Published online 24 February 2006
Published in Crop Sci 46:758-762 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
PLANT GENETIC RESOURCES
Genetic Diversity in the Core Subset of the U.S. Red Clover Germplasm
J. A. Mosjidis* and
K. A. Klingler
Dep. of Agronomy and Soils, Alabama Agric. Experiment Station, Auburn Univ., Auburn, AL 36849-5412
* Corresponding author (mosjija{at}auburn.edu)
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ABSTRACT
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Biochemical diversity present in accessions of the red clover (Trifolium pratense L.) U.S. core subset is unknown. Its isozyme characterization would facilitate the study of relationship among accessions and assist in the identification of unique accessions and duplicates and in the detection of genetic differences among accessions. This study was undertaken to assess genetic diversity in the core subset using the isozymes esterase, ß-glucosidase, phosphoglucomutase, peroxidase, diaphorase, phosphoglucoisomerase, and superoxide dismutase. Ten isozyme loci with 30 alleles were detected. All loci were polymorphic in at least one accession. Percentage polymorphic loci within populations ranged from 50 to 100% with an average of 77.5%. At the species level, the number of alleles per polymorphic locus was 3.00, and the effective number of alleles per locus was 1.81. Within accession averages were 2.75 and 1.66, respectively. The core subset had a large amount of genetic diversity. Most accessions were different from each other except PI 207972 and PI 251564, PI 217507, and PI 229799 that were alike. One accession, PI 235867, was not a red clover. Most accessions (56) were in Hardy-Weinberg equilibrium, and thus they are more likely to maintain genotypic and allelic frequencies when increased. Genetic diversity at the species level was high and there was nearly twice as much variability among the wild populations as among the cultivars or landraces included in the core subset.
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INTRODUCTION
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RED CLOVER is one of the most important forage species in the USA and the world. It is grown alone or in mixture with grasses in a wide range of soil and environmental conditions. The adaptability of red clover and its capacity to fix nitrogen in association with Rhizobium makes it useful for hay, silage, pasture, and soil improvement (Smith et al., 1985).
Red clover originated in Eurasia where wild-type populations are found in the Caucasus Mountains. In its natural habitat it is found in meadows, forest margins, and field borders (Gillett and Taylor, 2001). The U.S. National Germplasm System (NPGS) has a collection of about 1300 accessions of this species. A core subset was developed for red clover using morphological and physiological descriptors (Kouame and Quesenberry, 1993). This core subset is expected to represent the different patterns of phenotypic diversity present in the collection. However, the biochemical diversity present in the core subset is unknown. Characterization of the NPGS red clover core subset using isozymes would facilitate the study of relationship among accessions and assist in the identification of unique accessions. This information will help in the detection of genetic differences among accessions and in the identification of duplicate accessions (Bretting and Wilrlechner, 1995). Furthermore, information on genetic diversity serves as a basis to design strategies that capture and maintain a high proportion of the variability of the species (Hamrick and Godt, 1997).
Several diversity studies have been conducted in red clover using isozymes in recent years. 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. Yu et al. (2001) conducted a more extensive study on diversity of North American red clover cultivars. Percentage polymorphic loci within cultivars ranged from 61.5 to 84.6% with an overall mean of 74.0%. 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 cultivars. Most of the genetic diversity (98.4–99.7%) was distributed within the cultivars. Mosjidis et al. (2004) assessed genetic diversity in 15 wild red clover populations from Russia. They reported that 90% of the loci were polymorphic in at least one population. Percent polymorphic loci within populations ranged between 50 and 90%. At the species level, the number of alleles per polymorphic locus was 2.89 and effective number of alleles per locus was 1.84. Within-population averages were 2.75 and 1.70, respectively. Genetic diversity was 0.353 at the species level and the mean value for the populations was 0.323. The objective of this study was to assess genetic diversity in the core subset (Available online at http://www.ars-grin.gov/cgi-bin/npgs/html/eval.pl?424001) (verified 28 November 2005) of the USA red clover germplasm based on isozyme data.
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MATERIAL AND METHODS
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Seeds of 81 red clover accessions (Table 1) were obtained from the USDA-ARS Plant Introduction Stations at Pullman (W-6), Washington. 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 the core subset of T. pratense and at the species level. Parameters calculated on the basis of 10 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 from each individual plant. 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%) 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%) 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 (Yu et al., 2001). 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.
Population genetic parameters calculated for the species as a whole and on a population basis (indicated by subscripts s or p, 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 expected heterozygosity (He) under random mating also called genetic diversity (Weir, 1989).
Percentage polymorphic loci at the population level (PP) was calculated by dividing the number of loci polymorphic within a population 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 population by the total number of loci analyzed.
The mean number of alleles per polymorphic locus at the population level (APP) was determined by summing all the alleles detected at polymorphic loci in a population 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 population level (AeP) was calculated for each locus by 1/
fi2 (Hartl and Clark 1997) where fi is the frequency of the ith allele in each population. At the species level (AeS), the effective number of alleles was calculated for each locus using the mean frequency of the ith allele (fi) pooled across all populations. 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 – fi2. For species values (HeS) fi is the mean frequency of the ith allele (fi) pooled across all populations. For population values (HeP) fi is the frequency of the ith allele in each population. Genetic parameters at the population level represent population means, whereas at the species level, parameters represent overall genetic diversity within the species. Mean population parameters were obtained by averaging individual population'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. Smouse's multilocus test (Smouse et al., 1983) for single populations was used to test each population for Hardy-Weinberg disequilibrium.
Population divergence was examined by Nei's genetic identity (Nei, 1978) and by modified Rogers' distance parameters (Wright, 1978) for all pairs of populations. Dendrograms based on modified Rogers' distance were constructed via the unweighed pair group method with arithmetic averages (UPGMA) using the program NTSYS version 2.1 (Rohlf, 2000). The same program was employed to calculate the cophenetic value matrix from the tree matrix and to perform the Mantel test (Mantel, 1967) to determine the correspondence between the two matrices. Furthermore, the NTSYSpc program was also used to perform a principal components analysis (PCA) on the same data. The program PHYLIP- Phylogeny Inference Package (Version 3.6a) (Felsenstein, 1993) was used to obtain 1000 data sets by resampling the data using bootstrap. A consensus tree resulting of those 1000 data sets was built using the majority rule, i.e., the tree consisted of all accessions that occurred in more than 50% of the trees was included.
Populations in the core subset were classified as cultivars, landraces and wild. Genetic diversity statistics P, AP, Ae, Ho, He and GST were calculated for each of these groups. Nei's GST (coefficient of gene differentiation) values for each group were calculated according to the formula GST = DsP /HT = (HT – 
eP)/HT, where HT is total genetic diversity for the group estimated with Nei's genetic diversity statistics (Nei, 1978) and 
eP is mean diversity within populations.
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RESULTS AND DISCUSSION
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The core subset included 81 accessions. Initial isozyme analysis of accession PI 235867 indicated that it was much different from the rest of the accessions in the core subset suggesting that PI 235867 could be a different species. Field observations by N. Taylor and R. Smith (personal communication) also cast doubts on this accession. N. Taylor (personal communication) later classified PI 235867 as a Trifolium alexandrinum L. Consequently, PI 235867 was excluded from further analysis.
A total of 10 isozyme loci with 30 alleles were detected by seven enzyme systems in the 80 red clover accessions in the core subset. All loci (100%) were polymorphic in at least one accession. The percentage polymorphic loci within populations ranged from 50% in PI 234836, PI 250899, and PI 315533 to 100% in PI 171870, PI 230229, PI 314487, and PI 449326 with an average of 77.5% for all the accessions. Percentage polymorphic loci at the species level (PP) was 100%. The number of alleles per polymorphic locus was 3.00 at the species level and the average within accessions was 2.75. They ranged from 2.20 in PI 314487 to 3.80 in PI 234836. The effective number of alleles per locus (Ae) was 1.81 at the species level and the average within accessions was 1.66. The Ae values ranged from 1.29 in PI 207520 to 1.82 in PI 171870. These results agree with previous reports indicating that genetic diversity in red clover is high. Yu et al. (2001) measured average values for cultivated red clovers of P = 74, AP = 2.71, Ae = 1.59, and He = 0.285 and Mosjidis et al. (2004) reported that wild populations had P = 74, AP = 2.75, Ae = 1.70, and He = 0.323. The reported average values for naturalized red clovers were P = 68, AP = 2.52, Ae = 1.45, and He = 0.250 (Hagen and Hamrick 1998), whereas the mean values for the accessions in the core subset were higher. A comparison of the average values for isozyme variation of many plant species that are cross-pollinated by animals (P = 35.9, AP = 1.54, Ae = 1.17, and He = 0.124) reported by Hamrick and Godt (1989) shows that the mean values for the core subset populations were much higher.
Genetic diversity He was 0.342 at the species level and the mean value for the accessions was 0.316. The He value ranged from 0.178 in PI 207520 to 0.400 in PI 314487. Mean observed heterozygosity within accessions, Ho = 0.335, was slightly higher than expectation He = 0.316, suggesting that the populations as a whole were in Hardy-Weinberg disequilibrium (Table 1). However, Smouse's multilocus test for single populations detected that 56 of 80 accessions did not deviate from random union of gametes; thus, these populations should be in Hardy-Weinberg equilibrium. These accessions will more likely maintain genotypic and allelic frequencies over generations. The 24 accessions in Hardy-Weinberg disequilibrium were PI 120105, PI 171870, PI 179146, PI 184960, PI 217507, PI 228365, PI 230229, PI 237705, PI 251564, PI 255894, PI 266047, PI 271627, PI 293591, PI 294797, PI 306185, PI 307948, PI 311492, PI 315538, PI 318887, PI 345673, PI 376880, PI 401469, PI 419565, and PI 440737. Allele frequencies were significantly different among populations for nine of the 10 polymorphic loci (P < 0.001). The exception was Prx-2.
Mean observed heterozygosity tended to be lower than expected heterozygosity in 22 populations, suggesting that there is a deficiency of heterozygotes in those populations, as would be the case when there has been inbreeding. The populations with some degree of inbreeding were G 21245, PI 220856, PI 226952, PI 234836, PI 235870, PI 245141, PI 286222, PI 294481, PI 294797, PI 295355, PI 304842, PI 306677, PI 311492, PI 315522, PI 315533, PI 318887, PI 318888, PI 371959, PI 376880, PI 401469, PI 419565, and PI 440737.
The range in Nei's genetic identity values between pairs of accessions was wide. The highest value was 1.000 for accessions PI 179146 and PI 255894, PI 207972 and PI 251564, PI 217507 and PI 229799, PI 234448 and PI 318888, PI 235870 and PI 345673, PI 251564 and PI 384058, PI 253583 and PI 310465, PI 293591 and PI 314487, PI 293591 and PI 318888, and PI 294797 and PI 315538 and the lowest values were for PI 120105 and PI 251564 (0.7943), PI 207520 with PI 310465 (0.8154), and for PI 311492 with PI 315533 (0.8160). The average genetic identity was 0.9548.
The cophenetic correlation, i.e., the correlation between the cophenetic matrix and the matrix based on modified Rogers' distance, was r = 0.80, indicating a good fit to the dendrogram derived from the cluster analysis. The dendrogram of genetic distance indicated that although most of the accessions were rather unique (except PI 207972 and PI 251564 and PI 217507 and PI 229799 that were alike), there were seven accessions that clustered separate from the other 73.
Those were PI 120105, PI 207520, PI 250899, PI 293591, PI 314487, PI 315534, and PI 376880. However, these observations were not supported by bootstrap values, which were below 50%, i.e., isozyme variability did not have the power to discriminate the accessions into major related groups. The exceptions were a few branches that included two accessions each, namely, PI 371959 and PI 235870, PI 229799 and PI 217507, PI 418889 and PI 187008, PI 315522 and PI 294481, PI 419295 and PI 266047, and PI 253583 and PI 311492 which had bootstrap values between 55 and 84%. Principal component analysis indicated that 92.3% of the variation was accounted by the first two dimensions. Plotting of those two dimensions indicated that there were three groups. One was constituted by PI 120105, PI 207520, PI 250899, and PI 314487 that were apart from the rest of the populations, which agrees in part with the genetic distance dendrogram. These four accessions had first principal component scores smaller than 0.87 and positive second principal component scores, whereas the other accessions had first principal component scores larger than 0.87 and positive or negative second principal component scores. A second set of accessions that grouped apart from the other accessions were PI 315534, PI 286222, and PI 306677, which agrees in part with the genetic distance dendrogram. They had first principal component scores larger than 0.87 and second principal component scores smaller than –0.26 (Fig. 1
). The third group was the remainder 73 accessions that clustered together.
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Fig. 1. Two-dimensional representation of genetic relationships among 80 red clover accessions from the U.S. National Germplasm System core subset determined on the basis of principal component analysis of isozyme data.
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The NPGS classified the red clover populations on the basis of improvement status into cultivars, landraces, and wild populations when data were available. Thus, 30 populations in the core subset were classified as cultivars, 11 as landraces and 13 as wild. Genetic diversity statistics were calculated for each of these groups (Table 2). Values for P, AP, Ae, Ho, and He were similar, whereas GST values were remarkably different. Results indicated that 6.7% of the genetic diversity in cultivars resided among populations, 5.7% in landraces, and 10.6% in wild populations. These results point out that there was nearly twice as much variability among the wild populations as among cultivars or landraces. Furthermore, the GST values indicate that the level of among-populations variability for each group was much higher than those reported by Yu et al. (2001) for North American red clover cultivars. This indicates that each group is a good source of variability; consequently, all groups and their respective populations need to be represented in the core subset. It would be up to the user to decide which group (cultivars, landraces, or wild populations) to utilize.
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Table 2. Mean gene diversity statistics for the cultivars, landraces and wild populations that make up the red clover core subset. Parameters calculated for each group were percentage 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, the core subset had a large amount of genetic diversity as measured by isozymes. Most accessions were different from each other except PI 207972 and PI 251564 and PI 217507 and PI 229799 that were alike. One accession, PI 235867, was not a red clover. Most of the accessions (56) were in Hardy-Weinberg equilibrium; thus, they are more likely to maintain genotypic and allelic frequencies when increased. Genetic diversity at the species level was high and there was nearly twice as much variability among the wild populations as among the cultivars or landraces included in the core subset. Nevertheless, there was a large amount of among-populations variability in each of the above mentioned groups.
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NOTES
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This research was partially funded by the Clover and Special Purpose Legume Crop Germplasm Committee.
Received for publication May 20, 2005.
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REFERENCES
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- Bretting, P.K., and M.P. Wilrlechner. 1995. Genetic markers and plant genetic resource management. Plant Breed. Rev. 13:11–86.
- Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) version 3.63. Distributed by the author. Department of Genetics, University of Washington, Seattle. [Online]. Available at http://evolution.genetics.washington.edu/phylip/phylipweb.html (verified 28 November 2005).
- Gillett, J.M., and N.L. Taylor. 2001. The world of clovers CD-ROM. Iowa State Univ. Press, Ames, IA.
- Hagen, M.J., and J.L. Hamrick. 1998. Genetic variation and population genetic structure in Trifolium pratense. J. Hered. 89:178–181.[Abstract/Free Full Text]
- Hamrick, J.L., and M.J.W. Godt. 1989. Allozyme diversity in plant species. p. 43–63. In A.H.D. Brown et al. (ed.) Plant population genetics, breeding, and genetic resources. Sinauer Associates, Sunderland, MA.
- Hamrick, J.L., and M.J.W. Godt. 1997. Allozyme diversity in cultivated crops. Crop Sci. 37:26–30.[Abstract/Free Full Text]
- Hartl, D.L., and A.G. Clark. 1997. Principles of population genetics. Sinauer, Sunderland, MA.
- Kouame, C.N., and K.H. Quesenberry. 1993. Cluster analysis of a world collection of red clover germplasm. Genet. Resour. Crop Evol. 40:39–47.[CrossRef]
- Mantel, N.A. 1967. The detection of disease clustering and a generalized regression approach. Cancer Res. 27:209–220.[Abstract/Free Full Text]
- Mosjidis, J.A., S.L. Greene, K.A. Klingler, and A. Afonin. 2004. Isozyme diversity in wild red clover populations from the Caucasus. Crop Sci. 44:665–670.[Abstract/Free Full Text]
- Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590.[Abstract/Free Full Text]
- Rohlf, F.J. 2000. NTSYS: Numerical taxonomy and multivariate analysis system, v. 2.1. Exeter Software, Setauket, NY.
- Smith, R.R., N.L. Taylor, and S.R. Bowley. 1985. Red clover. p. 457–470. In N.L. Taylor (ed.) Clover science and technology. Agron. Monogr. 25. ASA, CSSA, and SSSA, Madison, WI.
- Smouse, P.E., J.V. Neel, and W. Liu. 1983. Multiple-locus departures from panmictic equilibrium within and between village gene pools of Amerindian tribes at different stages of agglomeration. Genetics 104:133–153.[Abstract/Free Full Text]
- Weir, B.S. 1989. Sampling properties of gene diversity. p. 23–42. In A.H.D. Brown et al. (ed.) Plant population genetics, breeding, and genetic resources. Sinauer Associates, Sunderland, MA.
- Wendel, J.F., and N.F. Weeden. 1989. Visualization and interpretation of plant isozymes. In D.E. Soltis and P.S. Soltis (ed.) Isozymes in plant biology. Dioscorides Press, Portland, OR.
- Wright, S. 1978. Evolution and genetics of populations. Volume 4: Variability within and among natural populations. University of Chicago Press, Chicago.
- Yeh, F.C., and T.J.B. Boyle. 1997. Population genetic analysis of co-dominant and dominant markers and quantitative traits. Bel. J. Bot. 129:157.
- Yu, J., J.A. Mosjidis, K.A. Klingler, and F.M. Woods. 2001. Isozyme diversity in North American cultivated red clover. Crop Sci. 41:1625–1628.[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
