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Background The plant hormone auxin is widely involved in plant growth, development, and morphogenesis, and the TIR1/AFB and AUX/IAA proteins are closely linked to rapid auxin response and signal transmission. However, their evolutionary history, historical patterns of expansion and contraction, and changes in interaction relationships are still unknown. Results Here, we analyzed the gene duplications, interactions, and expression patterns of TIR1/AFBs and AUX/IAAs to understand their underlying mechanisms of evolution. The ratios of TIR1/AFBs to AUX/IAAs range from 4:2 in Physcomitrium patens to 6:29 in Arabidopsis thaliana and 3:16 in Fragaria vesca. Whole-genome duplication (WGD) and tandem duplication have contributed to the expansion of the AUX/IAA gene family, but numerous TIR1/AFB gene duplicates were lost after WGD. We further analyzed the expression profiles of TIR1/AFBs and AUX/IAAs in different tissue parts of Physcomitrium patens, Selaginella moellendorffii, Arabidopsis thaliana and Fragaria vesca, and found that TIR1/AFBs and AUX/IAAs were highly expressed in all tissues in P. patens, S. moellendorffii. In A. thaliana and F. vesca, TIR1/AFBs maintained the same expression pattern as the ancient plants with high expression in all tissue parts, while AUX/IAAs appeared tissue-specific expression. In F. vesca, 11 AUX/IAAs interacted with TIR1/AFBs with different interaction strengths, and the functional specificity of AUX/IAAs was related to their ability to bind TIR1/AFBs, thus promoting the development of specific higher plant organs. Verification of the interactions among TIR1/AFBs and AUX/IAAs in Marchantia polymorpha and F. vesca also showed that the regulation of AUX/IAA members by TIR1/AFBs became more refined over the course of plant evolution. Conclusions Our results indicate that specific interactions and specific gene expression patterns both contributed to the functional diversification of TIR1/AFBs and AUX/IAAs.

Presence and absence polymorphisms (PAPs) exist extensively and have been investigated in different organisms. However, PAPs have rarely been detected between strawberry species at the genome level. This study identified the presence and absence genes (P/A genes) between wild strawberry species (Fragaria vesca) and octoploid cultivated species (F. × ananassa) under a relatively strict criterion. In total, 333 P/A genes present in the wild strawberry but absent in the cultivated strawberry were detected. Of the P/A genes, 91.89% (306/333) were single genes, and only 8.11% were confirmed as multi-genes. The majority of the identified P/A genes in Fragaria were generated by tandem duplications. The P/A genes were unevenly distributed on the seven chromosomes of woodland strawberry, and they clustered preferentially near the telomeric regions of the chromosomes. The P/A genes tended to encode proteins with domains closely associated with responses to varying ecological factors, such as PPR, Protein kinases (PKs), NB-ARC, F-box and EF-hand domains. This indicated that the P/A genes were associated with coping with biotic and abiotic stresses to improve the adaptability of plants to changing environments.

Fragaria vesca, commonly known as wild or woodland strawberry, is the most widely distributed diploid Fragaria species and is native to Europe and Asia. Because of its small plant size, low heterozygosity, and relative ease of genetic transformation, F. vesca has been a model plant for fruit research since the publication of its Illumina-based genome in 2011. However, its genomic contribution to octoploid cultivated strawberry remains a long-standing question. Here, we de novo assembled and annotated a telomere-to-telomere, gap-free genome of F. vesca ‘Hawaii 4’, with all seven chromosomes assembled into single contigs, providing the highest completeness and assembly quality to date. The gap-free genome is 220 785 082 bp in length and encodes 36 173 protein-coding gene models, including 1153 newly annotated genes. All 14 telomeres and seven centromeres were annotated within the seven chromosomes. Among the three previously recognized wild diploid strawberry ancestors, F. vesca, F. iinumae, and F. viridis, phylogenomic analysis showed that F. vesca and F. viridis are the ancestors of the cultivated octoploid strawberry F. × ananassa, and F. vesca is its closest relative. Three subgenomes of F. × ananassa belong to the F. vesca group, and one is sister to F. viridis. We anticipate that this high-quality, telomere-to-telomere, gap-free F. vesca genome, combined with our phylogenomic inference of the origin of cultivated strawberry, will provide insight into the genomic evolution of Fragaria and facilitate strawberry genetics and molecular breeding.

The various monochromatic Light Emitting Diode (LED) lights are widely used in growth facility for cultivating various plants, particularly horticultural crops because of their higher luminous efficiency, lower radiation and power consumption than the traditional white fluorescent lamp light. However, considerable inconsistent effects have been reported in literature. We conducted a meta-analysis to assess the effects of different colors of LED light on plant growth, development and various traits. Compared to the light from white fluorescent lamps, the red LED light significantly changed 4 out 26 plant characteristics by at least 37%, and blue LED light significantly increased 5 of 26 assessed characteristics by 37% or more. The combination of red/blue LED lights only significantly increased dry weight by 161% among 25 plant characteristics analyzed. Compared to the white LED light, red LED light significantly decreased 2 of 9 plant characteristics by at least 36%, and blue LED light significantly decreased only 1 of 9 plant characteristics, total chlorophyll content, by 42%. In the moderators analyzed, plant taxonomic families significantly influenced the effects of LED lights on shoot dry weight, and plant life cycles and plant taxonomic families significantly affected the effect on stomatal conductance. Through systematic meta-analysis, we found that the effect of LED on plant growth and quality traits was species-specific, and the effect was affected by the cultivation conditions. Therefore, we suggest that researchers be more targeted to experiment, and collect traits associated with practical production, especially related to the quality of product data, such as carotenoids, anthocyanin and other antioxidant compounds. This article is to provide more data with practical application, guide the application of LED in horticultural plant factory.

R2R3-MYB gene family play important roles in plants development, metabolism, and responses to various biotic and abiotic stresses. In this study, 838 R2R3-MYB genes were identified from six Rosaceae species, including 105 in woodland strawberry (Fragaria vesca), 173 in European pear (Pyrus communis), 219 in apple (Malus domestica), 121 in peach (Prunus persica), 121 in Chinese rose (Rosa chinensis), and 99 in black raspberry (Rubus occidentalis). All R2R3-MYB genes in the six Rosaceae species were clustered into 51 species-specific duplicated clades with 109 genes and 50 lineage-specific duplicated clades with 242 genes according to phylogenetic analysis. R2R3-MYB genes were distributed on all chromosomes in each of the six species, with a small amount of tandem duplication events. The proportion of tandem repeat genes ranged from 0 to 25.1%. The R2R3-MYB protein was conserved in a clade and likely to share similar functions. The distribution of Ks showed the duplication times of R2R3-MYB genes in six Rosaceae species. Furthermore, most of the R2R3-MYB genes had Ka/Ks values less than 1, which indicated they were driven by purifying selection during the evolutionary processes. The GO term enrichment analysis revealed that R2R3-MYB genes in strawberry and black raspberry were more divergent than in other Rosaceae species. Analysis of transcriptomes of 42 different tissues and development stages of woodland strawberry showed that high expression levels of R2R3-MYB suggested that the R2R3-MYB genes in strawberry played a key role in growth and development of both vegetative tissues and fruits. The strawberry R2R3-MYB genes in sub-group of S1, S2, S11, S20, and S22 had high expression levels both in young leaves (YL) and old leaves (OL) strawberry tissues under drought treatments.

There is an increasing demand for fresh fruits such as strawberry enriched in antioxidants and vitamins. Strawberry fruit quality is highly affected by genotype and environmental conditions. In thi...

Plants with crassulacean acid metabolism (CAM) generally utilize water 20%–80% more efficiently than non-CAM plants. The whole genomes of several CAM plants have been sequenced or are being sequenced. For effective genome characterization and genome editing of CAM plants, an efficient transformation system is essential. In this study, we developed an Agrobacterium-mediated transformation protocol for Kalanchoe laxiflora, an obligate CAM plant,by optimizing several factors affecting the transformation efficiency. Agrobacterium strains AGL1, C58, EHA105, and GV3101 were all suitable for K. laxiflora transformation. Fifty-nine percent of the leaf explants yielded kanamycin-resistant and GUS-positive shoots. Polymerase chain reaction and quantitative real-time PCR (qRT-PCR) using gusA-, gusPlus-, nptII- and hpt-specific primers confirmed that the transgenes were integrated into K. laxiflora genome and expressed. This efficient transformation system will allow effective functional characterization of genes through over- or down-expression, knockout, or genome editing. Keywords: Kalanchoe laxiflora, Agrobacterium-mediated transformation, CAM plants

Apple blotch caused by Alternaria alternata apple pathotype is a severe disease of apple (Malus ×domestica Borkh) occurring throughout the world, especially in eastern Asia. Phenotypic and genetic information about resistance/susceptibility of apple germplasm to this disease will be extremely valuable for selecting and developing new disease resistant cultivars. In this study, 110 apple cultivars obtained from the USDA apple germplasm in Geneva, NY, were evaluated for their resistance/susceptibility to apple blotch by field surveys, and inoculation of detached leaves with a suspension of germinated conidia of A. alternata apple pathotype. Disease incidence were different among the cultivars and categorized into resistant (R), moderately resistant (MR), or susceptible (S). Two molecular markers, S428, a random amplified polymorphic DNA (RAPD) marker associated with disease resistance, and a simple sequence repeat (SSR or microsatellite) marker CH05g07, linked to susceptibility were used to correlate the phenotypes expressed in field surveys and laboratory inoculations. The detection using either the S428 marker or the CH05g07 marker in 50 common breeding cultivars was consistent with R or S traits except for ‘Bisbee’ and ‘Priscilla’. These two cultivars were MR to apple blotch through phenotyping. However, SSR markers were detected, but RAPD markers were not and therefore were considered susceptible. Combined with the record of resistance to fire blight from Germplasm Resources Information Network (GRIN), ‘Dayton’, ‘Mildew Immune Seedling’, ‘Puregold’, and ‘Pumpkin Sweet’ were highly resistant to both diseases and considered as the best choices of parents for stacking resistance to multiple diseases in breeding program.

SQUAMOSA promoter binding protein-like (SPL) proteins is a class of plant specific transcription factors that play important roles during plant development. However, the majority of SPL genes in strawberry are functionally uncharacterized. In this study, three SPL genes, i.e. FvSPL1, FvSPL2, and FvSPL11 (FvSPL1/2/11), from woodland strawberry were cloned and characterized. Phylogenetic analysis with SPL genes from Arabidopsis, tomato and chrysanthemum indicated that FvSPL1/2/11 were clustered into the same group with those of miR156 target site located at the 3’-untranslated region (UTR). Further biochemical analysis indicated that FvSPL1 was exclusively localized in the nucleus. Electrophoretic mobility shift assay demonstrated FvSPL1 could specifically recognized the GTAC motif. Transcriptional activity analysis showed FvSPL1 was a transcriptional activator that could activate the expression of FvAP1 gene. Finally, all of the transgenic Arabidopsis that overexpression the three FvSPL genes were exhibited significantly early flowering phenotype. Taken together, our study indicated that FvSPL1/2/11 similar to their orthologs in Arabidopsis mainly functions in regulating plant flowering. These results enriched our understanding to the functions of SPL genes in strawberry and might be utilized for strawberry flowering time manipulation in the future.

In this study, genome-wide identification, phylogenetic relationships, duplication time and selective pressure of the NBS-LRR genes, an important group of plant disease-resistance genes (R genes), were performed to uncover their genetic evolutionary patterns in the six Prunus species. A total of 1946 NBS-LRR genes were identified; specifically, 589, 361, 284, 281, 318, and 113 were identified in Prunus yedoensis, P. domestica, P. avium, P. dulcis, P. persica and P. yedoensis var. nudiflora, respectively. Two NBS-LRR gene subclasses, TIR-NBS-LRR (TNL) and non-TIR-NBS-LRR (non-TNL), were also discovered. In total, 435 TNL and 1511 non-TNL genes were identified and could be classified into 30/55/75 and 103/158/191 multi-gene families, respectively, according to three different criteria. Higher Ks and Ka/Ks values were detected in TNL gene families than in non-TNL gene families. These results indicated that the TNL genes had more members involved in relatively ancient duplications and were affected by stronger selection pressure than the non-TNL genes. In general, the NBS-LRR genes were shaped by species-specific duplications, and lineage-specific duplications occurred at recent and relatively ancient periods among the six Prunus species. Therefore, different duplicated copies of NBS-LRRs can resist specific pathogens and will provide an R-gene library for resistance breeding in Prunus species.

Background In plant genomes, high proportions of duplicate copies reveals that gene duplications play an important role in the evolutionary processes of plant species. A series of gene families under positive selection after recent duplication events in plant genomes indicated the evolution of duplicates driven by adaptive evolution. However, the genome-wide evolutionary features of young duplicate genes among closely related species are rarely reported. Results In this study, we conducted a systematic survey of young duplicate genes at genome-wide levels among six Rosaceae species, whose whole-genome sequencing data were successively released in recent years. A total of 35,936 gene families were detected among the six species, in which 60.25% were generated by young duplications. The 21,650 young duplicate gene families could be divided into two expansion types based on their duplication patterns, species-specific and lineage-specific expansions. Our results showed the species-specific expansions advantaging over the lineage-specific expansions. In the two types of expansions, high-frequency duplicate domains exhibited functional preference in response to environmental stresses. Conclusions The functional preference of the young duplicate genes in both the expansion types showed that they were inclined to respond to abiotic or biotic stimuli. Moreover, young duplicate genes under positive selection in both species-specific and lineage-specific expansions suggested that they were generated to adapt to the environmental factors in Rosaceae species.

Transcription factors regulate gene expression in response to various external and internal cues by activating or suppressing downstream genes. Significant progress has been made in identifying and characterizing the Cysteine3Histidine (C3H) gene family in several dicots and monocots. They are characterized by their signature motif of three cysteine and one histidine residues, and reportedly play important roles in regulation of plant growth, developmental processes and environmental responses. In this study, we performed genome-wide and deep analysis of putative C3H genes, and a total of 117 PbeC3H members, were identified in P. betulaefolia and classified into 12 groups. Results were supported by the gene structural characteristics and phylogenetic analysis. These genes were unevenly distributed on 17 chromosomes. The gene structures of the C3H genes were relatively complex but conserved in each group. The C3H genes experienced a WGD event that occurred in the ancestor genome of P. betulaefolia and apple before their divergence based on the synonymous substitutions (Ks) values. There were 35 and 37 pairs of paralogous genes in the P. betulaefolia and apple genome, respectively, and 87 pairs of orthologous genes between P. betulaefolia and apple were identified. Except for one orthologous pairs PbeC3H66 and MD05G1311700 which had undergone positive selection, the other C3H genes had undergone purifying selection. Expression profiles showed that high salinity stress could influence the expression level of C3H genes in P. betulaefolia. Four members were responsive to salt stress in roots, nine were responsive to salt stress in leaves and eight showed inhibited expression in leaves. Results suggested important roles of PbeC3H genes in response to salt stress and will be useful for better understanding the complex functions of the C3H genes, and will provide excellent candidates for salt-tolerance improvement.

Adventitious root (AR) formation is critical for the efficient propagation of elite horticultural and forestry crops. Despite decades of research, the cellular processes and molecular mechanisms underlying AR induction in woody plants remain obscure. We examined the details of AR formation in apple (Malus domestica) M.9 rootstock, the most widely used dwarf rootstock for intensive production, and investigated the role of polar auxin transport in postembryonic organogenesis. AR formation begins with a series of founder cell divisions and elongation of the interfascicular cambium adjacent to vascular tissues. This process is associated with a relatively high indole acetic acid (IAA) content and hydrolysis of starch grains. Exogenous auxin treatment promoted this cell division, as well as the proliferation and reorganization of the endoplasmic reticulum and Golgi membrane. In contrast, treatment with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) inhibited cell division in the basal region of the cuttings and resulted in abnormal cell divisions during the early stage of AR formation. In addition, PIN-FORMED (PIN) transcripts were differentially expressed throughout the whole AR development process. We also detected upregulation of MdPIN8 and MdPIN10 during induction; upregulation of MdPIN4, MdPIN5, and MdPIN8 during extension; and upregulation of all MdPINs during AR initiation. This research provides an improved understanding of the cellular and molecular underpinnings of the AR process in woody plants.

SQUAMOSA-promoter binding protein-like (SPL) proteins are plant-specific transcript factors that play essential roles in plant growth and development. Although many SPL genes have been well characterized in model plants like Arabidopsis, rice and tomato, the functions of SPLs in strawberry are still largely elusive. In the present study, we cloned and characterized FvSPL10, the ortholog of AtSPL9, from woodland strawberry. Subcellular localization shows FvSPL10 localizes in the cell nucleus. The luciferase system assay indicates FvSPL10 is a transcriptional activator, and both in vitro and in vivo assays indicate FvSPL10 could bind to the promoter of FvAP1 and activate its expression. Ectopic expression of FvSPL10 in Arabidopsis promotes early flowering and increases organs size. These results demonstrate the multiple regulatory roles of FvSPL10 in plant growth and development and lay a foundation for investigating the biological functions of FvSPL10 in strawberry.

The ABA/water stress/ripening-induced (ASR) gene family exists universally in higher plants, and many ASR genes are up-regulated during periods of environmental stress and fruit ripening. Although a considerable amount of research has been performed investigating ASR gene response to abiotic stresses, relatively little is known about their roles in response to biotic stresses. In this report, we identified five ASR genes in apple (Malus × domestica) and explored their phylogenetic relationship, duplication events, and selective pressure. Five apple ASR genes (Md-ASR) were divided into two clades based on phylogenetic analysis. Species-specific duplication was detected in M. domestica ASR genes. Leaves of ‘Golden delicious’ and ‘Starking’ were infected with Alternaria alternata f. sp. mali, which causes apple blotch disease, and examined for the expression of the ASR genes in lesion areas during the first 72 h after inoculation. Md-ASR genes showed different expression patterns at different sampling times in ‘Golden delicious’ and ‘Starking’. The activities of stress-related enzymes, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia lyase (PAL), and polyphenoloxidase (PPO), and the content of malondialdehyde (MDA) were also measured in different stages of disease development in two cultivars. The ASR gene expression patterns and theses physiological indexes for disease resistance suggested that Md-ASR genes are involved in biotic stress responses in apple.

Pyracantha coccinea is a thorny evergreen shrub native to southeast Europe to southeast Asia. It is a popular ornamental plant because of its showy bright red fruits and small white flowers. However, in vitro vegetative propagation of P. coccinea has not been studied. Nodal segments with one or two axillary buds (1 to 1.5 cm in length) were cut and disinfected in a solution of 0.1% (v/v) mercuric chloride (HgCl2) for 5 minutes, and proliferated on Murashige and Skoog (MS) basal medium supplemented with various concentrations 6-benzylaminopurine (6-BA). After 4 weeks, newly formed shoots were transferred to proliferation and rooting media containing various concentrations of indole-3-butyric acid (IBA). Establishment of axillary buds was significantly better with an establishing rate of 67% on basal MS medium augmented with 6.6 µm 6-BA. The best medium for proliferation of shoots was three-fourth basal MS supplemented with 1.5 µm IBA, with a proliferation rate of 3.4 axillary bud. The optimum rooting medium was one-fourth MS basal medium containing 93 µm IBA. Rooting of shoots was as much as 77%. Rooted plantlets were transferred to pots containing vermiculite:perlite:peat (6:1:2) and acclimatized to ambient greenhouse conditions with a 95% survival rate. This protocol can be used for in vitro propagation of P. coccinea.

Superoxide dismutase (SOD) is an essential enzyme of the plant antioxidant system that responds to oxidative damage caused by adverse conditions. However, little is known about the SOD gene family in Vitis vinifera (Vv). In the present study, ten SOD genes, including 6 copper/zinc SODs, 2 iron SODs, and 2 manganese SODs, were identified in the grapevine genome where they were unevenly distributed on 12 chromosomes. Ten VvSOD genes were divided into three main groups based on phylogenetic analysis, subcellular localization, and the distribution of conserved protein motifs. Additionally, many cis-elements related to different stresses were found in the promoters of the 10 VvSOD genes. Syntenic analysis revealed that VvMSD1 and VvMSD2 were derived from segmental duplication, and VvCSD4 and VvCSD5 belong to a pair of tandemly duplicated genes. Gene expression analysis based on microarray data showed that the 10 VvSOD genes were expressed in all the tested tissues. Interestingly, the segmentally duplicated gene pair (VvMSD1 and VvMSD2) exhibited differential expression patterns in various organs. In contrast, the tandemly duplicated gene pair (VvCSD4 and VvCSD5) displayed similar expression patterns in the tested organs. Our results provide a basis for further functional research on the SOD gene family in grapevine.

Dehydration-responsive element binding proteins are transcription factors that play a critical role in plant response to temperature stress. Over-expression of DREB genes has been demonstrated to enhance temperature stress tolerance. A series of physiological and biochemical modifications occur in a complex and integrated way when plants respond to temperature stress, which makes it difficult to assess the mechanism underlying the DREB enhancement of stress tolerance. A meta-analysis was conducted of the effect of DREB overexpression on temperature stress tolerance and the various parameters modulated by overexpression that were statistically quantified in 75 published articles. The meta-analysis was conducted to identify the overall influence of DREB on stress-related parameters in transgenic plants, and to determine how different experimental variables affect the impact of DREB overexpression. Viewed across all the examined studies, 7 of the 8 measured plant parameters were significantly (p ≤ 0.05) modulated in DREB-transgenic plants when they were subjected to temperature stress, while 2 of the 8 parameters were significantly affected in non-stressed control plants. The measured parameters were modulated by 32% or more by various experimental variables. The modulating variables included, acclimated or non-acclimated, type of promoter, stress time and severity, source of the donor gene, and whether the donor and recipient were the same genus. These variables all had a significant effect on the observed impact of DREB overexpression. Further studies should be conducted under field conditions to better understand the role of DREB transcription factors in enhancing plant tolerance to temperature stress.

Background Plant disease resistance (R) genes are evolving rapidly and play a critical role in the innate immune system of plants. The nucleotide binding sites-leucine rich repeat (NBS-LRR) genes are one of the largest classes in plant R genes. Previous studies have focused on the NBS-LRR genes from one or several species of different genera, and the sequenced genomes of the genus Fragaria offer the opportunity to study the evolutionary processes of these R genes among the closely related species. Results In this study, 325, 155, 190, 187, and 133 NBS-LRRs were discovered from F. x ananassa, F. iinumae, F. nipponica, F. nubicola, and F. orientalis, respectively. Together with the 144 NBS-LRR genes from F. vesca, a total of 1134 NBS-LRRs containing 866 multi-genes comprised 184 gene families across the six Fragaria genomes. Extremely short branch lengths and shallow nodes were widely present in the phylogenetic tree constructed with all of the NBS-LRR genes of the six strawberry species. The identities of the orthologous genes were highly significantly greater than those of the paralogous genes, while the Ks ratios of the former were very significantly lower than those of the latter in all of the NBS-LRR gene families. In addition, the Ks and Ka/Ks values of the TIR-NBS-LRR genes (TNLs) were significantly greater than those of the non-TIR-NBS-LRR genes (non-TNLs). Furthermore, the expression patterns of the NBS-LRR genes revealed that the same gene expressed differently under different genetic backgrounds in response to pathogens. Conclusions These results, combined with the shared hotspot regions of the duplicated NBS-LRRs on the chromosomes, indicated that the lineage-specific duplication of the NBS-LRR genes occurred before the divergence of the six Fragaria species. The Ks and Ka/Ks ratios suggested that the TNLs are more rapidly evolving and driven by stronger diversifying selective pressures than the non-TNLs. Electronic supplementary material The online version of this article (10.1186/s12864-018-4521-4) contains supplementary material, which is available to authorized users.

The basic leucine zipper (bZIP) transcription factors are the most diverse members of dimerizing transcription factors. In the present study, 50, 116, and 47bZIPgenes were identified inMalus domestica(apple),Prunus persica(peach), andFragaria vesca(strawberry), respectively. Species-specific duplication was the main contributor to the large number ofbZIPsobserved in apple. After WGD in apple genome, orthologousbZIPgenes corresponding to strawberry on duplicated regions in apple genome were retained. However, in peach ancestor, these syntenic regions were quickly lost or deleted. Maybe the positive selection contributed to the expansion of clade S to adapt to the development and environment stresses. In addition, purifying selection was mainly responsible forbZIPsequence-specific DNA binding. The analysis of orthologous pairs between chromosomes indicates that these orthologs derived from one gene duplication located on one of the nine ancient chromosomes in the Rosaceae. The comparative analysis ofbZIPgenes in three species provides information on the evolutionary fate ofbZIPgenes in apple and peach after they diverged from strawberry.

VQ motif-containing proteins play crucial roles in plant growth, development, and stress responses. However, no information of VQ motif-containing proteins has been studied at the microevolutionary level in species of Fragaria. In this study, a total of 19, 21, 23, 23, 23, and 25 genes containing the VQ motif were identified from the genomes of F. nipponica, F. iinumae, F. orientalis, F. vesca, F. nubicola, and F. x ananassa, respectively. We classified the VQ genes into 15 clades with grapevine VQ genes, which indicated that at least 15 ancient VQ genes existed before the divergence of the six studied species of Fragaria. Phylogenetic analysis indicated that 28 gene duplication events have occurred in the evolutionary process of the six species of Fragaria. Structural analysis showed that most of the VQ genes have no introns and that VQ proteins in each clade have a similar motif composition. The majority of gene pairs had Ka/Ksratios less than 1, which illustrated that most of the VQ genes underwent purifying selection in the six species of Fragaria. Four types of cis-elements in promoters of VQ genes were detected, which is an important basis for further studies about plant stress responses. Furthermore, the expression analysis of FvVQ genes indicated that these genes are expressed differentially in the examined organs and tissues. The identification of VQ genes and the analysis of VQ gene duplication and polyploidization events in the six species of Fragaria provide important information on the evolutionary fate of VQ genes during the divergence of the six species of Fragaria.

Alternaria blotch, caused by Alternaria alternata f. sp. mali, is a destructive defoliating disease that affects apple worldwide, especially in Asia. Despite previous efforts in characterizing this disease, few have reported on nucleotide binding sites (NBS)-encoding genes in response to pathogen infection. In this study, we characterized the morphological and gene expression response of ‘Golden Delicious’ and ‘Starking’ apples challenged by A. alternata. Inoculation of detached leaves of ‘Golden Delicious’ resulted in a rapid initiation of disease symptoms but a slow spread of the infection from the point of inoculation. In contrast, detached leaves of ‘Starking’ exhibited a delayed visibility of infection but a rapid spread of the infection once it was established. Based upon quantitative reverse transcription polymerase chain reaction data, we characterized the expression of 12 NBS-encoding genes in response to infection by A. alternata. We further characterized the structure, chromosomal location, and...

Fruit skin coloration is a unique phase in the life cycle of fruiting plants and is mainly attributed to anthocyanin pigments. Anthocyanins are the largest and most diverse group of plant pigments derived from the phenyl propanoid pathway. They are water-soluble phenolic compounds that form part of a large and common group of plant flavonoids. Coloration encompasses several physiological and biochemical changes that happen through differential expression of various developmentally regulated genes. Due to research importance and economic value, Arabidopsis thaliana (chromosome no. = 5) and Vitis vinifera (chromosome no. = 19) have been used for investigations of the structural genes involved in anthocyanin biosynthesis. Thus for this review, V. vinifera is used as a model crop. In anthocyanin biosynthesis, a wide range of constructive genes including phenylalanine ammonia lyase, chalcone synthase and anthocyanidin synthase that are regulated by MYB transcription factors are involved. These genes are coordinately expressed and their levels of expression are positively related to the anthocyanin concentrations. Expression or suppression of the constructive genes contributes to a variety of changes that make fruits visually attractive and edible. Transgenic approaches also have discovered a strong relationship between phenyl propanoid/flavonoid gene expressions for fruit skin coloration. In this study, various developments that have taken place in the last decade with respect to identifying and altering the function of color-related genes are described.

Basic leucine zipper (bZIP) genes are known to play a crucial role in response to various processes in plant as well as abiotic or biotic stress challenges. We have performed an identification and characterization of 50 bZIP genes across the woodland strawberry (Fragaria vesca) genome, which were divided into 10 clades according to the phylogenetic relationship of the strawberry bZIP proteins with those in Arabidopsis and rice. Five categories of intron patterns were observed within basic and hinge regions of the bZIP domains. Some additional conserved motifs have been found with the group specificity. Further, we predicted DNA-binding specificity of the basic and hinge regions as well as dimerization properties of leucine zipper regions, which was consistent with our phylogenetic clade and classified into 20 subfamilies. Across the different developmental stages of 15 organs and two types of fruits, the clade A bZIP members showed different tissue-specific expression patterns and the duplicated genes were differentially regulated, indicating a functional diversification coupled with the expansion of this gene family in strawberry. Under normal growth conditions, mrna11837 and mrna30280 of clade A showed very weak expression levels in organs and fruits, respectively; but higher expression was observed with different set of genes following drought and heat treatment, which may be caused by the separate response pathway between drought and heat treatments.

Alternaria alternata apple pathotype (previously A. mali) causes alternaria blotch disease of apple (Malus ×domestica), which may result in leaf spots and up to 70% premature leaf drop in serious cases. This disease is of worldwide importance but is most serious in eastern Asia (Japan, Korea, and China) and in parts of the United States. The excessive use of fungicides not only adds cost to apple growers, but also pollutes the environment. In this study, we characterized a 5-year F1 population from a cross of a resistant cultivar (Huacui) and a susceptible cultivar (Golden Delicious) consisting of 110 individuals along with 14-year-old parent trees (10 each). A field evaluation of disease severity was conducted in 2008 and 2009 under the natural conditions in Liaoning, China (lat. 40°37′ N, long. 120°44′ E). Based on the field data, 110 F1 plants were divided into five groups. Artificial inoculation was carried out both on the living trees and on the detached leaves in 2009 to ensure that A. alternata apple pathotype was the causative agent. Eighty primer pairs of simple sequence repeat (SSR) were screened against the four genomic DNA pools, respectively, from six highly susceptible F1 plants, six most resistant F1 plants, one tree of the seed parent, and the one tree of the pollen parent. One pair of primers (CH05g07) was shown to be linked to the DNA pools of susceptible F1 and the parent tree, but not to the DNA pools of resistant F1 and parent trees. This primer pair was then used to screen all individual 110 F1 progenies and two parent trees. The differentiation of 103 individuals (97.3%) with the marker matched the field disease resistance rating. This marker was further screened with 20 cultivars with known susceptibility or resistance to A. alternata apple pathotype and its linkage to susceptibility was validated. These results suggest that this marker can be used in marker-assisted selection for resistance/susceptibility to alternaria blotch disease in apple.

Reliable micropropagation protocols were developed for Japanese honeysuckle (Lonicera japonica Thunb.) and Amur honeysuckle [L. maackii (Rupr.) Maxim], two horticulturally important invasive woody plants. Shoot tips were collected from mature plants and established in vitro on Murashige and Skoog (MS) medium and cultured in various media. Plant growth regulators and basal media had significant effects on proliferation rates. Proliferation of Japanese honeysuckle was highest on Driver and Kuniyuki walnut (DKW) medium with 5 μM (1.1 ppm) 6-benzyladenine (BA), yielding an average of 14 axillary microshoots in 12 weeks, whereas Amur honeysuckle produced 38 axillary microshoots in 12 weeks on MS medium containing 2.5 μM (0.6 ppm) BA with or without 1.25 μM (0.3 ppm) indole-3-butyric acid (IBA). Microcuttings of Japanese honeysuckle were rooted ex vitro with or without treatment with 980 μM (200 ppm) IBA, whereas microcuttings of Amur honeysuckle required treatment with 980 μM (200 ppm) IBA to root. These protocols can be used to continuously produce plant materials for various biotechnological modifications, such as in vitro mutations and genetic engineering for noninvasiveness.

COBRA proteins have been shown to be involved in both cell wall expansion and/or cellulose deposition. In this paper, we analyzed all 18 COBRA genes (PtCOBRA) from the completely sequenced Populus trichocarpa genome. The 14-member PtCOBRA subfamily I proteins have high similarities to the Arabidopsis (At) COB subfamily, and members with full length sequences were predicted to possess significant potentials for a GPI-anchor site. The 4-member PtCOBRA subfamily II proteins are 45% longer than subfamily I proteins and lack ω-attachment sites at the C terminus, and are more similar to AtCOBL7 subfamily. The expression of the Populus COBRA family genes were regulated in a tissue-specific manner, and were shown to also respond differentially to inductions of hormones and environmental stimuli which affect plant cell expansion. The high levels of expressions, particularly in shoot tip and young root organs, suggests that at least some Populus COBRA genes are likely involved in regulating cell expansion.

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene structure, expression and function. Modified genes and consequently protein/enzymes can bridge genomics and proteomics research or facilitate commercial applications of gene and protein technologies. In this review, we will summarize various strategies, designing softwares and error correction methods for chemical gene synthesis, particularly for the synthesis and assembly of long DNA molecules based on polymerase cycling assembly. Also, we will briefly discuss some of the major applications of chemical synthesis of DNA sequences in basic research and applied areas.

To achieve a thermostable beta-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70 degrees C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80 degrees C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100 degrees C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.

Background Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs or multiple transformation events that is often restricted by a lack of available transformation markers. Results Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. Conclusions We present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering. Electronic supplementary material The online version of this article (doi:10.1186/s13007-016-0116-8) contains supplementary material, which is available to authorized users.

The disease resistance (R) genes play an important role in protecting plants from infection by diverse pathogens in the environment. The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of genes is one of the largest R gene families. Chinese chestnut (Castanea mollissima) is resistant to Chestnut Blight Disease, but relatively little is known about the resistance mechanism. We identified 519 NBS-encoding genes, including 374 NBS-LRR genes and 145 NBS-only genes. The majority of Ka/Ks were less than 1, suggesting the purifying selection operated during the evolutionary history of NBS-encoding genes. A minority (4/34) of Ka/Ks in non-TIR gene families were greater than 1, showing that some genes were under positive selection pressure. Furthermore, Ks peaked at a range of 0.4 to 0.5, indicating that ancient duplications arose during the evolution. The relationship between Ka/Ks and Ks indicated greater selective pressure on the newer and older genes with the critical value of Ks = 0.4–0.5. Notably, species-specific duplications were detected in NBS-encoding genes. In addition, the group of RPW8-NBS-encoding genes clustered together as an independent clade located at a relatively basal position in the phylogenetic tree. Many cis-acting elements related to plant defense responses were detected in promoters of NBS-encoding genes.

The mitotic inhibitors, colchicine and oryzalin, were evaluated for their effects on callus, adventitious shoot formation, and tetraploid induction of Euphorbia pulchurrima `Winter Rose'. In vitro grown leaf sections were placed on various media supplemented with either colchicine or oryzalin at various concentrations for 1 to 4 days. Colchicine was less damaging to leaf tissues than oryzalin. On various colchicine-containing media, prolific calluses were produced and adventitious shoot formation was observed. Regenerated shoots were found to be diploid as determined by flow cytometry. On media supplemented with oryzalin (28.9 μm to 144 μm), leaf tissues produced callus but failed to form adventitious shoots. Samples of calluses produced on oryzalin-containing media were subject to analysis using flow cytometry and were found to be diploid. These results suggest that the colchicine is less toxic on poinsettia tissues and shoot induction than oryzalin. Additional experiments are needed to establish a protocol for in vitro induction of poinsettia tetraploid with colchicine and oryzalin.

It is widely accepted that gene duplication is a primary source of genetic novelty. However, the evolutionary fate of duplicated genes remains largely unresolved. The classical Ohno's Duplication-Retention-Non/Neofunctionalization theory, and the recently proposed alternatives such as subfunctionalization or duplication-degeneration-complementation, and subneofunctionalization, each can explain one or more aspects of gene fate after duplication. Duplicated genes are also affected by epigenetic changes. We constructed a phylogenetic tree using Dof (DNA binding with one finger) protein sequences from poplar (Populus trichocarpa) Torr. & Gray ex Brayshaw, Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). From the phylogenetic tree, we identified 27 pairs of paralogous Dof genes in the terminal nodes. Analysis of protein motif structure of the Dof paralogs and their ancestors revealed six different gene fates after gene duplication. Differential protein methylation was revealed between a pair of duplicated poplar Dof genes, which have identical motif structure and similar expression pattern, indicating that epigenetics is involved in evolution. Analysis of reverse transcription-PCR, massively parallel signature sequencing, and microarray data revealed that the paralogs differ in expression pattern. Furthermore, analysis of nonsynonymous and synonymous substitution rates indicated that divergence of the duplicated genes was driven by positive selection. About one-half of the motifs in Dof proteins were shared by non-Dof proteins in the three plants species, indicating that motif co-option may be one of the forces driving gene diversification. We provided evidence that the Ohno's Duplication-Retention-Non/Neofunctionalization, subfunctionalization/duplication-degeneration-complementation, and subneofunctionalization hypotheses are complementary with, not alternative to, each other.

Mannans are widespread hemicellulosic polysaccharides in plant cell walls. Hydrolysis of the internal beta-1,4-D: -mannopyranosyl linkage in the backbone of mannans is catalyzed by endo-beta-mannanase. Plant endo-beta-mannanase has been well studied for its function in seed germination. Its involvement in other plant biological processes, however, remains poorly characterized or elusive. The completed genome sequences of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and poplar (Populus trichocarpa) provide an opportunity to conduct comparative genomic analysis of endo-beta-mannanase genes in these three species. In silico sequence analysis led to the identification of eight, nine and 11 endo-beta-mannanase genes in the genomes of Arabidopsis, rice, and poplar, respectively. Sequence comparisons revealed the conserved amino acids and motifs that are critical for the active site of endo-beta-mannanases. Intron/exon structure analysis in conjunction with phylogenetic analysis implied that both intron gain and intron loss has played roles in the evolution of endo-beta-mannanase genes. The phylogenetic analysis that included the endo-beta-mannanases from plants and other organisms implied that plant endo-beta-mannanases have an ancient evolutionary origin. Comprehensive expression analysis of all Arabidopsis and rice endo-beta-mannanase genes showed divergent expression patterns of individual genes, suggesting that the enzymes encoded by these genes, while carrying out the same biochemical reaction, are involved in diverse biological processes.