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Gene therapy is likely to be the most promising way to tackle cancer, while defects in molecular strategies and delivery systems have led to an impasse in clinical application. Here, it is found that onco-miRNAs of the miR-515 and -449 families were upregulated in hepatocellular carcinoma (HCC), and the sponge targeting miR-515 family had a significant probability to suppress cancer cell proliferation. Then, we constructed non-toxic sponge-loaded magnetic nanodroplets containing 20% C6F14 (SLMNDs-20%) that are incorporated with fluorinated superparamagnetic iron oxide nanoparticles enhancing external magnetism-assisted targeting and enabling a direct visualization of SLMNDs-20% distribution in vivo via magnetic resonance imaging monitoring. SLMNDs-20% could be vaporized by programmable focused ultrasound (FUS) activation, achieving ∼45% in vitro sponge delivery efficiency and significantly enhancing in vivo sponge delivery without a clear apoptosis. Moreover, the sponge-1-carrying SLMNDs-20% could effectively suppress proliferation of xenograft HCC after FUS exposure because sponge-1-suppressing onco-miR-515 enhanced the expression of anti-oncogenes (P21, CD22, TIMP1, NFKB, and E-cadherin) in cancer cells. The current results indicated that ultrasonic cavitation-inducing sonoporation enhanced the intracellular delivery of sponge-1 using SLMNDs-20% after magnetic-assisted accumulation, which was a therapeutic approach to inhibit HCC progression.

Using ultrasound activating contrast agents to induce sonoporation is a potential strategy for effective lesion-targeted gene delivery. Previous reports have proven that submicron nanodroplets have a better advantage than microbubbles in that they can pass through tumor vasculature endothelial gaps by passive targeting; however, they cannot achieve an adequate dose in tumors to facilitate ultrasound-enhanced gene delivery. Additionally, a few studies focused on delivering macromolecular genetic materials (i.e. overexpression plasmid and CRISPR plasmid) have presented more unique advantages than small-molecular genetic materials (i.e. miRNA mimics, siRNA and shRNA etc.), such as enhancing the expression of target genes with long-term effectiveness. Thereby, we constructed novel plasmid-loadable magnetic/ultrasound-responsive nanodroplets, where superparamagnetic iron oxide nanoparticle dispersed perfluoropentane was encapsulated with lipids to which plasmids could be adhered, and branched polyethylenimine was used to protect the plasmids from enzymolysis. Furthermore, in vitro and in vivo studies were performed to verify the magnetic tumor-targeting ability of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets and focused ultrasound enhanced intracellular plasmid delivery. The plasmid-loadable magnetic/ultrasound-responsive nanodroplets, carrying 16-19 plasmids per droplet, had desirable diameters less than 300 nm, and integrated the merits of excellent magnetic targeting capabilities and phase transition sensitivity to focused ultrasound. Under programmable focused ultrasound exposure, the plasmid-loadable magnetic/ultrasound-responsive nanodroplets underwent a phase-transition into echogenic microbubbles and the subsequent inertial cavitation of the microbubbles achieved an ∼40% in vitro plasmid delivery efficiency. Following intravenous administration, T2-weighted magnet resonance imaging, scanning electron microscopy and inductively coupled plasma optical emission spectrometry of the tumors showed significantly enhanced intratumoral accumulation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets under an external magnetic field. And a GFP ELISA assay and immunofluorescence staining indicated that focused ultrasound-induced inertial cavitation of the plasmid-loadable magnetic/ultrasound-responsive nanodroplets significantly enhanced the intracellular delivery of plasmids within the tumor after magnet-assisted accumulation, while only lower GFP levels were observed in the tumors on applying focused ultrasound or an external magnet alone. Taken together, utilizing the excellent plasmid-loadable magnetic/ultrasound-responsive nanodroplets combined with magnetism and ultrasound could efficiently deliver plasmids to cancer cells, which could be a potential strategy for macromolecular genetic material delivery in the clinic to treat cancer.

Pear is a major fruit tree crop distributed worldwide, yet its breeding is a very time-consuming process. To facilitate molecular breeding and gene identification, here we have performed genome-wide association studies (GWAS) on eleven fruit traits. We identify 37 loci associated with eight fruit quality traits and five loci associated with three fruit phenological traits. Scans for selective sweeps indicate that traits including fruit stone cell content, organic acid and sugar contents might have been under continuous selection during breeding improvement. One candidate gene, PbrSTONE, identified in GWAS, has been functionally verified to be involved in the regulation of stone cell formation, one of the most important fruit quality traits in pear. Our study provides insights into the complex fruit related biology and identifies genes controlling important traits in pear through GWAS, which extends the genetic resources and basis for facilitating molecular breeding in perennial trees., Studies of fruit quality traits in pears are lagging behind the other major fruit trees. Here, the authors conduct GWAS of fruit quality and phenological traits in a panel of 312 sand pear accessions using SNPs called from resequencing data, and reveal the involvement of a lignin formation-related protein in regulating stone cell development.

Summary Pear (Pyrus; 2n = 34), the third most important temperate fruit crop, has great nutritional and economic value. Despite the availability of many genomic resources in pear, it is challenging to genotype novel germplasm resources and breeding progeny in a timely and cost‐effective manner. Genotyping arrays can provide fast, efficient and high‐throughput genetic characterization of diverse germplasm, genetic mapping and breeding populations. We present here 200K AXIOM ® PyrSNP, a large‐scale single nucleotide polymorphism (SNP) genotyping array to facilitate genotyping of Pyrus species. A diverse panel of 113 re‐sequenced pear genotypes was used to discover SNPs to promote increased adoption of the array. A set of 188 diverse accessions and an F1 population of 98 individuals from ‘Cuiguan’ × ‘Starkrimson’ was genotyped with the array to assess its effectiveness. A large majority of SNPs (166 335 or 83%) are of high quality. The high density and uniform distribution of the array SNPs facilitated prediction of centromeric regions on 17 pear chromosomes, and significantly improved the genome assembly from 75.5% to 81.4% based on genetic mapping. Identification of a gene associated with flowering time and candidate genes linked to size of fruit core via genome wide association studies showed the usefulness of the array in pear genetic research. The newly developed high‐density SNP array presents an important tool for rapid and high‐throughput genotyping in pear for genetic map construction, QTL identification and genomic selection.

Red skin colour is an important target trait in various pear breeding programmes. In this study, the genetic control of red skin colour was investigated in an interspecific population derived using the descendants of the red sport European pear cultivar ‘Max Red Bartlett’ (MRB) and the red-blushed Chinese pear cultivar ‘Huobali’. Approximately 550 seedlings from nine families were phenotyped for red skin over-colour coverage (Ocolcov) and the intensity of red over-colour (Ocolint) on a 0–9 scale, and genotyped using genotyping-by-sequencing. Genome-wide association analyses were conducted using 7500 high-quality single nucleotide polymorphisms (SNPs). Genomic regions on linkage groups (LG) 4 and 5 were found to be associated, and the best SNP (S578_25116) on LG4 accounted for ~15% of phenotypic variation in Ocolcov and Ocolint. The association of S578_25116 with Ocolcov and Ocolint was successfully validated in a sample of ~200 European and Asian pear accessions. The association with red skin at locus S578_25116 was not present in Asian pear accessions, suggesting its close proximity to the MRB’s Cardinal gene. Several putative candidate genes, including MYB transcription factors (PCP027962 and PCP027967), were identified in the quantitative trait locus region on LG4 and await functional validation., Crop genetics: What makes a pear red? Researchers in New Zealand have produced a map of genetic variants linked with red skin color in pears, opening the door to identifying the genes responsible. Satish Kumar and others at the New Zealand Institute for Plant and Food Research Limited measured the skin color of 550 hybrid pear seedlings and sequenced their genomes. Combining these data produced a map of 7,500 variants throughout the genome and identified those associated with red skin color. The most significant variant accounted for about 15% of the color variation. Further analysis of that genomic region revealed several genes which might be related to red skin color. The genomic map produced by this study will improve breeding efficiency by making it possible to screen seedlings for fruit color, but further research is necessary to characterize the candidate genes.

Pear, belonging to the genus Pyrus, is one of the most economically important temperate fruit crops. Pyrus is an important genus of the Rosaceae family, subfamily Maloideae, and has at least 22 different species with over 5000 accessions maintained or identified worldwide. With the release of draft whole-genome sequences for Pyrus, opportunities for pursuing studies on the evolution, domestication, and molecular breeding of pear, as well as for conducting comparative genomics analyses within the Rosaceae family, have been greatly expanded. In this review, we highlight key advances in pear genetics, genomics, and breeding driven by the availability of whole-genome sequences, including whole-genome resequencing efforts, pear domestication, and evolution. We cover updates on new resources for undertaking gene identification and molecular breeding, as well as for pursuing functional validation of genes associated with desirable economic traits. We also explore future directions for “pear-omics”.

BackgroundThe NBS disease-related gene family coordinates the inherent immune system in plants in response to pathogen infections. Previous studies have identified NBS-encoding genes inPyrus bretschneideri(‘Dangshansuli’, an Asian pear) andPyrus communis(‘Bartlett’, a European pear) genomes, but the patterns of genetic variation and selection pressure on these genes during pear domestication have remained unsolved.ResultsIn this study, 338 and 412 NBS-encoding genes were identified from Asian and European pear genomes. This difference between the two pear species was the result of proximal duplications. About 15.79% orthologous gene pairs had Ka/Ks ratio more than one, indicating two pear species undergo strong positive selection after the divergence of Asian and European pear. We identified 21 and 15 NBS-encoding genes under fire blight and black spot disease-related QTL, respectively, suggesting their importance in disease resistance. Domestication caused decreased nucleotide diversity across NBS genes in Asian cultivars (cultivated 6.23E-03; wild 6.47E-03), but opposite trend (cultivated 6.48E-03; wild 5.91E-03) appeared in European pears. Many NBS-encoding coding regions showed Ka/Ks ratio of greater than 1, indicating the role of positive selection in shaping diversity of NBS-encoding genes in pear. Furthermore, we detected 295 and 122 significantly different SNPs between wild and domesticated accessions in Asian and European pear populations. Two NBS genes (Pbr025269.1andPbr019876.1) with significantly different SNPs showed >5x upregulation between wild and cultivated pear accessions, and > 2x upregulation inPyrus calleryanaafter inoculation withAlternaria alternata. We propose that positively selected and significantly different SNPs of an NBS-encoding gene (Pbr025269.1) regulate gene expression differences in the wild and cultivated groups, which may affect resistance in pear againstA. alternata.ConclusionProximal duplication mainly led to the different number of NBS-encoding genes inP. bretschneideriandP. communisgenomes. The patterns of genetic diversity and positive selection pressure differed between Asian and European pear populations, most likely due to their independent domestication events. This analysis helps us understand the evolution, diversity, and selection pressure in the NBS-encoding gene family in Asian and European populations, and provides opportunities to study mechanisms of disease resistance in pear.

Flowering time plays a vital role in determining the life-cycle period, yield, and seed quality of rapeseed (Brassica napus L.) in certain environments. Quantitative trait locus (QTL) mapping to identify the genetic architecture of genes controlling flowering time helps accelerate the early maturity breeding process. In this study, simple sequence repeats (SSR) and specific-locus amplified fragment sequencing (SLAF-seq) technologies were adopted to map the QTLs for flowering time in four environments. As a result, three target intervals, FTA09, FTA10, and FTC05 were identified. Among this, FTA09 was considered as a novel interval, FTA10 and FTC05 as stable regions. Based on the parental re-sequencing data, 7,022 single nucleotide polymorphisms (SNPs) and 2,195 insertion-deletions (InDels) between the two parents were identified in these three target regions. A total of 186 genes possessed genetic variations in these intervals, 14 of which were related to flowering time involved in photoperiod, circadian clock, vernalization, and gibberellin pathways. Six InDel markers linked to flowering time were developed in the three target intervals, indicating that the results were credible in this study. These results laid a good foundation for further genetic studies on flowering-time regulation in B. napus L.

Additional file 1: Figure S1. A phylogenetic tree of P. bretschneideri NBS proteins constructed by ML (Maximum likelihood) method using IQ-TREE. Fig. S2: A phylogenetic tree of P. communis NBS proteins constructed by ML method using IQ-TREE. ① Two subfamilies are shown. Red represents non-TIR group and Blue represents TIR group. ② The six classes of NBS-encoding genes are marked by different colors. Green means CC-NBS-LRR type, light yellow means TIR-NBS-LRR type, yellow means NBS type, light blue means TIR-NBS type, orange means CC-NBS type and purple means NBS-LRR type. ③ Domains of NB-ARC, LRR, and TIR are displayed on the tree (CC domain was not shown). Yellow means TIR domain, red means NB-ARC domain and blue means LRR domain.

Lobed leaf is a common trait, which is related with photosynthesis and plant stress resistance in crops. In order to fine map and isolate the lobed-leaf gene in Brassica napus, an F2:3 population derived from 2205 (salt tolerance) and 1423 (salt sensitive) was constructed, and the quantitative trait locus (QTL) technology was adopted to identify the QTLs related to lobed leaf formation. As a result, one major QTL was identified on LG10, and two intron polymorphic (IP) markers and one sequence characterized amplified region (SCAR) marker were successfully developed in QTL region. The lobed-leaf gene was mapped to a region from 15.701 to 15.817 M on A10. In light of annotations of the genes in candidate region, a leaf morphological development related gene, Bra009510, was primary identified as the candidate gene. The full length of the candidate gene was 1390 bp containing three exons and two introns in the two parents. The open reading frame (ORF) was 693 bp and encoded a protein of 229 amino acids. Eight amino acid differences between the two parents in CDS (coding sequences) region were identified. qRT-PCR analysis showed that the expression of the candidate gene was significantly different between the two parents under salt stress. These results showed that the candidate gene might be related to leaf morphological development and abiotic stresses. Our study will lay a solid foundation for studying lobed leaf mechanism in B. napus L.

Clubroot is an economically important disease affecting plants in the family Cruciferae worldwide. In this study, a collection of 50 Cruciferae accessions was screened using Plasmodiophora brassicae pathotype 4 in China. Eight of these demonstrated resistance, including three Chinese cabbages, two cabbages, one radish, one kale, and one Brassica juncea. The three clubroot-resistant Chinese cabbages (1003, 1007 and 1008) were then used to transfer the clubroot resistance genes to B. napus by distant hybridization combined with embryo rescue. Three methods including morphological identification, cytology identification, and molecular marker-assisted selection were used to determine hybrid authenticity, and 0, 2, and 4 false hybrids were identified by these three methods, respectively. In total, 297 true hybrids were identified. Clubroot resistance markers and artificial inoculation were utilized to determine the source of clubroot resistance in the true hybrids. As a result, two simple sequence repeat (SSR) and two intron polymorphic (IP) markers linked to clubroot resistance genes were identified, the clubroot resistance genes of 1007 and 1008 were mapped to A03. At last, 159 clubroot-resistant hybrids were obtained by clubroot resistance markers and artificial inoculation. These intermediate varieties will be used as the 'bridge material' of clubroot resistance for further B. napus breeding.

Currently, cold temperatures are one of the main factors threatening rapeseed production worldwide; thus, it is imperative to identify cold-resistant germplasm and to cultivate cold-resistant rapeseed varieties. In this study, the cold resistance of four Brassica rapa varieties was analyzed. The cold resistance of Longyou6 and Longyou7 was better than that of Tianyou2 and Tianyou4. Thus, an F2 population derived from Longyou6 and Tianyou4 was used to study the correlation of cold resistance and physiological indexes. Our results showed that the degree of frost damage was related to the relative conductivity and MDA content (r1 = 0.558 and r2 = 0.447, respectively). In order to identify the markers related to cold resistance, 504 pairs of SSR (simple sequence repeats) primers were used to screen the two parents and F2 population. Four and five SSR markers had highly significant positive correlation to relative conductivity and MDA, respectively. In addition, three of these SSR markers had a highly significant positive correlation to both of these two indexes. These three SSR markers were subsequently confirmed to be used to distinguish between cold-resistant and non-cold-resistant varieties. The results of this study will lay a solid foundation for the mapping of cold-resistant genes and molecular markers assisted selection for the cold-resistance.

Interspecific pear (Pyrus spp.) hybrid populations are often used to develop novel cultivars. Pear cultivar breeding is a lengthy process because of long juvenility and the subsequent time required for reliable fruit phenotyping. Molecular techniques such as genome-wide association (GWA) and genomic selection (GS) provide an opportunity to fast-forward the development of high-value cultivars. We evaluated the genetic architecture of 10 pear fruit phenotypes (including sensory traits) and the potential of GS using genotyping-by-sequencing of 550 hybrid seedlings from nine interrelated full-sib families. Results from GWA suggested a complex polygenic nature of all 10 traits as the maximum variance explained by each marker was less than 4% of the phenotypic variance. The effect-size of SNPs for each trait suggested many genes of small effect and few of moderate effect. Some genomic regions associated with pear sensory traits were similar to those reported for apple – possibly a result of high synteny between the apple and pear genomes. The average (across nine families) GS accuracy varied from 0.32 (for crispness) to 0.62 (for sweetness), with an across-trait average of 0.42. Further efforts are needed to develop larger genotype-phenotype datasets in order to predict fruit phenotypes of untested seedlings with sufficient efficiency.

The PIK3 CA gene encodes the p110α protein subunit and is one of the most efficient cancer genes in solid and hematological tumors including hepatocellular carcinoma (HCC). There are currently ongoing therapies against tumors based on PIK3 CA inhibition. Because microRNAs (miRNAs) play an important role in post-transcriptional regulation and are also involved in the inhibition of PIK3 CA expression to suppress cancer cell proliferation, overexpression of tumor-suppressive miRNA is a promising therapeutic approach for HCC therapy. The successful and localized delivery of miRNA overexpression vectors (pre-miRNA plasmids) is very important in improving the therapeutic efficacy of this miRNA therapy strategy. In the study described here, submicron acoustic phase-shifted nanodroplets were used to efficiently deliver pre-miRNA plasmid in vitro and in vivo for HCC therapy under focused ultrasound (US) activation. Briefly, six miRNAs, inhibiting PIK3 CA and downregulated in HCC, were selected through summary and analysis of the currently existing literature data. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot and cell apoptosis assay revealed that pre-miR-139, -203a, -378a and -422a plasmids among the six miRNA overexpression vectors could suppress growth of the hepatoma cell line SMMC-7721. These four pre-miRNA plasmids were then electrostatically adhered to positively charged lipid-shelled nanodroplets to obtain plasmid-loaded nanodroplets (PLNDs). The PLND-generated microbubbles oscillated and even collapsed under US exposure to release the loaded pre-miRNA plasmids and enhance their cellular uptake through consequent sonoporation, that is, formation of small pores on the cell membrane induced by the mechanical effects of PLND cavitation. Fluorescence microscopy results revealed that PLNDs could effectively deliver the aforementioned four pre-miRNA plasmids into SMMC-7721 cells in vitro under 1.2-MHz 60-cycle sinusoid US exposure with a peak negative pressure >5.5 MPa at a 40-Hz pulse repetition frequency. Plasmid delivery efficiency and cell viability positively correlated with the inertial cavitation dose that was determined mainly by peak negative pressure. Furthermore, PLNDs combined with US were evaluated in vivo to deliver these four pre-miRNAs plasmids and verify their therapeutic efficacy in subcutaneous tumor of the mouse xenograft HCC model. The results revealed that the PLNDs loaded with pre-miR-139 and -378a plasmids could effectively suppress tumor growth after US treatment. Thus, combination of pre-miRNA PLNDs with US activation seems to constitute a potential strategy for HCC therapy.

The past three decades have witnessed the development of genetic linkage maps and use of DNA markers in mapping agronomic traits in many crops. In comparison with other plants, linkage mapping in pear has been initiated rather late, not until the year 2001. Pear is characterized by a typical self-incompatibility, and it has a long generation cycle. Therefore, genetic maps have been constructed using F1 populations. This may lead to the development of linkage maps of lower resolutions due to the lack of sufficient genetic variations. Fortunately, the development of next-generation sequencing technology has allowed for detection of high-quality genome-wide DNA markers, such as simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), in larger size populations, thus greatly improving the quality of genetic linkage maps. Overall, linkage maps are highly useful for dissecting complex agronomic traits, and for identifying either quantitative trait loci (QTL) or key genes regulating a target trait of interest. Furthermore, they contribute to efforts to pursue marker-assisted breeding (MAB) in pear.

Pyrus pyrifolia is considered one of the most important cultivated Pyrus species. Hundreds of landraces and bred cultivars have been developed through the natural and artificial hybridizations necessary due to self-incompatibility. In this study, the genetic diversity of 478 Pyrus accessions, including Chinese landraces, bred cultivars, and wild samples, as well as introduced pear cultivars from Japan and Korea, was investigated with a set of 17 simple sequence repeat (SSR) markers distributed across all 17 linkage groups of the pear genome. A total of 121 alleles were detected, including 4 rare alleles with a frequency lower than 5%. Diversity statistics indicated a high level of genetic variation as quantified by the average values of the observed heterozygosity, the expected heterozygosity, and Wright’s fixation index, at 0.76, 0.78, and 0.02, respectively. Population structure and discriminant analysis of principal component analysis implied extensive genetic communication between sand pears in China and revealed four contiguous geographical clusters with overlapping geographical regions. The diversity of the four clusters and approximate Bayesian computation (ABC) indicated that sand pear spread from west to east along the Pearl River and Yangtze River valleys. High diversity and polyphyletic genetic components of cultivars in southwestern China further support southwestern China as the probable center of divergence for Pyrus species. A core collection of 80 out of 470 cultivars was selected, accounting for about 17% of accessions, and capturing 91% of all alleles, including all rare alleles. Our research provides a comprehensive understanding of sand pear germplasm in East Asia and constructs a preliminary core collection, which will be useful for association genetics studies, germplasm conservation, and breeding programs.

Cold stress is one of the major abiotic stresses that seriously limit rapeseed production worldwide. However, few studies on the mechanism of cold resistance in Brassica napus have been reported. In this study, an F2:3 population including 147 lines was developed to identify the quantitative trait loci (QTLs) related to cold resistance in B. napus. As a result, a genetic linkage map based on 333 simple sequence repeat (SSR) markers covering 1317.70 cM was constructed. Up to 11 QTLs for four indicators were identified in the two locations. These QTLs accounted for 1.09% to 42.50% of the phenotypic variations, and six major QTLs accounted for more than 10% of the phenotypic variations. Three QTLs, qSPADYL-6, qSPADYS-6, and qMDAYS-6, were mapped to the same region of linkage group 6 (LG6). Blast analysis indicated that the sequences of the markers related to these three QTLs showed great collinearity with those on the A08 chromosome of Brassica rapa, and that the target genes might exist in the region from 1.069 to 15.652 M on A08. Two genes, BnaA08g05330D and BnaA08g15470D, encoding the respective cold-regulated proteins in B. napus, were identified. They exhibited high similarity with Bra039858 and Bra010579 (stress-responsive proteins) in the candidate region. RT-qPCR analysis showed a significant difference in gene expression between the two parents. These two genes were hence identified as the genes responsible for cold resistance.

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. Electronic supplementary material The online version of this article (10.1186/s13059-018-1452-y) contains supplementary material, which is available to authorized users.

Clubroot is an economically important disease affecting plants in the family Cruciferae worldwide. In this study, a collection of 50 Cruciferae accessions was screened using Plasmodiophora brassicae pathotype 4 in China. Eight of these demonstrated resistance, including three Chinese cabbages, two cabbages, one radish, one kale, and one Brassica juncea. The three clubroot-resistant Chinese cabbages (1003, 1007 and 1008) were then used to transfer the clubroot resistance genes to B. napus by distant hybridization combined with embryo rescue. Three methods including morphological identification, cytology identification, and molecular marker-assisted selection were used to determine hybrid authenticity, and 0, 2, and 4 false hybrids were identified by these three methods, respectively. In total, 297 true hybrids were identified. Clubroot resistance markers and artificial inoculation were utilized to determine the source of clubroot resistance in the true hybrids. As a result, two simple sequence repeat (SSR) and two intron polymorphic (IP) markers linked to clubroot resistance genes were identified, the clubroot resistance genes of 1007 and 1008 were mapped to A03. At last, 159 clubroot-resistant hybrids were obtained by clubroot resistance markers and artificial inoculation. These intermediate varieties will be used as the ‘bridge material’ of clubroot resistance for further B. napus breeding.

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.

Pear (Pyrus L.) is one of the most important fruits in temperate regions. Even in the mountainous border region of Tibet, Sichuan, and Yunnan Provinces (China), a variety of highly adapted pear landraces were discovered in Tibetan Autonomous Prefecture and were named “Zangli pears.” Here, we analyzed genetic diversity of 29 genotypes of Zangli distributed in Tibetan Region, together with 30 genotypes of sand pear (P. pyrifolia) in border regions, and 8 genotypes of P. ussuriensis as out groups, using simple sequence repeats (SSR) markers. Twenty-eight SSR markers displayed high polymorphism with a total of 202 alleles. Cluster analysis revealed a close genetic relationship between Zangli and sand pears, while population structure analysis revealed that gene flow between the western and eastern side of the Jin-sha River was restricted by geographic isolation. Diversity statistics for five geographic groups implied a migration event from Sichuan and Yunnan Provinces to the border region of western Sichuan, eastern Tibet, and northwestern Yunnan Provinces. Six specific genetic alleles in Zangli pear landraces might be related to their adaption to severe environmental conditions.

SWEET genes are a recently identified plant gene family that play an indispensable role in sugar efflux. However, no systematic study has been performed in pear. In this research, 18 SWEET transporters identified in pear, almost twice the number found in woodland strawberry and Japanese apricot, were divided into four clades. Conserved motifs and six exons of the SWEET transporters were found in six species. SWEET transporters contained seven transmembrane segments (TMSs) that evolved from an internal duplication of an ancestral three-TMSs unit, connected by TMS4. This is the first direct evidence identifying internal repeats through bioinformatics analysis. Whole-genome duplication (WGD) or segmental duplication and dispersed duplication represent the main driving forces for SWEET family evolution in six species, with former duplications more important in pear. Gene expression results suggested that PbSWEET15 and PbSWEET17 have no expression in any tissues because of critical lost residues and that 62.5% of PbSWEET duplicate gene pairs have functional divergence. Additionally, PbSWEET6, PbSWEET7 and PbSWEET14 were found to play important roles in sucrose efflux from leaves, and the high expression of PbSWEET1 and PbSWEET2 might contribute to unloading sucrose from the phloem in the stem. Finally, PbSWEET5, PbSWEET9 and PbSWEET10 might contribute to pollen development. Overall, our study provides important insights into the evolution of the SWEET gene family in pear and four other Rosaceae, and the important candidate PbSWEET genes involved in the development of different tissues were identified in pear.

The transcriptomes of five pear cultivars, ‘Hosui’ (P. pyrifolia), ‘Yali’ (P. bretschneideri), ‘Kuerlexiangli’ (P. sinkiangensis), ‘Nanguoli’ (P. ussuriensis) and ‘Starkrimson’ (P. communis) were sequenced at seven key fruit developmental stages, from fruit setting to maturation and fruit senescence after harvesting. In total, 33,136 genes that could be mapped by reads, were analyzed. Most gene expression cluster models showed a steadily decreasing trend. Gene expression patterns had obvious differences according to maturity type, that is, post-ripening cultivars were still vigorous at maturity and showed a higher proportion of up-regulated genes; non post-ripening cultivars had a gradually decreasing tendency during fruit maturation. Meanwhile, differentially expressed genes related to fruit quality and development, such as stone cells, sugar, acid and hormones, were identified. Co-expression analysis revealed that several ethylene synthesis genes and polyphenoloxidase-related genes interacted with each other directly and an indirect relationship was reflected between ethylene synthesis genes and ethylene response genes. In addition, the highly diverse SNPs represented the great differences between oriental and occidental pears. Understanding how RNA-seq based gene-expression patterns and differential gene expression contribute to fruit quality allows us to build models for gene-expression for fruit development of Pyrus species.

Supplementary data and analysis. (DOC 12492Â kb)

Supplementary data and analysis. (DOC 12492Â kb)