Jimena B. Alvarez and Dr. P. Jeff Maughan, Plant and Animal Sciences
Quinoa (Chenopodium quinoa Willd) is an important crop originally from the Andes of South America. Quinoa grows well in high altitude and high saline conditions, for which it is used as a substantial food source by the farmers of the Altiplano (high plains) in Peru, Bolivia, and Ecuador. Quinoa has great nutritional value, as its seeds contain high quality protein in vast amounts and other nutrients such as carbohydrates, lipids, and minerals1. However, quinoa has a seed coat with high levels of saponin that limits its nutritional uptake in humans. In addition, saponins are bitter-tasting compounds, thus making quinoa unpalatable. Consequently, a saponin removal process exists to make quinoa more palatable, but this increases the cost of the grain, and thus creates a barrier to expanding production of the crop.
For these reasons, studying the differential mRNA expression of saponin-containing (bitter) and non saponin-containing (sweet) quinoa has become a main goal of this project. This will help in the isolation of a few genes that will possibly be involved in the synthesis of saponin in quinoa. To start the project, seeds were collected from 46 different Chenopodium quinoa plants, plus the parent lines (LP and 0654), each of which were planted 4 times (total=192 plants). These seeds were collected at different stages of development, which are aqueous (~14 day post anthesis (dpa)), milky (~21 dpa), and pasty (~28 dpa). It is important to note that the aqueous stage was collected even though previous studies done in Bolivia and in our laboratory have shown that saponin has only been observed beginning in the milky stage. This was done in order to collect data that could be useful in determining with more precision the stage in which saponin biosynthesis begins.
While seed collection was taking place, the design of the oligonucleotides to be spotted on the microarrays was performed using eArray, an online design program by Agilent Technologies. These RNA products from the seed extractions are to be bound to the oligonuclotides on the microarrays. For the RNA extraction, we used the Qiagen RNeasy RNA extraction protocol. This protocol consists of freezing the samples with liquid nitrogen, and after the samples are ground in a mortar, a set of extractions is performed in order to isolate and purify the RNA.
At the present stage of the project, we have finished the extractions of all 192 plants. However, we ran out of some of the collected seeds during the extraction process. For this reason, we planted a second batch of the same 46 plants and the 2 parents. This second batch will be used to replace the lost seeds from the first batch, as well as serve as a back up if needed in the future. After extraction is fully completed using the second batch of plants, the next step will be cDNA amplification. This process is done to transform the RNA collected into cDNA, and then transcribe the cDNA back into cRNA incorporated with Cyanine-3 or Cyanine-5 fluorescent dyes. As a result, the transcribed regions only are joined together in the RNA end product. The cDNA amplification will be done using the Two-Color Microarray-Based Gene Expression Analysis Kit by Agilent Technologies.
Finally, the microarrays will be run using the designed oligonucleotides joined to the mRNA end product in the hopes of determining patterns of differential gene expression during saponin biosynthesis between the sweet and bitter quinoa samples and between the three different developmental stages. This will help to determine a certain number of genes that are active during biosynthesis of saponin in quinoa.
This microarray analysis will enable future researches to discover the true gene responsible for saponin biosynthesis and allow plant breeders in countries from the Altiplano to cultivate a better and more economically favorable crop. Consequently, consumers will be more able to benefit from the nutritional properties of quinoa.
Working on this project has helped me to become familiar with the cutting-edge technology that is related to the research field and to my career. I was also able to improve my working, group, and problem-solving skills. I also believe this experience has helped me to develop the abilities necessary to continue on my personal and career projects.
References
- P. J. Maughan, et al, 2004. A genetic linkage map of quinoa (Chenopodium quinoa) based on AFLP, RAPD, and SSR markers. Theor Appl Genet 109: 1188-1195.
- Ward, S. M., 2001. A recessive allele inhibiting saponin synthesis in two lines of Bolivian quinoa (Chenopodium quinoa Willd.). J Heredity 92: 83-86.
- Ward, S. M., 2000. Response for selection for reduced grain saponin content in quinoa (Chenopodium quinoa Willd.). Field Crops Research 68: 157-163.
- Hedge, P., et al, 2000. A concise guide to cDNA microarray analysis). BioTechniques 29: 548-562.