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PLANT GENETIC RESOURCES

Genetic Diversity in Natural Populations and Corresponding Seed Collections of Little Bluestem as Revealed by AFLP Markers

^a Plant Gene Resources of Canada, Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada
^b Canadian Grain Commission, 600-303 Main Street, Winnipeg, MB R3C 3G8, Canada

^* Corresponding author (fuy{at}agr.gc.ca)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Little bluestem [Schizachyrium scoparium (Michx.) Nash] is one of the most widespread native grasses in North America. Little is known about the genetic diversity of this species in natural populations and in seed collections. The amplified fragment length polymorphism (AFLP) technique was applied to assess the comparative genetic diversity of six natural populations of little bluestem in Manitoba and Saskatchewan and their corresponding seed collections. Five AFLP primer pairs were employed to screen a total of 180 samples representing about 15 tillers per population and 15 seeds per collection, and 158 polymorphic AFLP bands were scored for each sample. Analyses of these scored bands revealed that >91% of the total AFLP variation was present within the natural populations and within the seed collections. The among-population and among-collection variation components, although relatively small (7–9%), were statistically significant from zero. Comparisons of AFLP profiles between the seed and tiller samples revealed the seed samples had fewer polymorphic bands, higher average band frequencies, and more bands with extremely high or low frequencies. A significant association of AFLP variation with geographical origin was detected in the seed, but not the tiller samples. These results indicate collecting seeds may not be as effective as collecting tillers in sampling genetic diversity from natural populations for the improvement of little bluestem germplasm for rangeland seeding.

Abbreviations: AFLP, amplified fragment length polymorphism • AMOVA, analysis of molecular variance • RAPD, random amplified polymorphic DNA

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
DURING THE LAST 30 yr, efforts to develop native grass cultivars with improved seed production have increased in the USA and Canada (Crowle, 1970; Smoliak and Johnson, 1980, 1983; Jones et al., 2002). Many improved germplasm lines have been released and made commercially available for rangeland restoration and large-scale revegetation (May et al., 1997; Englert et al., 2002). These improved plant materials, however, may not maintain a sufficiently high level of genetic diversity necessary for adaptation in dynamic nonlocal environments (Knapp and Rice, 1996; Roundy, 1999; Larson et al., 2000). Such concern has its roots not only in adaptation, but also in genetic diversity. First, information on genetic diversity of the released germplasm is not available (Huff, 1997; Fu et al., 2004), as diversity assessments of the released seeds have rarely been performed, particularly in relation to adaptation to different environments. Second, little is known about genetic diversity of the germplasm collected and used for cultivar development (Phan et al., 2003), as the effectiveness of sampling germplasm from natural populations has rarely been questioned. Third, general knowledge about genetic diversity of many grass species native to North America is largely lacking (Huff et al., 1998; Larson et al., 2001a), as few molecular diversity studies on these species have been made. Thus, informative diversity studies on native grasses with molecular techniques are warranted.

Little bluestem is a native, warm-season, highly outcrossing, tetraploid, perennial bunchgrass (Archer and Bunch, 1953; Gould, 1956) that inhabits a wide ecological range in North America where it is used for reclamation and ground cover (Hitchcock, 1950). While breeding efforts to improve little bluestem seed production have been made in North America (Phan and Smith, 2000) and more than 10 germplasms have been released (Englert et al., 2002), challenges remain in improving seed production while simultaneously maintaining genetic diversity for adaptation to nonlocal environments (Smith and Phan, 1999; Smith and Whalley, 2002). Little is known about the molecular diversity of this grass species, even with the records of extensive phenological and morphological variation observed throughout the species' range (McMillan, 1964, 1965). Huff et al. (1998) made the first attempt to assess the genetic diversity of four little bluestem populations in New Jersey and Oklahoma using random amplified polymorphic DNA (RAPD) markers, and found most of the RAPD variation (95%) resided within the populations with no pronounced groupings of the populations with respect to fertility or biome. While these findings are informative to germplasm sampling for improvement and conservation, it remains unclear how general these patterns of genetic variation are with respect to little bluestem in the northern fringe of its distribution. Also, the RAPD study was based on collected tillers, not the sampled seeds that could show differentiation from the parental populations. Diversity assessments of little bluestem germplasm collected in western Canada would facilitate the development of cultivars specifically adapted to this region (Smith and Phan, 1999; Phan and Smith, 2000).

The objective of this study was to assess the comparative genetic diversity of little bluestem germplasm of both tillers and seeds sampled from six sites in Manitoba and Saskatchewan using AFLP markers. The AFLP method (Vos et al., 1995) is a robust, highly informative DNA fingerprinting technique and has been applied to detect genetic variation in bluebunch wheatgrass [Pseudoroegneria spicata (Pursh) A. Love] (Larson et al., 2000), bluegrasses (Poa spp.) (Larson et al., 2001b), smooth bromegrass (Bromus inermis Leyss.), meadow bromegrass (Bromus riparius Rehmann) (Ferdinandez and Coulman, 2002), crested wheatgrass (Agropyron spp.) (Mellish et al., 2002), and blue grama [Bouteloua gracilis (Willd. ex Kunth) Lag. ex Griffiths] (Fu et al., 2004).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Plant Materials
Little bluestem germplasm used in this study was collected on 29 to 31 July 2002, by staff of Native Plant Solutions of Ducks Unlimited Canada from six sites (Brandon, MB; Carberry, MB; Glenboro, MB; Narcisse, MB; Winnipeg, MB; and Saskatoon, SK) (Table 1). At each site, 30 single plants at least 5 m apart were randomly selected and the tillers of these single plants were collected, representing the tiller sampling for the natural population. On the same day, another 30 single plants, also at least 5 m apart, were randomly selected. The seeds of these single plants were collected separately for the seed collection.

DNA Extraction and AFLP Analysis
Tissue fragments from the collected leaf tissue were randomly sampled into 96-well plates for DNA extraction. Genomic DNA was extracted from 180 tiller and seed samples using the DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacturer's directions. Extracted DNA was quantified by fluorometry using Hoechst 33258 stain (Sigma Chemical Co., St. Louis, MO), followed by dilution to 25 ng µL^–1 for AFLP analysis. The AFLP analysis was performed using the AFLP Analysis System 1 (Life Technologies, Burlington, ON, Canada) following the protocol described by Vos et al. (1995). The protocol included four main steps: (i) restriction digestion of 250 ng genomic DNA with EcoRI and MseI restriction enzymes and ligation of adapters to the restriction fragments to create primary templates; (ii) preamplification of the primary templates with AFLP primers with an additional single nucleotide at the 3' end; (iii) selective amplification of the preamplified fragments with MseI and [³³P]-labeled EcoRI primers, both having three selective nucleotides at the 3' end; and (iv) separation of the amplification products on a 5% denaturing polyacrylamide gel for 2.5 h at 90 W. After electrophoresis, the gel was transferred to Whatman 3-mm paper, dried on a gel dryer for 2 h at 80°C, and exposed to Kodak BIOMAX film at –80°C for 1 to 7 d, depending on the signal intensity. The sizes of amplification products were determined by comparison with a 330-30 bp AFLP DNA ladder (Promega, Madison, WI). The polymerase chain reaction profile for step (iii) was performed in a PTC-200 DNA Engine thermocycler (MJ Research, Watertown, MA) using the following amplification protocol: a denaturing step of 30 s at 94°C, followed by an annealing step of 30 s at 65°C, and an extension step of 60 s at 72°C. Next, 12 cycles followed a touchdown format decreasing the annealing temperature by 0.7°C after each cycle until a final annealing temperature of 56°C was attained. The last cycle was repeated 23 times and was performed using the profile: 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s. Ten EcoRI:MseI primer pairs were initially screened on six tiller samples and five most informative ones (Table 2) were selected for this AFLP analysis.

For each sample type, analysis of molecular variance (AMOVA; Excoffier et al., 1992) was performed using Arlequin version 2.001 (Schneider et al., 2002) to assess AFLP variations across the six populations (or collections). This analysis not only allows the partition of the total AFLP variation into within- and among- population (or collection) components, but also provides a measure of interpopulation distances as the proportion of the total AFLP variation residing between any two populations or collections. Significance of resulting variance components and interpopulation distances was tested with 5000 permutations. To assess the differences in AFLP variation between the two sample types from a given population or collection, AMOVA was also performed. This population- (or collection-) specific AMOVA analysis not only allows the partition of the total AFLP variation into within- and among- sample components from single populations or collections, but also provides tests on the significance of the difference between the two types of sampling with 5000 permutations.

To assess the genetic relationships of the individual samples assayed for each sample type, the interpopulation distance matrices were analyzed using NTSYS-pc 2.01 (Rohlf, 1997) and clustered with the algorithm of Unweighted Pair-Group Methods Using Arithmetic Averages. Associations of these distance matrices with the corresponding geographical distances of their collection sites for each type of sampling were also examined with the MXCOMP program of NTSYS-pc with 5000 permutations.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
AFLP Variation
Five AFLP primer pairs amplified a total of 854 AFLP bands for the tiller samples and 653 bands for the seed samples (Table 2). The number of observable bands per primer pair ranged from 136 to 195 for the tiller samples and from 114 to 162 for the seed samples. None of the observable bands were monomorphic in the tiller samples, and only three of them were monomorphic in the seed samples, indicating the presence of large AFLP variation in this native grass species. Such a high level of polymorphism was expected for an outcrossing grass species like little bluestem, and this is consistent with the RAPD variation previously reported for this species (Huff et al., 1998). These results are also compatible with those AFLP findings reported for other highly outcrossing grass species such as bluebunch wheatgrass (Larson et al., 2000), bluegrasses (Larson et al., 2001b), smooth and meadow bromegrasses (Ferdinandez and Coulman, 2002), crested wheatgrass (Mellish et al., 2002) and blue grama (Fu et al., 2004).

Since a majority of the detected amplified polymorphic bands lacked clarity and robustness, only 158 of them were scored for further analyses. The frequencies of the scored bands in all six collection sites averaged 0.61 (0.03 to 1.00) for the tiller samples and 0.67 (0.03 to 0.98) for the seed samples. A larger proportion of the scored bands with frequencies of either <0.10 or >0.90 occurred in the seed, rather than tiller, samples (Fig. 1) . For each primer pair, statistics (mean, minimum, and maximum) of the band frequencies in all six collection sites are given in Table 2. Clearly, the lowest mean frequencies were found from the primer pair E + ACT/M + CGC. These results indicated the AFLP technique was effective in detection of molecular genetic variation in little bluestem.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Numbers of polymorphic amplified fragment length polymorphism (AFLP) bands with respect to their frequencies of occurrence in each of the six little bluestem populations, as reflected in two types of sampling (tiller and seed).

Partitioning of the total AFLP variation into within- and among-population components by AMOVA showed that 92.8% of the total variation was present within the natural populations and 7.2% was present among the six populations. The among-population variation, although small, was significantly different from zero (P = 0.0069) based on the permutation test. These results not only indicate the existence of differential genotypes in some natural populations, but also confirm the highly outcrossing nature of this grass species. The largest between-population difference measured by interpopulation distance was observed between the Brandon and Narcisse populations (0.229), followed by the three other significant population pairs of Glenboro vs. Narcisse (0.140), Brandon vs. Winnipeg (0.132), and Carberry vs. Narcisse (0.119) (Table 3). These differences can also be visualized in the inferred genetic relationships of the six populations given in a dendrogram (Fig. 2) . Clearly, the populations at Brandon, Glenboro, and Carberry were clustered together as a group, while the other populations (Narcisse, Winnipeg, and Saskatoon) formed another group from tiller sampling. However, these groupings were not significantly associated (r = 0.02; P = 0.5244) with geographical origin of the populations, when assessed by a Mantel test. This result indicates the genetic divergence among these original natural populations with respect to geographical distance is weak.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Unweighted Pair-Group Methods Using Arithmetic Averages clustering of the six little bluestem populations based on interpopulation distances obtained from AMVOA on the scored polymorphic amplified fragment length polymorphism bands, as reflected in two types of sampling [(A) tiller, and (B) seed].

The large variation residing within single natural populations implies that substantial genetic variation could be captured by sampling only a small number of tillers from single sites, but exact numbers of tillers to be sampled from one site remain to be determined empirically. The observation of small between-population variation implies a high genetic similarity between nearby native stands, and thus distant populations should be considered for sampling genetic diversity for little bluestem germplasm improvement.

AFLP Diversity in Seed Collections
The seed collection at Glenboro had the highest number (150) of polymorphic bands, and the Saskatoon collection had the lowest (92) (Table 1). The Glenboro collection also had the highest mean band frequency (0.70), and the Carberry collection had the lowest (0.58) (Table 1). When considering both the number of bands and their frequencies in each collection, the Carberry collection had the highest within-collection variation (54.9), followed by the collections at Brandon (54.4), Glenboro (42.3), Narcisse (37.2), Winnipeg (32.5), and Saskatoon (26.0) (Table 1). These results indicate seed collections from Carberry and Brandon had relatively more AFLP variation than the others.

On the basis of AMOVA, 91.5% of the total variation was present within the seed collections and 8.5% among the six collections. The among-collection variation was highly significantly different from zero (P < 0.0001), indicating differential genotypes existed among these seed collections. The largest between-collection difference was observed between the Brandon and Saskatoon collections (0.165), followed by the significant collection pairs of Carberry vs. Saskatoon (0.135) and Narcisse vs. Saskatoon (0.134) (Table 3). These differences were significantly correlated (r = 0.79; P = 0.0289) with geographical distances of the collection sites. The inferred genetic relationships of the six seed collections are displayed in Fig. 2. Clearly, the Saskatoon collection was distinguished from the other collections. Also, the Brandon and Carberry collections displayed no marked difference in variation, and thus grouped together. Another group consisted of the collections from Glenboro, Narcisse, and Winnipeg, although some levels of difference existed within the group.

The significant association of AFLP variation with geographical origin, observed in the seed but not the tiller samples, is not surprising, given the smaller within-collection AFLP variation and the larger differentiation among seed collections than those detected in the tiller samples. However, questions remain about the generality of this association in the seed samples, as this finding may reflect only those seed collections from the northern fringe of the species' range, not those in southern latitudes, and the seed variations in these natural populations for the year 2002 only, not other years. Thus, further assessment of this association is desirable. If the generality holds, caution is needed in sampling seeds and selecting distant seed sources for the development of genetically broad-based germplasm, particularly for seeding in nonlocal environments.

Comparisons of AFLP Variation between Tiller and Seed Samples
It is evident that the seed samples displayed less AFLP variation than the tiller samples. First, the seed samples had a smaller number of polymorphic bands (Table 1) and a larger proportion of the scored bands with occurrence frequencies of either <0.10 or >0.90 (Fig. 1) than the tiller samples. These AFLP differences between two types of sampling were significant, as revealed by the heterogeneity ² tests (Table 4). Second, when considering the variations in both the number of scored bands and their frequencies, the seed samples had smaller within-sample AFLP variation than the tiller samples (Table 1). Third, the interpopulation distances obtained from the tiller samples varied more widely and fewer were statistically significant from zero than those from the seed samples (Table 3). Significant difference in AFLP distance between two sampling types was observed for each collection site (Table 4). Fourth, significant associations of among-sample distance with geographical origin were found in the seed but not the tiller sampling. Larger differentiation among the collection sites was observed in the seed than the tiller samples (Fig. 2).

The findings of sampling differences have significant consequences for sampling little bluestem from natural populations for germplasm improvement and conservation. First, more genetic variability revealed by tiller sampling appears to support the practice of using tillers, rather than seeds, as the means of collecting little bluestem germplasm for cultivar development. However, using tillers will limit the number of genotypes sampled from a site, digging tillers may be disruptive for populations of small size, and maintaining tiller collections is more challenging than seed collections. Second, given sampled seeds may poorly reflect genetic diversity in natural populations, the need for establishment of some in situ little bluestem reserves is obvious (Brown, 1995). For long-term ex situ germplasm conservation programs, such as those in national seed gene banks, adjustment to some germplasm sampling procedures by increasing sample size and having multiple sampling per year and/or multiple-year seed collections may be warranted to minimize possible initial loss of variability. Third, these sampling differences may not be applicable to other native grass species with different phenology and mating systems, and thus further assessments on the generality of sampling difference in other native grass species are needed to increase the effectiveness of sampling native grass germplasm.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
This AFLP analysis revealed that >91% of total AFLP variation was present within single little bluestem populations or collections. The among-population (or collection) variation components, although relatively small (7–9%), were significantly different from zero. Seed sampling displayed fewer polymorphic bands, higher average band frequencies, and more scored bands with extremely high or low frequency than did tiller sampling. Significant association of AFLP variation with geographical origin was observed in the seed, but not the tiller samples. These findings are significant not only for the establishment of effective guidelines on sampling plant germplasm from natural populations, but also for the improvement of little bluestem germplasm for rangeland restoration and revegetation.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the financial support of Native Plant Solutions/Ducks Unlimited Canada and AAFC MII on this research. We thank Tamara Horechko, Launi Riediger, Kim Strut, and Darryl MacNeil for their help with plant collection. We also thank Yasas Ferdinandez, Lasantha Ubayasena, Angela Taylor, and Gregory W. Peterson for their technical assistance on the AFLP analysis, and Yasas Ferdinandez and Daniel Schoen for their helpful comments on the early version of the manuscript.

Received for publication January 27, 2004.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Related articles in Crop Science Similar articles in this journal Similar articles in Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (10) Citing Articles via Google Scholar Google Scholar Articles by Fu, Y.-B. Articles by Richards, K. W. Search for Related Content PubMed Articles by Fu, Y.-B. Articles by Richards, K. W. Agricola Articles by Fu, Y.-B. Articles by Richards, K. W. Related Collections Other Forage Crops Crop Genetics