The Journal of Heredity 2002:93(1)
© 2002 The American Genetic Association 93:52-55
Brief Communication |
Genetics of Qualitative Traits in Domesticated Chia (Salvia hispanica L.)
From the Department of Botany and Plant Sciences, University of California, Riverside, Riverside, CA 92521.
Address correspondence to J. P. Cahill at the address above or e-mail: jpca{at}citrus.ucr.edu.
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Abstract |
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In Salvia hispanica L., several changes in qualitative characters, including seed coat color, stem pigmentation, and shattering, have evolved with cultivation and domestication. Three F2 segregating generations from crosses between wild and domesticated parents were scored for three qualitative traits. A single recessive gene, designated scc, was found to govern the white seed characteristic. A single dominant gene, designated SSP, was found to control striated stem pigmentation. A complete dominance of open calyx over closed calyx was observed in F1 generations and small numbers of plants with closed calyxes were observed in F2 generations, not conforming to Mendelian ratios. For this nonshattering trait, a complementation test was conducted between two lines representative of geographically and morphologically divergent domesticated varieties. Complementary gene action was not observed in any F1 plants, and all F2 plants were homogeneous with respect to the trait, suggesting the same genetic control for nonshattering among domesticated varieties. An analysis of limited data for linkage of SSP and scc indicated that the two loci segregate independently.
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Introduction |
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Chia (Salvia hispanica L.) has a long history of plant-human interaction. In pre-Columbian Mesoamerica the annual crop species was a major commodity and its seeds were valued for food, medicine, and oil (Sahagun 1950–1982). Economic historians have suggested S. hispanica was as important as maize in pre-Columbian Mexico, and in some areas more important (Harvey 1991; Rojas-Rabiela 1988). After Spanish contact and colonization, cultivation of the species plummeted, however, highly productive domesticates are still grown in a few areas.
Basic biological knowledge pertaining to the species is minimal, and published genetic information is limited to chromosome number, 2n = 12 (Haque and Ghoshal 1980). In the southwestern United States, field trials have demonstrated the species has great potential as a future crop plant. Applied research has focused on seed oil (Ayerza 1995), seed polysaccharide mucilage (Lin et al. 1994), leaf essential oil (Ting et al. 1996), and nutritional composition for human and animal food (Weber et al. 1991). Recent development of the crop has involved selection from domesticated varieties (Coates and Ayerza 1996; Estilai et al. 1990), but has not included breeding efforts. Comparative morphological analyses of flowers of Salvia species indicate that the small flowers (3–4 mm) of S. hispanica are reflective of a highly self-pollinated breeding system (Haque and Ghoshal 1981). The small corollas, fused flower parts, and propensity for self-pollination have hampered the establishment of breeding programs. This analysis of the inheritance of qualitative domesticated traits will provide a genetic basis for ongoing breeding efforts at the University of California, Riverside and elsewhere.
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Materials and Methods |
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The origins and characteristics of five inbred parental lines chosen for this study are shown in Table 1. All parental lines were inbred for at least three generations, and those originating from Mexico for at least five generations. For each parental line, no morphological variation was observed among the selfed generations. Parental, F1, and F2 generations were established in a greenhouse from 1998 to 2000, with parental accessions grown each year. Reciprocal crosses were made by hand-pollinating. The tubular corollas of male parent plants were first sectioned longitudinally, then sections with adnate stamens and corolla portions were removed with a forceps. Newly opened flowers on female parental plants were examined with a hand lens. Stigmas which appeared to lack pollen grains were contacted with the anthers of the male parent and reexamined to ensure pollen had been transferred.
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The first cross made was between JC09001 (dark seeds, nonpigmented stems, closed calyxes) and JC15001 (white seeds, pigmented stems, open calyxes). The second cross was between JC20001 (dark seeds, nonpigmented stems, and closed calyxes) and JC10001 (dark seeds, pigmented stems, and open calyxes). The third cross was between JC09001 and JC19001 (dark seeds, nonpigmented stems, and closed calyxes).
F1 plants were verified as successful crosses by a combination of dominant inheritance of stem pigmentation and dominant loci generated with the random amplified polymorphic DNA (RAPD) primer G15 (Operon Technologies Inc., Alameda, CA). The RAPD procedure followed that of Jones and Sutton (1997). The segregating F2 generations from each cross were harvested and scored for three qualitative traits: calyx type, seed coat color (scc), and stem pigmented (SSP). For seed color, F1 generation refers to the seeds derived from F1 plants. Supplemental data were included in the analysis for the calyx type and seed coat color traits. These data originate from F2 generations derived from the same JC09001 and JC15001 crosses in a separate agronomic experiment conducted in irrigated field plots at the University of California, Riverside Agricultural Operations located at N 33°57' W 117°20'. For all greenhouse and field generations, seeds were sown on the first of June, flowered 2–3 weeks after the autumnal equinox, and were harvested throughout November and December. Backcrosses were not performed because neither morphological markers nor the hand-crossing method could be relied on to confirm or produce crosses with any consistency. Attempts to improve the hand-crossing method through manual emasculation failed due to small flower size and the high degree of fusion of floral organs. To determine goodness-of-fit to various genetic ratios, the observed segregation ratios for calyx type, seed coat color, and stem pigmentation were subjected to chi-squared tests.
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Results and Discussion |
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The three RAPD loci, between 1.0 and 1.6 kb in size, produced dominant inheritance patterns in F1 plants, and together with dominant inheritance of stem pigmentation, confirmed the success of some crosses. The results indicated that only 60.7% efficacy was achieved with the hand-crossing method. The low success and absence of an effective emasculation procedure will undoubtedly remain an obstacle for future examination of inheritance.
Subsequent to fertilization, the calyxes close in all varieties of S. hispanica, thereby protecting the seeds during development. Seed maturity coincides with cell death in the calyx walls. As the cells die and dehydrate, the calyx opens, allowing seed dispersal in wild types. In all domesticated parental lines, the cells of the calyx walls die, however, the function of opening upon dehydration has been lost. Human selection has resulted in plants incapable of dispersing seed, and therefore unable to survive outside of cultivation. This characteristic is a hallmark of plant domestication. The parent plants from domesticated accessions JC09001 (27 plants), JC19001 (25 plants), and JC20001 (8 plants) exhibited closed calyxes, while the parent plants of JC15001 (24 plants) and JC10001 (4 plants) had open calyxes. Though a small number of plants served as parents for JC10001 and JC15001, about 100 plants were grown during each selfing generation for each accession and consistent morphological traits were maintained, giving us the confidence to work with small numbers of parents. All 62 F1 plants derived from the parental crosses JC09001 x JC15001 and 12 from JC20001 x JC10001 had open calyxes, indicating a complete dominance of open calyx over closed calyx. For the JC20001 x JC10001 cross, the F2 generation segregated into 136 open calyx and 19 closed calyx plants, not fitting a 3:1 or 15:1 ratio. For the JC09001 x JC15001 cross, the F2 greenhouse generation segregated into 125 open calyx and 23 closed calyx plants, not fitting to a 3:1 ratio. In the field generation, the 3077 open calyx and 146 closed calyx plants clearly did not fit a 3:1 or 15:1 ratio. These results suggest that more than a single gene controls closed calyxes; however, the number of genes could not be determined. Given the stages of calyx development and their reliance on cell death, environmental factors may account for the lack of conformity of this trait to a Mendelian ratio in segregating generations.
The difference in humidity between the greenhouse and field conditions offers a possible explanation for the observed difference in ratios. The results indicated ratios of approximately 9:1 for greenhouse generations and 21:1 for the field generation. Since F2 inflorescences from both growing conditions were thoroughly dried in an exhaust hood before scoring, a humidity-dependent developmental process could play a role in the drying and opening of dead calyx walls. However, differences in water availability, temperature, and plant stress also existed, and may have contributed to the difference in ratios. Parental lines in greenhouse and field conditions exclusively produced their respective phenotypes without exception, and no plants in the F1 or F2 generations produced any intermediate forms with respect to this trait. Given the results, our hypothesis is that the human selected trait of closed calyxes exhibits recessive inheritance and that the gene(s) controlling this trait likely have expression patterns that are somewhat dependent on environmental factors. From an evolutionary perspective, if a single loss-of-function mutation resulted in plants that did not consistently produce closed calyxes, then subsequent human selection for modifier genes to ensure consistent closure of the calyxes might result in more than one gene controlling the trait.
Further analysis of inheritance of calyx states was conducted using domesticated varieties from Mexico, which are distinct from those cultivated in Central America and exhibit numerous morphological and physiological differences. Accessions JC09001 and JC19001 were crossed in a complementation test. All 12 F1 and 130 F2 plants exhibited closed calyxes, indicating the same gene or set of genes controls the recessive trait of closed calyxes.
After about 6 weeks of growth in a long-day environment, vertical striations of anthocyanin pigmentation began to appear on the stems of all parental plants of accessions JC15001 and JC10001, and were lacking in JC0900, JC20001, and JC19001. Some domesticated varieties have solid purple stems, but this trait was never observed in any of the parental lines used in this study and should not be confused with the striated stem pigmentation trait being analyzed in this study. All F1 plants derived from the parental crosses had striated stem pigmentation, indicating a complete dominance over nonpigmented stem. The segregation patterns for SSP in greenhouse generations are presented in Table 2. For the JC20001 x JC10001 cross, the F2 generation segregated into 117 pigmented:38 non pigmented plants, fitting very closely to a 3:1 ratio. For the JC09001 x JC15001 cross, the F2 greenhouse generation segregated into 113 pigmented:35 nonpigmented plants, also fitting a 3:1 ratio.
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The results indicate that striated stem pigmentation in JC15001 and JC10001 is controlled by a single dominant gene, SSP. Since all domesticated varieties lack the striated stem pigmentation trait that is present in most wild types, the trait can be successfully applied as a morphological marker when crossing wild and domesticated varieties. This knowledge is applicable in breeding efforts directed toward the incorporation of the beneficial characteristics of wild types into highly productive domesticated lines. Characteristics of wild plants that have the potential to increase the agronomic and commercial value of domesticated varieties include their greater variation in flowering time, leaf chemotypes, and growth habit.
Parental accession JC09001 from Nicaragua produced only dark-seeded plants, while parental accession JC15001 produced exclusively white-seeded plants. White- and dark-seeded cultivars were first described and illustrated by the Spanish monastic Fray Bernardino de Sahagun working in Mexico in 1575 (Sahagun 1950–1982). Both seed coat types still exist today and are easily distinguished from each other (Figure 1). While JC15001 is cultivated in mixed crop settings, it does not meet the criteria for domestication and has retained its wild morphological characters with the exception of the white seed coat trait. The segregation patterns for both field and greenhouse generations are presented in Table 3. The F2 greenhouse generation segregated 115 dark-seeded:33 white-seeded plants, fitting closely to a 3:1 ratio. The field generation included 2630 dark-seeded and 832 white-seeded plants, also fitting a 3:1 ratio. The results indicate that the cultivated material originating from Guerrero, Mexico (JC15001) has a single recessive gene controlling the white-seeded trait. Whether human or natural selection has resulted in exclusively white-seeded generations remains unresolved. Since S. hispanica has many uses similar to Sesamum, the white seed coat may also have some modern commercial preference.
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Because scc and SSP are controlled by single genes, linkage analysis of the two loci is possible. Unfortunately parental accession JC09001 was the only dark-seeded parental line with a flowering time that overlapped the late-flowering white-seeded JC15001, subsequently linkage analysis was limited to one F2 generation (JC09001 x JC15001) grown under greenhouse conditions. The test resulted in a chi-squared value of 1.69 and P = .64, indicating SSP and scc segregate independently of each other and fit a 9:3:3:1 ratio. If late-flowering varieties homozygous for dark seed coat are developed, further crosses can be made to confirm the observed independent segregation.
The recessive inheritance of nonshattering combined with the lack of effective crossing and backcrossing techniques may present several problems for future research. Our observations in F2 generations showed small numbers of nonshattering plants, presenting an additional problem for plant breeders. If the goal is to incorporate desired wild characters into productive domesticated lines, large F2 generations will have to be screened to identify desirable nonshattering plants. The knowledge that single genes govern seed coat color and stem pigmentation should serve to simplify applied research, particularly efforts to select parents for breeding, given the great difficulty in performing crosses, and the present lack of described molecular markers for the species. Our basic research is continuing with the development of isozyme and RAPD markers with the intent of mapping the six linkage groups of S. hispanica. The qualitative characters described here will prove to be valuable as genetic map markers, contribute to a greater understanding of the genes involved in the traits of closed calyxes, and contribute to our general understanding of the domestication process.
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Acknowledgments |
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Thanks are extended to Dr. Arturo Gomez-Pompa, Dr. Giles Waines, J. A. Hernandez Gomez, P. McCrowan, and R. Gentry for support and providing seeds of chia accessions.
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Footnotes |
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Corresponding editor: Susan Gabay-Laughnan
Received January 23, 2001
Accepted September 5, 2001
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References |
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