Crop Science 42:634-636 (2002)
© 2002 Crop Science Society of America
PLANT GENETIC RESOURCES
Frequency of the No Mark Leaflet Allele in Red Clover
Robin Bortnem* and
Arvid Boe
Plant Science Dep., South Dakota State Univ., Brookings, South Dakota 57007-2141. South Dakota Agric. Exp. Stn. Journal Series No. 3240
* Corresponding author (Robin_Bortnem{at}sdstate.edu)
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ABSTRACT
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Red clover (Trifolium pratense L.) is known for trifoliolate leaves with light marks (i.e., variegation) on the leaflets. However, red clover plants without leaf marks occur in some wild and cultivated populations. The no mark (NM) phenotype is due to a homozygous recessive genotype. This study was conducted to evaluate the core collection of red clover for frequency of the recessive allele that imparts the NM phenotype. Ninety-eight seeds from 82 accessions in the core collection and one selected population (Blankleaf, NSL 303023) were planted in individual containers in the greenhouse. Seedlings were evaluated weekly for leaf markings. If a plant had no mark on any of its leaves from emergence to flowering, it was classified as a NM phenotype. We assumed that all evaluated accessions were diploid and in Hardy-Weinberg equilibrium for the leaf mark locus. Estimates of gene frequencies for the NM trait ranged from 0.0 for 21 out of the 82 accessions to 0.67 for PI 419294 from Greece. The grand mean was 0.20. Our data indicated the recessive allele was absent or at least extremely rare in about 25% of the accessions. Assuming the core collection represents the genetic variability in the species, our data provide a basis for designing additional studies to determine if natural and/or artificial selection are responsible for variation among populations for this conspicuous feature of red clover.
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INTRODUCTION
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RED CLOVER is known for trifoliolate leaves with light marks (i.e., variegation) on the leaflets. The marks result from a low concentration of chloroplasts in the palisade tissue (Liang, 1965) and vary in size, shape, position, and color intensity. Although the leaf mark is typically thought of as a characteristic of the species, both wild and cultivated red clover plants without leaf marks occur frequently in some populations (Wexelsen, 1932). Fergus and Hollowell (1960) and McLennan et al. (1960) described Dollard, a cultivar developed in Canada, as typically having leaves without the leaf mark.
The NM phenotype is due to a homozygous recessive genotype (Wexelsen, 1932; Fergus and Hollowell, 1960; McLennan et al., 1960; Liang, 1965; Taylor, 1980, 1982). A leaf marking descriptor has been developed for the red clover core collection (Smith et al., 2000), but the frequency of the recessive gene that produces NM phenotypes hasn't been determined for individual accessions. Our objective was to evaluate accessions from the core collection of red clover for frequency of the recessive allele that produces the NM phenotype.
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MATERIALS AND METHODS
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The core collection of red clover (more than 80 accessions from 36 different countries) and one selected population (Blankleaf, NSL 303023) were obtained from the Western Regional Plant Introduction Station (USDA-ARS) at Pullman, WA. Ninety-eight seeds from each accession were planted individually in 40 mm diameter by 205 mm long plastic cones in the greenhouse. No auxiliary light was used. Temperature inside the greenhouse generally ranged from about 20°C at night to as high as 30°C during the day, depending on ambient weather conditions. Seedlings were evaluated weekly for leaf markings, and those that displayed leaf marks were counted and discarded. For some plants a leaf mark appeared on the first trifoliolate leaf. If a plant had no marks on any of its leaves from emergence to flowering, it was classified as a NM phenotype.
Sixty-two accessions and ‘Blankleaf’ were evaluated from mid-Feb. through mid-June 1999, and 24 accessions were evaluated from mid-Feb. through mid-June 2000. Space restrictions in the greenhouse precluded evaluating all accessions during the same time period. However, four accessions were evaluated during both years to determine repeatability of frequencies of phenotypic expression. Twelve accessions planted during 2000 had seedlings (range was 1 to 32 plants accession-1) without leaf marks that did not flower after 160 d in the greenhouse. However, all but one accession (PI 419294) had less than 10 seedlings that did not flower. The majority of those 12 accessions had already produced plants that flowered and had no leaf marks. Two of the accessions evaluated during both years also had a few seedlings that we classified prior to flowering.
Frequency of the NM allele for each accession was estimated by q = 
, where x was number of NM plants and y was total number of plants evaluated. The standard error of frequency was calculated as 
(Li, 1961). Accessions were assumed to be diploid (K.H. Quesenberry, personal communication, 1999) and in Hardy-Weinberg equilibrium for the leaf mark locus. Kouamé and Quesenberry (1993) pointed out that although several recently released European cultivars are tetraploid, wild introductions of red clover are diploid. The core collection contains many cultivars. However, virtually all were developed prior to 1970.
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RESULTS AND DISCUSSION
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Chi-squared analyses of four accessions evaluated both years showed no significant (P > 0.05) differences between years, indicating estimates for the frequency of the recessive allele were highly repeatable (Table 1).
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Table 1. Estimated gene frequencies (q) and standard errors for the no mark allele for core accessions of red clover in the U.S. National Plant Germplasm System evaluated during 1999 and 2000.
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Extensive variability occurred among accessions within the core collection for frequency of the recessive allele that controls the NM leaf trait in red clover. We did not observe homozygous recessive individuals in approximately 25% of the accessions, and the highest frequency of NM phenotypes in any accession was 45% for PI 419294 from Greece. Sixty-five percent of the accessions had gene frequencies between 0.10 and 0.40 for the NM allele. In addition, only 61% of the plants of ‘Blankleaf’, which was presumably selected for the NM phenotype, were homozygous recessive. However, Bortnem and Boe (1998) fixed the homozygous recessive condition in a population with one cycle of recurrent phenotypic selection.
Although accessions that lacked the recessive allele for the NM phenotype were more common from the Central and Southern Europe regions, no conspicuous regional trends occurred for frequency of the recessive allele. For example, every one of the 240 plants from the three Italian accessions had leaf marks. In contrast, the five Swedish accessions had gene frequencies from 0.0 to 0.50. Overall, the range of the mean gene frequencies for countries (0.00 to 0.36) was smaller than the ranges among accessions within some countries (Table 2). Canada, Greece, and Turkey had overall mean NM gene frequencies greater than 0.25, but each also showed substantial within-country variability (Table 2).
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Table 2. Estimated gene frequencies (q) and standard errors for the no mark allele for core accessions of red clover in the US National Plant Germplasm System.
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Wexelsen (1932) found plants without leaf marks were more frequent in wild Norwegian red clover (38%) compared with local cultivated strains (9%). His observation leads us to speculate that some of the intraregional variation we observed was due to differences among accessions for selection pressure. If random genetic drift due to sampling accidents associated with small population size was involved and there was no selective advantage for the dominant allele, we would expect comparable numbers of accessions with loss or fixation of the recessive and dominant alleles. However, since we examined less than 100 plants accession-1, more plants need to be evaluated from those that had no homozygous recessive phenotypes before it can be concluded with a high degree of certainty that the leaf mark locus is monomorphic. Nevertheless, our data strongly suggested the recessive allele was rare in a substantial number of the accessions that compose the core collection.
Assuming the core collection represents the genetic variability in the species, our data provide: (1) a basis for designing additional studies after Wexelsen (1932) to determine the relative importance of natural and artificial selection in creating variation among populations for this conspicuous trait, and (2) rationale for comparing marked and nonmarked plants from the same populations/cultivars for agronomic traits.
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ACKNOWLEDGMENTS
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We would like to acknowledge helpful suggestions and advice given by K.H. Quesenberry, R.R. Smith, and N.L. Taylor.
Received for publication April 6, 2001.
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REFERENCES
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- Bortnem, R., and A. Boe. 1998. Leaf variegation in red clover. p. 70. In 1998 Agronomy abstracts. ASA, Madison, WI.
- Fergus, E.N., and E.A. Hollowell. 1960. Red clover. Adv. Agron. 12: 365– 436.
- Kouamé, C.N., and K.H. Quesenberry. 1993. Cluster analysis of a world collection of red clover germplasm. Genet. Resour. Crop Evol. 40:39–47.
- Li, C.C. 1961. Human genetics: Principles and methods. McGraw-Hill Book Company, Inc., NY.
- Liang, G.H. 1965. The inheritance of leaf marking and the effects of x-rays and ethyl methanesulfonate treatment as measured by M1-generation response in red clover (Trifolium pratense L.). Ph.D. diss. University of Wisconsin, Madison.
- McLennan, H.A., J.E.R. Greenshields, and R.M. MacVicar. 1960. A genetic analysis of population shifts in pedigree generations of Lasalle red clover. Can. J. Plant Sci. 40:509–515.
- Smith, R.R., N.L. Taylor, and K.H. Quesenberry. 2000. Variability of agronomic characteristics (descriptors) in red clover core collection. In Proc. 16th Trifolium Conf. 20–22 June, Pipestem, WV.
- Taylor, N.L. 1980. Clovers. p. 261–271. In W.R. Fehr and H. Hadley (ed.) Hybridization of crop plants. ASA, CSSA, and SSSA, Madison, WI.
- Taylor, N.L. 1982. Registration of gene marker germplasm for red clover. Crop Sci. 22:1269.[Free Full Text]
- Wexelsen, H. 1932. Segregations in red clover (Trifolium pratense L.). Hereditas (Lund, Swed.) 16:219–240.
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ABSTRACT
INTRODUCTION
