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Broomcorn millet (Panicum miliaceum L.) was domesticated in northern China at least 7,000 years ago and was subsequentially adopted as a cereal in many areas throughout Eurasia. One such locale is Areni-1 an archaeological cave site in Southern Armenia, a region that has an important history in crop domestication. The rich botanical material found at Areni-1 includes grains identified by morphology as broomcorn millet that were 14C dated to the medieval era (873 ± 36 CE and 1118 ± 35 CE). To retrace the demographic history of these broomcorn millet samples, we used ancient DNA extraction and hybridization capture enrichment to sequence and assemble three chloroplast genomes from the Areni-1 grains and then compared these sequences to 50 modern chloroplast genomes. Overall, the chloroplast genomes contained a low amount of diversity and little inference on broomcorn demography could be made. However, in a phylogeny the chloroplast genomes separated into two clades with strong bootstrap support, similar to what has been reported for nuclear DNA from broomcorn millet. In a haplotype network, the chloroplast genomes of two accessions of wild (undomesticated) broomcorn millet contained a relatively large number of variants, 11 SNPs. These SNPs were not present in the domesticated varieties, suggesting these wild accessions may not be directly related to the lineages that underwent domestication or that broomcorn millet may have undergone a domestication bottleneck resulting in lost diversity in the chloroplast genome. These results demonstrate that broomcorn millet from archaeological sites can preserve DNA for at least 1000 years and serve as a genetic resource to study the domestication of this cereal crop.

Genome fractionation (also known as diploidization) frequently occurs following paleopolyploidization events. Biased fractionation between subgenomes has been found in some paleo-allopolyploids, while this phenomenon is absent in paleo-autopolyploids. Pear (Pyrus bretschneideri Rehd.) experienced a recent whole-genome duplication (WGD, ~30 million years ago); however, the evolutionary fate of the two subgenomes derived from this WGD event is not clear. In this study, we identified the two paleo-subgenomes in pear using peach (Prunus persica) as an outgroup and investigated differences in the gene loss rate, evolutionary rate, gene expression level, and DNA methylation level between these two subgenomes. Fractionation bias was not found between the two pear subgenomes, which evolved at similar evolutionary rates. The DNA methylation level of the two subgenomes showed little bias, and we found no expression dominance between the subgenomes. However, we found that singleton genes and homeologous genes within each subgenome showed divergent evolutionary patterns of selective constraints, expression and epigenetic modification. These results provide insights into subgenome evolution following paleopolyploidization in pear.

Broomcorn millet (Panicum miliaceum L.) is the most water-efficient cereal and one of the earliest domesticated plants. Here we report its high-quality, chromosome-scale genome assembly using a combination of short-read sequencing, single-molecule real-time sequencing, Hi-C, and a high-density genetic map. Phylogenetic analyses reveal two sets of homologous chromosomes that may have merged ~5.6 million years ago, both of which exhibit strong synteny with other grass species. Broomcorn millet contains 55,930 protein-coding genes and 339 microRNA genes. We find Paniceae-specific expansion in several subfamilies of the BTB (broad complex/tramtrack/bric-a-brac) subunit of ubiquitin E3 ligases, suggesting enhanced regulation of protein dynamics may have contributed to the evolution of broomcorn millet. In addition, we identify the coexistence of all three C4 subtypes of carbon fixation candidate genes. The genome sequence is a valuable resource for breeders and will provide the foundation for studying the exceptional stress tolerance as well as C4 biology., Broomcorn millet is one of the earliest domesticated plants and has the highest water use efficiency among cereals. Here, the authors report its genome assembly and annotation, which provides a valuable resource for breeders and paves the way for studying plant drought tolerance and C4 photosynthesis.

Epidermal fragments enriched in guard cells (GCs) were isolated from the halophyte quinoa (Chenopodium quinoa Wild.) species, and the response at the proteome level was studied after salinity treatment of 300 mM NaCl for 3 weeks. In total, 2147 proteins were identified, of which 36% were differentially expressed in response to salinity stress in GCs. Up and downregulated proteins included signaling molecules, enzyme modulators, transcription factors and oxidoreductases. The most abundant proteins induced by salt treatment were desiccation-responsive protein 29B (50-fold), osmotin-like protein OSML13 (13-fold), polycystin-1, lipoxygenase, alpha-toxin, and triacylglycerol lipase (PLAT) domain-containing protein 3-like (eight-fold), and dehydrin early responsive to dehydration (ERD14) (eight-fold). Ten proteins related to the gene ontology term &ldquo, response to ABA&rdquo, were upregulated in quinoa GC, this included aspartic protease, phospholipase D and plastid-lipid-associated protein. Additionally, seven proteins in the sucrose&ndash, starch pathway were upregulated in the GC in response to salinity stress, and accumulation of tryptophan synthase and L-methionine synthase (enzymes involved in the amino acid biosynthesis) was observed. Exogenous application of sucrose and tryptophan, L-methionine resulted in reduction in stomatal aperture and conductance, which could be advantageous for plants under salt stress. Eight aspartic proteinase proteins were highly upregulated in GCs of quinoa, and exogenous application of pepstatin A (an inhibitor of aspartic proteinase) was accompanied by higher oxidative stress and extremely low stomatal aperture and conductance, suggesting a possible role of aspartic proteinase in mitigating oxidative stress induced by saline conditions.

Soil salinity is a major environmental constraint affecting crop growth and threatening global food security. Plants adapt to salinity by optimizing the performance of stomata. Stomata are formed by two guard cells (GCs) that are morphologically and functionally distinct from the other leaf cells. These microscopic sphincters inserted into the wax-covered epidermis of the shoot balance CO2 intake for photosynthetic carbon gain and concomitant water loss. In order to better understand the molecular mechanisms underlying stomatal function under saline conditions, we used proteomics approach to study isolated GCs from the salt-tolerant sugar beet species. Of the 2088 proteins identified in sugar beet GCs, 82 were differentially regulated by salt treatment. According to bioinformatics analysis (GO enrichment analysis and protein classification), these proteins were involved in lipid metabolism, cell wall modification, ATP biosynthesis, and signaling. Among the significant differentially abundant proteins, several proteins classified as &ldquo, stress proteins&rdquo, were upregulated, including non-specific lipid transfer protein, chaperone proteins, heat shock proteins, inorganic pyrophosphatase 2, responsible for energized vacuole membrane for ion transportation. Moreover, several antioxidant enzymes (peroxide, superoxidase dismutase) were highly upregulated. Furthermore, cell wall proteins detected in GCs provided some evidence that GC walls were more flexible in response to salt stress. Proteins such as L-ascorbate oxidase that were constitutively high under both control and high salinity conditions may contribute to the ability of sugar beet GCs to adapt to salinity by mitigating salinity-induced oxidative stress.

Summary The complexity of polyploid Saccharum genomes hindered progress of genome research and crop improvement in sugarcane. To understand their genome structure, transcriptomes of 59 F1 individuals derived from S. officinarum LA Purple and S. robustum Molokai 5829 (2n = 80, x = 10 for both) were sequenced, yielding 11 157 and 8998 SNPs and 83 and 105 linkage groups, respectively. Most markers in each linkage group aligned to single sorghum chromosome. However, 71 interchromosomal rearrangements were detected between sorghum and S. officinarum or S. robustum, and 24 (33.8%) of them were shared between S. officinarum and S. robustum, indicating their occurrence before the speciation event that separated these two species. More than 2000 gene pairs from S. spontaneum, S. officinarum and S. robustum were analysed to estimate their divergence time. Saccharum officinarum and S. robustum diverged about 385 thousand years ago, and the whole‐genome duplication events occurred after the speciation event because of shared interchromosomal rearrangements. The ancestor of these two species diverged from S. spontaneum about 769 thousand years ago, and the reduction in basic chromosome number from 10 to 8 in S. spontaneum occurred after the speciation event but before the two rounds of whole‐genome duplication. Our results proved that S. officinarum is a legitimate species in its own right and not a selection from S. robustum during the domestication process in the past 10 000 years. Our findings rejected a long‐standing hypothesis and clarified the timing of speciation and whole‐genome duplication events in Saccharum.

Background Leucine-rich repeat receptor-like protein kinase (LRR-RLK) is the largest gene family of receptor-like protein kinases (RLKs) and actively participates in regulating the growth, development, signal transduction, immunity, and stress responses of plants. However, the patterns of LRR-RLK gene family evolution in the five main Rosaceae species for which genome sequences are available have not yet been reported. In this study, we performed a comprehensive analysis of LRR-RLK genes for five Rosaceae species: Fragaria vesca (strawberry), Malus domestica (apple), Pyrus bretschneideri (Chinese white pear), Prunus mume (mei), and Prunus persica (peach), which contained 201, 244, 427, 267, and 258 LRR-RLK genes, respectively. Results All LRR-RLK genes were further grouped into 23 subfamilies based on the hidden Markov models approach. RLK-Pelle_LRR-XII-1, RLK-Pelle_LRR-XI-1, and RLK-Pelle_LRR-III were the three largest subfamilies. Synteny analysis indicated that there were 236 tandem duplicated genes in the five Rosaceae species, among which subfamilies XII-1 (82 genes) and XI-1 (80 genes) comprised 68.6%. Conclusions Our results indicate that tandem duplication made a large contribution to the expansion of the subfamilies. The gene expression, tissue-specific expression, and subcellular localization data revealed that LRR-RLK genes were differentially expressed in various organs and tissues, and the largest subfamily XI-1 was highly expressed in all five Rosaceae species, suggesting that LRR-RLKs play important roles in each stage of plant growth and development. Taken together, our results provide an overview of the LRR-RLK family in Rosaceae genomes and the basis for further functional studies. Electronic supplementary material The online version of this article (10.1186/s12864-017-4155-y) contains supplementary material, which is available to authorized users.

Hexokinase genes (HXK) encode enzymes that play an important role in fruit quality and plant development because they participate in the first step of the glycolytic pathway for sugar accumulation and metabolism. In this study, the authors conducted analysis of the phylogeny, gene structure, and expression of HXK genes in pear (Pyrus bretschneideri Rehd.), comparing them with 7 other plant species. A total of 10 HXK protein sequences from pear and 33 HXKs from 7 other species were identified and clustered into 4 groups, matching their motif classification. Duplication events and motif structure variation attributed the diversification of HXK genes, supported by differential expression pattern of PbrHXKs from pear fruit transcriptome data and qRT – PCR verification. Combining pear fruit soluble sugar content data and analyses above, PbrHXK1and PbrHXK3 were identified as important candidate genes in sorbitol, fructose, and glucose accumulation during pear fruit development. PbrHXK1and PbrHXK3 were i...

Background The sharp increase of plant genome and transcriptome data provide valuable resources to investigate evolutionary consequences of gene duplication in a range of taxa, and unravel common principles underlying duplicate gene retention. Results We survey 141 sequenced plant genomes to elucidate consequences of gene and genome duplication, processes central to the evolution of biodiversity. We develop a pipeline named DupGen_finder to identify different modes of gene duplication in plants. Genes derived from whole-genome, tandem, proximal, transposed, or dispersed duplication differ in abundance, selection pressure, expression divergence, and gene conversion rate among genomes. The number of WGD-derived duplicate genes decreases exponentially with increasing age of duplication events—transposed duplication- and dispersed duplication-derived genes declined in parallel. In contrast, the frequency of tandem and proximal duplications showed no significant decrease over time, providing a continuous supply of variants available for adaptation to continuously changing environments. Moreover, tandem and proximal duplicates experienced stronger selective pressure than genes formed by other modes and evolved toward biased functional roles involved in plant self-defense. The rate of gene conversion among WGD-derived gene pairs declined over time, peaking shortly after polyploidization. To provide a platform for accessing duplicated gene pairs in different plants, we constructed the Plant Duplicate Gene Database. Conclusions We identify a comprehensive landscape of different modes of gene duplication across the plant kingdom by comparing 141 genomes, which provides a solid foundation for further investigation of the dynamic evolution of duplicate genes.

The MYB superfamily is large and functionally diverse in plants. To date, MYB family genes have not yet been identified in Chinese white pear (Pyrus bretschneideri), and their functions remain unclear. In this study, we identified 231 genes as candidate MYB genes and divided them into four subfamilies. The R2R3-MYB (PbrMYB) family shared an R2R3 domain with 104 amino acid residues, including five conserved tryptophan residues. The Pbr MYB family was divided into 37 functional subgroups including 33 subgroups which contained both MYB genes of Rosaceae plants and AtMYB genes, and four subgroups which included only Rosaceae MYB genes or AtMYB genes. PbrMYB genes with similar functions clustered into the same subgroup, indicating functional conservation. We also found that whole-genome duplication (WGD) and dispersed duplications played critical roles in the expansion of the MYB family. The 87 Pbr MYB duplicated gene pairs dated back to the two WGD events. Purifying selection was the primary force driving Pbr MYB gene evolution. The 15 gene pairs presented 1-7 codon sites under positive selection. A total of 147 expressed genes were identified from RNA-sequencing data of fruit, and six Pbr MYB members in subgroup C1 were identified as important candidate genes in the regulation of lignin synthesis by quantitative real-time PCR analysis. Further correlation analysis revealed that six PbrMYBs were significantly correlated with five structural gene families (F5H, HCT, CCR, POD and C3'H) in the lignin pathway. The phylogenetic, evolution and expression analyses of the MYB gene family in Chinese white pear establish a solid foundation for future comprehensive functional analysis of Pbr MYB genes.

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.

Pear is an important fruit crop of the Rosaceae family and has experienced two rounds of ancient whole-genome duplications (WGDs). However, whether different types of gene duplications evolved differently after duplication remains unclear in the pear genome. In this study, we identified the different modes of gene duplication in pear. Duplicate genes derived from WGD, tandem, proximal, retrotransposed, DNA-based transposed or dispersed duplications differ in genomic distribution, gene features, selection pressure, expression divergence, regulatory divergence and biological roles. Widespread sequence, expression and regulatory divergence have occurred between duplicate genes over the 30-45 million years of evolution after the recent genome duplication in pear. The retrotransposed genes show relatively higher expression and regulatory divergence than other gene duplication modes. In contrast, WGD genes underwent a slower sequence divergence and may be influenced by abundant gene conversion events. Moreover, the different classes of duplicate genes exhibited biased functional roles. We also investigated the evolution and expansion patterns of the gene families involved in sugar and organic acid metabolism pathways, which are closely related to the fruit quality and taste in pear. Single-gene duplications largely account for the extensive expansion of gene families involved in the sorbitol metabolism pathway in pear. Gene family expansion was also detected in the sucrose metabolism pathway and tricarboxylic acid cycle pathways. Thus, this study provides insights into the evolutionary fates of duplicated genes.

Quantitative real-time PCR (qRT-PCR) is the preferred method for accurate analysis of targeted gene expression. The aim of this study was to validate optimal reference genes for qRT-PCR analyses in pear pollen. Ten candidate reference genes were chosen and examined in 36 pear samples. Among these, PbrEF1α, PbrGAPDH and PbrAPT were developed to detect the purity of cDNA. Three qRT-PCR analysis programs, geNorm, NormFinder and BestKeeper, were applied for systematic determinations. The results showed that all the candidate reference genes could be used for qRT-PCR normalization in pear pollen. PbrEF1α was the most stable reference gene across all abiotic stresses of pollen samples, especially for high temperature, N-butyl alcohol and NaCl treatments, and also during pollen development. PbrTUB was the optimal reference gene for pear tissue expression. PbrGAPDH was suitable for low temperature treatment and PbrPP2A was suitable for abscisic acid and also for NaCl treatment. For CuCl2 treatment, either of reference genes PbrCYP and PbrUBI was suitable. To illustrate the suitability of these reference genes, the expression profile of PbrVHA-a was used to analyze the expression patterns in different pear tissues, pollen developing stages and NaCl treatment. The results provide a valuable resource for gene expression characterization in pear pollen.

MADS-boxtranscription factors play significant roles in plant developmental processes such as floral organ conformation, flowering time, and fruit development. Pear (Pyrus), as the third-most crucial temperate fruit crop, has been fully sequenced. However, there is limited information about theMADSfamily and its functional divergence in pear. In this study, a total of 95MADS-boxgenes were identified in the pear genome, and classified into two types by phylogenetic analysis. Type IMADS-boxgenes were divided into three subfamilies and type II genes into 14 subfamilies. Synteny analysis suggested that whole-genome duplications have played key roles in the expansion of theMADSfamily, followed by rearrangement events. Purifying selection was the primary force drivingMADS-boxgene evolution in pear, and one gene pairs presented three codon sites under positive selection. Full-scale expression information forPbrMADSgenes in vegetative and reproductive organs was provided and proved by transcriptional and reverse transcription PCR analysis. Furthermore, thePbrMADS11(12)gene, together with partnersPbMYB10andPbbHLH3was confirmed to activate the promoters of the structural genes in anthocyanin pathway of red pear through dual luciferase assay. In addition, thePbrMADS11andPbrMADS12were deduced involving in the regulation of anthocyanin synthesis response to light and temperature changes. These results provide a solid foundation for future functional analysis ofPbrMADSgenes in different biological processes, especially of pigmentation in pear.

Background Cysteine-rich peptides (CRPs) are gaining recognition as regulators of cell–cell communication in plants. Results We identified 9556 CRPs in 12 plant species and analysed their evolutionary patterns. In most angiosperm plants, whole genome duplication and segmental duplication are the major factors driving the expansion of CRP family member genes, especially signal peptides. About 30% of the CRP genes were found clustered on the chromosomes, except in maize (Zea mays). Considerable collinearities between CRP genes between or within species reveal several syntenic regions on the chromosomes. Different subfamilies display diverse evolutionary rates, suggesting that these subfamilies are subjected to different selective pressures. CRPs in different duplication models also show contrasting evolutionary rates, although the underlying mechanism is unclear because of the complexity of gene evolution. The 1281 positively selected genes identified are probably generated within a certain period of time. While most of these belonged to maize and sorghum (Sorghum bicolor), new CRP functions would also be expected. Up-regulation of 10 CRPs was observed in self-pollinated pear pistils and pollen tubes under self S-RNase treatments in vitro. The expression divergence between different CRP gene duplication types suggests that different duplication mechanisms affected the fate of the duplicated CRPs. Conclusion Our analyses of the evolution of the CRP gene family provides a unique view of the evolution of this large gene family. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3948-3) contains supplementary material, which is available to authorized users.

Red fruits are popular and widely accepted by consumers because of an enhanced appearance and enriched anthocyanins. The molecular mechanism of anthocyanin regulation in red-skinned pear (Pyrus) has been studied, and the genes encoding the biosynthetic steps and several transcription factors (TFs) have been characterized. In this study, a candidate R2R3 MYB TF, PyMYB114, was identified by linkage to the quantitative trait loci (QTL) for red skin color on linkage group 5 in a population of Chinese pear (Pyrus bretschneideri). The function of PyMYB114 was verified by transient transformation in tobacco (Nicotinana tabacum) leaves and strawberry (Fragaria) and pear fruits, resulting in the biosynthesis of anthocyanin. Suppression of PyMYB114 could inhibit anthocyanin biosynthesis in red-skinned pears. The ERF/AP2 TF PyERF3 was found to interact with PyMYB114 and its partner PybHLH3 to co-regulate anthocyanin biosynthesis, as shown by a dual luciferase reporter system and a yeast two-hybrid assay. In addition, the transcript abundance of PyMYB114 and PyMYB10 were correlated, and co-transformation of these two genes into tobacco and strawberry led to enhanced anthocyanin biosynthesis. This interaction network provides insight into the coloration of fruits and the interaction of different TFs to regulate anthocyanin biosynthesis.

Summary A highly saturated pear genetic map was constructed using over 3000 SNP markers developed by RADseq integrated with anchored SSR markers. The reliable QTLs of several fruit traits were also identified., Pear (Pyrus spp) is an important fruit crop, grown in all temperate regions of the world, with global production ranked after grape and apples among deciduous tree crops. A high-density linkage map is a valuable tool for fine mapping quantitative trait loci (QTL) and map-based gene cloning. In this study, we firstly constructed a high-density linkage map of pear using SNPs integrated with SSRs, developed by the rapid and robust technology of restriction-associated DNA sequencing (RADseq). The linkage map consists of 3143 SNP markers and 98 SSRs, 3241 markers in total, spanning 2243.4 cM, with an average marker distance of 0.70 cM. Anchoring SSRs were able to anchor seventeen linkage groups to their corresponding chromosomes. Based on this high-density integrated pear linkage map and two years of fruit phenotyping, a total of 32 potential QTLs for 11 traits, including length of pedicel (LFP), single fruit weight (SFW), soluble solid content (SSC), transverse diameter (TD), vertical diameter (VD), calyx status (CS), flesh colour (FC), juice content (JC), number of seeds (NS), skin colour (SC), and skin smooth (SS), were identified and positioned on the genetic map. Among them, some important fruit-related traits have for the first time been identified, such as calyx status, length of pedicel, and flesh colour, and reliable localization of QTLs were verified repeatable. This high-density linkage map of pear is a worthy reference for mapping important fruit traits, QTL identification, and comparison and combination of different genetic maps.

As an important fruit crop that is widely grown commercially in temperate regions of the world, pear (Pyrus) is a target for pursuing efficient breeding strategies. Construction of a reliable and dense genetic linkage map is critical for undertaking marker-assisted breeding. In this study, a population of 56 F1 seedlings of ‘Bayuehong’ × ‘Dangshansuli’ was used to construct a high-density simple sequence repeat (SSR)-based genetic linkage map. A total of 1,756 SSR markers, including 1,341 newly designed SSRs based on whole-genome sequencing of an Asiatic pear along with 415 previously reported SSRs, were first evaluated for polymorphism. Based on 894 SSRs demonstrating polymorphism, a consensus genetic map consisting of 734 loci distributed along all 17 linkage groups (LG) was constructed, with a total length of 1,661.4 cM and with an average marker interval of 2.26 cM. Comparisons among different maps of pear and apple were then made based on positions of previously mapped SSR markers on the consensus map. As a result, homologous linkage groups LG3 and LG11, LG5 and LG10, LG9 and LG17, LG13 and LG16, LG8 and LG15 have been identified. This high-density SSR map along with a set of SSR markers covering the whole genome of pear will greatly facilitate integration of independent maps, aid in pursuing comparative genome studies, and in evaluation of different germplasm in future genetic and breeding studies.

Cassandra transposable elements belong to a specific group of terminal-repeat retrotransposons in miniature (TRIM). Although Cassandra TRIM elements have been found in almost all vascular plants, detailed investigations on the nature, abundance, amplification timeframe, and evolution have not been performed in an individual genome. We therefore conducted a comprehensive analysis of Cassandra retrotransposons using the newly sequenced pear genome along with four other Rosaceae species, including apple, peach, mei, and woodland strawberry. Our data reveal several interesting findings for this particular retrotransposon family: 1) A large number of the intact copies contain three, four, or five long terminal repeats (LTRs) (~20% in pear); 2) intact copies and solo LTRs with or without target site duplications are both common (~80% vs. 20%) in each genome; 3) the elements exhibit an overall unbiased distribution among the chromosomes; 4) the elements are most successfully amplified in pear (5,032 copies); and 5) the evolutionary relationships of these elements vary among different lineages, species, and evolutionary time. These results indicate that Cassandra retrotransposons contain more complex structures (elements with multiple LTRs) than what we have known previously, and that frequent interelement unequal recombination followed by transposition may play a critical role in shaping and reshaping host genomes. Thus this study provides insights into the property, propensity, and molecular mechanisms governing the formation and amplification of Cassandra retrotransposons, and enhances our understanding of the structural variation, evolutionary history, and transposition process of LTR retrotransposons in plants.

Summary Neural progenitors with distinct potential to generate progeny are associated with a spatially distinct microenvironment. Neocortical intermediate progenitors (IPs) in the subventricular zone (SVZ) of the developing brain generate neurons for all cortical layers and are essential for cortical expansion. Here, we show that spatial control of IP positioning is essential for neocortical development. We demonstrate that HDAC1 and HDAC2 regulate the spatial positioning of IPs to form the SVZ. Developmental stage-specific depletion of both HDAC1 and HDAC2 in radial glial progenitors results in mispositioning of IPs at the ventricular surface, where they divide and differentiate into neurons, thereby leading to the cortical malformation. We further identified the proneural gene Neurogenin2 as a key target of HDAC1 and HDAC2 for regulating IP positioning. Our results demonstrate the importance of the spatial positioning of neural progenitors in cortical development and reveal a mechanism underlying the establishment of the SVZ microenvironment.

Glutamate-like receptors (GLRs) is a highly conserved family of ligand-gated ion channels, which have been associated with various physiological and developmental processes. Here, we investigated the evolutionary pattern of GLRs in plants. We observed that tandem duplications occupied the largest proportion of the plant GLR gene family expansion. Based on a phylogenetic tree, we suggested a new subfamily, GLR4, which is widespread in angiosperm but absence on Brassicales. Meanwhile, because GLR1 and GLR2 subfamilies were potential sister clades, we combined them into the GLR1&2 subfamily. A comparative analysis of plant GLR subfamilies revealed that selective forces shaped the GLR1&2 repertoires in the stems of eudicotyledons with distinct functional preferences. Moreover, GLR1&2 formed a species-specific highwoody-expanded subfamily, with preferential expression in the cambial-enriched and shoot apical meristem fractions of the highwood species. Together, these findings lay the foundation for evolutionary analysis of plant GLRs over the entire plant timescale and identified unique targets for manipulating the woody-growth behaviours of plant GLRs.

Pear, the third most important temperate fruit species after grape and apple, belongs to the subfamily Pomoideae in the family Rosaceae. The majority of cultivated pears are functional diploids (2n = 34). As a popular fruit in the world market, pear has widespread cultivation on six continents, with major production in China, the United States, Italy, Argentina, and Spain (Supplemental Fig. 1). Pears are among the oldest of the world's fruit crops, with >3000 yr of cultivation history (Lombard and Westwood 1987), likely originating during the Tertiary period (65–55 million years ago [MYA]) in the mountainous regions of southwestern China and, from there, spreading on to both the East and West (Rubtsov 1944; Zeven and Zhukovsky 1975). Central Asia and eastern China are identified as two subcenters of genetic diversity for pear (Vavilov 1951). The Pyrus genus is genetically diverse with thousands of cultivars, but it can be divided into two major groups, Occidental pears (European pears) and Oriental pears (Asiatic pears). At least 22 primary species are well-recognized in Pyrus; however, only a few species, including Pyrus bretschneideri, Pyrus pyrifolia, Pyrus ussuriensis, Pyrus sinkiangensis, and Pyrus communis, have been utilized for fruit production. Herein, we report on a high-quality draft genome sequence of the diploid P. bretschneideri Rehd. cv. ‘Dangshansuli’ (also known as ‘Suli’), the most important commercial Asiatic pear cultivar grown in the world (>4 million tons per year), having >500 yr of cultivated history in China. Pear is highly heterozygous due to self-incompatibility and interspecies compatibility. The genome is known to have an abundance of repetitive DNA sequences. In this study, a novel combination of BAC-by-BAC (bacterial artificial chromosome) strategy, with Illumina sequencing technology, is used for the first time for de novo assembly of a highly heterozygous genome of this size with highly repetitive DNA sequences. This has demonstrated that a complex plant genome sequence can be assembled and characterized using these technologies without the availability of a physical reference. Additionally, we also report on primary factors contributing to genome size differences between pear and apple, both belonging to the subfamily Pomoideae; chromosomal evolution of Rosaceae; and genes controlling valuable traits of pear, including self-incompatibility, lignified stone cells in flesh of fruit (unique to pear), sugar, and aroma.

The basic helix-loop-helix (bHLH) transcription factors are involved in arrays of physiological and biochemical processes. However, knowledge concerning the functions of bHLHs in cold tolerance remains poorly understood. In this study, a PubHLH1 gene isolated from Pyrus ussuriensis was characterized for its function in cold tolerance. PubHLH1 was upregulated by cold, salt, and dehydration, with the greatest induction under cold conditions. PubHLH1 had the transactivational activity and localized in the nucleus. Ectopic expression of PubHLH1 in transgenic tobacco conferred enhanced tolerance to cold stress. The transgenic lines had higher survival rates, higher chlorophyll, higher proline contents, lower electrolyte leakages and MDA when compared with wild type (WT). In addition, transcript levels of eight genes associated with ROS scavenging, regulation, and stress defense were higher in the transgenic plants relative to the WT under the chilling stress. Taken together, these results demonstrated that PubHLH1 played a key role in cold tolerance and, at least in part, contributed to activation of stress-responsive genes.

Inter-retrotransposon amplified polymorphisms (IRAP) are one of the main components of the retrotransposon-based molecular marker system, but currently have no applications in pear. In this context, new IRAP markers were developed and used to study the genetic polymorphism of pear cultivars and bud mutants. In total, eight polymorphic IRAP markers were selected in ten genotypes of pear with a wide variation in genetic backgrounds and for further genotypic identification of cultivars and bud sports. A total of 76 alleles with an average of 9.5 per locus were amplified and 96.05 % showed polymorphism. Through genetic structure analysis, 62 pear cultivars were divided into two groups, i.e., Oriental and Occidental pears, with a few samples showing a distinct admixed genetic background. When polymorphic IRAP markers were tested on a total of 33 bud sports and their corresponding parent cultivars, the genetic similarity coefficient ranged from 0.54 to 0.96. Each bud mutation was separated from its original variety, and 92.3 % of bud sports clustered perfectly with their original varieties. Furthermore, the transferability of pear IRAP to apples and other Rosaceae species was very high, ranging from 87.5 to 100 %. Our results demonstrate that IRAP markers are an excellent tool for the study of genetic relationships and comparative genomics analysis in pear and Rosaceae species.

Background Heat shock transcription factors (Hsfs), which act as important transcriptional regulatory proteins in eukaryotes, play a central role in controlling the expression of heat-responsive genes. At present, the genomes of Chinese white pear (‘Dangshansuli’) and five other Rosaceae fruit crops have been fully sequenced. However, information about the Hsfs gene family in these Rosaceae species is limited, and the evolutionary history of the Hsfs gene family also remains unresolved. Results In this study, 137 Hsf genes were identified from six Rosaceae species (Pyrus bretschneideri, Malus × domestica, Prunus persica, Fragaria vesca, Prunus mume, and Pyrus communis), 29 of which came from Chinese white pear, designated as PbHsf. Based on the structural characteristics and phylogenetic analysis of these sequences, the Hsf family genes could be classified into three main groups (classes A, B, and C). Segmental and dispersed duplications were the primary forces underlying Hsf gene family expansion in the Rosaceae. Most of the PbHsf duplicated gene pairs were dated back to the recent whole-genome duplication (WGD, 30–45 million years ago (MYA)). Purifying selection also played a critical role in the evolution of Hsf genes. Transcriptome data demonstrated that the expression levels of the PbHsf genes were widely different. Six PbHsf genes were upregulated in fruit under naturally increased temperature. Conclusion A comprehensive analysis of Hsf genes was performed in six Rosaceae species, and 137 full length Hsf genes were identified. The results presented here will undoubtedly be useful for better understanding the complexity of the Hsf gene family and will facilitate functional characterization in future studies. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0401-5) contains supplementary material, which is available to authorized users.

Genetic maps are essential tools for pear genetics and genomics research. In this study, we first constructed an integrated simple sequence repeat (SSR) and single nucleotide polymorphism (SNP)-based consensus genetic map for pear based on common SSR markers between nine published maps. A total of 5,085 markers, including 1,232 SSRs and 3,853 SNPs, were localized on a consensus map spanning 3,266.0 cM in total, with an average marker interval of 0.64 cM, which represents the highest density consensus map of pear to date. Using three sets of high-density SNP-based genetic maps with European pear genetic backgrounds, we anchored a total of 291.5 Mb of the ‘Bartlett’ v1.0 (Pyrus communis L.) genome scaffolds into 17 pseudo-chromosomes. This accounted for 50.5% of the genome assembly, which was a great improvement on the 29.7% achieved originally. Intra-genome and inter-genome synteny analyses of the new ‘Bartlett’ v1.1 genome assembly with the Asian pear ‘Dangshansuli’ (Pyrus bretschneideri Rehd.) and apple (Malus × domestica Borkh.) genomes uncovered four new segmental duplication regions. The integrated high-density SSR and SNP-based consensus genetic map provided new insights into the genetic structure patterns of pear and assisted in the genome assembly of ‘Bartlett’ through further exploration of different pear genetic maps.

The sugar transporter (ST) plays an important role in plant growth, development and fruit quality. In this study, a total of 75 ST genes were identified in the pear (Pyrus bretschneideri Rehd) genome based on systematic analysis. Furthermore, all ST genes identified were grouped into eight subfamilies according to conserved domains and phylogenetic analysis. Analysis of cis-regulatory element sequences of all ST genes identified the MYBCOREATCYCB1 promoter in sucrose transporter (SUT) and monosaccharide transporter (MST) genes of pear, while in grape it is exclusively found in SUT subfamily members, indicating divergent transcriptional regulation in different species. Gene duplication event analysis indicated that whole-genome duplication (WGD) and segmental duplication play key roles in ST gene amplification, followed by tandem duplication. Estimation of positive selection at codon sites of ST paralog pairs indicated that all plastidic glucose translocator (pGlcT) subfamily members have evolved under positive selection. In addition, the evolutionary history of ST gene duplications indicated that the ST genes have experienced significant expansion in the whole ST gene family after the second WGD, especially after apple and pear divergence. According to the global RNA sequencing results of pear fruit development, gene expression profiling showed the expression of 53 STs. Combined with quantitative real-time PCR (qRT-PCR) analysis, two polyol/monosaccharide transporter (PLT) and three tonoplast monosaccharide transporter (tMT) members were identified as candidate genes, which may play important roles in sugar accumulation during pear fruit development and ripening. Identification of highly expressed STs in fruit is important for finding novel genes contributing to enhanced levels of sugar content in pear fruit.

Coffee, tea, and chocolate converge Caffeine has evolved multiple times among plant species, but no one knows whether these events involved similar genes. Denoeud et al. sequenced the Coffea canephora (coffee) genome and identified a conserved gene order (see the Perspective by Zamir). Although this species underwent fewer genome duplications than related species, the relevant caffeine genes experienced tandem duplications that expanded their numbers within this species. Scientists have seen similar but independent expansions in distantly related species of tea and cacao, suggesting that caffeine might have played an adaptive role in coffee evolution. Science , this issue p. 1181 ; see also p. 1124

The cyclic nucleotide-gated channel (CNGC) family is involved in the uptake of various cations, such as Ca(2+), to regulate plant growth and respond to biotic and abiotic stresses. However, there is far less information about this family in woody plants such as pear. Here, we provided a genome-wide identification and analysis of the CNGC gene family in pear. Phylogenetic analysis showed that the 21 pear CNGC genes could be divided into five groups (I, II, III, IVA and IVB). The majority of gene duplications in pear appeared to have been caused by segmental duplication and occurred 32.94-39.14 million years ago. Evolutionary analysis showed that positive selection had driven the evolution of pear CNGCs. Motif analyses showed that Group I CNGCs generally contained 26 motifs, which was the greatest number of motifs in all CNGC groups. Among these, eight motifs were shared by each group, suggesting that these domains play a conservative role in CNGC activity. Tissue-specific expression analysis indicated that functional diversification of the duplicated CNGC genes was a major feature of long-term evolution. Our results also suggested that the P-S6 and PBC & hinge domains had co-evolved during the evolution. These results provide valuable information to increase our understanding of the function, evolution and expression analyses of the CNGC gene family in higher plants.

Background 'Kuerlexiangli’ (Pyrus sinkiangensis Yu), a native pear of Xinjiang, China, is an important agricultural fruit and primary export to the international market. However, fruit with persistent calyxes affect fruit shape and quality. Although several studies have looked into the physiological aspects of the calyx abscission process, the underlying molecular mechanisms remain unknown. In order to better understand the molecular basis of the process of calyx abscission, materials at three critical stages of regulation, with 6000 × Flusilazole plus 300 × PBO treatment (calyx abscising treatment) and 50 mg.L-1GA3 treatment (calyx persisting treatment), were collected and cDNA fragments were sequenced using digital transcript abundance measurements to identify candidate genes. Results Digital transcript abundance measurements was performed using high-throughput Illumina GAII sequencing on seven samples that were collected at three important stages of the calyx abscission process with chemical agent treatments promoting calyx abscission and persistence. Altogether more than 251,123,845 high quality reads were obtained with approximately 8.0 M raw data for each library. The values of 69.85%-71.90% of clean data in the digital transcript abundance measurements could be mapped to the pear genome database. There were 12,054 differentially expressed genes having Gene Ontology (GO) terms and associating with 251 Kyoto Encyclopedia of Genes and Genomes (KEGG) defined pathways. The differentially expressed genes correlated with calyx abscission were mainly involved in photosynthesis, plant hormone signal transduction, cell wall modification, transcriptional regulation, and carbohydrate metabolism. Furthermore, candidate calyx abscission-specific genes, e.g. Inflorescence deficient in abscission gene, were identified. Quantitative real-time PCR was used to confirm the digital transcript abundance measurements results. Conclusions We identified candidate genes that showed highly dynamic changes in expression during the calyx abscission process. These genes are potential targets for future functional characterization and should be valuable for exploration of the mechanisms of calyx abscission, and eventually for developing methods based on small molecule application to induce calyx abscission in fruit production.

© 2013 Ming et al. Background: Sacred lotus is a basal eudicot with agricultural, medicinal, cultural and religious importance. It was domesticated in Asia about 7,000 years ago, and cultivated for its rhizomes and seeds as a food crop. It is particularly noted for its 1,300-year seed longevity and exceptional water repellency, known as the lotus effect. The latter property is due to the nanoscopic closely packed protuberances of its self-cleaning leaf surface, which have been adapted for the manufacture of a self-cleaning industrial paint, Lotusan. Results: The genome of the China Antique variety of the sacred lotus was sequenced with Illumina and 454 technologies, at respective depths of 101× and 5.2×. The final assembly has a contig N50 of 38.8 kbp and a scaffold N50 of 3.4 Mbp, and covers 86.5% of the estimated 929 Mbp total genome size. The genome notably lacks the paleo-triplication observed in other eudicots, but reveals a lineage-specific duplication. The genome has evidence of slow evolution, with a 30% slower nucleotide mutation rate than observed in grape. Comparisons of the available sequenced genomes suggest a minimum gene set for vascular plants of 4,223 genes. Strikingly, the sacred lotus has 16 COG2132 multi-copper oxidase family proteins with root-specific expression; these are involved in root meristem phosphate starvation, reflecting adaptation to limited nutrient availability in an aquatic environment. Conclusions: The slow nucleotide substitution rate makes the sacred lotus a better resource than the current standard, grape, for reconstructing the pan-eudicot genome, and should therefore accelerate comparative analysis between eudicots and monocots.

A set of 120 simple sequence repeats (SSRs) was developed from the newly assembled pear sequence and evaluated for polymorphisms in seven genotypes of pear from different genetic backgrounds. Of these, 67 (55.8 %) primer pairs produced polymorphic amplifications. Together, the 67 SSRs detected 277 alleles with an average of 4.13 per locus. Sequencing of the amplification products from randomly picked loci NAUPy31a and NAUpy53a verified the presence of the SSR loci. When the 67 primer pairs were tested on 96 individual members of eight species in the Rosaceae family, 61.2 % (41/67) of the tested SSRs successfully amplified a PCR product in at least one of the Rosaceae genera. The transferability from pear to different species varied from 58.2 % (apple) to 11.9 % (cherry). The ratio of transferability also reflected the closer relationships within Maloideae over Prunoideae. Two pear SSR markers, NAUpy43c and NAUpy55k, could distinguish the 20 different apple genotypes thoroughly, and UPGMA cluster analysis grouped them into three groups at the similarity level of 0.56. The high level of polymorphism and good transferability of pear SSRs to Rosaceae species indicate their promise for application to future molecular screening, map construction, and comparative genomic studies among pears and other Rosaceae species. Electronic supplementary material The online version of this article (doi:10.1007/s11105-013-0586-z) contains supplementary material, which is available to authorized users.

Background Sugarcane is a major sugar and biofuel crop, but genomic research and molecular breeding have lagged behind other major crops due to the complexity of auto-allopolyploid genomes. Sugarcane cultivars are frequently aneuploid with chromosome number ranging from 100 to 130, consisting of 70–80 % S. officinarum, 10–20 % S. spontaneum, and 10 % recombinants between these two species. Analysis of a genomic region in the progenitor autoploid genomes of sugarcane hybrid cultivars will reveal the nature and divergence of homologous chromosomes. Results To investigate the origin and evolution of haplotypes in the Bru1 genomic regions in sugarcane cultivars, we identified two BAC clones from S. spontaneum and four from S. officinarum and compared to seven haplotype sequences from sugarcane hybrid R570. The results clarified the origin of seven homologous haplotypes in R570, four haplotypes originated from S. officinarum, two from S. spontaneum and one recombinant.. Retrotransposon insertions and sequences variations among the homologous haplotypes sequence divergence ranged from 18.2 % to 60.5 % with an average of 33.7 %. Gene content and gene structure were relatively well conserved among the homologous haplotypes. Exon splitting occurred in haplotypes of the hybrid genome but not in its progenitor genomes. Tajima’s D analysis revealed that S. spontaneum hapotypes in the Bru1 genomic regions were under strong directional selection. Numerous inversions, deletions, insertions and translocations were found between haplotypes within each genome. Conclusions This is the first comparison among haplotypes of a modern sugarcane hybrid and its two progenitors. Tajima’s D results emphasized the crucial role of this fungal disease resistance gene for enhancing the fitness of this species and indicating that the brown rust resistance gene in R570 is from S. spontaneum. Species-specific InDel, sequences similarity and phylogenetic analysis of homologous genes can be used for identifying the origin of S. spontaneum and S. officinarum haplotype in Saccharum hybrids. Comparison of exon splitting among the homologous haplotypes suggested that the genome rearrangements in Saccharum hybrids after hybridization. The combined minimum difference at 19.5 % among homologous chromosomes in S. officinarum would be sufficient for proper genome assembly of this autopolyploid genome. Retrotransposon insertions and sequences variations among the homologous haplotypes sequence divergence may allow sequencing and assembling the autopolyploid Saccharum genomes and the auto-allopolyploid hybrid genomes using whole genome shotgun sequencing. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2817-9) contains supplementary material, which is available to authorized users.

Background Lipoxygenases (LOXs), a type of non-haem iron-containing dioxygenase, are ubiquitous enzymes in plants and participate in the formation of fruit aroma which is a very important aspect of fruit quality. Amongst the various aroma volatiles, saturated and unsaturated alcohols and aldehydes provide the characteristic aroma of the fruit. These compounds are formed from unsaturated fatty acids through oxidation, pyrolysis and reduction steps. This biosynthetic pathway involves at least four enzymes, including LOX, the enzyme responsible for lipid oxidation. Although some studies have been conducted on the LOX gene family in several species including Arabidopsis, soybean, cucumber and apple, there is no information from pear; and the evolutionary history of this gene family in the Rosaceae is still not resolved. Results In this study we identified 107 LOX homologous genes from five Rosaceous species (Pyrus bretschneideri, Malus × domestica, Fragaria vesca, Prunus mume and Prunus persica); 23 of these sequences were from pear. By using structure analysis, phylogenic analysis and collinearity analysis, we identified variation in gene structure and revealed the phylogenetic evolutionary relationship of this gene family. Expression of certain pear LOX genes during fruit development was verified by analysis of transcriptome data. Conclusions 23 LOX genes were identified in pear and these genes were found to have undergone a duplication 30–45 MYA; most of these 23 genes are functional. Specific gene duplication was found on chromosome4 in the pear genome. Useful information was provided for future research on the evolutionary history and transgenic research on LOX genes. Electronic supplementary material The online version of this article (doi: 10.1186/1471-2164-15-444) contains supplementary material, which is available to authorized users.

Background Lotus is a diploid plant with agricultural, medicinal, and ecological significance. Genetic linkage maps are fundamental resources for genome and genetic study, and also provide molecular markers for breeding in agriculturally important species. Genotyping by sequencing revolutionized genetic mapping, the restriction-site associated DNA sequencing (RADseq) allowed rapid discovery of thousands of SNPs markers, and a crucial aspect of the sequence based mapping strategy is the reference sequences used for marker identification. Results We assessed the effectiveness of linkage mapping using three types of references for scoring markers: the unmasked genome, repeat masked genome, and gene models. Overall, the repeat masked genome produced the optimal genetic maps. A high-density genetic map of American lotus was constructed using an F1 population derived from a cross between Nelumbo nucifera ‘China Antique’ and N. lutea ‘AL1’. A total of 4,098 RADseq markers were used to construct the American lotus ‘AL1’ genetic map, and 147 markers were used to construct the Chinese lotus ‘China Antique’ genetic map. The American lotus map has 9 linkage groups, and spans 494.3 cM, with an average distance of 0.7 cM between adjacent markers. The American lotus map was used to anchor scaffold sequences in the N. nucifera ‘China Antique’ draft genome. 3,603 RADseq markers anchored 234 individual scaffold sequences into 9 megascaffolds spanning 67% of the 804 Mb draft genome. Conclusions Among the unmasked genome, repeat masked genome and gene models, the optimal reference sequences to call RADseq markers for map construction is repeat masked genome. This high density genetic map is a valuable resource for genomic research and crop improvement in lotus.