Theron J Baker and Dr. Eric N Jellen, Plant and Animal Sciences
Quinoa (Chenopodium quinoa) is an Andean cereal chenopod known for its ideal nutritional characteristics along with its abilities to grow in drought conditions and in soils with high salt content (Fairbanks 2002). Until recently, quinoa has received little attention from the scientific community; but due to recognition of its nutritional and agronomic potential, science is now beginning to take interest (Fleming 1995). Among the few genetic studies published, Giusti (1970) established the base chromosome number of the species to be eight or nine with varying ploidy levels. More recently, Kolano (2001) mapped the 45S ribosomal DNA (rDNA) sequences using fluorescence in situ hybridization (FISH) in two lines of C. quinoa: PIQ-1 and PIQ-8; both were tetraploid accessions having 36 chromosomes. Parkinson (2001) also mapped 45S rDNA using FISH in two other quinoa lines: ‘Surimi’ and ‘Real’. Two signals were observed in all for genotypes, indicating there is only one pair of chromosomes harboring a single 45S locus in spite of the allotetraploid composition of the quinoa genome (Ward 2000).
Our objective was to identify the direct ancestral diploids of C. quinoa using FISH. Parkinson (2001) had recently developed improved protocols for examining quinoa chromosomes cytologically. Our proposed research was to begin physical mapping of five Chenopodium species thought to be potential progenitors of C. quinoa: C album, C. botrys, C. murale, C. pallidicaule, and C. berlandieri var. nuttalliae. We selected highly repetitive quinoa DNA sequences compiled by Brian Gardunia that show homology to known genes of various species (see Table 1).
The chromosome preparation protocol developed by Parkinson proved to be ineffective with the diploid Chenopodium species. This is most likely due to seed dormancy in the weedy species, which included all of the ones examined except C. pallidicaule and C. berlandeieri var. nuttalliae. By taking root tips from young greenhouse plants we were able to overcome this problem. This was done by excising root tips from young greenhouse plants and treating them in 0.1% colchicine for 4-6 hours. Next the root tips were fixed in 3:1 ethanol:glacial acetic acid for at least 16 hours. We then treated the root tips in 4% cellulase, 1% pectolyase in 30 mM citrate buffer (pH 4.5) for 15 minutes at 37° C. Next we gently washed the root tips in distilled water and squashed them in 60% acetic acid under a glass cover slip. Slides were then stored at –20° C until needed. This protocol needs some adjustments, as the cellular mitotic index was low; however, the cells in Figs. 1 and 2 were obtained by this method.
Labeling the probe DNA was difficult and time-consuming. Our proposed protocol was to use standard nick translation with Alexa fluor 488-5-dUTP and Alexa fluor 595-5-dUTP direct-label Probe Description 18-18L Microsatellite sequence showing homology to rire retrotransposon DNA 22-E21 Minisatellite sequence showing homology to Beta corolliflora 16-12P Copia-like pTA 71 45S ribosomal DNA (rDNA) Table 1. FISH probes fluorophores (Molecular Probes, Eugene, OR). Instead we decided to attempt PCR labeling using the fluorescent labels listed above. This was ineffective, perhaps due to steric hindrance during incorporation of hapten-labeled nucleotides. It was decided that we should use non-enzymatic probe incorporation using Oregon Green 488. This was done using the Ulysis Nucleic Acid Labeling Kit and protocol (Molecular Probes, Eugene, OR). Figures 1 and 2 both used probes labeled in this manner.
We successfully identified four hybridization sites for pTA71 in C. berlandieri (two loci) and two sites (one locus) each in C. murale and C. pallidicaule (see Figures 1 and 2). These results indicate that if C. berlandieri and C. quinoa have a monophyletic origin that the former species is likely the progenitor of the latter. Also, the two diploids successfully hybridized are both candidate progenitors of the New World tetraploids. I am confident with the protocols we have used and developed over the course of this process and that they will eventually lead us to the information we need to expand our knowledge of quinoa and its diploid ancestors.
References
- Fairbanks DJ (2002) The story of Quinoa: An ancient food gives new hope to an impoverished people. The Ezra Taft Benson Institute Review, Spring 2002: 12-17.
- Fleming JE, Galwey NW (1995) Quinoa (Chenopodium quinoa). In: Williams JT (ed) Cereals and pseudocereals. Chapman & Hall, London, pp. 3-83.
- Giusti L (1970) El genero Chenopodium en Argentina: I. Numeros de cromosomas. Darwiniana 16: 98-105.
- Kolano B, Pando LG, Maluszynska (2001) Molecular cytogenetic studies in Chenopodium quinoa and Amaranthus caudatus. Acta Societatis Botanicorum Poloniae, 70: 85-90.
- Parkinson SE (2001) Cytogenetic studies and construction of bacterial artificial chromosome library for Chenopodium quinoa (Willd.). M.S. Thesis, Brigham Young University.
- Ward SM (2000) Allotetraploid segregation for single-gene morphological characters in quinoa (Chenopodium quinoa Willd.). Euphytica 116:11-16