Four glycosyltransferase genes are responsible for synthesis and accumulation of different flavonol glycosides in apple tissues.

Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species.

Self S-RNase inhibits ABF-LRX signaling to arrest pollen tube growth to achieve self-incompatibility in pear.

Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S-RNase in GSI species are limited. In the present study, two leucine-rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self-compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self-incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S-RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S-RNase.

PbHsfC1a-coordinates ABA biosynthesis and H2O2 signalling pathways to improve drought tolerance in Pyrus betulaefolia.

Abiotic stresses have had a substantial impact on fruit crop output and quality. Plants have evolved an efficient immune system to combat abiotic stress, which employs reactive oxygen species (ROS) to activate the downstream defence response signals. Although an aquaporin protein encoded by PbPIP1;4 is identified from transcriptome analysis of Pyrus betulaefolia plants under drought treatments, little attention has been paid to the role of PIP and ROS in responding to abiotic stresses in pear plants. In this study, we discovered that overexpression of PbPIP1;4 in pear callus improved tolerance to oxidative and osmotic stresses by reconstructing redox homeostasis and ABA signal pathways. PbPIP1;4 overexpression enhanced the transport of H2O2 into pear and yeast cells. Overexpression of PbPIP1;4 in Arabidopsis plants mitigates the stress effects caused by adding ABA, including stomatal closure and reduction of seed germination and seedling growth. Overexpression of PbPIP1;4 in Arabidopsis plants decreases drought-induced leaf withering. The PbPIP1;4 promoter could be bound and activated by TF PbHsfC1a. Overexpression of PbHsfC1a in Arabidopsis plants rescued the leaf from wilting under drought stress. PbHsfC1a could bind to and activate AtNCED4 and PbNCED4 promoters, but the activation could be inhibited by adding ABA. Besides, PbNCED expression was up-regulated under H2O2 treatment but down-regulated under ABA treatment. In conclusion, this study revealed that PbHsfC1a is a positive regulator of abiotic stress, by targeting PbPIP1;4 and PbNCED4 promoters and activating their expression to mediate redox homeostasis and ABA biosynthesis. It provides valuable information for breeding drought-resistant pear cultivars through gene modification.

Two adjacent NAC transcription factors regulate fruit maturity date and flavor in peach.

Although maturity date (MD) is an essential factor affecting fresh fruit marketing and has a pleiotropic effect on fruit taste qualities, the underlying mechanisms remain largely unclear. In this study, we functionally characterized two adjacent NAM-ATAF1/2-CUC2 (NAC) transcription factors (TFs), PpNAC1 and PpNAC5, both of which were associated with fruit MD in peach. PpNAC1 and PpNAC5 were found capable of activating transcription of genes associated with cell elongation, cell wall degradation and ethylene biosynthesis, suggesting their regulatory roles in fruit enlargement and ripening. Furthermore, PpNAC1 and PpNAC5 had pleiotropic effects on fruit taste due to their ability to activate transcription of genes for sugar accumulation and organic acid degradation. Interestingly, both PpNAC1 and PpNAC5 orthologues were found in fruit-producing angiosperms and adjacently arranged in all 91 tested dicots but absent in fruitless gymnosperms, suggesting their important roles in fruit development. Our results provide insight into the regulatory roles of NAC TFs in MD and fruit taste.

The transcription factor PbrMYB24 regulates lignin and cellulose biosynthesis in stone cells of pear fruits.

Lignified stone cell content is a key factor used to evaluate fruit quality, influencing the economic value of pear (Pyrus pyrifolia) fruits. However, our understanding of the regulatory networks of stone cell formation is limited due to the complex secondary metabolic pathway. In this study, we used a combination of co-expression network analysis, gene expression profiles, and transcriptome analysis in different pear cultivars with varied stone cell content to identify a hub MYB gene, PbrMYB24. The relative expression of PbrMYB24 in fruit flesh was significantly correlated with the contents of stone cells, lignin, and cellulose. We then verified the function of PbrMYB24 in regulating lignin and cellulose formation via genetic transformation in homologous and heterologous systems. We constructed a high-efficiency verification system for lignin and cellulose biosynthesis genes in pear callus. PbrMYB24 transcriptionally activated multiple target genes involved in stone cell formation. On the one hand, PbrMYB24 activated the transcription of lignin and cellulose biosynthesis genes by binding to different cis-elements [AC-I (ACCTACC) element, AC-II (ACCAACC) element and MYB-binding sites (MBS)]. On the other hand, PbrMYB24 bound directly to the promoters of PbrMYB169 and NAC STONE CELL PROMOTING FACTOR (PbrNSC), activating the gene expression. Moreover, both PbrMYB169 and PbrNSC activated the promoter of PbrMYB24, enhancing gene expression. This study improves our understanding of lignin and cellulose synthesis regulation in pear fruits through identifying a regulator and establishing a regulatory network. This knowledge will be useful for reducing the stone cell content in pears via molecular breeding.

MADS-box protein PpDAM6 regulates chilling requirement-mediated dormancy and bud break in peach.

Bud dormancy is crucial for winter survival and is characterized by the inability of the bud meristem to respond to growth-promotive signals before the chilling requirement (CR) is met. However, our understanding of the genetic mechanism regulating CR and bud dormancy remains limited. This study identified PpDAM6 (DORMANCY-ASSOCIATED MADS-box) as a key gene for CR using a genome-wide association study analysis based on structural variations in 345 peach (Prunus persica (L.) Batsch) accessions. The function of PpDAM6 in CR regulation was demonstrated by transiently silencing the gene in peach buds and stably overexpressing the gene in transgenic apple (Malus × domestica) plants. The results showed an evolutionarily conserved function of PpDAM6 in regulating bud dormancy release, followed by vegetative growth and flowering, in peach and apple. The 30-bp deletion in the PpDAM6 promoter was substantially associated with reducing PpDAM6 expression in low-CR accessions. A PCR marker based on the 30-bp indel was developed to distinguish peach plants with non-low and low CR. Modification of the H3K27me3 marker at the PpDAM6 locus showed no apparent change across the dormancy process in low- and non-low- CR cultivars. Additionally, H3K27me3 modification occurred earlier in low-CR cultivars on a genome-wide scale. PpDAM6 could mediate cell-cell communication by inducing the expression of the downstream genes PpNCED1 (9-cis-epoxycarotenoid dioxygenase 1), encoding a key enzyme for ABA biosynthesis, and CALS (CALLOSE SYNTHASE), encoding callose synthase. We shed light on a gene regulatory network formed by PpDAM6-containing complexes that mediate CR underlying dormancy and bud break in peach. A better understanding of the genetic basis for natural variations of CR can help breeders develop cultivars with different CR for growing in different geographical regions.

Auxin inhibits lignin and cellulose biosynthesis in stone cells of pear fruit via the PbrARF13-PbrNSC-PbrMYB132 transcriptional regulatory cascade.

Stone cells are often present in pear fruit, and they can seriously affect the fruit quality when present in large numbers. The plant growth regulator NAA, a synthetic auxin, is known to play an active role in fruit development regulation. However, the genetic mechanisms of NAA regulation of stone cell formation are still unclear. Here, we demonstrated that exogenous application of 200 µM NAA reduced stone cell content and also significantly decreased the expression level of PbrNSC encoding a transcriptional regulator. PbrNSC was shown to bind to an auxin response factor, PbrARF13. Overexpression of PbrARF13 decreased stone cell content in pear fruit and secondary cell wall (SCW) thickness in transgenic Arabidopsis plants. In contrast, knocking down PbrARF13 expression using virus-induced gene silencing had the opposite effect. PbrARF13 was subsequently shown to inhibit PbrNSC expression by directly binding to its promoter, and further to reduce stone cell content. Furthermore, PbrNSC was identified as a positive regulator of PbrMYB132 through analyses of co-expression network of stone cell formation-related genes. PbrMYB132 activated the expression of gene encoding cellulose synthase (PbrCESA4b/7a/8a) and lignin laccase (PbrLAC5) binding to their promotors. As expected, overexpression or knockdown of PbrMYB132 increased or decreased stone cell content in pear fruit and SCW thickness in Arabidopsis transgenic plants. In conclusion, our study shows that the 'PbrARF13-PbrNSC-PbrMYB132' regulatory cascade mediates the biosynthesis of lignin and cellulose in stone cells of pear fruit in response to auxin signals and also provides new insights into plant SCW formation.

Two loss-of-function alleles of the glutathione S-transferase (GST) gene cause anthocyanin deficiency in flower and fruit skin of peach (Prunus persica).

Flower and fruit colors are important agronomic traits. To date, there is no forward genetic evidence that the glutathione S-transferase (GST) gene is responsible for the white flower color in peach (Prunus persica). In this study, genetic analysis indicated that the white-flower trait is monogenetic, is recessive to the non-white allele, and shows pleiotropic effects with non-white-flowered types. The genetic locus underpinning this trait was mapped onto chromosome 3 between 0.421951 and 3.227115 Mb by using bulked segregant analysis in conjunction with whole-genome sequencing, and was further mapped between 0 and 1.178149 Mb by using the backcross 1 (BC1 ) population. Finally, the locus was fine-mapped within 535.974- and 552.027-kb intervals by using 151 F2 individuals and 75 individuals from a BC1 self-pollinated (BC1 S1 ) population, respectively. Pp3G013600, encoding a GST that is known to transport anthocyanin, was identified within the mapping interval. The analysis of genome sequence data showed Pp3G013600 in white flowers has a 2-bp insertion or a 5-bp deletion in the third exon. These variants likely render the GST non-functional because of early stop codons that reduce the protein length from 215 amino acids to 167 and 175 amino acids, respectively. Genetic markers based on these variants validated a complete correlation between the GST loss-of-function alleles and white flower in 128 peach accessions. This correlation was further confirmed by silencing of Pp3G013600 using virus-induced gene silencing technology, which reduced anthocyanin accumulation in peach fruit. The new knowledge from this study is useful for designing peach breeding programs to generate cultivars with white flower and fruit skin.

Transposon insertions regulate genome-wide allele-specific expression and underpin flower colour variations in apple (Malus spp.).

Allele-specific expression (ASE) can lead to phenotypic diversity and evolution. However, the mechanisms regulating ASE are not well understood, particularly in woody perennial plants. In this study, we investigated ASE genes in the apple cultivar 'Royal Gala' (RG). A high quality chromosome-level genome was assembled using a homozygous tetra-haploid RG plant, derived from anther cultures. Using RNA-sequencing (RNA-seq) data from RG flower and fruit tissues, we identified 2091 ASE genes. Compared with the haploid genome of 'Golden Delicious' (GD), a parent of RG, we distinguished the genomic sequences between the two alleles of 817 ASE genes, and further identified allele-specific presence of a transposable element (TE) in the upstream region of 354 ASE genes. These included MYB110a that encodes a transcription factor regulating anthocyanin biosynthesis. Interestingly, another ASE gene, MYB10 also showed an allele-specific TE insertion and was identified using genome data of other apple cultivars. The presence of the TE insertion in both MYB genes was positively associated with ASE and anthocyanin accumulation in apple petals through analysis of 231 apple accessions, and thus underpins apple flower colour evolution. Our study demonstrated the importance of TEs in regulating ASE on a genome-wide scale and presents a novel method for rapid identification of ASE genes and their regulatory elements in plants.

The effect of different carbon sources on biofouling in membrane fouling simulators: microbial community and implications.

Biofouling is a problem affecting the operation of nanofiltration systems due to the complexity of the carbon matrix affecting bacteria and biofilm growth. This study used membrane fouling simulators to investigate the effects of five different carbon sources on the biofouling of nanofiltration membranes. For all the carbon sources analyzed, the increase in pressure drop was most accelerated for acetate. The use of acetate as the single carbon source produced less adenosine triphosphate but more extracellular polymers than glucose. The microbial community was analyzed using 16 s rRNA. The use of more than a single carbon source produced an increase in bacteria diversity even at similar concentrations. The relative abundance of proteobacteria was the highest at the phylum level (95%) when a single carbon source was added. Additionally, it was found that the use of different carbon sources produced a shift in the microbial community, affecting the biofouling and pressure drop on membranes.

Reduced expression of a subunit gene of sucrose non-fermenting 1 related kinase, PpSnRK1ßγ, confers flat fruit abortion in peach by regulating sugar and starch metabolism.

BACKGROUND: Fruit abortion is a major limiting factor for fruit production. In flat peach, fruit abortion is present in the whole tree of some accessions during early fruit development. However, the physiological factors and genetic mechanism underlying flat fruit abortion remain largely elusive. RESULTS: In this study, we have revealed that the fertilization process was accomplished and the reduction of sucrose and starch contents might result in flat fruit abortion. By combining association and gene expression analysis, a key candidate gene, PpSnRK1ßγ, was identified. A 1.67-Mb inversion co-segregated with flat fruit shape altered the promoter activity of PpSnRK1ßγ, resulting in much lower expression in aborting flat peach. Ectopic transformation in tomato and transient overexpression in peach fruit have shown that PpSnRK1ßγ could increase sugar and starch contents. Comparative transcriptome analysis further confirmed that PpSnRK1ßγ participated in carbohydrate metabolism. Subcellular localization found that PpSnRK1ßγ was located in nucleus. CONCLUSIONS: This study provides a possible reason for flat fruit abortion and identified a critical candidate gene, PpSnRK1ßγ, that might be responsible for flat fruit abortion in peach. The results will provide great help in peach breeding and facilitate gene identification for fruit abortion in other plant species.

PbrmiR397a regulates lignification during stone cell development in pear fruit.

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.

PbrMYB169 positively regulates lignification of stone cells in pear fruit.

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.

Cit1,2RhaT and two novel CitdGlcTs participate in flavor-related flavonoid metabolism during citrus fruit development.

Neohesperidosides are disaccharides that are present in some flavonoids and impart a bitter taste, which can significantly affect the commercial value of citrus fruits. In this study, we identified three flavonoid-7-O-di-glucosyltransferase (dGlcT) genes closely related to 1,2-rhamnosyltransferase (1,2RhaT) in citrus genomes. However, only 1,2RhaT was directly linked to the accumulation of neohesperidoside, as demonstrated by association analysis of 50 accessions and co-segregation analysis of an F1 population derived from Citrus reticulata × Poncirus trifoliata. In transgenic tobacco BY2 cells, over-expression of CitdGlcTs resulted in flavonoid-7-O-glucosides being catalysed into bitterless flavonoid-7-O-di-glucosides, whereas over-expression of Cit1,2RhaT converted the same substrate into bitter-tasting flavonoid-7-O-neohesperidoside. Unlike 1,2RhaT, during citrus fruit development the dGlcTs showed an opposite expression pattern to CHS and CHI, two genes encoding rate-limiting enzymes of flavonoid biosynthesis. An uncoupled availability of dGlcTs and substrates might result in trace accumulation of flavonoid-7-O-di-glucosides in the fruit of C. maxima (pummelo). Past human selection of the deletion and functional mutation of 1,2RhaT has led step-by-step to the evolution of the flavor-related metabolic network in citrus. Our research provides the basis for potentially improving the taste in citrus fruit through manipulation of the network by knocking-out 1,2RhaT or by enhancing the expression of dGlcT using genetic transformation.

Identification of the defense-related gene VdWRKY53 from the wild grapevine Vitis davidii using RNA sequencing and ectopic expression analysis in Arabidopsis.

BACKGROUND: Grapevine is an important fruit crop grown worldwide, and its cultivars are mostly derived from the European species Vitis vinifera, which has genes for high fruit quality and adaptation to a wide variety of climatic conditions. Disease resistance varies substantially across grapevine species; however, the molecular mechanisms underlying such variation remain uncharacterized. RESULTS: The anatomical structure and disease symptoms of grapevine leaves were analyzed for two grapevine species, and the critical period of resistance of grapevine to pathogenic bacteria was determined to be 12 h post inoculation (hpi). Differentially expressed genes (DEGs) were identified from transcriptome analysis of leaf samples obtained at 12 and 36 hpi, and the transcripts in four pathways (cell wall genes, LRR receptor-like genes, WRKY genes, and pathogenesis-related (PR) genes) were classified into four co-expression groups by using weighted correlation network analysis (WGCNA). The gene VdWRKY53, showing the highest transcript level, was introduced into Arabidopsis plants by using a vector containing the CaMV35S promoter. These procedures allowed identifying the key genes contributing to differences in disease resistance between a strongly resistant accession of a wild grapevine species Vitis davidii (VID) and a susceptible cultivar of V. vinifera, 'Manicure Finger' (VIV). Vitis davidii, but not VIV, showed a typical hypersensitive response after infection with a fungal pathogen (Coniella diplodiella) causing white rot disease. Further, 20 defense-related genes were identified, and their differential expression between the two grapevine species was confirmed using quantitative real-time PCR analysis. VdWRKY53, showing the highest transcript level, was selected for functional analysis and therefore over-expressed in Arabidopsis under the control of the CaMV35S promoter. The transgenic plants showed enhanced resistance to C. diplodiella and to two other pathogens, Pseudomonas syringae pv. tomato DC3000 and Golovinomyces cichoracearum. CONCLUSION: The consistency of the results in VID and transgenic Arabidopsis indicated that VdWRKY53 might be involved in the activation of defense-related genes that enhance the resistance of these plants to pathogens. Thus, the over-expression of VdWRKY53 in transgenic grapevines might improve their resistance to pathogens.

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.

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.

A microRNA allele that emerged prior to apple domestication may underlie fruit size evolution.

The molecular genetic mechanisms underlying fruit size remain poorly understood in perennial crops, despite size being an important agronomic trait. Here we show that the expression level of a microRNA gene (miRNA172) influences fruit size in apple. A transposon insertional allele of miRNA172 showing reduced expression associates with large fruit in an apple breeding population, whereas over-expression of miRNA172 in transgenic apple significantly reduces fruit size. The transposon insertional allele was found to be co-located with a major fruit size quantitative trait locus, fixed in cultivated apples and their wild progenitor species with relatively large fruit. This finding supports the view that the selection for large size in apple fruit was initiated prior to apple domestication, likely by large mammals, before being subsequently strengthened by humans, and also helps to explain why signatures of genetic bottlenecks and selective sweeps are normally weaker in perennial crops than in annual crops.

Apple SEPALLATA1/2-like genes control fruit flesh development and ripening.

Flowering plants utilize different floral structures to develop flesh tissue in fruits. Here we show that suppression of the homeologous SEPALLATA1/2-like genes MADS8 and MADS9 in the fleshy fruit apple (Malus x domestica) leads to sepaloid petals and greatly reduced fruit flesh. Immunolabelling of cell-wall epitopes and differential staining showed that the developing hypanthium (from which the apple flesh develops) of MADS8/9-suppressed apple flowers lacks a tissue layer, and the remaining flesh tissue of fully developed apples has considerably smaller cells. From these observations, it is proposed that MADS8 and MADS9 control the development of discrete zones within the hypanthium tissue, and therefore fruit flesh, and also act as foundations for development of different floral organs. At fruit maturity, the MADS8/9-suppressed apples do not ripen in terms of both developmentally controlled ripening characters, such as starch degradation, and ethylene-modulated ripening traits. Transient assays suggest that, like the RIN gene in tomato, the MADS9 gene acts as a transcriptional activator of the ethylene biosynthesis enzyme, 1-aminocyclopropane-1-carboxylate (ACC) synthase 1. The existence of a single class of genes that regulate both flesh formation and ripening provides an evolutionary tool for controlling two critical aspects of fleshy fruit development.

Hypermethylation in the promoter regions of flavonoid pathway genes is associated with skin color fading during 'Daihong' apple fruit development.

Apple fruit skin color fading is not well understood although the molecular mechanism of skin color formation is well known. The red-fleshed apple cultivar 'Daihong' (DH) exhibited fading skin color during fruit development despite having a heterozygous R6 allele but lacking Red-TE for red fruit skin. In this study, transcriptomic analysis revealed the expression level of MdMYB10 increased with fruit development whereas reduced expression levels of MdMYBPA1, MdCHS, MdANS, MdUFGT, MdLAR, and MdANR were observed, consistent with decreased levels of chalcone, anthocyanin, catechin, epicatechin, and procyanidin B2. Whole-genome bisulfite sequencing (WGBS) indicated a global gain in cytosine methylation levels and increased methylation in 5' and 3' flanking regions of genes and transposable elements (TEs), and in TE bodies in all CG, CHG and CHH contexts, especially the mCHH context, during fruit development. The increased DNA methylation was attributed to reduced expression levels of DNA demethylase genes, including MdDME1, MdROS1, and MdROS2. Association analysis revealed a significant negative correlation between promoter methylation levels of MdCHS, MdCHI, MdMYBPA1, and their respective transcript levels, as well as a negative correlation between promoter methylation levels of MdCHS, MdCHI, MdANR, and MdFLS, and the content of chalcones, naringenin-7-glucoside, epicatechin, and quercetin. Treatment with the DNA demethylation agent 5-aza-2'-deoxycytidine verified the negative correlation between DNA methylation and gene expression within the flavonoid pathway. These findings suggest that hypermethylation in promoter regions of genes of the flavonoid biosynthesis pathway is associated with the reduction of gene expression and flavonoid content, and fruit skin color fading during DH apple development.