Crop Science 41:1625-1628 (2001)
© 2001 Crop Science Society of America
PLANT GENETIC RESOURCES
Isozyme Diversity in North American Cultivated Red Clover
J. Yua,
J. A. Mosjidis*,a,
K. A. Klinglera and
F. M. Woodsb
a Dep. of Agronomy and Soils, Alabama Agric. Exp. Stn., Auburn Univ., Auburn, AL 36849-5412
b Dept. of Horticulture, Alabama Agric. Exp. Stn., Auburn Univ., Auburn, AL 36849-5412
* Corresponding author (mosjija{at}auburn.edu)
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ABSTRACT
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Red clover (Trifolium pratense L.) is a forage legume with considerable economic importance in world agriculture. Knowledge of the amount and distribution of genetic variability within a species is vital to breeders and geneticists when selecting breeding germplasm. The objectives of this study were to assess genetic diversity in 34 North American red clover cultivars by means of isozymes. Isozymes assayed were esterase, ß-glucosidase, phosphoglucomutase, peroxidase, diaphorase, phosphoglucoisomerase, and superoxide dismutase. Eleven of the 13 loci (84.62%) were polymorphic in at least one cultivar. Percentage polymorphic loci within cultivar ranged from 61.54% in cv Morred, Persist, and Redland II to 84.62% in cv Arlington, Ram, and Red Baron, with an overall mean of 73.98%. At the species level, the number of alleles per polymorphic locus was 2.55 and effective number of alleles per locus was 1.64. Within-cultivar averages were 2.71 and 1.59, respectively. Genetic diversity was 0.292 at the species level and 0.285 for within populations. Most of the genetic diversity (98.4–99.7%) was distributed within the cultivars. Grouping the 34 cultivar on the basis of genetic distance indicated that their isozyme variability could be represented by a few cultivar of each of four groups plus the cv Morred and Redon.
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INTRODUCTION
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RED CLOVER is a short-lived herbaceous forage crop thought to have originated in southeastern Europe and Asia Minor near the Mediterranean Sea (Taylor and Quesenberry, 1996, p. 1). It is a short-lived perennial diploid species with seven-pairs of chromosomes (2n = 2x = 14) that is able to grow in a wide range of soil types, pH levels, and environmental conditions (Smith et al., 1985). It is insect pollinated and selfincompatible, thus red clover populations are heterogeneous and consist of heterozygous individuals. Naturalized populations of red clover occur along roadsides and in old fields as well as in grasslands.
Knowledge of the amount and distribution of genetic variability within a species is vital to plant breeders because it is an important consideration when selecting germplasm to be included in a breeding program. Also, it is helpful to geneticists managing plant genetic resources and provides information for designing sampling protocols (Bretting and Wilrlechner, 1995). There is a paucity of isozyme diversity studies in red clover. Hagen and Hamrick (1998) measured high levels of genetic diversity within nine naturalized red clover populations and low levels of genetic divergence among the red clover populations. Hickey et al. (1991) compared genetic variation in naturalized red clover and alsike clover (Trifolium hybridum L.) populations with native running buffalo clover (T. stoloniferum Muhlenberg ex Eaton) and buffalo clover (T. reflexum L.). Their results indicated that the red clover populations had 1.93 alleles per locus, 53.3% polymorphic loci, and the amount of among-population genetic variation was much smaller than the within-population variation. The objective of this study was to assess genetic diversity in North American red clover cultivars on the basis of isozyme data.
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MATERIALS AND METHODS
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Seeds of 34 red clover cultivars (Table 1) were obtained from the USDA-ARS Plant Introduction Stations at Pullman (W-6), WA, and Griffin (S-9), GA. Eighteen plants of each accession were grown in pots filled with potting soil in a greenhouse.
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Table 1. Summary of isozyme variation within 34 cultivars of red clover and at the species level. Parameters calculated on the basis of 13 loci were percentage of polymorphic loci (P), mean number of alleles per polymorphic locus (AP), effective number of alleles per locus (Ae), observed heterozygosity (Ho), and expected heterozygosity (He).
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Isozyme assays were conducted on young leaf tissue. The youngest fully expanded leaf (about 230 mg) was homogenized with 90 µL extraction buffer [sucrose 16.7% (w/v) and sodium ascorbate 8.3% (w/v) in 50 mM Tris-HCl, pH 7.4] at -20°C. Crude extracts were centrifuged for 5 min at 8160 x g. Supernatant (5.5 µL/well) was loaded onto precast, agarose isoelectric focusing (IEF) gels (Isolab, Akron, OH). Gels with pH gradients 3 to 5 (50%), 3 to 7 (25%), and 3 to 10 (25%) were used for esterase (EST; E.C. 3.1.1.-), ß-glucosidase (GLU; E.C. 3.2.1.21), phosphoglucomutase (PGM; E.C. 5.4.2.2), and peroxidase (PRX; E.C. 1.11.1.7); gels with pH gradients 3 to 7 (50%) and 4 to 5 (50%) were used for diaphorase (DIA; E.C. 1.6.99.1) and phosphoglucoisomerase (PGI; E.C. 5.3.1.9); and gels with pH gradients 3 to 10 (75%) and 3 to 7 (25%) were used for superoxide dismutase (SOD; E.C. 1.15.1.1). Staining procedures were those of Wendel and Weeden (1989) with minor concentration, pH, and ingredient modifications; namely, ACP, 0.1 M sodium acetate, and Na-1-napthyl phosphate; DIA, 0.2 M Tris-HCl and 3 mg 2,6-dichloroindolphenol; EST, 0.6 M Na phosphate buffer pH 6.1, 50 mg 
-naphthyl acetate and omitted ß-naphthyl acetate; GLU, 500 mg PVP-40 decreased, 25 mg 6-bromo-2-naphthyl-ß-D-glucoside, 2.5 mL N,N'-dimethyl formamide; MDH, 0.2 M Tris-HCl, 20 mg NAD and 3 mg PMS; ME, 15 mg NADP, 3 mg PMS, 100 mg malic acid; 6PGD, 10 mg NADP, 3 mg PMS increased; SOD, 50 mL 0.5 M Tris-HCl, pH 8.5, 15 mg NAD+, 15 mg MTT, 1 mg PMS; PRX, 0.1 M Na-acetate buffer, 0.4 mL hydrogen peroxide, 40 mg 3-amino-9-ethylcarbazole. The IEF gels were run at constant power and voltage limited to 1500. The first run was 60 min at 40 W and the second was 20 min at 60 W.
Data Analysis
Population genetic parameters calculated for the species as a whole and on a cultivar basis (indicated by subscripts s or cv, respectively) were percentage of polymorphic loci (P), mean number of alleles per polymorphic locus (AP), effective number of alleles per locus (Ae, observed heterozygosity (Ho), and genetic diversity (He) which is the expected heterozygosity when populations are allowed to mate randomly (Weir, 1989; Hagen and Hamrick, 1998). Percent polymorphic loci at the cultivar level (PCV) was calculated by dividing the number of loci polymorphic within a cultivar by the total number of loci analyzed. Percent polymorphic loci, at the species level (PS), was calculated by dividing the number of loci polymorphic in at least one cultivar by the total number of loci analyzed.
The mean number of alleles per polymorphic locus at the cultivar level (APCV) was determined by summing all the alleles detected at polymorphic loci in a cultivar and dividing by the number of polymorphic loci. The mean number of alleles per polymorphic locus at the species level (APS) was determined by summing all the alleles detected at polymorphic loci and dividing by the total number of polymorphic loci. The effective number of alleles at the cultivar level (AeCV) was calculated for each locus by 1/
f2i (Hartl and Clark 1997) where fi is the frequency of the ith allele in each cultivar. At the species level, the effective number of alleles was calculated for each locus using the mean frequency of the ith allele (fi) pooled across all cultivars. These values were then averaged across loci to obtain AeS.
Genetic diversity was calculated for each locus (including monomorphic and polymorphic loci) by He = 1 - f2i. For species values (HeS) fi is the mean frequency of the ith allele (fi) pooled across all cultivars. For cultivar values (HeCV) fi is the frequency of the ith allele in each cultivar. Genetic parameters at the cultivar level represent cultivar means, whereas at the species level, parameters represent overall genetic diversity within the species. Mean cultivar parameters were obtained by averaging individual cultivar's P, AP, Ae, Ho, and He.
The population genetics software package POPGENE (Yeh and Boyle, 1997) was used to calculate P, Ae, Ho, and He and test genetic population parameters. We calculated AP values on the basis of A (mean number of alleles per locus). Expected heterozygosity was estimated by Nei's (1978) unbiased heterozygosity procedure. Deviations from Hardy-Weinberg equilibrium at each locus in each population and heterogeneity in allele frequencies among cultivars were tested by chi-square (
2). Smouse's multilocus test (Smouse et al., 1983) for single populations was used to test each cultivar for Hardy-Weinberg disequilibrium.
Population divergence was examined by Nei's genetic identity and distance parameters (Nei, 1978) for all pairs of cultivars. Nei's genetic identity between populations X and Y with frequencies xi and yi of the ith allele at a particular locus is
where Jxy is the arithmetic mean across loci of xiyi and n is sample size. Genetic Distance (D) is D = -ln (I). Dendrograms based on Nei's genetic distances were constructed via the unweighed pair group method with arithmetic averages (UPGMA).
Data from cultivars grouped based on the dendrogram of Nei's genetic distance were evaluated for within- and among-cultivars isozyme diversity components. Total genetic diversity (HT) estimated with Nei's genetic diversity statistics (Nei, 1978) was partitioned into within-cultivar (HeCV, mean diversity within cultivars) and among-cultivar components (DsCV), i.e., HT = HeCV + DsCV. To estimate the proportion of variation distributed among cultivars, the among-cultivar component was used to calculate Nei's GST (coefficient of gene differentiation) values averaged over all polymorphic loci where GST = DsCV/HT = (HT - HeCV)/HT. The proportion of total genetic diversity found within cultivars was calculated as GwCV = 1 - GST.
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RESULTS AND DISCUSSION
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A total of 13 isozyme loci with 28 alleles was detected by means of seven enzyme systems in the 34 red clover cultivars. Eleven of the 13 loci (84.6%) were polymorphic in at least one cultivar. The two phosphoglucomutase loci were monomorphic. The percent polymorphic loci within cultivars ranged from 61.54% in cultivars Morred, Persist, and Redland II to 84.62% in cultivars Arlington, Ram, and Red Baron, with an overall mean of PCV = 73.98% (Table 1). At the species level, Ps was 84.62%. The number of alleles per polymorphic locus was 2.55 at the species level and the average within cultivars (APCV) was 2.71. The APCV values ranged from 2.45 for Arlington and Red Baron to 3.13 for Redland II. The effective number of alleles per locus was 1.65 at the species level (AeS) and the average within cultivars (AeCV) was 1.59. The AeCV values ranged from 1.48 for Marathon and 1.70 to Lakeland. Genetic diversity was 0.292 at the species level (HeS) and the mean value within cultivars (HeCV) was 0.285. The HeCV values ranged from 0.248 to Ruby for 0.316 for Cherokee and Lakeland (Table 1). These values are similar to those measured in naturalized populations of red clover (Hagen and Hamrick, 1998) and larger than those reported for many outcrossing cultivated species (Hamrick and Godt, 1997). The same parameters calculated using many outcrossing crop species were Ps = 64.3% and Hes = 0.205 at the species level, and PP = 37.3%, and HeP = 0.127 at the within-populations level (Hamrick and Godt, 1997). These results confirm the report of Hagen and Hamrick (1998) indicating that red clover genetic diversity is high. This high level of genetic diversity as measured by the expected heterozygosity may be due to the obligated outcrossing mode of reproduction of red clover. It also may be in part caused by a large number of recessive alleles concealed in the heterozygotes. This is evidenced by the severe inbreeding depression that can be experienced in red clover progenies derived from crossing closely related lines or sister lines (Taylor and Quesenberry, 1996, p. 141). Another factor to consider is that red clover is a widely distributed species and widespread species maintain more allozyme variation than endemic species (Hamrick and Godt, 1989).
Hardy-Weinberg expectations for genotypic frequencies were not observed in 144 of the 442 chi-square tests. Considering that 22.1 significant deviations at the P = 0.05 significance level would be expected by chance alone, we conclude that the 34 cultivars as a whole were not in Hardy-Weinberg equilibrium. Mean observed heterozygosity within cultivars, Ho = 0.321, was slightly higher than expectation He = 0.285 supporting again that the populations were in Hardy-Weinberg disequilibrium (Table 1). Smouse's multilocus test for single populations detected that 14 of 34 cultivars did not deviate from random union of gametes, thus, these cultivars should be in Hardy-Weinberg equilibrium. The cultivars were Atlas, Cherokee, Dollard, Florex, Kenstar, Lakeland, Letcher, Morred, Ram, Redland II, Redon, Red Star, Ruby, and Wildcat. These cultivars would be more likely to maintain genotypic and allelic frequencies over generations; i.e., these cultivars will tend to maintain genetic integrity thus they will be less likely to experience loss of variation caused by evolution during cultivation. Allele frequencies were significantly different among populations for seven of the 11 polymorphic loci (P < 0.001).
Genetic identity among pairs of cultivars was relatively high, ranging from 0.955 to 1.000, with a mean of 0.985. The dendrogram of UPGMA clustering of the 34 red clover cultivars based on Nei's genetic distance indicated that cultivar pairs to be identical to each other were Lakeland and Midland, Chesapeake and Red Baron, Cinnamon and Renegade, and Redland and Red Star (Fig. 1). The rest of the cultivars appeared distinct on the dendrogram despite the high genetic identity values. The dendrogram of genetic distance also indicated that cultivars Morred and Redon were unique and the rest of the cultivars could be divided into four groups (Fig. 1). Population genetic parameters for the four groups of cultivars were similar (Table 2). Allele frequencies were similar among cultivars for the 11 polymorphic loci (P < 0.001) in each of the groups, except for one of the PGM loci in group 2. Variability in each group was located mostly within cultivars (98.4–99.7%). Among-cultivar differentiation (GST) was negligible ranging from 0.3 to 1.6% of the total variability of the respective group. Hence, isozyme variability in the 34 cultivars studied in a breeding program could be represented by a few cultivars of each of the four groups and the cultivars Morred and Redon. Grouping of the cultivars by Nei's genetic distance on the basis of isozymes did not correspond to the origin and/or breeding pedigree of the cultivars as shown by Rumbaugh (1991) and Taylor and Quesenberry (1996). For example, Tensas, a southern cultivar of unknown pedigree, was in group one together with mostly northen cultivars and Cherokee, also a southern cultivar with about 14% Tensas (Quesenberry et al., 1993), was in group two. A more extreme example is Morred, a selection from Arlington (Moutray et al., 1985), that was found to be unique and distant from Arlington which was in group one.
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Table 2. Mean gene diversity statistics for four groups of red clover cultivars. Parameters calculated for each group were polymorphic loci (P), mean number of alleles per polymorphic locus (AP), effective number of alleles per locus (Ae), observed heterozygosity (Ho), expected heterozygosity (He), and Nei's coefficient of gene differentiation (GST).
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In summary, red clover is a highly variable species at the enzyme level. Most of the genetic diversity in red clover cultivars released in North America is within the cultivars. Grouping the cultivars using genetic distance indicated that the isozyme variability in the 34 cultivars studied could be represented by a few cultivars of each of four groups plus the cultivars Morred and Redon.
Received for publication October 20, 2000.
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ABSTRACT
INTRODUCTION
