Dear readers,
Thank you for taking the time to follow along with me on this experience which I can say has already impacted me greatly. I never would have imagined that I would be able to step outside my comfort zone and develop a work-life balance within only 2 weeks of time.
June 12, 1898, is the date on which the Philippines declared its independence from Spain. Also, on July 4, 1946, the Philippines declared its independence from the United States. Not only is there a long history of colonial powers within the Philippines but there is also a long history of Filipino resistance. Many Filipinos on Monday, June 12 take the day off and spend time with their loved ones.
The IWU group started the week off on an unsteady foot. Since we did not have to go into the office or laboratory on Monday, we decided to visit Makiling Botanic Gardens in order to take a calm stroll. There was a bit of confusion about where to get off the shuttle but we ended up making it to the entrance. As we were walking forward, we noticed a sign which stated “Eco Trail”. I guess we simply came to the conclusion that even though we were not prepared for a hike, we would just explore the trail for a bit. In my mind, this was not going to be a hike but rather a simple stroll. Unfortunately, I was very wrong. When we noticed that this trail was actually a hike, there was a small moment in which we questioned if we should turn back. But we decided to continue forward because we were practically halfway to the end. About 30 minutes later, as I was attempting to catch up to the group, I suddenly felt my foot sliding forward upon the ground. I quickly held onto a tree trunk with all my force. Yet, still, I ended up falling flat onto my back! Luckily I did not hit my head. I got up fast in order to pretend like nothing happened but sadly the rest of the group were looking my way. I laughed, mentioned I was ok, and then we continued on. Apparently, my fall was contagious because everyone started slipping and sliding after that. Thankfully no one was injured! As we came to the end of the trail, I was exhausted but glad for the experience. We went down a couple of stairs to see a beautiful waterfall and then headed back to the IRRI campus.
On Tuesday, I spent the day reading articles recommended by Ate Hsu. You may remember that Ate Hsu is a Ph.D. student analyzing the effects of heat stress in various strains of rice. These readings gave me a better insight into the complex research Ate Hsu is conducting. The first paper I read was titled “Mapping QTL for heat tolerance at flowering stage in rice using SNP markers”. I will attempt to give a brief overview of the main points this article proposed. Rice productivity is limited by various abiotic factors such as drought, submergence, and high temperature. As we have and continue to see with global warming, these factors have only been heightened. For each 1 °C increase in global temperature, rice grain yield is expected to decline by 10%. Thus, biofortification is a vital tool that through selective breeding, resistance to abiotic stresses can be introgressed into already established and well-known lines of rice. Rice grain yield is impacted by several components which often vary in where they are present within rice stages. Some examples include pollen/spikelet sterility and anther indehiscence (tight closure of the locules and a delay in locule opening). These effects ultimately lead to a low harvest index and low grain quality. However, these negative effects which are brought about oftentimes due to heat stress do not impact all strains the same. To better explain, some strains like Indica and Japonica have been identified to be heat tolerant. But what causes this tolerance? Well, that is where Quantitative Trait Loci (QTL) come in. QTL are functionally important region(s) of the genome and within these regions are single nucleotide polymorphisms (SNPs). SNPs are a type of marker or rather a location within the genome that can be used to distinguish between individuals because there is a difference present if a comparison is conducted. These locations can be linked to important genes and can be inherited with genes of interest. Consequently, they can be useful in breeding if associations between polymorphisms and favorable traits are detected accurately. Therefore if a strain is demonstrated within the field that it is tolerant to an imposed heat stress, through QTL analysis, one can identify the region that is associated with a particular gene that is causing such tolerance. For instance, QTL for heat tolerance at flowering (the most sensitive stage to high temperature) has been mapped on all chromosomes except 6 and 7. From crossing IR64 (susceptible female, Indica) x N22 (heat tolerant male, Indica), and then through self-pollination as well as backcrossing, this study was able to find 4 SNPs associated with heat tolerance in BC1F1. Phenotyping, DNA extraction, PCR amplification, and hybridization, genotyping using SNP markers, confirmation of QTL on chromosome 4 allowed for the understanding of how spikelet fertility is controlled within the genome.
During this second week, I have also been able to learn about the research conducted by Ate Muditha Kumari and Ate Nirusha Dissanayake who are PhD students. To start off, Ate Muditha is looking at the introgression of the beta carotene (provitamin A) compound into popular rice varieties. From Golden Rice analyses, it is known that the endosperm of rice which contains beta carotene has a yellow hue. Thus, by looking at seed color, one can phenotypically select for such a trait in terms of breeding. When the harvest comes, these samples will then be sent for character analysis. Ate Nirusha is identifying lines with high yield and high zinc through the analysis of collected MAGIC (Multi-parent advanced generation inter-cross) population data from previous seasons of field experiments. Both of their research is focused on the nutrient content of rice. There are also similarities in the methodology toward how their data sets/tables are being analyzed. To better understand, both Ate Muditha and Ate Nirusha are analyzing phenotypic and genotypic data for the establishment of a selection index, the ranking of genes, and the analysis of allelic effect (how much of the total variation is contributed by a particular locus). This compilation of data surveys is for the purpose of identifying QTLs that are in some way related to the maintaining of high levels of beta carotene and/or zinc in rice. Programs such as R and RStudio are utilized in both research studies as they pertain to statistical computing and the construction of graphics.
Also, this week Ate Muditha and Ate Nirusha met with Ate Mona, an associated scientist within the biofortification division. Ate Mona explained that since they are both nearing the finalization of tables and graphics, it is time to find R-squared values. These values measure the proportion of phenotypic variation explained by molecular markers. Phenotypic variance refers to how much of the total variation is contributed by a particular locus. So, if there is a high phenotypic variance, it is more likely that a major QTL will be found at that locus. Furthermore, LD pruning of the data sets also has to occur. This signifies the reduction of markers or rather deciding how many SNIPS to use from a particular segment. Oftentimes, SNIPS located closely near one another reveal the same information thus instead of studying all, it can be more time efficient to study a few of those or even 1 of them. Also, when having to look into a lot of markers, it is helpful to group based on the genotype of each marker. Thus, all plants with allele A, will group and take the mean and this will then be compared with a different group to determine the significance of a marker. Software such as PLINK, GaPit, and Farmcpu aid in such genome-wide analysis methods. Farrmcpu in particular will only show you unique markers.
On Thursday, June 15 I was able to join an experiment conducted by Ate Eva Maghirang in the genotyping services Laboratory (GSL). QTL 6.2 had been previously identified to increase zinc content in rice strains. 4 donors contain this QTL and there are 6 parents in total. This experiment is the confirmation to confirm the true heterozygotes through comparison and use of 10 primers. The first step was to extract the DNA of the 6 parents. This process began by grinding the leaf tissue of each parent using a GENO machine. After this, CTAB buffer was added to each sample. Then, the washing process with ethanol was conducted. After, the samples were centrifuged twice. These samples were stored until Monday.
At IRRI’s experimental station, I met Ate Lesela Anoidem, Ate Mhyla Mantala, Ronald Adolfo Mantala, and Ate Nora. This team conducts the scooping, threshing, and transplanting of seeds/plants which resulted from specifically biofortification experiments.
During our meryenda, we all had sapin-sapin and coffee. After this break, I was able to join the team in the scooping of seeds. Each small brown bag contains the seeds of a single plant which is further identified by a number.
Ate Hsu’s screen house research is conducted in BG06 within the experimental station. During my first visit, plants of IR 2006 lines with a flowering date of 88 were transplanted 21 days after they were planted. Transplanting is the process of moving a plant from one location to another 15-40 days after seeding (all dependent on flowering or heading date). This process ensures the growth of a more uniform plant. Some key procedures to follow when transplanting include transplanting only the strongest. When pulling the plants out of their previous locations, it is vital to be cautious to not rip their roots. The strongest/healthiest plant was placed in the middle of the new container and the other was placed near the side of this container. All roots were covered with moist soil and the plants were soaked with water.
I was also introduced to the fertilizer that is mixed with the soil used in this experiment. It is a mixture of ammonium nitrate, phosphate (solophos), and potassium nitrate (nitrate of potash).
Also, Ate Hsu had previously replicated this screen house experiment but in the field a season prior. Therefore, the yield from this harvest is being analyzed in regard to the amount of filled and unfilled grains per every 10 particles. Filled grain weight is also being recorded.
Furthermore, this day I had the chance to practice seeding. I placed around 18 Swarna Sub-1 seeds, with the aid of tweezers, into a container filled with very fine and damp soil.
This week, Kuya Froilan organized an orientation for newly arrived interns. We took a trip to the Internation Rice Genebank! This is where more than 132,000 accessions are tested for viability and saved for preserving wild rice species’ biodiversity. These accessions can be requested by countries. Also within the gene bank, there are 2 divisions. The active short-term unit which is maintained at 2-4 degrees Celsius, contains varieties that are available for distribution. The viability of these seeds is monitored every 5 years.
On the other hand, the long-term storage unit which is maintained at -20 degrees Celsius contains samples not being withdrawn for distribution. The viability of these seeds is monitored every 10 years. Seeds are within vacuumed aluminum cans. If viability is found to be below 85% then those seeds will be planted and then the fresh seeds would be stored.
After visiting the gene bank, I headed upstairs to attend 2 biofortification presentations. The first was titled” Boosting genetic grain through genomic selection, speed breeding, and CRISPR CAS”. Presenter Kuya Gaurav emphasized the importance of maximizing genetic gain in order to increase rice yield. The following are some practices presented to have a role in the maximizing of genetic gain:
- Speed breeding: Manipulation of environmental conditions during breeding
- CRISPER: Transgene insertion
- GEEN: The insertion, deletion, and replacement of DNA in the gene of living organisms using engineered nucleases
The second presentation was titled “Major abiotic stresses and impact screening”. Presenter Dr. Bala explained how characteristics of interest/analysis in rice (panicle length, spikelet fertility, etc.) can be negatively impacted by poor water quality, drought, salinity, land degradation, uneven rainfall patterns, and flooding. He proposed the benefit of engaging in screening in order to better understand how specific strains react differently to these stresses. Through screening, breeders are able to isolate which strains are resistant to certain stresses and then map genes that cause such resistance.
On Saturday, the IWU group as well as other IRRI staff (11 people in total) took a trip to Villa Escudero. There, we participated in activities such as eating delicious coconut flavored ice-cream, bamboo rafting, swimming, and having dinner near a waterfall!
One major takeaway from this week would be how the production, testing, and an analysis of bio-fortified rice is a process that involves many roles and perspectives. Being able to look into and join in on each step has allowed me to meet amazing, strong, and passionate individuals.