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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.

Key messagePp4ERF24 and Pp12ERF96 fine tune blue light-induced anthocyanin biosynthesis via interacting with PpMYB114 and promoting the interaction between PpMYB114 and PpbHLH3, which enhances the expression of PpMYB114-induced PpUFGT.The red coloration of pear fruit is attributed to anthocyanin accumulation, which is transcriptionally regulated by the MYB-bHLH-WD40 complex. A number of ethylene response factors (ERF) have been identified to regulate anthocyanin biosynthesis in different plants. In pear, several ERF transcription factor genes were identified to be potentially involved in the light-induced anthocyanin biosynthesis according to transcriptome data. But the molecular mechanism of these ERFs underlying the regulation of anthocyanin accumulation is unknown. In this study, exposure of ‘Red Zaosu’ pear, a mutant of ‘Zaosu’ pear, to blue light significantly induced the anthocyanin accumulation by increasing the expression levels of anthocyanin biosynthetic genes. Gene expression analysis confirmed that the expression of Pp4ERF24 and Pp12ERF96 genes were up-regulated in the process of blue light-induced anthocyanin biosynthesis. Yeast two-hybrid and bimolecular fluorescence complementation assay revealed that Pp4ERF24 and Pp12ERF96 interacted with PpMYB114, but not with PpMYB10. Bimolecular fluorescence complementation assay demonstrated that the interaction between these two ERFs and PpMYB114 enhanced the interaction between PpMYB114 and PpbHLH3. Further analysis by dual luciferase assay verified that these two ERFs increased the up-regulation of PpMYB114-mediated PpUFGT expression. Furthermore, co-transformation of Pp12ERF96 with PpMYB114 and PpbHLH3 in tobacco leaves led to enhanced anthocyanin accumulation. Transient overexpression of Pp4ERF24 or Pp12ERF96 alone in ‘Red Zaosu’ pear fruit also induced anthocyanin biosynthesis in pear peel. Our findings provide insights into a mechanism involving the synergistic interaction of ERFs with PpMYB114 to regulate light-dependent coloration and anthocyanin biosynthesis in pear fruits.

Background 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. Results 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. Conclusions 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.

Summary WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report the identification and functional characterization of PbrWRKY53 isolated from Pyrus betulaefolia. PbrWRKY53 was greatly up‐regulated by drought and abscisic acid, but slightly induced by salt and cold. Subcellar localization analyses showed that PbrWRKY53 was located in the nucleus. Ectopic expression of PbrWRKY53 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to drought stress. The transgenic plants exhibited better water status, less reactive oxygen species generation and higher levels of antioxidant enzyme activities and metabolites than the wild type. In addition, overexpression of PbrWRKY53 in transgenic tobacco resulted in enhanced expression level of PbrNCED1, and led to the increase in larger amount of vitamin C accumulation in comparison to WT. Knock‐down of PbrWRKY53 in P. ussuriensis down‐regulated PbrNCED1 abundance, accompanied by compromised drought tolerance. Yeast one‐hybrid assay, EMSA and transient expression analysis demonstrated that PbrWRKY53 could bind to the W‐box element in the promoter region of PbrNCED1. Taken together, these results demonstrated that PbrWRKY53 plays a positive role in drought tolerance, which might be, at least in part, promoting production of vitamin C via regulating PbrNCED1 expression.

Stone cells negatively affect fruit quality because of their firm and lignified cell walls, so are targets for reduction in pear breeding programmes. However, there is only limited knowledge of the molecular mechanisms underlying the formation of stone cells. Here, we show that PbrMYB169, an R2R3 MYB transcription factor, of Pyrus bretschneideri positively regulates lignification of stone cells in pear fruit. PbrMYB169 was shown to be co-expressed with lignin biosynthesis genes during pear fruit development, and this co-expression pattern was coincident with stone cell formation in the fruit of Pyrus bretschneideri ‘Dangshansuli’. The PbrMYB169 expression level was also positively correlated with stone cell content in 36 pear cultivars tested. PbrMYB169 protein significantly activated the promoter of lignin genes C3H1, CCR1, CCOMT2, CAD, 4CL1, 4CL2, HCT2, and LAC18 via binding to AC elements [ACC(T/A)ACC] in these promoters. Furthermore, overexpression of PbrMYB169 in transgenic Arabidopsis plants enhanced the expression of lignin genes, and increased lignin deposition and cell wall thickness of vessel elements, but did not change the ratio of syringyl and guaiacyl lignin monomers. In conclusion, PbrMYB169 appears to be a transcriptional activator of lignin biosynthesis and regulates secondary wall formation in fruit stone cells. This study advances the understanding of the regulation of lignin biosynthesis and provides valuable molecular genetic information for reducing stone cell content in pear fruit.

Pear ( Pyrus L.) is a significant commercial fruit globally, with diverse species exhibiting variations in their flowering periods due to environmental factors. CONSTANS-like ( COL ) genes, known from previous studies in Arabidopsis , are key regulators of flowering time by sensing photoperiod. However, the evolutionary history and functions of COL genes in different pear species remain unclear. In this study, we identified a total of 79 COL genes in different pear species, including 12 COL genes in Pyrus bretschneideri ‘DangshanSuli’, 9 in Pyrus ussuriensis  ×  hybrid ‘Zhongai 1’, 11 in Pyrus communis ‘Bartlett’, 13 in Pyrus betulifolia , 18 in Pyrus pyrifolia ‘Cuiguan’, 16 in Pyrus pyrifolia ‘Nijisseiki’. Analysis of gene structure, phylogenetic tree, and multiple sequences provided valuable insights into the fundamental understanding of COL genes in pear. The impact of selection pressure on the PbrCOLs in Chinese white pear was assessed using Ka / Ks , revealing that the evolution rate of PbrCOLs was influenced by purification selection factors. The study also revealed different tissue-specific expression patterns of PbrCOLs under varying light quality. Real-time quantitative PCR revealed that under natural light conditions, the expression patterns of PbrCOL2 , PbrCOL3 , and PbrCOL4 are similar to previous studies on CONSTANS gene in Arabidopsis , with increased expression levels during the day and decreased levels at night. However, PbrCOL1 , PbrCOL6 , and PbrCOL9 exhibit different expression patterns, with decreased expression levels both during the day and at night. After red light treatment, high expression of PbrCOL3 and PbrCOL4 was observed at night, while the expression patterns of the other four genes did not show significant changes. Following blue light treatment, the expression peaks of PbrCOL1 and PbrCOL6 occurred during the night, showing opposite expression patterns compared to the study in Arabidopsis . The overexpression of PbrCOL3 significantly increase the chlorophyll content in pear seedlings, and its expression significantly affected the expression of other key flowering-related genes. Also, overexpression of PbrCOL3 resulted in a late-flowering phenotype in Arabidopsis. These findings indicate diverse responsive mechanisms and functions of PbrCOL genes on flowering time in pear. In conclusion, this study established a foundation for a deeper understanding of the specific roles of PbrCOLs in regulating the reproductive development of pear, particularly in the context of the photoperiodic flowering process.

Sugar content is an important trait of fleshy fruit, and elevating Suc levels is a major goal in horticultural crop breeding. Here, we examined the sugar content in two varieties of the Ussurian pear (Pyrus ussuriensis), 'Nanguo' (NG) and its bud sport (BNG), and we found that Suc content was higher in BNG fruit than in NG fruit. We compared the transcriptomes of the two varieties using RNA sequencing and identified a SWEET (Sugars Will Eventually be Exported Transporter) gene, PuSWEET15, expressed at higher levels in BNG fruit. Heterologous expression of PuSWEET15 in a SUSY7/ura yeast (Saccharomyces cerevisiae) strain showed that PuSWEET15 is an active Suc transporter. Overexpression of PuSWEET15 in NG pear fruit increased Suc content, while silencing of PuSWEET15 in BNG fruit decreased Suc content. The WRKY transcription factor PuWRKY31 was also expressed more highly in BNG fruit than in NG fruit, and we found that PuWRKY31 bound to the PuSWEET15 promoter and induced its transcription. The histone acetylation level of the PuWRKY31 promoter was higher in BNG fruit, suggesting a mechanism by which Suc levels can be elevated.

Peel color is an economically important characteristic that influences the appearance quality of red pear, whose red color is due to anthocyanin accumulation. The process of coloration in the fruit peel is strongly influenced by light. However, how light quality influences color development remains unclear. In this study, we analyzed the effects of different light qualities on color development in the red pear ‘Red Zaosu’, a mutant of the hybrid cultivar ‘Zaosu’ of Pyrus pyrifolia and P. communis. The results showed that blue light increased anthocyanin accumulation after 72 h of light treatment, while red light had almost no effect. The expression of anthocyanin biosynthesis-related genes showed a similar trend to the anthocyanin accumulation. To clarify the mechanism of blue-light induced coloration, PpCRYs, PpCOP1 and PpHY5 genes were cloned. Gene expression analysis showed that their transcript abundance did not correlate with the expression of anthocyanin-related genes or anthocyanin content, but the yeast two-hybrid system revealed conserved physical interactions among these proteins. In addition, PpHY5 directly bound to the promoters of the anthocyanin biosynthesis genes PpCHS, PpDFR, PpANS and PpMYB10, and activated the transcription of PpCHS in a Nicotiana benthamiana-based dual-luciferase assay. In summary, our results preliminarily revealed that the conserved blue light signal transduction module CRY–COP1–HY5 contributed to the anthocyanin biosynthesis induced by blue light in red pear. However, our results did not provide evidence for why red light had no effect on anthocyanin accumulation, which needs further study.

Summary Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell‐based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl‐ and guaiacyl‐lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild‐type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.

Summary The red coloration of pear (Pyrus pyrifolia) results from anthocyanin accumulation in the fruit peel. Light is required for anthocyanin biosynthesis in pear. A pear homolog of Arabidopsis thaliana BBX22, PpBBX16, was differentially expressed after fruits were removed from bags and may be involved in anthocyanin biosynthesis. Here, the expression and function of PpBBX16 were analysed. PpBBX16's expression was highly induced by white‐light irradiation, as was anthocyanin accumulation. PpBBX16's ectopic expression in Arabidopsis increased anthocyanin biosynthesis in the hypocotyls and tops of flower stalks. PpBBX16 was localized in the nucleus and showed trans‐activity in yeast cells. Although PpBBX16 could not directly bind to the promoter of PpMYB10 or PpCHS in yeast one‐hybrid assays, the complex of PpBBX16/PpHY5 strongly trans‐activated anthocyanin pathway genes in tobacco. PpBBX16's overexpression in pear calli enhanced the red coloration during light treatments. Additionally, PpBBX16's transient overexpression in pear peel increased anthocyanin accumulation, while virus‐induced gene silencing of PpBBX16 decreased anthocyanin accumulation. The expression patterns of pear BBX family members were analysed, and six additional BBX genes, which were differentially expressed during light‐induced anthocyanin biosynthesis, were identified. Thus, PpBBX16 is a positive regulator of light‐induced anthocyanin accumulation, but it could not directly induce the expression of the anthocyanin biosynthesis‐related genes by itself but needed PpHY5 to gain full function. Our work uncovered regulatory modes for PpBBX16 and suggested the potential functions of other pear BBX genes in the regulation of anthocyanin accumulation, thereby providing target genes for further studies on anthocyanin biosynthesis.