Salmonberry Genomics

Sample Collection

2018-06-24T00:00:00+00:00

On May 20th, myself and other volunteers for the Salmonberry project met in a residential area in Bellevue, Washington, where one of the volunteers reported a specimen of Rubus spectabilis growing near their house.

First, we came to a consensus as to how we would safely, cheaply, and temporarily store any collected samples of the specimen at -20 degrees Celsius, until we found a freezer which could store the samples indefinitely at -80 degrees Celsius. We agreed to store the samples of Rubus spectabilis inside of test tubes, within a heavily insulated box filled with dry ice, within a conventional freezer. However, this will be temporary while we look for alternative indefinite means of storage.

We mixed isopropyl alcohol with dry ice to make a bath for the vials. We went out to collect samples of the specimen, ripping out leaves, stems, flowers, and berries with our gloved hands, and removed the roots with a shovel provided by a Salmonberry volunteer. Aside from minor complications such as the difficulty of accessing the specimen, or the painful thorns on it, collection of the samples went without incident.

In order to prepare the samples for storage and sequencing, and remove any potential contaminants from them, we submerged all of the samples underwater at least once. For particuarly dirty samples such as the roots and stem, we had to put them under running water and submerge them three times. We used tweezers to remove the petals and scissors that had been wiped with alcohol to cut up the stems and the root pieces.

Black Raspberry Genome Paper

2018-06-20T00:00:00+00:00

Overview

This paper outlines the experiment of sequencing the genome and the RNA of a black raspberry. When considering the steps our own experiment will take, it is important to examine past related experiments and take into account their processes and decisions.

This paper, by VanBuren et. al, covers the RNA sequencing of a kin of salmonberry, the black raspberry. Black raspberry, or Rubus occidentalis, shares the same genus as salmonberry, or Rubus spectabilis. There haven’t been many papers published on sequencing of species of the Rubus genus, especially not their RNA, and so this is an important paper to examine for our potential experiment.

Procedure

In this experiment, the RNA of the black raspberry was extracted from several parts of its plant: leaves, stems, canes, green fruit, red fruit and ripe fruit. The RNA was also extracted from both healthy roots and those treated with disease (specifically, the fungus Verticillium dahliae), in order to study black raspberry’s immune response and potential for human health.

The extraction of the black raspberry’s RNA consisted of the following steps: First, the part of the black raspberry was frozen in liquid nitrogen. It was then taken out and grounded by a mixer mill (Mixer Mill MM 301) and suspended in a RNA reagent solution (PureLink Plant RNA Reagent) to extract the RNA. The RNA was then precipitated from the solution using 2-propanol. The extracted RNA was suspended in another RNA reagent solution (RNAsecure reagent) in order to extract it completely.

The extracted RNA was then sequenced using Illumina HiSeq2000, and analyzed using various libraries and models in order to understand its gene properties.

Technology

After our team receives the sequencing of the RNA sample, it is critical to understand how to analyze the sequencing readings once they return. VanBuren and his team utilized a variety of libraries to assemble and annotate the readings, which may be helpful to examine.

Once the black raspberry RNA sequencing returned, the readings were trimmed down using Trimmomatic (V0.32). The genes were predicted using a program called MAST, or Motif Alignment and Scanning Tool (v.4.10.0). Trinity was then used to assemble the RNA readings into a complete RNA.

In order to compare and analyze the assembled readings, the following programs were used: A program called MAKER (v2.28) was used to compare the assembled readings to reference gene models. Annotating of the protein-encoding sequences – predicting the proteins encoded for by certain sequences of the assembled RNA – was done using the program InterProScan (5-RC6) and NCBI BLASTP. Our team has experience in using BLASTP, although using InterProScan along with it might provide for better accuracy.

These models used by VanBuren and his team are just a sample of potential programs available to assemble, annotate and analyze RNA sequences. However, his team’s use of a spectrum of potential programs allows our team to examine their uses and potential benefits.

Accuracy and Error Prevention

As noted in its procedure, VanBuren’s team took a few measures in order to ensure the accuracy of the black raspberry RNA sequencing and analysis. For example, before assembling the RNA sequences, each sequence’s individual RNA integrity was measured using the program Bioanalyzer. The team also took other steps in order to eliminate any potentially erroneous readings.

The following discards were made by VanBuren and his team to ensure accuracy: A sequence with a Bioanalyzer integrity number less than or equal to 6 was removed. For clusters of protein-encoding genes that were at least 95% similar, only the longest sequence was kept. Genes predicted to encode proteins with less than 33 amino acids were discarded. Only sequences with an error value from BLASTP less than or equal to 10-5 were kept. Protein sequences with MAKER AED of 1 and no functional annotation from InterProScan or BLASTP were removed.

These steps were taken by VanBuren and his team in order to optimize their experimental results. If our team were to use any of the listed technologies or resources, it is worth considering following the same steps in order to prevent experimental errors.

References

Lastly, just like our team, the black raspberry research team also followed the examples of past experiments, which it noted in its paper. The following papers could potential benefit our team when we consider which steps to follow, which other sequences are possible to compare the salmonberry reads to, or simply how a genome assembly research paper is organized.

Past genome reads used by VanBuren et al.: Apple (Velasco et al, 2010), Peach (Verde et al, 2013), Pear (Wu et al, 2011; Chagne et al, 2014), Strawberry (Shulaev et al, 2011), Chinese plum (Zhang et al, 2012).

Protein sequences used by VanBuren et al. when annotating black raspberry readings: Arabidopsis (Swarbreck et al, 2008), Brachypodium (Vogel, 2010), Rice (Project IRGS, 2005), Poplar (Tuskan, 2006), Sorghum (Paterson, 2009), Maize (Schnable, 2009).

Conclusion

In conclusion, VanBuren et al. successfully sequenced a black raspberry RNA as well as its genome.

It proved its close relationship to the woodland strawberry (Fragaria vesca) using the sequencing technologies noted above. It also managed to identify “290 very recent small-scale gene duplicates enriched for sugar metabolism, fruit development, and anthocyanin related genes” through comparing the history of black raspberries, as well as the locations from which the genes were extracted. Lastly, the team investigated black raspberry’s fruit development and disease response from sequencing the RNA from the corresponding parts of the plant.

Following the black raspberry research team’s example, our team might be able to also successfully sequence and analyze a salmonberry’s RNA. The technologies VanBuren’s team used, as well as the process by which and locations from which the RNA was extracted are worth considering for our own experiment.

Sequencing Centers

2018-04-02T00:00:00+00:00

One of the many steps in our salmonberry project entails getting our samples sequenced. Sequencing determines the exact order of nucleotides within a certain genetic molecule. We will then analyze those sequences using bioinformatics and relate what we find to salmonberry’s health benefits.

Under the umbrella of “sequencing”, there is a plethora of sequencing options to choose from. In general, there is RNA sequencing and DNA sequencing. For different uses, one option can be more advantageous over the other. Regarding the salmonberry and the work we plan to do, getting insight into the functionality of the active genes themselves is a priority. Consequently, RNA sequencing (RNAseq) is the better option for our project both because of its lower cost and because it will give us more useful information on salmonberry genes than RNA sequencing.

There are various sequencing centers across the country that offer these services, so we needed to contact them to figure out what the best rates would be. We used a procedure from a paper on the blackberry genome as a guide for our rate inquiries.

Duke Center for Genomics and Computational Biology has a online estimator (DUGSIM) where you can figure out the cost of several different sequencing services. However, those prices are for much larger services than what we need.

Instead, centers such as the Northwest Genomics Center at the University of Washington have systems more appropriate for our sample sizes: 100 bp (sample length) reads on an Illumina NovaSeq 6000 S2 flow cell (a type of sequencing machine). For 50 million reads per sample, the cost is $320.

In the bigger scheme of things, we’ll use this estimate to plan our crowdfunding amount accordingly.

Flavonoids

2018-03-25T00:00:00+00:00

Flavonoids can be found in all parts of a plant: its roots, its stem, its bark, as well as in its fruit. The production of flavonoids is dependent on the amount of sunlight, heat, ozone, and hydration. They are repsonsible for numerous health benefits from different fruits.

For instance, there is a process called oxidation that happens in every human’s body. Oxidation is the metabolizing of oxygen, which creates things called free radicals and essentially causes us to age. Flavonoids are antioxidants, meaning they slow down this process of oxidation in the body, which in turn creates less free radicals and slows down aging!

This general property of most flavonoids has made them the center of fruit advertisement and scientific research. You may see blueberries advertised as ‘antioxidants’ or ‘anti-inflammatory’ - this is because of the flavonoids they contain.

Other responsibilities of flavonoids include the color and taste of the fruit, as well as the production of enzymes and vitamins in a plant. In addition to slowing down oxidation, flavonoids can also help prevent damage to the liver, so several flavonoids such as catechin, apigenin, and quercetin can aid in preventing diabetes.

Blackberries, like salmonberries, are part of the Rubus genus, so it would be reasonable to infer that salmonberries and blackberries contain similar flavonoids and health benefits. For example, blackberries have a high content of myricetin, a flavonoid that is supposed to have antithrombic and anticarcinogenic effects, meaning it might help prevent cancer and stops coagulation in one’s blood. Thus, salmonberries may also have these health effects.

By finding out the health benefits of each fruit and vegetable, we can start being more mindful of what we eat and its effects on our body.

Salmonberry Reproduction

2018-03-18T00:00:00+00:00

The salmonberry, or the Rubus spectabilis, is a member of the Rosacea family, the same family as strawberries, blackberries, raspberries, and many more fruits. The salmonberry shares a similar structure to blackberries and raspberries, but has a bright orange color like salmon roe. These berries grow almost exclusively in the West of North America, on the coasts of Washington, Oregon, and California.

Reproduction

Salmonberries attract hummingbirds and various insects, which usually land on the flower and take some pollen. They then travel to another salmonberry plant where the pollen that they acquired from the first plant rubs off and pollinates the plant, initiating salmonberry reproduction.

Salmonberries also reproduce through consumption by mammals. Each drupelet of a salmonberry has a seed, which is protected enough to withstand the digestive tract of an animal. The seed is later dropped in the hopes that it grows into a plant. Not very many seeds germinate immediately after being dropped since they often stay dormant for decades. They do this to avoid trying to grow in adverse conditions. A triggering factor for germination could be a low-intensity fire, which causes a seed to break out of its cotyledon and start sprouting. A high-intensity fire, on the other hand, can be detrimental and inhibit the growth of a salmonberry.

Salmonberries also extensively use an alternate form of reproduction: vegetative. An example of this is the usage of rhizomes. Rhizomes are a part of the salmonberry plant’s extensive root system, which grow horizontally and create buds. These buds then grow into flowering plants. Rhizomes can grow rapidly and a single network has the potential to grow hundreds of thousands of buds per acre.

Another form of reproduction is layering. This is when one of the stems is pushed over by something, causing it to make contact with the ground. When there is a bud on the fallen stem, a new plant can sprout from the single bud, and layering occurs. This is another example of interconnected salmonberry plants growing off one another.

The last method of reproduction is basal sprouting, when multiple buds grow off of the base of the stem. This is particularly useful when the main aerial stems are damaged, as it can create a new set of stems for the plant to reproduce through.

In a world where chronic hunger is normal for a sixth of the global population, plant reproduction is important to study to maximize the output of food.

Introducing Salmonberry

2018-03-11T00:00:00+00:00

What is a salmonberry? How is it scientifically related to other plants?

Contrary to public perception, salmonberry does not refer to salmon mixed with berries, although the name could’ve come from observation that natives enjoyed eating the berries with salmon roe. In essence, the salmonberry is an edible berry grown on large bushes from southern Alaska to the northern California coast. Most note, however, that they thrive in the Pacific Northwest because of the rain and mild temperatures. Appearance-wise, the salmonberry looks like a raspberry, but its taste is quite tart. Its colors range from bright orange to a deep red. These berries are also known to possess large seeds and a high water content.

Their scientific name is Rubus spectabilis. Rubus refers to a large genus of 450-750 species. The name itself is derived from the Latin word, ruber, which means red. Spectabilis refers to the term ‘spectacular’, because of the conspicuous flowers and fruits. Salmonberries are classified as eudicots, which are plants that produce seeds in their flowers, later to be enclosed in fruits. Most eudicots share the following characteristics: leaves that have a netted venation, flower parts in 2’s, 4’s, and 5’s (or multiples of 4 and 5), seeds that have 2 seed leaves (cotyledons) and stem vascular bundles arranged around the pith in a ring. Common eudicots include the dandelion, apple, buttercup, and macadamia.

Delving further into the phylogeny of salmonberries, one will notice that they are also classified as Rosids. About 70,000 species belong to this clade, some of which include fruits and flowers of the balsam apple and the rowan (mountain-ash). Some of the salmonberry’s closest relatives include thimbleberries, blackberries, and raspberries, because they are all part of the rose family.