UPGMA cluster analysis. CONCLUSIONS: The results of the present study showed that the use of next-generating sequencing to develop SSR markers is fast and effective, and the developed SSR markers can be utilized by researchers and breeders for future pear improvement.

Chromosome level high-density integrated genetic maps improve the Pyrus bretschneideri 'DangshanSuli' v1.0 genome.

BACKGROUND: Chromosomal level reference genomes provide a crucial foundation for genomics research such as genome-wide association studies (GWAS) and whole genome selection. The chromosomal-level sequences of both the European (Pyrus communis) and Chinese (P. bretschneideri) pear genomes have not been published in public databases so far. RESULTS: To anchor the scaffolds of P. bretschneideri 'DangshanSuli' (DS) v1.0 genome into pseudo-chromosomes, two genetic maps (MH and YM maps) were constructed using half sibling populations of Chinese pear crosses, 'Mantianhong' (MTH) × 'Hongxiangsu' (HXS) and 'Yuluxiang' (YLX) × MTH, from 345 and 162 seedlings, respectively, which were prepared for SNP discovery using genotyping-by-sequencing (GBS) technology. The MH and YM maps, each with 17 linkage groups (LGs), were constructed from 2606 and 2489 SNP markers and spanned 1847 and 1668 cM, respectively, with average marker intervals of 0.7. The two maps were further merged with a previously published genetic map (BD) based on the cross 'Bayuehong' (BYH) × 'Dangshansuli' (DS) to build a new integrated MH-YM-BD map. By using 7757 markers located on the integrated MH-YM-BD map, 898 scaffolds (400.57 Mb) of the DS v1.0 assembly were successfully anchored into 17 pseudo-chromosomes, accounting for 78.8% of the assembled genome size. About 88.31% of them (793 scaffolds) were directionally anchored with two or more markers on the pseudo-chromosomes. Furthermore, the errors in each pseudo-chromosome (especially 1, 5, 7 and 11) were manually corrected and pseudo-chromosomes 1, 5 and 7 were extended by adding 19, 12 and 14 scaffolds respectively in the newly constructed DS v1.1 genome. Synteny analyses revealed that the DS v1.1 genome had high collinearity with the apple genome, and the homologous fragments between pseudo-chromosomes were similar to those found in previous studies. Moreover, the red-skin trait of Asian pear was mapped to an identical locus as identified previously. CONCLUSIONS: The accuracy of DS v1.1 genome was improved by using larger mapping populations and merged genetic map. With more than 400 MB anchored to 17 pseudo-chromosomes, the new DS v1.1 genome provides a critical tool that is essential for studies of pear genetics, genomics and molecular breeding.

Identification of candidate genes involved in the sugar metabolism and accumulation during pear fruit post-harvest ripening of 'Red Clapp's Favorite' (Pyrus communis L.) by transcriptome analysis.

BACKGROUND: Pear (Pyrus spp.) is a popular fruit that is commercially cultivated in most temperate regions. In fruits, sugar metabolism and accumulation are important factors for fruit organoleptic quality. Post-harvest ripening is a special feature of 'Red Clapp's Favorite'. RESULTS: In this study, transcriptome sequencing based on the Illumina platform generated 23.8 - 35.8 million unigenes of nine cDNA libraries constructed using RNAs from the 'Red Clapp's Favorite' pear variety with different treatments, in which 2629 new genes were discovered, and 2121 of them were annotated. A total of 2146 DEGs, 3650 DEGs, 1830 DEGs from each comparison were assembled. Moreover, the gene expression patterns of 8 unigenes related to sugar metabolism revealed by qPCR. The main constituents of soluble sugars were fructose and glucose after pear fruit post-harvest ripening, and five unigenes involved in sugar metabolism were discovered. CONCLUSIONS: Our study not only provides a large-scale assessment of transcriptome resources of 'Red Clapp's Favorite' but also lays the foundation for further research into genes correlated with sugar metabolism.

The genetic locus underlying red foliage and fruit skin traits is mapped to the same location in the two pear bud mutants 'Red Zaosu' and 'Max Red Bartlett'.

BACKGROUND: Red-skinned pears are attractive to consumers because of their aesthetic appeal and the antioxidant-associated health benefits provided by the anthocyanins in their red skin. In China, the 'Red Zaosu' (RZS) red bud mutation of the Zaosu (ZS) pear has been used as a parent in Asian pear breeding to generate new cultivars with crispy red fruit and red tender shoots resembling those of the 'Max Red Bartlett' (MRB) pears. RESULTS: In this study, a segregation ratio of 1:1 was observed between plants with red or green shoots in four families with RZS as the only red shoot gene donor parent, suggesting that the red shoot trait of RZS is associated with a dominant gene. Three markers, In1400-1, In1579-1 and In1579-3, were chosen from 22 pairs of indel primers targeting regions in the vicinity of the previously identified red fruit skin locus of MRB and were able to effectively distinguish the eight red shoot plants from the eight green shoot plants. Linkage analysis indicated that the genetic distance between the two marker loci (In1579-1 and In1579-3) and the red shoot locus of RZS were both 1.4 cM, while the genetic distance between the In1400-1 marker and the red shoot locus was 2.1 cM. The physical position of the red locus in RZS should be in the 368.6 kb candidate interval at the bottom of LG4. CONCLUSIONS: The genetic locus responsible for the red tender shoots of RZS was located in the same interval of the red fruit skin gene of MRB, meaning that the bud mutation loci of RZS and MRB may be the same or adjacent to each other, and the red shoot trait and the red fruit skin trait in RZS may be controlled by the same, or a closely linked locus. As a result, breeders could use red shoots as a morphological marker to select for the red-skinned hybrids from RZS families.

Transcriptome and Physiological Analysis Highlight Lignin Metabolism of the Fruit Dots Disordering during Postharvest Cold Storage in 'Danxiahong' Pear.

Pear (Pyrus L.) is one of the most important fruits in the world. Fruit dots are an important trait that affects pear quality. Abnormal fruit dots usually reduce the merchantability of pears. In this research, during cold storage, 'Danxiahong' pear fruit exhibited protrudent fruit dots on the peels. Microscopy system measurement showed that fruit dots size and height on the abnormal fruit peel were bigger and higher than the normal ones. Likewise, scanning electron microscopy observations indicated that the abnormal fruit peel, in contrast to the normal pear peel, exhibited an abnormal cell structure and fruit dots. Physiological analysis showed that the lignin content in abnormal fruit peel was significantly higher than in normal fruit peel. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the top-enriched pathways were mainly associated with lignin synthesis and metabolism. The transcripts of lignin biosynthesis-associated genes were analyzed, and the results showed that the expression of a cascade of structural genes, including PpyPAL, PpyCCR, PpyC3H, PpyC4H, PpyHCT, PpyCAD, PpyLAC, and PpyPOD, was significantly induced in the protrudent peels. Furthermore, the expression of regulatory genes involved in lignin biosynthesis, especially the NAC-MYB-based gene regulatory network, was significantly upregulated in the abnormal peels. Real-time quantitative PCR (RT-qPCR) analysis confirmed the induction of lignin biosynthesis genes. Overall, this research revealed that the abnormal fruit surface was caused by fruit dots disorder during cold storage. This research provides insights into the fine regulation pathways in the prevention of fruit dots protrusion, especially in modulating lignin synthesis and metabolism during postharvest storage.

Transcriptome Analysis Reveals Roles of Sucrose in Anthocyanin Accumulation in 'Kuerle Xiangli' (Pyrus sinkiangensis Yü).

Pear (Pyrus L.) is one of the most important temperate fruit crops worldwide, with considerable economic value and significant health benefits. Red-skinned pears have an attractive appearance and relatively high anthocyanin accumulation, and are especially favored by customers. Abnormal weather conditions usually reduce the coloration of red pears. The application of exogenous sucrose obviously promotes anthocyanins accumulation in 'Kuerle Xiangli' (Pyrus sinkiangensis Yü); however, the underlying molecular mechanism of sucrose-mediated fruit coloration remains largely unknown. In this study, comprehensive transcriptome analysis was performed to identify the essential regulators and pathways associated with anthocyanin accumulation. The differentially expressed genes enriched in Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes items were analyzed. The transcript levels of some anthocyanin biosynthetic regulatory genes and structural genes were significantly induced by sucrose treatment. Sucrose application also stimulated the expression of some sugar transporter genes. Further RT-qPCR analysis confirmed the induction of anthocyanin biosynthetic genes. Taken together, the results revealed that sucrose promotes pear coloration most likely by regulating sugar metabolism and anthocyanin biosynthesis, and this study provides a comprehensive understanding of the complex molecular mechanisms underlying the coloration of red-skinned pear.

Interval mapping for red/green skin color in Asian pears using a modified QTL-seq method.

Pears with red skin are attractive to consumers and provide additional health benefits. Identification of the gene(s) responsible for skin coloration can benefit cultivar selection and breeding. The use of QTL-seq, a bulked segregant analysis method, can be problematic when heterozygous parents are involved. The present study modified the QTL-seq method by introducing a |Δ(SNP-index)| parameter to improve the accuracy of mapping the red skin trait in a group of highly heterozygous Asian pears. The analyses were based on mixed DNA pools composed of 28 red-skinned and 27 green-skinned pear lines derived from a cross between the 'Mantianhong' and 'Hongxiangsu' red-skinned cultivars. The 'Dangshansuli' cultivar genome was used as reference for sequence alignment. An average single-nucleotide polymorphism (SNP) index was calculated using a sliding window approach (200-kb windows, 20-kb increments). Nine scaffolds within the candidate QTL interval were in the fifth linkage group from 111.9 to 177.1 cM. There was a significant linkage between the insertions/deletions and simple sequence repeat markers designed from the candidate intervals and the red/green skin (R/G) locus, which was in a 582.5-kb candidate interval that contained 81 predicted protein-coding gene models and was composed of two subintervals at the bottom of the fifth chromosome. The ZFRI 130-16, In2130-12 and In2130-16 markers located near the R/G locus could potentially be used to identify the red skin trait in Asian pear populations. This study provides new insights into the genetics controlling the red skin phenotype in this fruit.

Short-chain fatty acids act as antiinflammatory mediators by regulating prostaglandin E(2) and cytokines.

AIM: To investigate the effect of short-chain fatty acids (SCFAs) on production of prostaglandin E(2) (PGE(2)), cytokines and chemokines in human monocytes. METHODS: Human neutrophils and monocytes were isolated from human whole blood by using 1-Step Polymorph and RosetteSep Human Monocyte Enrichment Cocktail, respectively. Human GPR41 and GPR43 mRNA expression was examined by quantitative real-time polymerase chain reaction. The calcium flux assay was used to examine the biological activities of SCFAs in human neutrophils and monocytes. The effect of SCFAs on human monocytes and peripheral blood mononuclear cells (PBMC) was studied by measuring PGE(2), cytokines and chemokines in the supernatant. The effect of SCFAs in vivo was examined by intraplantar injection into rat paws. RESULTS: Human GPR43 is highly expressed in human neutrophils and monocytes. SCFAs induce robust calcium flux in human neutrophils, but not in human monocytes. In this study, we show that SCFAs can induce human monocyte release of PGE(2) and that this effect can be enhanced in the presence of lipopolysaccharide (LPS). In addition, we demonstrate that PGE(2) production induced by SCFA was inhibited by pertussis toxin, suggesting the involvement of a receptor-mediated mechanism. Furthermore, SCFAs can specifically inhibit constitutive monocyte chemotactic protein-1 (MCP-1) production and LPS-induced interleukin-10 (IL-10) production in human monocytes without affecting the secretion of other cytokines and chemokines examined. Similar activities were observed in human PBMC for the release of PGE(2), MCP-1 and IL-10 after SCFA treatment. In addition, SCFAs inhibit LPS-induced production of tumor necrosis factor-alpha and interferon-gamma in human PBMC. Finally, we show that SCFAs and LPS can induce PGE(2) production in vivo by intraplantar injection into rat paws (P < 0.01). CONCLUSION: SCFAs can have distinct antiinflammatory activities due to their regulation of PGE(2), cytokine and chemokine release from human immune cells.

Identification and characterization of a novel RF-amide peptide ligand for orphan G-protein-coupled receptor SP9155.

Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide.