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Published in Crop Sci. 44:283-288 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

PLANT GENETIC RESOURCES

AFLP Variation in Four Blue Grama Seed Sources

Yong-Bi Fu*,a, Yasas S. N. Ferdinandeza, Anh T. Phanb, Bruce Coulmana and Ken W. Richardsa

a Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada
b Native Plant Solutions, Ducks Unlimited Canada, 1255 Clarence Avenue, Winnipeg, MB, R3T 1T4, Canada

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Blue grama [Bouteloua gracilis (Willd. ex Kunth) Lag. ex Griffiths] is one of the most widespread native grasses in western North America. Several blue grama seed sources are currently used for rangeland seeding, but little is known about the genetic diversity of these seed sources. Amplified fragment length polymorphism (AFLP) technique was applied to compare the genetic diversity among four blue grama seed sources (a precultivar germplasm [balanced multisite composite, BMSC], the ecotype Bad River, a Minnesota ecotype, and a native Manitoba seed collection) and to assess the genetic shift during two generations of BMSC seed multiplication. Germplasm BMSC was a balanced multisite composite of 99 clones selected from 495 live plants collected from 11 sites across Manitoba. Six AFLP primer pairs were employed to screen a total of 176 individual plants sampled from both the first three generations of BMSC and the other three seed sources and 167 polymorphic AFLP bands were scored for each plant. Large AFLP variation was observed within the four seed sources. Greater AFLP variation was detected in the BMSC than Bad River, Minnesota ecotype, and the Manitoba native harvest. No genetic shift in the BMSC was found across the two seed multiplications. These results indicate a balanced composite of multisite blue grama germplasm can maintain high genetic diversity with little genetic shift in a few generations of seed multiplication.

Abbreviations: AFLP, amplified fragment length polymorphism • AMOVA, analysis of molecular variance • BMSC, balanced multisite composite


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
BLUE GRAMA is a native warm-season, highly outcrossing tetraploid perennial grass (Riegel, 1941; Miller, 1967; McGinnies et al., 1988) that inhabits a wide ecological range in North America where it is used for reclamation and groundcover (Hitchcock, 1950). These adaptive features have stimulated interest in cultivar development of this species (May et al., 1997; Smith and Phan, 1999). Since 1992, breeding efforts in blue grama have been made in Canada and shown the potential of generating an additional source of abundant, high-quality seed specifically adapted to western Canada (Phan, 2000; Phan and Smith, 2000). However, challenges remain in improving seed production while simultaneously maintaining genetic diversity for adaptation in nonlocal environments (Smith and Whalley, 2002).

Seeds harvested directly from remnant blue grama plants (i.e., native harvest) still represent an important seed source for rangeland seeding in western North America, but such seed supply is sporadic and prohibitively expensive for large-scale plantings. Releases of a commercial Minnesota ecotype in 1995 and the USDA ecotype Bad River originating from South Dakota in 1996 have improved the seed supply of blue grama. As these ecotypes represented germplasm selected from single native sites, however, concern has been raised that these plant materials may lack the genetic diversity to maintain adaptation in dynamic, nonlocal environments (Knapp and Rice, 1996; Roundy, 1999; Larson et al., 2000). To address this concern, a BMSC was developed in 2000 from 495 live plants collected from 11 sites across Manitoba (Phan, 2000). While the BMSC is expected to maintain higher genetic diversity than the other seed sources, no comparison of genetic diversity has been made among them. Even though the BMSC had been shown to capture most of the RAPD variations present in its source populations (Phan et al., 2003), it still remains unknown if genetic shift has occurred during successive generations of the BMSC seed multiplication.

The objective of this study was to compare the genetic diversity among four blue grama seed sources (BMSC, Bad River, the Minnesota ecotype, and a Manitoba harvest) and to assess the genetic shift across two generations of the BMSC seed multiplication with AFLP markers. The AFLP method (Vos et al., 1995) is a robust, highly informative DNA fingerprinting technique and has been applied to detect genetic variations in bluebunch wheatgrass [Pseudoroegneria spitcata (Pursh) A. Love] (Larson et al., 2000); bluegrasses (Poa spp.) (Larson et al., 2001); smooth bromegrass (Bromus inermis Leyss.) and meadow bromegrass (Bromus riparius Rehmann) (Ferdinandez and Coulman, 2002); and crested wheatgrass (Agropyron spp. Gaertn) (Mellish et al., 2002).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Plant Materials
Plant materials used in this study consisted of four blue grama seed sources. The Manitoba harvest was a native harvest at a remnant site near Brandon, MB, Canada, which was conducted in 2001 by staff of Native Plant Solutions of Ducks Unlimited Canada, Winnipeg, MB. The Minnesota ecotype was a commercial increase of native blue grama plants collected by Mr. Oscar Carlson from Lake Bronson, MN, and made commercially available in 1995. Bad River was an ecotype developed from a single seed collection of blue grama along the Bad River in Haakan County, SD, and released in 1996 by the USDA Natural Resource Conservation Service and Plant Materials Center at Bismark, ND. Germplasm BMSC was a balanced multisite composite of 99 plant selections made from an original base population consisting of 495 live plants collected in 1993 from 11 sites across Manitoba (Phan, 2000). Open-pollination in isolation among the 99 selections produced the prebreeder seed generation (designated here as G0), and this germplasm has been multiplied without selection to two more generations (G1 and G2) according to the Canadian pedigreed seed production regulations (Canadian Seed Growers' Association, 1994). With one more multiplication, the BMSC will be commercially released by Native Plant Solutions of Ducks Unlimited Canada.

Seed samples for G0, G1, and G2 of BMSC and the other three seed sources were obtained from Native Plant Solutions of Ducks Unlimited Canada. Fifty seeds were randomly selected from each seed sample and germinated on filter papers in Petri dishes moistened with 0.5% KCl solution under a 16-h/8-h day/night photoperiod at 25°C in a growth chamber. Seedlings were transplanted into individual pots and grown in the greenhouse at the Saskatoon Research Centre of Agriculture and Agri-Food Canada. Thirty individual seedlings were randomly selected for each seed sample, except for the Manitoba harvest sample with only 26 individual seedlings available. Young leaf tissue was harvested separately from each seedling into small envelopes, freeze dried, and stored at –20°C.

DNA Extraction and AFLP Analysis
Genomic DNA was extracted from 176 individual leaf samples with DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON, Canada) according to the manufacture's directions. Extracted DNA was quantified by fluorimetry with Hoechst 33258 stain (Sigma Chemical Co., St. Louis, MO, USA), followed by dilution to 25 ng µL–1 for AFLP analysis. The AFLP analysis was performed with 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 [{gamma}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 3MM 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, USA). The polymerase chain reaction profile for step iii was performed in a PTC-200 DNA Engine thermocycler (MJ Research, Watertown, MA, USA) with 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. The 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 with the profile 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s. Six informative EcoRI:MseI primer pairs (Table 1) were applied.


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  		Table 1. Amplified fragment length polymorphism (AFLP) variation patterns in blue grama for each primer pair.

 
Data Analysis
For each gel generated from each primer pair, the numbers of observable and monomorphic AFLP bands were counted. Selective polymorphic AFLP bands with clarity for all the samples were manually scored as 1 (present) or 0 (absent). The scored bands were first analyzed for AFLP polymorphism by counting the number of polymorphic bands and calculating their frequencies with respect to primer pair, sample, and seed source. To compare the AFLP polymorphism among different seed sources, the numbers of polymorphic bands were plotted against their frequencies of occurrence in each seed source.

Average AFLP similarity was calculated with the Dice coefficient (Dice, 1945) across all pairwise samples within each seed source. Average AFLP similarity across all paired samples between various seed sources was also estimated and tested for its significance with an approximate t test. Bias-corrected variance of similarity was estimated with the method of Leonard et al. (1999). An approximate F test was performed to test heterogeneity of average AFLP similarity among different seed sources. This was done specifically with a SAS program (SAS Institute, 1996) written in SAS IML. To confirm both t test and F test with AFLP similarity, analysis of molecular variance (AMOVA; Excoffier et al., 1992) on AFLP variation among different seed sources was also performed. The AFLP variation residing within and between seed sources (Huff, 1997) were generated and tested with 5040 random permutations.

To assess further the significance of genetic shift in BMSC among the two generations of seed multiplication, the numbers of polymorphic bands with lower or higher frequencies of occurrence in G1 and G2 of BMSC than those in G0 of BMSC were calculated. For each polymorphic band, {chi}2 test of significance was made for the difference in band frequency between G0 and G1 of BMSC and between G0 and G2 of BMSC. Heterogeneity {chi}2 test was applied to all the polymorphic bands. To assess the genetic relationships of the plants of BMSC and other seed sources, the similarity matrix obtained for 83 individual plants of BMSC and average similarity estimates among the four seed sources were separately analyzed with NTSYS-PC 2.01 (numerical taxonomy and multivariate analysis system; Rohlf, 1997) and clustered with the algorithm of unweighted pair-group methods by arithmetic averages. To help assess the clustering of the BMSC plants further, a principal component analysis was also conducted with SAS PROC PRINCOMP (SAS Institute, 1996) treating AFLP data as exploratory variables. Plots of the first three resulting principal components were made to assess the associations of three BMSC samples.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSION
 REFERENCES
 
AFLP Variation
A total of 763 AFLP bands were amplified from six AFLP primer pairs (Table 1). The number of observable bands per primer pair ranged from 107 to 138 with an average of 127.1. Fifty-two of the observable bands were monomorphic and the others polymorphic, indicating the presence of large AFLP variation in this grass species. This result is consistent with the RAPD variations previously reported in blue grama (Phan et al., 2003) and also compatible with those AFLP findings reported for bluebunch wheatgrass (Larson et al., 2000), bluegrasses (Larson et al., 2001), smooth bromegrass and meadow bromegrass (Ferdinandez and Coulman, 2002), and crested wheatgrass (Mellish et al., 2002). No AFLP band was found to be specific to any seed source. As a majority of the amplified polymorphic bands detected lacked clarity and robustness, only 167 of them were scored and used in this analysis. Their band frequencies in all the assayed plants ranged from 0.012 to 0.951 with an average of 0.40 (Table 1). The scored bands from the primer pair E+ACG/M+CAT had the lowest average band frequencies. These results indicate the AFLP technique was effective in detection of molecular genetic variation in blue grama.

AFLP Diversity among Seed Sources
Assessment of the 167 scored bands among the seed sources revealed BMSC had the highest number of polymorphic bands. The number of polymorphic bands for G0 of BMSC was 165, G1 of BMSC was 163, G2 of BMSC was 162, Bad River was 158, Minnesota ecotype was 153, and Manitoba harvest was 141. The number of AFLP bands with respect to its frequency of occurrence in each seed source is presented in Fig. 1. A large proportion of the polymorphic bands were infrequently (f ≤ 0.3) detected in each seed source (Fig. 1), which is expected from Table 1. Comparison of the number of AFLP bands for the three generations of BMSC (Fig. 1A) showed there were more bands with low and high frequencies (f < 0.3 and f > 0.8), and fewer bands with frequencies of 0.6 to 0.8, in G1 or G2 of BMSC. This result implies some genetic change might have occurred in the BMSC seed multiplications. Comparison of the number of AFLP bands for different seed sources (Fig. 1B) did not reveal any clear patterns for each seed source, except the observation that the Manitoba harvest had an extremely large number (54) of polymorphic bands with occurrence frequency ≤ 0.1.



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  		Fig. 1. Numbers of amplified fragment length polymorphism (AFLP) bands with respect to their frequencies of occurrence in each seed source (A) for the three generations (G0, G1, G2) of the balanced mulitsite composite (BMSC) and (B) for G0 of the BMSC, Bad River (BR), Minnesota ecotype (ME), and Manitoba harvest (MH).

 
For AFLP similarity, G0 of BMSC had the lowest average similarity (0.5573 ± 0.0085), followed by G1 of BMSC (0.5583 ± 0.0092), G2 of BMSC (0.5620 ± 0.0105), Bad River (0.5815 ± 0.0060), Minnesota ecotype (0.5892 ± 0.0069), and Manitoba harvest (0.6308 ± 0.0169), indicating BMSC had the largest AFLP variation (Table 2). The lowest AFLP variation detected in the Manitoba harvest entry was expected, as the harvest was from one location, whereas plants which made up BMSC were harvested from 11 sites. The approximate F test revealed a highly significant (P < 0.005) difference among these average similarities, confirming BMSC had the higher genetic diversity than the other seed sources. However, t tests of similarity differences among the three generations of BMSC were not significant (Table 2), indicating no detectable genetic shift in the BMSC seed multiplications.


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  		Table 2. Average amplified fragment length polymorphism similarity within (at diagonal) and among (above diagonal) blue grama seed sources and the approximate t test of significance for the between-source difference (below diagonal).

 
On the basis of AMOVA analysis, the within-source AFLP variation ranged from 23.09 (Manitoba harvest) to 28.79 (G0 of BMSC) (Table 3), indicating G0 of BMSC had the largest AFLP variation. This is consistent with those similarity findings described above. However, the large between-sources AFLP variations were observed between BMSC and Manitoba harvest (Table 3). This unexpected result may reflect the insufficient coverage of Manitoba blue grama diversity in the germplasm sampling and/or the selection bias in the development of BMSC. The random permutation tests revealed nonsignificant differences among the three generations of BMSC, but highly significant (P < 0.0001) differences between BMSC and the other three seed sources.


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  		Table 3. Summary statistics from analysis of molecular variance of amplified fragment length polymorphism (AFLP) variations in blue grama seed sources. The within-source AFLP variation is given at the diagonal. The proportion of the AFLP variation residing between two seed sources ({Phi}st) is given above diagonal. Significance testing of each {Phi}st value is given below the diagonal as the probability obtained by 5040 random permutations that a random {Phi}st value was greater than the observed value.

 
The estimated genetic relationships among the seed sources were given in a dendrogram based on their average AFLP similarities (Fig. 2). Clearly, BMSC was not close to the three other seed sources. This result was consistent with the variation patterns revealed from the similarity and AMOVA analyses described above.



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  		Fig. 2. Genetic relationships of four blue grama seed sources reflected in a dendrogram based on their amplified fragment length polymorphism similarities. MN.Ecotype stands for Minnesota ecotype and MB.Harvest for Manitoba harvest.

 
Given BMSC was a balanced blue grama composite of multisite germplasm and the other seed sources represented the germplasm selection only from single sites, it was reasonable to expect higher genetic variation in BMSC than the other seed sources. The results presented here provide the first empirical confirmation of this expectation. Also, it is generally expected that more genetic diversity in a seed source would have the higher potential of being adapted to various nonlocal environments. To confirm this expectation, however, requires further adaptation assessments on these blue grama seed sources in different environments.

Genetic Changes across Seed Multiplications
Assessments of the changes in band frequency among the three BMSC generations revealed some level of genetic change, but overall no genetic shift, in the BMSC seed multiplications (Table 4). There were 14 polymorphic bands displaying significant changes from G0 to G1 of BMSC and 15 from G0 to G2 of BMSC, revealed from the {chi}2 tests for individual bands. However, these tests only considered either frequency increase or decrease. Heterogeneity {chi}2 tests took into account both types of genetic change and revealed nonsignificant (P > 0.05) changes in frequency of all the polymorphic bands between G0 and G1 of BMSC and between G0 and G2 of the BMSC. Although the average band frequencies for the three BMSC samples were roughly equal (0.381 to 0.390), there were more polymorphic bands with reduced than increased frequencies in G1 and G2 of the BMSC. For example, the frequency-reduced polymorphic bands were reduced in G2 of the BMSC from 95 to 87, while the frequency-increased bands increased from 75 to 79.


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  		Table 4. Comparison of band changes in the three generations (G0, G1, G2) of balanced multisite composite blue grama.

 
Assessment of the genetic relationships of the 83 individual plants of the BMSC reflected in a dendrogram based on their AFLP similarities (Fig. 3) showed there was no separate grouping for the three BMSC samples and most of these plants were mix-clustered into various subgroups. This result was consistent with the close genetic relationships detected among the individuals of the BMSC based on the average AFLP similarities (Fig. 2). These results also confirm the finding of a nonsignificant overall genetic shift described above.



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  		Fig. 3. Genetic relationships of the 83 individual plants assayed for the three generations (G0, G1, G2) of the balanced mutlisite composite reflected in a dendrogram based on their amplified fragment length polymorphism similarities. An individual plant of a generation was labeled with a dot and a number after its generation designation.

 
Three principal components were obtained and accounted for only 10.3% of the total AFLP variation (3.74, 3.29, and 3.23%, respectively). Although these components explained <11% of the total variation, the results of the principal component analysis were consistent with those obtained from the clustering analyses above. No distinct groupings for the three BMSC generations were observed in the biplots of the three principal components, although five individual plants appeared to display some degree of genetic distinctness (results not shown).

The finding of little genetic shift in the BMSC indicates natural selection was generally weak in the first few generations of seed multiplication. Genetic drift may mostly explain the genetic changes detected in some polymorphic bands. Founder effect, although cannot be completely excluded for the detected genetic changes, is expected to be relatively small given the large initial population size (99 out of 495 live plants) used in the development of the BMSC. Thus, it is important to have a large initial population size in the germplasm development. However, the finding for the BMSC may not be directly extrapolated to the other blue grama seed sources, as the initial population sizes used to establish them may differ. Further studies are needed to assess the genetic shift in seed multiplications of selected germplasm, particularly of small initial population sizes.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
REFERENCES
 
This AFLP assessment provided the first empirical confirmation that a balanced composite of multisite blue grama germplasm can maintain high genetic diversity with little genetic shift in the first few generations of seed multiplication. This confirmation should help to address the concern regarding the suitability of utilizing various seed sources in rangeland seeding (Roundy, 1999), to resolve the issue of maintaining captured genetic diversity in seed production (Larson et al., 2000), and to facilitate the registration of selected germplasm (Lombard et al., 2000). Further diversity assessments of selected blue grama germplasm will enhance the cultivar development and utilization of blue grama and other outcrossing grass species (Vogel and Pedersen, 1993; Smith and Whalley, 2002).


   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 Gregory W. Peterson and Steve Whitwill for their technical assistance on the AFLP analysis and Grant Mcleod and Ray Smith for their helpful comments on the early version of the manuscript.

Received for publication January 21, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 


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