The Journal of Heredity 2001:92(1)
© 2001 The American Genetic Association 92:83-86
Brief Communication |
A Recessive Allele Inhibiting Saponin Synthesis in Two Lines of Bolivian Quinoa (Chenopodium quinoa Willd.)
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Abstract |
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Quinoa cultivars currently grown in North America and Europe require removal of bitter-tasting saponins from the grain prior to human consumption. This need for postharvest processing is a barrier to expanding production of the crop outside its Andean area of origin. Grain saponin content in quinoa shows continuous variation and is considered to be a quantitative trait. However, segregation for the presence or absence of grain saponin in F2 generations derived from crosses between high- and low-saponin parents indicates a major gene effect, with plants homozygous for a recessive allele sp1 having no detectable grain saponin. Variation in saponin levels among F2 plants with detectable grain saponin was consistent with polygenic inheritance. It appears that grain saponin level in quinoa is both qualitatively and quantitatively controlled, with saponin production requiring at least one dominant allele at the Sp locus and the amount of grain saponin being determined by an unknown number of additional quantitative loci. Introgression of sp1 into day-neutral lines will facilitate the development of short-season "sweet" quinoa cultivars which do not require postharvest processing to remove grain saponin.
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Introduction |
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Quinoa (Chenopodium quinoa Willd.) is a traditional Andean grain with many useful attributes. The species is drought and frost tolerant, will grow in poor soils at high elevation, and the grain is rich in essential amino acids (Koziol 1992). For these reasons quinoa is attracting attention as an alternative crop outside its area of origin, especially in the western United States, Canada, and northern Europe (Jacobsen et al. 1996; Ortiz et al. 1998). A significant barrier to expanding quinoa production in these areas, however, is the grain saponin content of short-season day- neutral varieties which can be grown at higher latitudes. Plant saponins are triterpenoid glucoside compounds found in many genera; most plant saponins have an intensely bitter flavor and all are potentially toxic if ingested in large quantities (Koziol 1992). Mizui et al. (1988; 1990) identified 13 different saponins in quinoa bran, 10 of them previously unknown. Quinoa saponins are concentrated in the external layers of the grain, which is botanically a fruit with a tightly adhering pericarp covering two seed coat layers (Varriano-Marston and DeFrancisco 1984). The tissue containing saponins is therefore of maternal origin, and grain saponin content reflects the genotype of the plant from which the grain is harvested. Saponins are traditionally removed from quinoa by washing the grain before cooking it, although mechanical dehulling by abrasion is also effective (Chauhan et al. 1992; Reichert et al. 1986). This method is used by U.S. quinoa producers, but it increases costs and is a major deterrent to potential growers who do not have access to the appropriate equipment. The key to developing commercial quinoa cultivars for North America and Europe is to combine early maturity and high yield with a sufficiently reduced grain saponin content to eliminate the need for postharvest processing (Jacobsen et al. 1996; Johnson and Ward 1993).
Estimates of grain saponin content in different quinoa cultivars range from 0.00 to 11.2 mg/g (Koziol 1992; Wahli 1990). Some Ecuadorian and Bolivian cultivars have extremely low levels of saponin in the grain, which can be consumed directly without washing or milling; however, these low-saponin or "sweet" lines ("quinoas dulces") are late maturing and perform very poorly at higher latitudes. There has been little investigation of the genetic basis of quinoa saponin content. Gandarillas (1979) proposed that the trait was controlled by two alleles at a single locus, with "bitter" (high saponin) dominant to "sweet" (low saponin). More recently researchers have observed that grain saponin content in quinoa is a continuously distributed variable and is therefore more likely to be polygenically controlled and quantitatively inherited (Galwey et al. 1990; Jacobsen et al. 1996).
Gas chromatography and spectrophotometric techniques have been used to characterize and quantify some individual quinoa saponins (Ng et al. 1994). Such methods are unsuitable for screening total grain saponin content in large numbers of samples, however, as they are time consuming and expensive, and at least 13 different saponins must be quantified, not all of which have been fully characterized. Afrosimetric methods, based on the quantity of foam generated when saponins are shaken in water, cannot quantify the individual saponins present but do offer the advantage of providing a rapid and economical estimate of total grain saponin content. Koziol (1991) has described a standardized afrosimetric test for estimating grain saponin levels in quinoa. This method has some minor disadvantages: it underestimates very high levels of grain saponin, and results may be affected by the presence of other surfactants. However, the test readily identifies "sweet" quinoa lines that contain no detectable saponins, as these produce no measurable foam. Coefficients of variation obtained for repeated tests using this method are within acceptable limits, and the standardized test has proved satisfactory when screening for saponin content of individual plants within quinoa populations (Jacobsen et al. 1996; Ward 1994).
Genetic analysis of economically important traits in quinoa has been hampered by the difficulty of making controlled crosses. Quinoa is a predominantly self- pollinating allotetraploid with large numbers of very small (3 mm diameter) perfect flowers clustered on an inflorescence. This floral structure makes artificial hybridization very difficult and hinders the production of segregating generations large enough for analysis. The research described here used cytoplasmic male sterile (CMS) quinoa lines as female parents in four different crosses, two of which used low saponin "sweet" quinoa cultivars as pollen parents. Subsequent F1, F2, and F3 generations were screened for saponin content using Koziol's standardized afrosimetric test. The goal of this research was to characterize genetic control of grain saponin content in quinoa, especially in low-saponin lines.
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Materials and Methods |
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All quinoa plants used in this study were grown in a greenhouse in Fort Collins, Colorado, in 1998 and 1999. Five plants were grown in each 25 cm diameter pot, using commercial potting compost supplemented by liquid fertilizer. Greenhouse temperature was maintained at 25°C and plants were grown under broad-spectrum halogen lamps with a 16 h photoperiod maintained throughout the experiment. Four individual crosses were made using CMS quinoa plants as female parents (Table 1). The CMS lines were derived from a Peruvian accession (PI 510536) from the USDA- ARS Chenopodium collection; this CMS system and the associated maintainer and restorer lines have been described previously (Ward 1998). The pollen parents were selected from these restorer lines to generate fully fertile F1 generations. The four crosses were made by enclosing a male sterile inflorescence with exserted stigmas together with a male fertile inflorescence at anthesis within a single waxed paper pollination bag for 5 days; this also enabled simultaneous selfing of the pollen parent. Grain saponin content of all parent plants was determined by using Koziol's (1991) standardized afrosimetric test on the F1 seed harvested from the female parents and on the S1 seed harvested from the male parents. Twenty F1 progeny from each of the four crosses were grown and self-pollinated by enclosing the inflorescence in a waxed paper pollination bag throughout anthesis, and the grain saponin content of the F1 plants was determined by using Koziol's test on the F2 seed harvested from each F1 plant. Details of the four initial crosses are given in Table 1.
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Seed from a single F1 plant from each cross was planted to produce an F2 of at least 160 plants. Three of the four F2 generations segregated for presence or absence of the nuclear restorer allele, and in these cases all male sterile F2 plants were discarded at flowering. Each F2 plant was self-pollinated by enclosing the inflorescence in a waxed paper pollination bag throughout anthesis, and F2 grain saponin contents were determined by testing the F3 seed harvested from each F2 plant. F3 families of 40 plants were raised from each of 10 selected zero-saponin F2 parents, 5 of which were derived from cross 3 and 5 from cross 4. These F3 plants were self-pollinated and tested for grain saponin content as already described. Chi-square tests for goodness-of-fit were performed on the two F2 generations segregating for presence or absence of detectable levels of grain saponin, using Yates' continuous correction factor (Gomez and Gomez 1984).
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Results |
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All of the CMS plants used as female parents had high grain saponin levels, while two of the male parent plants had medium saponin levels and two were effectively zero saponin, with grain saponin levels below the limits of detection using afrosimetric methods (Table 1). All F1 plants were high saponin, although variation in saponin levels among plants within individual F1 generations was observed in three of the four crosses. All F2 offspring from cross 1 (Amachuma x Tango) and cross 2 (Calcha x Baer), where a medium- saponin male parent was used, had measurable levels of grain saponin, although considerable variation was observed between plants within F2 families. F2 progeny from cross 3 (Isluga x Sajama), where the male parent lacked saponin, segregated 148:41 for the presence or absence of saponin. This corresponds to a ratio of 3:1 (
2 = 1.02, P = .34). F2 progeny from cross 4 (Amachuma x Sayana), where the male parent also lacked saponin, segregated 139:27. This ratio falls between 3:1 (2 = 6.53, P = .013) and 15:1 (2 = 29.97, P < .01). In both cross 3 and cross 4, variation among F2 plants that contained measurable levels of grain saponin was observed. Frequencies of plants containing different levels of grain saponin are summarized for all four crosses in Figure 1. None of the 40 F3 progeny grown from each of the 10 selected zero-saponin F2 plants had detectable grain saponin levels.
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Discussion |
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Segregation for a continuous range of saponin levels was seen in the F2 populations from crosses 1 and 2, where both parents had either high or medium saponin levels. This is consistent with the hypothesis that grain saponin content in quinoa is a quantitative trait controlled at multiple loci. The high saponin levels in the F1 generations from all four crosses and the skewed frequency distributions for F2 progeny from crosses 1 and 2 also indicate that dominance effects may be significant contributors to the observed phenotypic variance. F1 saponin levels exceeded parental values in all four crosses, which could be a heterotic effect due to overdominance. This suggestion should be treated with caution, however: as a quantitative trait, grain saponin level may also be affected by the environment, and it is possible that even in a greenhouse growing the parent and F1 plants at different times of the year (parents in late summer 1998, F1 plants in winter 1998–1999) generational differences in grain saponin content may be observed. The nature and extent of environmental influences on grain saponin in quinoa and the possibility of heterosis for the trait clearly need further investigation. The amount of between-plant variation observed in three of the F1 generations indicates heterozygosity at the relevant multiple loci at one or both of the parents used in those crosses. This was not unexpected: although quinoa is largely self- pollinating, at least 1–2% outcrossing typically occurs between adjacent plants (Espindola G, personal communication). The male fertile restorer lines used in this study were maintained as individual bulk populations, isolated from other lines but without bagging individual plants, so complete homozygosity of all plants cannot be assumed. As an allotetraploid, quinoa may also be subject to fixed heterozygosity at some loci, even when self-pollinated.
The F2 progeny in crosses 3 and 4, where zero-saponin pollen parents were used, show frequency distributions for those plants with measurable quantities of grain saponin consistent with quantitative inheritance and similar to those from crosses 1 and 2. A striking difference in the F2 generations from crosses 3 and 4, however, is the clear segregation for presence or absence of grain saponin, with a ratio of 3:1 in cross 3 and a distorted segregation falling between 3:1 and 15:1 in cross 4. As quinoa is an allotetraploid, multiple genotypes are possible: the variation in F2 progeny ratios between crosses 3 and 4 reflects different genotypes at the Sp locus in the CMS female parents, and consequent differences in genotype between the F1 plants selfed to produce the segregating F2 generations. The F2 ratios observed here are typical of allotetraploid segregation at a single locus present in both genomes, where partial pairing between homologous chromosomes produces a range of erratic segregations. Similar segregation ratios have been observed for other single-gene traits in quinoa (Ward 2000). This segregation for presence or absence of grain saponin is consistent with the observation made by Gandarillas (1979) that a cross between "bitter" and "sweet" Bolivian quinoas produced a high- saponin or "bitter" F1 and an F2 generation that segregated for presence or absence of grain saponin ("bitter" or "sweet") in a 3: 1 ratio. Gandarillas did not report any F3 data; however, the 400 F3 plants derived from 10 zero-saponin F2 individuals in this experiment were all uniformly zero-saponin, indicating that both they and their F2 parents were homozygous at the relevant locus.
These results suggest that Sayana and Sajama, the Bolivian cultivars used as pollen parents in crosses 3 and 4, possess a recessive allele that inhibits saponin production when homozygous. This allele may be a mutated or null version of a gene encoding either an enzyme critical for saponin synthesis or a regulator of the saponin synthetic pathway. Crossing these zero-saponin cultivars with saponin-containing lines produces F1 plants in which saponin synthesis is restored, possibly because at least one functional copy of the key gene is now present. The between- plant variation in grain saponin content in three of the four F1 generations, and the more extensive variation seen among F2 plants containing saponin, indicate that in plants with at least one functional copy of the key gene, grain saponin levels will depend on alleles present at an unknown number of additional loci. Variation for this trait as seen in the F2 distributions (Figure 1) does not indicate simple additive effects, which is not surprising given the number of different saponins known to be present in quinoa. More extensive biochemical analysis is needed to determine the relative contributions of the different triterpenoid glucosides in quinoa to the total grain saponin content.
Similar combinations of qualitative and quantitative variation for a single trait have been reported elsewhere: in maize silks, for example, maysin concentration is controlled at a major locus p1 coding for a transcription activator, with additional minor loci only active in the presence of a functional p1 allele (Byrne et al. 1996). Another possibility is that zero-saponin quinoa plants result from the accumulation and fixation of null alleles at the relevant quantitative loci, regardless of any additional major gene effect. However, no zero-saponin plants were identified in a total of 263 segregating F2 plants from crosses 1 and 2, suggesting that if this does occur it is a relatively rare event.
An unresolved question is whether the "sweet" quinoa plants identified in this experiment are totally lacking in grain saponin or are failing to produce only certain types of saponin compounds. As described earlier, Mizui et al. (1988; 1990) identified more than 13 different saponins in quinoa bran. Ng et al. (1994) reported that using thin-layer chromatography and mass spectrometry they were able to detect saponins containing phytolaccagenic acid and oleanolic acid in three quinoa lines identified as "sweet," while "bitter" lines also contained the sapogenol hederagenin. However, the total saponin contents reported by these researchers for the "sweet" lines tested ranged from 1.3 to 3 mg/g, levels which would actually taste bitter to most humans and which are above the 1.1 mg/g threshold for "sweet" as defined by Koziol (1991). These results indicate that different types of saponins are found in varying proportions in different quinoa varieties, again suggesting that multiple loci are involved. As discussed earlier, Koziol's afrosimetric test cannot distinguish between different types of saponins, but it does distinguish effectively between quinoa plants containing measurable levels of some form of saponin and those which do not. Seed from all the plants classified as zero saponin in this experiment failed to generate any measurable foam when shaken with water, and such seed also had no detectable bitterness when tasted.
This recessive allele, provisionally designated sp1, offers a number of advantages to breeding programs developing commercial quinoa varieties for North America and Europe. Introduction of this allele into the early maturing day-neutral lines via conventional backcross techniques should be straightforward and less time consuming than attempting to manipulate grain saponin content as a quantitative trait. Plants that are homozygous for sp1 can be identified easily and inexpensively using afrosimetric methods, will produce uniformly zero-saponin progeny, and will eliminate the need for postharvesting processing of the grain. A potential disadvantage is that zero-saponin quinoa lines homozygous for sp1 must be grown in isolation if seed is to be saved, as even low levels of cross- pollination from saponin-containing lines will result in the loss of the "sweet" trait in subsequent generations. It is also likely that the presence of saponins in quinoa grain provides protection against bird and insect attacks, and such damage in the field may be a greater problem with zero- saponin quinoa lines. These disadvantages, however, are offset by eliminating the need for postharvest grain processing, which will increase the potential for quinoa as an alternative crop for non-Andean countries.
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Acknowledgments |
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From the Department of Soil and Crop Sciences, Colorado State University, Fort Collins, CO 80523. This research was supported by Colorado State University cooperative extension project no. 0168529.
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Footnotes |
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Corresponding Editor: David B. Wagner
Received March 12, 2000
Accepted August 31, 2000
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References |
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