Derrick J. Reynolds and Dr. P. Jeff Maughan, Plant and Animal Sciences
Quinoa (Chenopodium quinoa Willd) is an important crop for subsistence farmers in the Altiplano (high plains) of Peru, Bolivia, and Argentina. Quinoa has the potential to be an effective crop for many temperate and highland-tropical regions due to its ability to thrive in drought, saline and high-altitude conditions. The nutritional value of quinoa has been well documented. Several analyses have confirmed that quinoa grain has an excellent balance of carbohydrates, lipids, and proteins and provides an ideal balance of essential amino acids for human nutrition.1 Quinoa grain also has a seed coating consisting of various saponins. Saponins are waxy, soap-like substances that exhibit a wide range of properties and therefore are regarded as important biological compounds. Some saponins found in quinoa act as a natural pesticide for the plant by producing bitter compounds that deter insects and birds.
Bitter saponin, a major seed coating component found in quinoa, is responsible for bitterness and inhibits nutrient uptake in humans. A series of expensive washings must be performed before the seed is able to be cooked or ground into flour.3 Previous attempts using selective breeding to lower the saponin content of quinoa have failed. The crop had the desired “sweet” characteristics but had lost its ability to ward off pests and as a result birds consumed nearly the entire crop. It has since been discovered that some varieties of quinoa produce high levels of saponins, thus retaining the effect of a natural pesticide, but the saponins produced are non-bitter and do not decrease human palatability.3 However, breeding a high quality, pest-resistant, bitter saponin-free quinoa variety with the other desired traits (high yield, short growing season, etc.) through traditional breeding could take years or decades. A breeding program assisted through genetic knowledge of bitter saponins would shave years off the process.
First, we extracted and reverse transcribed mRNA directly from a bitter saponin variety (NL-6) quinoa seed using the SuperScript III One Step RT-PCR Platinum Taq HiFi Kit. (Invitrogen, California, USA). We reverse transcribed the mRNA at different stages of seed development to determine when the gene is expressed. We then used known β-amyrin synthase sequences previously identified in several other species, including model systems such as soybean (Glycine max) and thale cress (Arabidopsis thaliana), in order to identify homologous gene sequences. We designed consensus primers based on these homologues and used them to amplify fragments of β-amyrin synthase gene candidates from the reverse transcribed mRNA (cDNA) of the gene to confirm its presence in quinoa.
We subsequently took these PCR products and inserted them into plasmids using a pGEM-T Easy Vector kit (Promega Corp., Wisconsin, USA) The genes inserts will then be isolated, purified and sequenced using a GenElute Plasmid Miniprep Kit. (Sigma-Aldrich, Steinheim, Germany) The sequence data was analyzed using ContigExpress v3.1 (Invitrogen, California, USA) and compared to all known gene sequences in the NCBI GenBank database. The DNA sequences homologous to β-amyrin synthase sequences in other plants were saved and aligned in MEGA v3.1, a multi-pairwise alignment program (The Biodesign Institute, Arizona, USA). We found what seem to be 2 distinct copies of the β-amyrin synthase gene in quinoa. This makes sense as quinoa is an allotetraploid.
The partial sequence obtained only covered ~1400bp, while the β-amyrin synthase gene in other plants is ~2500bp. In order to get the full-length sequence we attempted to hybridize the partial sequence over a quinoa seed cDNA library using radioactive labeling (*32P) and a Prime-a-Gene Labeling System (Promega Corp., Wisconsin, USA). After multiple attempted hybridizations and even the construction of a new cDNA library followed by additional hybridizations we were unsuccessful in finding the full-length sequence. We have started the process of investigating alternative methods to elucidate the entire β-amyrin synthase gene, including the rapid amplification of cDNA ends (RACE) and hybridization over a quinoa BAC library.
In retrospect this project was more difficult and of a much larger scope than I had initially realized. However, the experience I have gained, as a result of working with this project has been a great way to learn about cutting-edge technologies in areas related to my fields of study. It has also been an excellent opportunity to make connections that will enable me to take the next step in my academic and professional careers with confidence. I also believe that it has provided me with tools to enable the realization of my personal goals in life and science.