Published online 6 May 2005
Published in Crop Sci 45:1064-1068 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP BREEDING, GENETICS & CYTOLOGY
Detecting Genetic Changes over Two Generations of Seed Increase in an Awned Slender Wheatgrass Population Using AFLP Markers
Yasas S. N. Ferdinandez,
Bruce E. Coulman* and
Yong-Bi Fu
Agric. and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK, Canada S7N 0X2
* Corresponding author (coulmanb{at}agr.gc.ca)
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ABSTRACT
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Diverse native grass populations are being developed for revegetation and land reclamation purposes, but little is known about the maintenance of the genetic diversity of these developed populations during the process of seed increase. The objectives of this study were to assess the genetic shift over two generations of seed increase in a multisite composite population (AC Pintail) of the self-pollinating awned slender wheatgrass [Elymus trachycaulus subsp. subsecundus (Link) Gould] and to compare its genetic variation with the released cultivar AEC Hillcrest. AC Pintail was formed by bulking seed of 200 plants collected from 60 sites across the prairie of western Canada. The amplified fragment length polymorphism (AFLP) technique was applied to assay 50 plants from each of four populations (AC Pintail G0, G1, and G2 and AEC Hillcrest breeder seed). For each sample, seven AFLP primer pairs were applied and 194 polymorphic bands were scored. AC Pintail revealed more polymorphic bands (74%) than AEC Hillcrest (47%), and most of the scored bands for AEC Hillcrest had occurrence frequencies approaching 1 or 0. The largest within-population AFLP variation observed resided within AC Pintail G0 (30.3), followed by G1 (29.7), G2 (27.9), and AEC Hillcrest (10.4). Significant differences were found among these seed sources and >95% of the total AFLP variation resided within the three AC Pintail populations. Fifty-three bands displayed significant changes from G0 to G1 and 76 from G0 to G2 of AC Pintail. These results indicate that AC Pintail harbored more genetic variation than AEC Hillcrest but could lose up to 8% of the original diversity in the first two generations of seed increase.
Abbreviations: AFLP, amplified fragment length polymorphism • AMOVA, analysis of molecular variance
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INTRODUCTION
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BREEDING EFFORTS to develop diverse native grass populations with improved seed production have increased in the USA and Canada (Crowle, 1970; Smoliak and Johnson, 1980, 1983; May et al., 1997; Jones et al., 2002; Phan et al., 2003). 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). Such achievement is encouraging, but challenges remain in the efforts to improve seed production while simultaneously capturing genetic diversity for adaptation in different environments (Smith and Whalley, 2002). Difficulties exist in the development of an effective selection protocol to capture genetic diversity from source populations (Larson et al., 2000; Phan et al., 2003). Little attention has been paid to the effectiveness of maintaining the genetic diversity during seed increases (Fu et al., 2004).
Awned slender wheatgrass is a native allotetraploid (2n = 28) with two genomes, designated St and H, and a self-fertile bunch-type perennial widely distributed across North America (Asay and Jensen, 1996). This plant is adapted to areas receiving as little as 350 mm annual precipitation, making it well suited to the revegetation programs on the rangelands of the western USA and Canada. To enhance the utilization of this species in revegetation and land reclamation, a multisite composite awned slender wheatgrass population, AC Pintail, was developed in Saskatoon, SK. To approximate the natural genetic diversity of the species, seed was collected from 200 plants sampled from 60 locations across Manitoba, Saskatchewan, and Alberta, and bulked equally for each sampled plant. Thus, AC Pintail so developed should have higher genetic diversity than released cultivars such as AEC Hillcrest which was selected from a single plant (Darroch and Acharya, 1996).
Natural selection due to environmental factors and/or unintended selection caused by management factors during seed increase could generate quantitative and qualitative changes in the genetic diversity of a forage crop population. However, such changes may not be always detectable. Morphological assessments revealed significant changes in morphological traits of red clover (Trifolium pratense L.), a highly heterozygous cross-pollinating species, when seed multiplication was performed in environments that were different from the original environment in which the cultivars were developed (Taylor et al., 1979, 1990). These changes were not observed in meadow fescue (Festuca pratensis Huds.), timothy (Phleum pratense L.), perennial ryegrass (Lolium perenne L.) (Simon and Kastenbauer, 1979), and Italian ryegrass (L. multiflorum Lam.) (Rincker et al., 1982) when they were increased in non-original environments. Using molecular markers, genetic shifts in populations of self-pollinating red beet (Beta vulgaris L.) were detected using RAPD markers (Eagen and Goldman, 1996) and in wheat (Triticum aestivum L.) using RFLP markers (Enjalbert et al., 1999), but not in a multisite composite population of blue grama [Bouteloua gracilis (Willd. ex Kunth) Lag. ex Griffiths], a cross-pollinating grass species of western North America, using AFLP markers (Fu et al., 2004).
The objectives of this study were to compare the genetic diversity of the composite population AC Pintail and cultivar AEC Hillcrest and to assess the genetic shift over two generations of AC Pintail seed multiplication by means of the AFLP technique (Vos et al., 1995). No applications of AFLP markers to this grass species have been made, but its effectiveness in detecting genetic variation has been well demonstrated in our genetic diversity studies of meadow bromegrass (Bromus riparius Rehmann) (Ferdinandez and Coulman, 2002), crested wheatgrass (Agropyron spp. Gaertn.) (Mellish et al., 2002), and blue grama (Fu et al., 2004).
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MATERIALS AND METHODS
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Plant Material
AC Pintail awned slender wheatgrass generation-0 (G0) seed was formed by compositing 50 g of seed from each of 200 collected plants from the provinces of Manitoba, Saskatchewan, and Alberta in Canada. Generation-0 seed was planted at the research farm of the Saskatoon Research Center to produce generation-1 (G1) seed. Generation-1 seed was planted by a commercial seed grower at Birsay, SK, in the same ecozone as Saskatoon, to produce generation-2 (G2) seed. Generation-0, G1, and G2 correspond to breeder, foundation, and certified seed in the Canadian pedigreed seed production system. Breeder seed of AEC Hillcrest awned slender wheatgrass, which was developed from the progeny of a single plant (Darroch and Acharya, 1996), was provided by Jay Woosaree of the Alberta Environmental Center in Vegreville, AB.
One hundred seeds from each of the four seed sources were germinated on filter paper moistened with a solution of 0.5% KCl. The seeds were incubated at room temperature until germination. Germinated seeds were transplanted into root trainers and grown in the greenhouse. Young leaves from each of the four seed sources were harvested from 50 individual plants, stored at –80°C for two days, and freeze-dried before DNA extraction.
DNA Extraction and AFLP Analysis
Genomic DNA was extracted from 200 individual leaf samples using DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON) according to the manufacturer's directions. Extracted DNA was quantified by fluorimetry using Hoechst 33258 stain (Sigma Chemical Co., St. Louis, MO), followed by dilution to 25 ng µL–1 for AFLP analysis. AFLP analysis was performed using the AFLP Analysis System 1 (Life Technologies, Burlington, ON) 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) pre-amplification of the primary templates with AFLP primers with an additional single nucleotide at the 3' end; (iii) selective amplification of the pre-amplified 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 3MM paper (Whatman International Ltd., Maidstone, UK), dried on a gel dryer for 2 h at 80°C, and exposed to Kodak BIOMAX film (Eastman Kodak Co., Rochester, NY) at –80°C for 1 to 7 d depending on the signal intensity. The sizes of amplification products were determined by comparison with a 30 to 330 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. 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 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 five samples, and the seven most informative ones (Table 1) were selected for this AFLP analysis.
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Table 1. Patterns of amplified fragment length polymorphism (AFLP) variation in AC Pintail G1 and ‘AEC Hillcrest’ awned slender wheatgrass with respect to AFLP primer pair.
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Statistical Analysis
For each gel generated from each primer pair, the numbers of observable and polymorphic 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 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.
Analysis of molecular variance (AMOVA) (Excoffier et al., 1992) was performed using Arlequin version 2.001 (Schneider et al., 2002) to assess AFLP variation within and among populations. This analysis not only allows the partition of the total AFLP variation into within- and among-population variation components, but it also provides a measure of genetic distance to assess population differentiation as the proportion of the total AFLP variation residing between any two populations (
st) (Excoffier et al., 1992). Significance of the resulting variance components and genetic distances from zero was tested with 5040 random permutations (Huff, 1997).
To assess further the significance of genetic shift in AC Pintail over the two generations of seed multiplication, the numbers of polymorphic bands with lower or higher frequencies of occurrence in G1 and G2 of AC Pintail than those in G0 were calculated. For each polymorphic band, a 
2 test of significance was made for the difference in band frequency between G0 and G1 and between G0 and G2 of AC Pintail. A heterogeneity 2 test was applied to all the polymorphic bands to assess the homogeneity of band changes over the seed increases. To assess the genetic relationships of all the assayed plants of AC Pintail, a principal component analysis was also conducted using SAS PROC PRINCOMP (SAS Institute, 1996), treating AFLP data as exploratory variables and plotting the first two resulting principal components.
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RESULTS AND DISCUSSION
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AFLP Variation
Seven AFLP primer pairs generated a total of 833 observable bands, and the number of bands amplified per primer combination ranged from 76 to 176 with an average of 119 (Table 1). Such high number of bands revealed by each primer combination is consistent with those reported for other grass species (Ferdinandez and Coulman, 2002; Mellish et al., 2002). On average, 71% of the bands amplified in AC Pintail populations were polymorphic, while only 48% were polymorphic in AEC Hillcrest. Since many of the amplified polymorphic bands lacked clarity and robustness, only 194 of them were scored for further analyses. Assessment on the polymorphism of the scored bands revealed more difference between AC Pintail G1 (98%) and AEC Hillcrest (44%) than those reflected in the observable bands (Table 1). Such difference is expected, as AC Pintail was formed by bulking seed of 200 plants, most, if not all, being distinct genotypes, and AEC Hillcrest was selected from a single plant. However, such high levels of polymorphism are not expected for a principally self-pollinating species. For example, self-pollinating wheat (Barrett et al., 1998) and tef [Eragrostis tef (Zucc.) Trotter] (Bai et al., 1999) showed only 12% and 18% of polymorphic bands, respectively, while cross-pollinating ryegrass (Lolium spp.) (Roldán-Ruiz et al., 2001) and Texas bluegrass (Poa arachnifera Torr.) (Renganayaki et al., 2001) showed 83% and 64% of polymorphic bands, respectively. Our results reflect the bulking of seed of different genotypes and may be partially the consequence of higher than expected outcrossing in this grass species, but this latter explanation would have to be further assessed.
Further assessment on the frequencies of the scored bands revealed that a majority of the bands for AEC Hillcrest had frequencies approaching 1.0 or 0.0 (Fig. 1A). In the three populations of AC Pintail, the majority of the AFLP band frequencies were less than 0.3 or greater than 0.8 (Fig. 1B). Thus, AC Pintail differed from AEC Hillcrest not only in the number of polymorphic bands but also in the occurrence frequency of the scored bands. These results clearly demonstrated the AFLP technique was effective in detection of molecular variation in awned slender wheatgrass.
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Fig. 1. Number of amplified fragment length polymorphism (AFLP) bands with respect to their frequencies of occurrence in each seed source of awned slender wheatgrass. (A) ‘AEC Hillcrest’ and (B) G0, G1, and G2 populations of AC Pintail.
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AFLP Variation among Populations
Based on the AMOVA analysis, AFLP variation detected among four populations was significantly different (P < 0.0001). AEC Hillcrest displayed highly significant differences from the three populations of AC Pintail. Excluding AEC Hillcrest, AFLP variation residing among the three AC Pintail populations explained <5% of the total variation, but it was statistically significant (P < 0.001). AFLP variation residing within each seed population varied from 10.4 for AEC Hillcrest to 30.3 for G0 of AC Pintail (Table 2). Generation-1 and G2 of AC Pintail showed a significant (P < 0.002) reduction in marker variation from G0 (29.7 and 27.9, respectively; Table 2). However, AC Pintail G2 still harbored 92% (= 27.9/30.3 in percentage) of the original AFLP variation observed in G0.
Based on their breeding histories as discussed above, it was not surprising to observe the differences between the AC Pintail and AEC Hillcrest populations. However, the reasons for the reduced diversity estimates of G1 and G2 compared to G0 are largely unknown. Such a reduction may largely reflect the effects of differential seed yields of the original component plants of AC Pintail. Other possible factors include variable seed germination and differential adaptation of the individual plants to the regeneration environments and random genetic drift and nonrepresentative sampling of seed to plant successive generations. Also unknown is the generality of such diversity reduction in different seed increase environments.
Capturing and maintaining high genetic diversity in native grass populations is important for their successful introduction to many diverse environments. This study has shown that the balanced multisite composite method used in the development of AC Pintail has generated a population with considerably higher genetic diversity than the cultivar AEC Hillcrest. Even with the diversity reduction observed in the successive seed-increase generations, the G2 population is still highly diverse. Thus, such awned slender wheatgrass composite populations should still maintain substantial genetic diversity for revegetation and land reclamation purposes.
Genetic Changes over Seed Increase
In AC Pintail, a number of AFLP bands displayed significant changes in band frequency in successive generations of seed increase from G0 (Table 3). The number of bands where the frequency was significantly reduced were similar from G0 to G1 (29) and G0 to G2 (28). A majority of the bands of reduced frequency from G0 to G1 displayed further reduction to G2. The number of bands where the frequency significantly increased was lower from G0 to G1 (24) than G0 to G2 (48). The heterogeneity 2 tests performed on all the markers between G0 and G1 and between G0 and G2 were highly significant (P < 0.001). This indicates a qualitative genetic shift occurred over the two cycles of seed increase from G0; that is, a large proportion of AFLP markers changed frequency. Further assessment on the bands with changed frequencies revealed more bands with frequencies > 0.8 in G2 than G0 or G1, suggesting a change in the direction of increased band frequency over the generations of seed increase. However, only one AFLP band that was present in G0 and G1 was lost in G2.
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Table 3. Comparison of band frequency changes over the two generations of seed increase in AC Pintail awned slender wheatgrass.
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To better understand the genetic change among the individuals of the three populations of AC Pintail, the data were further analyzed using principal component analysis (Fig. 2). Although individuals of all three generations were widely scattered, the degree of scatter was less for G2 than for G0, as measured by its variance on the figure. This supports the within-population variation estimates obtained by the AMOVA analysis (Table 2), where the within-source variation for G0 and G1 was somewhat higher than for G2. There was no distinct grouping of any of the three populations, further confirming that the population differentiation was mainly caused by the changes in band frequency, not the loss of polymorphic bands, and thus was relatively weak.
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Fig. 2. Plot of the individual plants of the G0, G1, and G2 populations of AC Pintail awned slender wheatgrass based on the first two principal components obtained from the analysis of amplified fragment length polymorphism (AFLP) data.
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Also using AFLP markers, Fu et al. (2004) found no genetic shift over two seed multiplications in a balanced multisite composite of the cross-pollinating blue grama. AC Pintail, like the blue grama, is a balanced multisite composite, but its self-pollinating habit would have made a difference in changing marker frequencies toward 0 or 1 in individual composite lines. When individual lines vary in seed yield, the overall marker frequencies of the composite population would change. Successive generations of seed-increase in some cross-pollinating grass species have shown some morphological differences compared to the original seed source, but the differences were generally too small to alter the productivity of the cultivars (Simon and Kastenbauer, 1979; Rincker et al., 1982). However, red clover yield reductions were found to be associated with genetic shift in successive generations of seed increase in an environment other than that in which the original population was developed (Taylor et al., 1979, 1990). It is not known whether such association would be found if AC Pintail was multiplied in different environments.
The finding of genetic shift has several implications for developing and utilizing diverse grass germplasm. First, the detected genetic shift consisted mainly of changes in band frequency, not the loss of AFLP bands; that is, only one was lost in the seed increases of AC Pintail. Thus, it is important to assess the nature of genetic shift in seed increase. Second, the genetic shift may have been caused by the differential seed production in individual lines, implying the genetic contribution of each component may not be equal. Thus, emphasis should be given to the selection of each component on equivalent seed-yielding ability, and assessments of seed-yielding ability of individual lines are needed before compositing. Third, if such a shift continues in further generations of seed increase and/or if the shift is detected in multiple environments of seed increase, larger reductions in diversity are possible. Thus, the need is obvious for a comprehensive assessment of genetic shift in seed increase of diverse grass germplasm. As demonstrated here, applications of molecular markers should facilitate the assessments of genetic changes in diverse grass populations.
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CONCLUSIONS
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The AFLP analysis presented here shows that a multisite composite of the awned slender wheatgrass, AC Pintail, harbors more genetic variation than the released cultivar AEC Hillcrest and that genetic shift has occurred in the seed increases of the composite population. Although the true causes for such genetic shift remain largely unknown, these findings not only demonstrate the effectiveness of applying molecular techniques to assess the genetic changes in an improved population but also to facilitate the development of diverse native grass populations. Despite this genetic shift, G2 of AC Pintail still harbors more than 92% of AFLP variation of its original composite and much more variation than AEC Hillcrest. Thus, AC Pintail is useful for revegetation and reclamation purposes.
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ACKNOWLEDGMENTS
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We thank Drs. Van Ripley and Kevin Falk for their helpful comments on the early version of the manuscript.
Received for publication May 30, 2004.
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ABSTRACT
INTRODUCTION
