Blackberries can make a nice addition to the home fruit garden as a beautiful living hedge with nice flowers and tasty fruits. Gardeners can also enjoy blackberries as fresh fruit, jam or cobbler. Maintaining a neat, clean blackberry planting can be a considerable challenge. A good weed, fertility and pest control program can promote plant growth and quality fruit.
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Blackberries Rubus spp. Interestingly, these compounds also play a role on plant physiology, being especially relevant their role in plant defense against biotic and abiotic stress.
Hence, we hypothesize that since blackberry fruits have high amounts of flavonols and anthocyanins, leaves would also have high amounts of these compounds, and can be studied as a source of active molecules; furthermore, leaf synthesis would support their high contents in fruits. To explore this hypothesis, the present study reports a de novo transcriptome analysis on field grown blackberry leaves and fruits at the same time point, to establish the metabolic relationship of these compounds in both organs.
Transcripts were aligned against Fragaria vesca genome, and genes were identified and annotated in different databases; tissue expression pattern showed 20, genes common to leaves and fruits, while 6, genes were significantly overexpressed only in fruits, while another 6, genes were significantly overexpressed in leaves, among which flavonol-anthocyanin transporter genes were present.
Bioactives characterization indicated that total phenolics in leaves were three-fold, and flavonols were six-fold than in fruits, while concentration of anthocyanins was higher in fruits; HPLC-MS analysis indicated different composition in leaves and fruits, with cyanidinglucoside as the only common compound identified. Interestingly, genes in the flavonol-anthocyanin pathway and flavonol-transport families were overexpressed in leaves, consistent with the higher bioactive levels.
On the other hand, transcription factors were overexpressed in fruits anticipating an active anthocyanin biosynthesis upon ripening. This suggests that, in addition to the biosynthesis taking place in the fruits during ripening, translocation of flavonols from leaves to fruits contributes to the high amounts of bioactives starting to accumulate in fruits. Blackberry fruits are an important source of bioactive compounds, among which are flavonols, catechins, and anthocyanins Garcia-Seco et al.
These molecules have been reported to have beneficial effects to prevent metabolic syndrome at different levels such as cardiovascular disease and type II diabetes; hence, including blackberry on human diet is beneficial for health Tavares et al.
On the other hand, they play a relevant role in plant defense against biotic and abiotic stress Daayf et al. Catechins have also proved their improving endothelial function, hypertension, coronary heart disease, obesity, insulin resistance, as well as glucose and lipid metabolism Kim,They have also a growth regulating role, as they can act as auxin controllers, so they are able to control the individual plant organs and development of the whole plant Agati et al.
Despite the agricultural and biological importance of the genus Rubus , knowledge of their genetics and genome is very limited. Rubus spp. However, and despite its high added economic value and as a source of bioactive compounds, its genome has not been sequenced yet.
Therefore, other strategies need to be used to gain knowledge to improve production and health related benefits. RNA-Seq has been recognized as a powerful tool to analyze the transcriptome of an organism that has not been completely sequenced Wang et al. The sequencing of RNA has long been recognized as an efficient method for gene discovery and remains the gold standard for annotation of both coding and non-coding genes Adams et al.
Many studies have addressed flavonoid synthesis, identification and subcellular accumulation mechanisms in different plant species as in grapevine Perez-Diaz et al. Long distance transport has been demonstrated in A.
The biosynthesis of flavonols and anthocyanins in blackberry fruits along maturation has already been described, demonstrating an increased biosynthesis of anthocyanins in the black ripen stage Garcia-Seco et al. On the other hand, it may be anticipated that blackberry leaves also have an active secondary metabolism, in which flavonols must be abundant to participate in adaptation to environmental changes. Based on this background, the aim of this study is focused on i the relevance of the transcriptome to envisage the specific situation of leaves and red fruits at the same time point, and ii to establish the relationship of the flavonol-anthocyanin pathway core and regulatory gene expression and their contents in two different organs, given the relevance of these metabolites for defense in leaves and for human health in fruits.
In this work, we describe the transcriptome of leaves and red fruits by alignment to Fragaria as a reference genome sequence from the same family Rosaceae ; then, both transcriptomes are compared, quantification of core and regulatory genes of the flavonol-anthocyanin pathway expression by RT-qPCR, and HPLC-MS analysis of leaves and fruits is carried out. Differential gene expression suggests that leaf-flavonols are translocated to fruits during the early stages of maturation to achieve the high amounts accumulated therein on ripe fruits.
The Rubus spp. Plants and greenhouses were handled according to regular agricultural practices Ramos-Solano et al. Blackberry cycle has three stages: vegetative, flowering and flowering-fruiting; the duration of these stages is variable depending on the transplant moment, and each average one third of the plant's life. In this experiment, plants were transplanted at the end of September, flowering in November and fruiting in January when flowers, green, red and black fruits are present in the plants at the same time.
Fruits were sampled in red stage. Leaves and fruits were immediately frozen with liquid nitrogen and brought to the lab. Three replicates were taken, being each one constituted from plant material of 20 plants; a total of 25 leaves and g of ripening fruits were randomly sampled and pooled constituting the replicates.
RNA obtained from the three biological replicates were pooled and separated in three analytical replicates for RNA-Seq analysis. RNA samples were DNase treated and extracted as described before.
A total of six libraries were done, three form leaves and three from fruits. During the library preparation, poly-A tailed transcripts were enriched, as mRNA sequencing targets this type of transcripts, using an Oligo-dT magnetic bead-based system were enriched. The library preparation also retains information of which of the two strands of DNA was used to transcribe the given RNA, which enables the detection of antisense transcript expression.
Mitochondrial poly-A tailed transcripts were bioinformatically filtered since they were considered to be high abundance sequences. The sequencing was paired end, which increases the mapping percentage to poorly annotated genomes, and identifies splice variants.
Two types of sequencing library quality controls were performed, firstly after the library preparation and bead based size selection, the size distribution of the library was evaluated using a Bioanalyzer high sensitivity DNA chip. Then, qPCR based quantification of each library was performed, and samples were normalized and pooled in equimolar ratios.
After pooling of sample libraries, qPCR based quantification was performed on the library pool to ensure optimal concentration for cluster generation on the flow cell. Mapping of the sequencing data represents a useful quality control step in the Next generation sequencing NGS data analysis pipeline as it can help to evaluate the quality of the samples.
For this purpose, reads were classified in the following classes i outmapped reads or high abundance reads: rRNA, mtRNA, polyA, and PolyC homopolymers; ii unmapped reads: no alignment to reference genome possible; iii mappable reads: alignment to reference genome possible.
However, this number depends upon the quality of the sample and the coverage of the relevant reference genome; if the sample is degraded, fewer reads will be mRNA specific and more material will be degraded rRNA. In this case where the reference genome is different from the host organism, the mapping fraction is expected to be significant lower.
To test the differences between the blackberry and strawberry, we have taken reads that align to blackberry contig sequences, these reads should thus be bona fide blackberry sequences, and aligned them to the strawberry genome. Then, cluster generation was performed on the appropriate flow cell using single molecule clonal amplification.
Finally, the high- throughput NGS was performed using the Illumina sequencing technology platform. The raw data cDNA was aligned to the reference sequence, using as reference the sequence of wild strawberry Fragaria vesca subesp. Vesca a species of same family Rosaceae , but from a different genus, Fragaria. By this method the identification of the genes was achieved. Gene sequences were further aligned by BLASTX to protein databases such as Swiss-Prot, and KEGG, retrieving proteins with the highest sequence similarity with the given genes along with the functional annotations for their proteins.
The Blast2GO software was then used to perform mapping and annotation mapping is used to look for associated GO terms to the Hit Blast, and annotation selected from this GOs those with good statistical support.
Annotation was made with an annotation cutoff of 55, and a GO weight of 5. The log 2 fold-change is the log-ratio of a gene's or a transcript's expression values in two different conditions. To describe the expression obtained in the analysis, cycle threshold Ct was used. Two reference genes were used, actin for fruits and histone for leaves.
A different reference gene was used for each organ, since no common gene was found meeting criteria for a reference gene. However, these genes have similar Ct, so they can be compared. Core and regulatory genes studied and the primers used for each appear in Table 1. Table 1. Phenolic acids including, gallic acid, caffeic acid, ferulic acid, and chlorogenic acid were purchased from Sigma St.
The standard solutions 10 ppm were prepared in methanol. Formic acid was purchased from Aldrich St. The data acquisition software employed was Esquire Control 5. Data analysis was performed using the Data Analysis 3. Total phenols were quantitatively determined with Folin-Ciocalteu agent Sigma. One milliliter of extract was mixed with 0. Aldrich, St Louis, MO and 0. After 30 min at room temperature, absorbance was measured at nm with an UV-Visible spectrophotometer Biomate 5.
Results are expressed in mg of gallic acid equivalents per g of fresh weigh FW. All samples were measured in triplicate. Total flavonols were quantitatively determined through the test described by Jia et al. One milliliter of extract was added to a flask of 10 mL with 4 mL of distilled water.
After that 0. The solution was mixed and measured at nm with an UV-Visible spectrophotometer Biomate 5. Results are expressed as mg of catechin equivalents per g of fresh weigh FW.
Total anthocyanins were quantitatively determined through the pH differential method described by Giusti and WrolstadExtracts were diluted in pH 1 buffer 0. After that, absorbance was measured at and nm respectively, in a UV-Visible spectrophotometer Biomate 5. Results are expressed in cyanidinglucoside equivalents per g of fresh weigh FW. To construct a de novo transcriptome database, six mRNA libraries were generated by Illumina sequencing, three from leaves and three from red fruits of Rubus spp.
Loch Ness. Table 2 summarizes mapping results. In addition to these, it also shows the total number of reads obtained for each sample. The uniformity of the sample's mapping indicates that samples are comparable. In this case, in which the reference genome is different from the host organism, the mapping fraction was expected to be significantly lower.
Mapping Alignment of paired-end reads to the genome was carried out against the Fragaria vesca Whole Genome v2. Figure 1. Length distribution of the unigene assembly in the Rubus leaves and fruits transcriptome.
After sequencing and mapping alignment, normalized expression and differential expression fruit vs. The heatmap diagram Figure 2 shows the result of the two-way hierarchical clustering of RNA transcripts and samples. It includes the genes that have the largest coefficient of variation based on FPKM counts. Each row represents one gene and each column represents one sample.
The color represents the relative expression level of a transcript across all samples. The color scale is shown below: red represents an expression level above the mean; green represents an expression level below the mean.
A popular bramble fruit, blackberries are often picked in late summer to autumn to make jams, jellies and pies. The leaves of this versatile plant also make a pleasant tea. Bramble is one of our most commonly used wild edibles. Trailing, tangled, thorny stems bearing five-petalled flowers of varying size and colour, with prickly and toothed green leaves that turn reddish-purple in autumn. The fruits turn from green to red to deep-purple black. Commonly found in hedgerows, roadsides and waysides across Britain and Ireland.
The leaves are normally made up of (sometimes 7) leaflets, The plant is generally smaller than Blackberry with rounded stems which have fewer.
Allegheny Blackberry is a native perennial shrubby plant with stems that are erect to arching, from 2 to 7 feet long with thorns that are large and nearly straight. Arching canes do not usually root at the tips if they touch the ground. Canes live for two years. They are green initially then becoming a dull reddish brown, without any whitish bloom. The leaves are alternate, 3 - 7 parted usually 3 to 5 , not lobed but with deep teeth, and on long stalks which have a pair of very short stipules at the base. Upper leaves on the fruiting branches will usually have three leaflets with only the terminal leaflet stalked; larger lower leaves will have 5 leaflets and all can be stalked. The terminal leaflet is always larger. The total leaf is no more than twice as long as wide. The upper surface is medium green, the lower surface much paler due to fine hair. There is glandular hair on the leaf stalks.
In early summer months, eastern forests yield many plants that can be eaten raw or cooked, and dozens of edible mushroom species. Searching for berries is an excellent way to get to know a piece of land and a great way to share the benefits of forests with friends and family. Here are five of the most common edible berries that are ripe in June and July in forests of the Chesapeake Bay watershed. They are much smaller than domestic fruits, but are quite sweet and very common. The plants have three leaves with toothed leaf margins, grow low to the ground, and spread by runners.
Blackberry, is a perennial shrub in the family Rosaceae that is grown for its aggregate black fruit of the same name. The name blackberry is used to describe several species, including Rubus fruticosis wild blackberry , Rubus ursinus and Rubus argutus , two species native to North America.
Blackberries and raspberries are one of the most popular fruits to grow and they are among the easiest for the home gardener to successfully produce. Blackberries and raspberries come as erect types no trellis required and trailing types trellis required , depending on the varieties selected. Certain varieties of erect and trailing blackberries do well in Georgia, while only the trailing raspberry Dormanred has proven itself for all of Georgia. Developed by Mississippi State, Dormanred is an ever-bearing trail-type raspberry well adapted for growing in southern states. Fruits are firm, red and have very good flavor.
Learn which plants thrive in your Hardiness Zone with our new interactive map! Wild blackberries Rubus spp. There are 11 species that grow in the wild throughout the United States. Several were introduced from Asia, and four species are considered weeds because of their invasive growth. Blackberries are a source of food to both humans and animals as well as a source of cover for birds and animals. Some varieties stand erect, others are semi-erect and a few are trailing with long canes that sprawl over the ground. Blackberries produce alternate leaves. They are either lobed or lobed and serrated.
Blackberry is the common name for any of the various perennial plants of the genus Rubus and subgenus Rubus (or Eubatus) with compound leaves and bearing.
New and improved varieties of blackberry plants make growing this plump and delicious summer fruit easier than ever. Erect blackberries have stiff canes that are upright and arching. They can be trellised or grown in the landscape as a hedge or shrub border.
Your blackberries are suffering from bramble rust Phragmidium violaceum. This is a type of leaf fungus. The upper sides of the leaves on infested bramble plants exhibit dark red spots. The coating that you described as 'pollen' on the underside of the leaves is the yellow-orange to orange-red hymenium of the fungus.
Top side of leaf left and bottom side of same leaf right. Necrotic spots will typically show on both sides of the same leaf.
A: There are those who suspect Wildflower Center volunteers are the culpable and capable culprits. Yet, others think staff members play some, albeit small, role. You can torture us with your plant questions, but we will never reveal the Green Guru's secret identity. Did you know you can access the Native Plant Information Network with your web-enabled smartphone? Ask Mr.
Every plant has the future potential for disease and insect damage. Factors such as location and weather will play a part in which issues your plants encounters. If available, disease-resistant varieties are the best option for easy care; and for all types of plants, proper maintenance such as watering, pruning, spraying, weeding, and cleanup can help keep most insects and diseases at bay.