Shanna Mason and Lucy Stewart with Dr. Craig E. Coleman, Botany and Range Science
Quinoa (Chenopoduim quinoa) is a pseudograin cultivated in the Altiplano region of Bolivia and other areas of South America, where it is well adapted to harsh local conditions including high elevation, drought, frost, and saline soils. In addition to its environmental adaptations, quinoa is unique among crops in its exceptional nutritional quality. Quinoa seeds not only have high protein content, but the amino acid composition of these proteins is nutritionally complete, including all of the essential amino acids for human dietary requirements (The Quinoa Project at Brigham Young University: A five-year proposal, unpublished). However, since quinoa use and cultivation is culturally localized to Andean South America, it has not had the advantage of extensive breeding programs to improve yields. While it is an important to crop to Bolivian subsistence farmers, and their principal source of protein, current yields are insufficient to provide them food year round, and protein deficiency is a serious problem in Bolivia (The Quinoa Project at Brigham Young University: A five-year proposal, unpublished).
The Bolivian government, in cooperation with the Ezra Taft Benson Agriculture and Food Institute, the U. S. Agency for International Development, and Brigham Young University, is working on improving quinoa production. The joint goal is to accelerate traditional crop breeding programs using modern molecular techniques in order to develop higher yielding varieties, while maintaining the high nutritional value already found in quinoa seeds. An important step in this undertaking is to sequence the genes corresponding to the major seed storage proteins in quinoa. Researchers at Brigham Young University have already identified seven major seed storage proteins in quinoa. By sequencing the genes that encode these proteins, and mapping these genes relative to amplified fragment length polymorphism (AFLP) markers being developed in another laboratory at Brigham Young University, researchers will be able to monitor and maintain protein quality as well as quantity in the breeding programs. In order to accomplish this, we planned to sequence the seed storage proteins from a quinoa seed cDNA library. Once the sequences are mapped relative to AFLP markers, they become useful selection tools termed expressed sequence tags (ESTs).
Our objectives for winter 2001 in this ongoing project were to complete construction of a cDNA library from quinoa seeds, sequence the cDNA from this library, and compare these sequences with those found in the GenBank online sequence database. This would allow us to determine the gene sequences of the seven major seed storage proteins, as well as other potentially interesting and useful genes. We made some progress on these objectives, although not to the extent anticipated.
To build a cDNA library from quinoa seed, we inserted quinoa cDNA (complementary DNA, previously synthesized from isolated quinoa seed mRNA using the enzyme reverse transcriptase) into plasmid vectors. We then transformed these plasmids into bacteria for replication and maintenance. We plated out these bacteria onto agar medium and selected the transformed colonies with blue-white screening. We placed each of these colonies in a glycerol solution so that the bacteria with the cDNA fragments could be frozen and stored indefinitely for culturing and retrieval. With the assistance of volunteers1, we constructed a cDNA library of approximately 11,000 such colonies, each with a single cDNA from quinoa seed messenger RNA.
We then submitted a sample of ten PCR (polymerase chain reaction) products from this library for sequencing at the BYU DNA Sequencing Center. Of these ten, we obtained four good sequences. Two of these do not show any significant similarity to plant genes in the GenBank database. The other two did have sequence homology to plant genes in the database. One sequence is very similar to a cDNA sequence of a lipid transferase in spinach (Spinacia oleracea), which, like quinoa, is a member the family Chenopodiaceae. The other, of greater interest, probably encodes a seed storage protein, having a sequence similar to that of a carrot (Daucus carota) storage protein. As we obtain more sequences, we will be able to determine if this is one of the seven major seed storage proteins in quinoa, since cDNAs for the most abundant seed proteins will probably be well represented in the cDNA library.
Even though sequencing the ten trial PCR products was mildly successful (but with a suboptimal number of good sequences), our attempts so far to sequence cDNA directly from plasmids purified from the bacteria cultures have been unsuccessful, and we have spent much of our effort attempting to troubleshoot this procedure. As a result of these difficulties, we have been unable to sequence and identify any significant number of cDNA clones. Once working and optimized, however, the plasmid sequencing prodecure should be rapid and efficient, since entire 96-well plates of cDNA clones may be prepared and sequenced simultaneously. This will provide the desired genes sequences of quinoa seed storage proteins, as well as other potentially interesting protein-encoding sequences. Mapped as expressed sequence tags, (ESTs), these sequences will contribute to the ongoing project to ultimately develop higher yielding quinoa varieties with maintained protein quality.
References
- Elizabeth Johnson, Steve Turley, and Jolynn Stevens aided considerably in the labor-intensive process of picking bacteria colonies.