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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Genetic Relationships among Smooth Bromegrass Cultivars of Different Ecotypes Detected by AFLP Markers

Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, Saskatchewan, Canada S7H 0X2

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Smooth bromegrass (Bromus inermis Leyss.), the most commonly cultivated perennial bromegrass species in North America, belong to three ecotypes; northern, intermediate, and southern. The objective of this study was to analyze the genetic relationships of cultivars belonging to these ecotypes by amplified fragment length polymorphic (AFLP) markers. Seven AFLP primer combinations produced 176 polymorphic markers, which occurred in different frequencies in different cultivars, with no ecotype or cultivar specific markers. Cluster analysis and principle component analysis grouped the cultivars according to their pedigrees rather than by ecotypes. The southern cultivars (Baylor, Lincoln, Beacon, and Blair), older cultivars developed in Iowa and Nebraska, grouped distantly to the rest of the cultivars. The recently developed southern type cultivars (Badger, Alpha, and Radisson) were grouped closely with the intermediate type cultivars (Magna and Signal). The northern type cultivars (Carlton, Jubilee, and S-7133) grouped with the recently developed southern and intermediate cultivars. The close association of the recently developed southern cultivars to the intermediate and the northern cultivars but distinct from the older southern cultivars may indicate introgression of northern ecotype germplasm into the former. Analysis of molecular variance (AMOVA) of the 14 cultivars indicated a higher within-population variation (85%) than the among-population variation (15%). Higher molecular variations observed in the recently developed southern and some northern cultivars reflect the diverse genetic backgrounds of the source populations. The older southern cultivars showed the lowest molecular variation due to the narrow genetic background of the source populations. This study shows that AFLP markers can be used to differentiate phenotypically similar smooth bromegrass cultivars according to their breeding history, which will aid in future smooth bromegrass breeding projects.

Abbreviations: AFLP, amplified fragment length polymorphism • AMOVA, analysis of molecular variance • NTSYS, numerical taxonomy and multivariate analysis system • UPGMA, unweighted paired group method of arithmetic averages

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
SMOOTH BROMEGRASS is the most commonly cultivated perennial bromegrass species in North America (Vogel et al., 1996). It is a drought tolerant, sod forming, cool season grass species grown mainly for hay production and land reclamation (Casler and Carlson, 1995). The cultivated smooth bromegrass species, which is an auto-allo-octoploid with 2n = 8x = 56 chromosomes (Armstrong, 1991), is native to Eastern Europe and temperate Asia (Vogel et al. 1996). It was introduced to North America in 1884 but did not receive widespread acceptance until the drought years of the 1930s (Casler et al., 2000).

The first detailed study on the ecotypes of smooth bromegrass conducted by Zerbina in the 1930s, and summarized by Knowles and White (1949), divided the species into two ecotypes on the basis of morphology and native adaptive regions. Two main ecological-geographical groups were identified: (i) the "meadow" group or northern ecotype, and (ii) the "steppe" group or southern ecotype. The southern ecotype had deeper roots and the canopy height of the vegetative tillers was one half of the reproductive tillers. For the northern ecotype, the vegetative tillers reached two-thirds of the height of the reproductive tillers. The southern ecotype had coarser leaves that were shorter, narrower, and more erect than the northern ecotype. Furthermore, stronger seedling pubescence was observed in southern ecotype cultivars (Knowles, 1980). In their native habitat, the northern ecotype was found in valleys and moist regions, whereas the southern ecotype occurred in the dry steppe areas. The term "intermediate type" has been used to describe some smooth bromegrass cultivars which were developed from germplasm of both southern and northern ecotypes. Some of these intermediate ecotype cultivars have performed better in forage and seed yield trials in Canada than the traditional southern type cultivars (Elliott and Bolton, 1970).

Since the release of the first smooth bromegrass cultivar in the 1940s, extensive cultivar development has occurred in Canada and the USA. Initially, cultivar development occurred within the two main ecotypes, resulting in cultivars that could be readily distinguished phenotypically into southern and northern ecotypes. However, there are several overlapping morphological characteristics of the two ecotypes that are influenced by environmental factors, which makes their differentiation difficult. More recently, integration of the two ecotypes has occurred in smooth bromegrass cultivar development to integrate desirable characteristics from both ecotypes into one cultivar. Thus, differentiation of cultivars by ecotype has become even more difficult. Differentiating populations or cultivars into their respective ecotypes are important in smooth bromegrass breeding projects to select diverse parental material to maximize heterozygosity in the progeny.

Since the release of the first registered cultivar, Lincoln, in 1942 the number of cultivars that has been released in North America is limited. A detailed description of registered grass cultivars in the USA by Alderson and Sharp (1995) lists 26 smooth bromegrass cultivars registered in USA and Canada. The overwhelming majority of these cultivars belong to the southern ecotype, whereas there are only five northern and three intermediate cultivars registered. The majority of the smooth bromegrass cultivars used in the USA belong to the southern ecotype (Vogel et al., 1996). In Canada cultivars of all three ecotypes are widely used.

There have been a limited number of studies on smooth bromegrass ecotype characterization and these have been largely based on morphological and agronomic differences (Knowles and White, 1949). Casler et al. (2000) conducted an extensive study on the agronomic and quality characteristics of commonly grown smooth bromegrass cultivars in North America that belong to the three ecotypes. The cluster analysis conducted in this study showed that grouping of some cultivars was along known pedigree information; however, most of the cultivars grouped together by phenotypic similarities, rather than similarity of genetic background.

Molecular markers can be used to overcome some of the limitations associated with the use of morphological characters in determining genetic relationships. There have been no studies conducted to study genetic relationships and variation of smooth bromegrass cultivars using molecular markers. The AFLP (Vos et al., 1995) marker system combines the restriction site recognition aspect of restriction fragment length polymorphism (RFLP) markers and the amplification aspect of the polymerase chain reaction (PCR) based markers. This combination provides a less labor intensive and more robust marker type than commonly used random amplified polymorphism DNA (RAPD) markers. Studies have shown that AFLP markers are an efficient marker system for detecting polymorphisms (Fuentes et al., 1999; Powell et al., 1996), as they show a higher multiplex ratio [number of bands (loci) that can be detected in each gel lane] than many other markers. As a result AFLP markers offer an opportunity to carry out a detailed assay of smooth bromegrass genotypes.

The objective of this study was to use AFLP markers to differentiate and determine the genetic relationships of smooth bromegrass cultivars of different ecotypes.

	MATERIALS AND METHODS

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Plant Material
Among the 26 registered smooth bromegrass cultivars (Alderson and Sharp, 1995) in North America, 18 are of the southern, four northern, and three intermediate ecotype. To characterize adequately the smooth bromegrass ecotypes, we have included cultivars representing each of the decades of bromegrass cultivar development, starting from the first registered cultivar Lincoln to the most recently released cultivar Alpha (1995). Furthermore, the selected cultivars were developed in Saskatoon, Wisconsin, and Iowa, major centers of smooth bromegrass cultivar development in North America. Eight cultivars representing the southern (Alpha, Badger, Baylor, Beacon, Blair, Lincoln, Radisson, and Rebound), four northern (Bravo, Carlton, Jubilee and S-7133), and two intermediate (Signal and Magna) smooth bromegrass cultivars were used in this study. The breeding histories of these cultivars can be found in Table 1.

AFLP Analysis
Seven EcoRI:MseI primer combinations were used in this study. The AFLP analysis was performed as described by Vos et al. (1995) with the AFLP Analysis System 1 provided by Life Technologies (Burlington, ON, Canada). Initially, a restriction digestion of 250 ng of genomic DNA with EcoRI and MseI restriction enzymes followed by ligation of adapters to the restriction fragments. This was followed by preamplification of the primary templates with AFLP primers with an additional single nucleotide at the 3' end. A selective amplification of the preamplified fragments with -³³P-labeled EcoRI primers having three selective nucleotides at the 3' end and MseI primers having three selective nucleotides at the 3' end was performed. The PCR profile was performed in a MJ Research PTC-200 DNA Engine thermocycler (Waltham, MA, USA) using the following amplification profile: 1 cycle with a denaturation step at 94°C for 30 s, an annealing step at 65°C for 30 s an extension step at 72°C for 60 s. The next 12 cycles emulated a Touchdown PCR format decreasing the annealing temperature by 0.7°C each cycle to 56°C. A further 23 cycles were performed using the profile: 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s. Finally the amplification products were separated on 5% (w/v) polyacrylamide gels for 2.30 h at 90 W. The gels were transferred to Whatman paper and dried on a gel dryer for 2 h at 80°C. Gels were exposed to Kodak BIOMAX film at –80°C for 1 to 7 d depending on the signal intensity.

Statistical Analysis
Polymorphic AFLP bands were transformed into a binary rectangular matrix. AFLP bands on the gel were scored as 1 (present) or 0 (absent) and missing data points were assigned a 9. The analysis of molecular variance (AMOVA) component in the ARLEQUIN 2.000 software (Schneider et al., 2000) was used to partition the total variance into within-cultivar and among-cultivar components and to calculate the interpopulation distances (phi-statistic = _st) as stated by Huff (1997). Using the interpopulation distance matrix, we constructed a dendrogram using NTSYS-PC 2.02C (Rohlf, 1997) with unweighted pair-group method of arithmetic averages (UPGMA). A total of 176 markers were used to construct the dendrogram. To determine whether this number of markers was sufficient, random subsets (88, 132, and 150 markers) were also used to construct dendrograms. There was consistency in the grouping of cultivars in all four dendrograms, suggesting that the genetic distances found in the present study are accurate estimates of true genetic distance.

Proportion of total molecular variation residing between populations (_st) was calculated by the analysis of molecular variance (AMOVA) (Excoffier et al., 1992). Significance of each _st value was tested as the probability that a random _st value was greater than the observed value by 1000 random permutations (Huff, 1997). The proportion of the total AFLP variation residing in each cultivar was calculated by dividing the mean square (from AMOVA sum of squares) of each cultivar by the total variation obtained from AMOVA.

A principle component analysis (PCA) was conducted with SAS PROC PRINCOMP (SAS Institute, 1996) using the binary matrix of all the individuals as the input matrix. The scatter plot was generated by the first two principal components.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
AFLP Polymorphism
A total of 15 AFLP primer combinations were screened for their ability to detect polymorphisms among the 14 cultivars. Following this initial primer screening, seven primer combinations that amplified profiles that contained clear and distinct polymorphic bands, and could be scored without any ambiguity, were used to genotype the entire population. The number of bands that were amplified by each primer combination varied from 97 to 184. These polymorphic bands ranged from 90 to 300 bp with majority of the bands within the 100- to 250-bp range. A key observation made in this study was that high percentages (90%) of the bands amplified by the AFLP analysis were polymorphic. However, only 176 polymorphic bands that could be scored without ambiguity were used in the analysis. The observed band frequencies ranged from 0.08 to 0.92 with an average of 0.52. Occurrence of a highly polymorphic band profile can be attributed to the fact that smooth bromegrass is a highly self-incompatible, cross-pollinating, polyploid grass species. Thus, individual plants are highly heterozygous, and as a result there were no two individuals with identical marker profiles. AFLP bands appeared in different frequencies among the cultivars. In terms of differentiating ecotypes or cultivars from each other, there were no ecotype or cultivar specific bands or band profiles generated by the AFLP analysis.

Molecular Variation of Cultivars
The analysis of molecular variance of the 14 cultivars indicated a higher within-population variation (85%) than the among-population variation (15%) estimate of the total variation (Table 2). This indicates a high level of heterozygosity and variation among the individual bromegrass plants. The high within-population variation estimation is indicative of out-crossing species as previously described for buffalograss [Buchloë dactyloides (Nutt.) Engelm.] (Huff et al. 1993); blue grama (Bouteloua gracilis [H.B.K.] Lag. ex. Steud.) (Phan, 2000); and crested wheatgrass (Agropyron spp.) (Mellish et al., 2002).

View this table: [in this window] [in a new window]   Table 3. Summary statistics from AMOVA of AFLP variation in 14 smooth bromegrass cultivars. The proportion of total AFLP variation residing within a cultivar is given at the diagonal. The proportion of total AFLP variation residing between cultivars (st) is given below the diagonal. Significant testing of each st value is given above diagonal as the probability obtained by 1000 random permutations that a random st value was greater than the observed value.

Genetic Relatedness of Cultivars
Previous studies used morphological characters to differentiate cultivars and assess their relationships in smooth bromegrass (Knowles and White, 1949). Recently, using morphological and agronomic characters, Casler et al. (2000) observed that North American cultivars separated along southern and northern ecotypes with most intermediate ecotype cultivars grouping with the northern ecotype cluster. It was also noted that cultivars of the same ecotype which were phenotypically indistinguishable, but had diverse pedigrees, were often closely associated in the cluster analysis. Thus, a major limitation in using morphological characteristic in relationship assessment studies is the inability to differentiate lines with diverse pedigrees having similar morphological characteristics.

The interpopulation distance (phi-statistic = _st) matrix was used to construct a dendrogram to determine the relationships among the 14 cultivars used in this experiment (Fig. 1). Alpha, Badger, and Radisson clustered closely together, with Magna and Signal. The three northern ecotype cultivars Jubilee, Carlton, and S7133 were more loosely grouped to the above six cultivars. The second main group consisted of the southern type cultivars Baylor, Lincoln, Beacon, and Blair.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Genetic relationship of smooth bromegrass cultivars developed by UPGMA cluster analysis using the phi-statistic (st) as the inter-population distance. S, southern type; N, northern type; I, intermediate type.

The intermediate ecotype cultivars Magna and Signal clustered closely to the more recently developed southern cultivars. Magna, the first reported intermediate ecotype of smooth bromegrass, was developed mainly from southern ecotype germplasm and other outcrossed sources. The parental material that was used to develop Signal and Radisson included individuals from Magna, explaining the close association of these three cultivars in this study. Casler et al. (2000) found that Signal and Magna were more closely grouped with northern ecotype cultivars and lines.

In the present study, the northern ecotype cultivars Jubilee, S-7133, and Carlton did not group closely to each other and were clustered to the intermediate and more recently developed southern ecotype cultivars. These three northern cultivars do not share any common ancestors and thus, they would not be expected to cluster closely. The cultivar Bravo is classified as a northern ecotype; however, the source population included individuals that belong to southern and northern ecotypes (Mederick, 1985). This may have resulted in Bravo being genetically closer to the intermediate and recently developed southern type ecotype cultivars. Thus, it may have been misclassified as a northern ecotype cultivar. The clustering of Rebound separately from the rest of the southern ecotype may be explained by its pedigree. It was selected from the cultivar Saratoga, a southern type cultivar which was itself selected from germplasm sources from breeders across the USA.

From the initial release of Lincoln in 1942, until the late 1970s, our analysis indicates that southern ecotype cultivars released during this period had little or no genome introgression with northern ecotypes (i.e., Lincoln, Blair, Beacon, and Baylor clustered together, separate from the northern ecotypes). This was not true for the recently developed southern ecotype cultivars (Badger, Alpha, and Radisson), which grouped closer to the intermediate and northern ecotype cultivars. This suggests some introgression of northern ecotype germplasm in more recently developed southern type smooth bromegrass cultivars. The source populations of the cultivars Badger and Alpha contained a diverse mix of individuals that belonged to several known cultivars and individuals of unknown origins (Table 1). It is possible that some of these individuals were from northern ecotype germplasms. Radisson clustered more closely to the intermediate ecotype cultivars than Badger and Alpha likely due to germplasm from Magna, an intermediate ecotype cultivar, used in its development.

To understand the relationships among the smooth bromegrass cultivars better, the data were further analyzed by PCA. The first two principal components accounted for 20% of the total variation (Fig. 2). The PCA analysis further supported the within-cultivar variation values observed by the AMOVA analysis. The majority of the genotypes of the cultivars Lincoln, Beacon, Baylor, and Blair were closely grouped with each other suggesting that these are genetically more similar than those of other cultivars. On the contrary, the genotypes of the newly developed southern cultivars, intermediate cultivars, and the northern cultivars were more scattered, indicating wider genetic differentiation.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Principle component analysis plot of 125 genotypes representing the 14 cultivars of southern, northern and intermediate ecotypes of smooth bromegrass. The first two principal components (PC1 and PC2) account for 20% of the variation observed.

	ACKNOWLEDGMENTS

 
The authors thank Drs. Yong-Bi Fu and Van Ripley of Agriculture and Agri-Food Canada, Saskatoon, for their critical review of the manuscript.

Received for publication November 29, 2002.

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